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chronic rhinosinusitis is a common disease , causing high social and economic costs , and heavily impacting on the patient s quality of life . imaging of chronic rhinosinusitis is often regarded as a difficult task , particularly by young residents , probably because of the complexity of the anatomy that makes the search for the key to unravelling such a labyrinth of air cells ( and its pathological features ) a tough task . however , first impressions often lie : the anatomy can be quite easily understood if described from the perspective of function ; likewise , imaging findings in chronic rhinosinusitis can be quite clearly interpreted by radiologists when they have sufficient background on the functional anatomy . the purpose of this review is to offer a handful of basic concepts that may guide the inexperienced radiologist towards an easier approach to imaging of the paranasal sinuses in his or her daily routine . although the paranasal sinuses are a complex framework of air cavities , widely variable in number and size , their anatomy may be simplified using a schematic approach based on drainage pathways . in this perspective , two functionally distinct compartments can be identified : first , the anterior compartment including the anterior ethmoid cells , the frontal and the maxillary sinus , draining into the middle meatus . second , the posterior compartment which is composed of the posterior ethmoid cells and the sphenoid sinus , draining into the superior meatus . the line of demarcation between the two compartments is the basal lamella of the middle turbinate , i.e. the attachment of the turbinate to the lateral nasal wall or medial orbital wall . although the basal lamella can be seen on axial and coronal images , sagittal reconstructions , easily available on multislice computed tomography ( msct ) examinations , are best suited to precisely delineate its obliquely oriented course ( fig . 1 ) . arrows outline the course of the basal lamella of the middle turbinate , which marks the border between anterior and posterior ethmoid air cells ( a agger nasi cell ) sagittal mpr reconstruction . arrows outline the course of the basal lamella of the middle turbinate , which marks the border between anterior and posterior ethmoid air cells ( a agger nasi cell ) for each of the aforementioned compartments some key anatomical landmarks can be defined . in the anterior one , all drainage pathways of the anterior ethmoid cells , and the frontal and maxillary sinus converge in an anatomical area located in the middle meatus ; this is referred to as the ostiomeatal complex ( fig . 2 ) . the ostiomeatal complex is composed of several structures : the uncinate process , a sickle - shaped bone structure projecting from the medial maxillary sinus wall up to the skull base ; the ethmoid bulla , generally the most prominent ethmoid air cell located above the uncinate process ; the ethmoid infundibulum , the thin channel in between the uncinate process and the ethmoid bulla ; the frontal recess , which is the obliquely orientated , funnel - shaped drainage pathway of the frontal sinus . the anatomy of the frontal recess is closely related to that of the uncinate process : the medial and lateral borders of the frontal recess depend upon the superior attachment of the uncinate process . as described by landsberg and friedman and lee et al . , a cranial insertion of the uncinate process on the ethmoid fovea ( the roof of the ethmoid ) or on the middle turbinate will result in a frontal recess coursing lateral to the uncinate process and emptying into the ethmoid infundibulum ; otherwise , if the superior insertion of the uncinate process is on the medial orbital wall , the frontal recess will run medial to the process , closer to the middle turbinate ( fig . 3 ) . the sphenoethmoid recess is the drainage pathway for the sphenoid sinus and posterior ethmoid cells , and is easily seen on axial images as a short and thin channel arising from the anterior sphenoid sinus wall and coursing just medial to the nasal septum . on coronal and sagittal images , the sphenoethmoid recess is less clearly seen ; on msct examinations , correct identification is improved by interactive navigation on 3d workstations ( fig . the ostiomeatal complex is the functional area in which the anterior ethmoid cells , and the maxillary and frontal sinus drain . it is composed of the middle turbinate ( pneumatised in this case , c ) , the uncinate process ( arrowhead ) , the ethmoid bulla ( b ) , the ethmoid infundibulum ( arrow ) , and the frontal recess ( asterisks)fig . 3coronal oblique mpr reconstruction showing the variable cranial attachment of the uncinate process . on the left side , the uncinate process attaches laterally to the medial orbital wall ( arrowheads ) , thus the frontal recess ( asterisks ) courses close to the middle turbinate ( mt ) . the ethmoid infundibulum is obstructed resulting in sinusitis with an infundibular pattern . on the right side , the uncinate process inserts on both the medial orbital wall and the skull base ( arrows ) ; the frontal recess ( not visible ) will drain into the middle meatusfig . 4the sphenoethmoid recess ( asterisks ) is generally well seen on the axial plane along the medial aspect of the anterior sphenoid sinus wall . identification on the coronal and sagittal planes can be simplified navigating through the volume as shown in b , c and d coronal msct . the ostiomeatal complex is the functional area in which the anterior ethmoid cells , and the maxillary and frontal sinus drain . it is composed of the middle turbinate ( pneumatised in this case , c ) , the uncinate process ( arrowhead ) , the ethmoid bulla ( b ) , the ethmoid infundibulum ( arrow ) , and the frontal recess ( asterisks ) coronal oblique mpr reconstruction showing the variable cranial attachment of the uncinate process . on the left side , the uncinate process attaches laterally to the medial orbital wall ( arrowheads ) , thus the frontal recess ( asterisks ) courses close to the middle turbinate ( mt ) . the ethmoid infundibulum is obstructed resulting in sinusitis with an infundibular pattern . on the right side , the uncinate process inserts on both the medial orbital wall and the skull base ( arrows ) ; the frontal recess ( not visible ) will drain into the middle meatus the sphenoethmoid recess ( asterisks ) is generally well seen on the axial plane along the medial aspect of the anterior sphenoid sinus wall . identification on the coronal and sagittal planes can be simplified navigating through the volume as shown in b , c and d overall , the number and frequency of anatomical variants is so high that at least one is to be expected in up to 60% of patients undergoing imaging . some are clinically irrelevant , some may be regarded as cofactors of chronic rhinosinusitis , as they narrow the drainage pathways for the paranasal sinuses ; others need to be reported as a potential source of iatrogenic damage during endoscopic sinus surgery . in the anterior compartment , agger nasi cells , for example , are the result of ethmoidal pneumatisation of the lacrimal bone ; they are the most anterior ethmoidal cells found on standard coronal images , visible anterior to the ethmoid fovea and the crista galli . when large , these cells may narrow the frontal recess , as best displayed by sagittal reformations ( fig . infraorbital and supraorbital cells are the extension of ethmoid pneumatisation to the orbital floor or roof , respectively . the former , more commonly called haller cells , may narrow the ethmoid infundibulum , whereas the relevance of the latter is more strictly related to the correct planning of endoscopic surgery . being located just posterior to the frontal sinus , these supraorbital cells may be easily overlooked on the coronal plane ; the axial and sagittal planes are far more accurate in the detection of these cells , which drain through an ostium located inferior and lateral to the frontal recess ( fig . 6 ) . concha bullosa , i.e. pneumatisation of the middle turbinate , is very frequently associated with contralateral septal deviation , although the developmental correlation between the two is unclear . both the variants may restrict the ostiomeatal complex , however no statistically significant correlation was found between the presence of these variants and any sinus disease . bulla frontalis is a group of variants basically indicating air cells located cranial to agger nasi cells and extending towards the frontal sinus : they can be isolated and protruding ( type iii ) or not ( type i ) ( fig . 7 ) in the frontal sinus , or they can be a chain of air cells ( type ii ) ; finally , type iv relates to isolated cells into the frontal sinus . the uncinate process itself may exhibit variants such as pneumatisation or medial bending of the tip . the most relevant variant of the uncinate process is hypoplasia , which results in occlusion of the ethmoid infundibulum ; this is associated with hypoplasia of the maxillary sinus , and commonly also chronic maxillary inflammation ( fig . the onodi cell is by far the most dreaded variant for the surgeon ; this is generally a rather huge posterior ethmoid cell , which expands on top or lateral to the sphenoid sinus . if overlooked on a presurgical image , it can increase the hazard of middle cranial fossa penetration during endoscopic surgery ( fig . similarly , the risk of iatrogenic damage may be amplified by other variants such as dehiscences and protrusions ( fig . dehiscences are more crucial when seen at the level of the ethmoid roof and medial orbital wall ; their classification as either of post - traumatic or of congenital origin is debated as the apparently congenital ones might also be the result of a neglected trauma . protrusions relate to bulging of the carotid canal , foramen rotundum , vidian canal , optic canal within an hyperpneumatised sphenoid sinus . 5msct coronal ( a , b ) and sagittal reformations obtained on the right ( c ) and left ( d ) sides . bilateral agger nasi cells ( asterisks ) are visible on the coronal images , but sagittal reformations better display their relationships with the frontal recesses ( arrows ) . a type i bulla frontalis ( arrowhead in b ) can be seen on top of the left agger nasi cell . inflammatory material with focal calcifications is retained within the right sphenoid sinus whose walls are densely sclerotic ( arrowheads in c ) : chronic inflammation , possibly a fungus ball ( see below)fig . 6anterior ( a ) and posterior ( p ) infraorbital cells seen in two different patients ; the former is strictly adjacent to the ethmoid infundibulum , the latter minimally displaces the orbital wall . the anterior one is also called a haller cellfig . i bulla frontalis ( bf ) , on top of an agger nasali cell ( asterisk ) , and its relationships with the frontal recess ( arrows ) , posteriorlyfig . the left uncinate process ( u ) is hypoplastic and tightly attached to the medial orbital wall ( compare with the contralateral side ) , hindering maxillary sinus drainage . the sinus cavity is shrunk and the orbital floor is slightly depressed because of chronic hypopressurefig . 9a huge onodi cell extends above and lateral to the right sphenoid sinus ( arrowhead points to the sphenoethmoid recess ) , the arrow points to the protrusion of the optic canal within the air cellfig . 10two focal bone dehiscences are seen along the medial orbital wall , through which a small amount of orbital fat tissue herniates . this condition increases the risks of endoscopic sinus surgery , particularly if extrinsic ocular muscles are entrapped in the bone gap msct coronal ( a , b ) and sagittal reformations obtained on the right ( c ) and left ( d ) sides . bilateral agger nasi cells ( asterisks ) are visible on the coronal images , but sagittal reformations better display their relationships with the frontal recesses ( arrows ) . a type i bulla frontalis ( arrowhead in b ) can be seen on top of the left agger nasi cell . inflammatory material with focal calcifications is retained within the right sphenoid sinus whose walls are densely sclerotic ( arrowheads in c ) : chronic inflammation , possibly a fungus ball ( see below ) anterior ( a ) and posterior ( p ) infraorbital cells seen in two different patients ; the former is strictly adjacent to the ethmoid infundibulum , the latter minimally displaces the orbital wall . the anterior one is also called a haller cell coronal and sagittal msct reformations showing type i bulla frontalis ( bf ) , on top of an agger nasali cell ( asterisk ) , and its relationships with the frontal recess ( arrows ) , posteriorly axial msct and coronal reconstructions of a patient suffering silent sinus syndrome . the left uncinate process ( u ) is hypoplastic and tightly attached to the medial orbital wall ( compare with the contralateral side ) , hindering maxillary sinus drainage . the sinus cavity is shrunk and the orbital floor is slightly depressed because of chronic hypopressure a huge onodi cell extends above and lateral to the right sphenoid sinus ( arrowhead points to the sphenoethmoid recess ) , the arrow points to the protrusion of the optic canal within the air cell two focal bone dehiscences are seen along the medial orbital wall , through which a small amount of orbital fat tissue herniates . this condition increases the risks of endoscopic sinus surgery , particularly if extrinsic ocular muscles are entrapped in the bone gap the rationale for imaging of patients affected with chronic rhinosinusitis , simplified to the extreme , consists in demonstrating any impairment of mucus clearance through the aforementioned pathways , along with any anatomical variant that may either incite rhinosinusitis or raise the risks of endoscopic surgery . given its superb detail in depicting the thinnest bone structures , msct is the most suitable technique for the task ; the inherent contrast displayed on bone algorithm reconstructions among bone , air and soft tissues allows the radiation dose to be decreased to a minimum , thus allowing all information to be obtained with limited biological invasiveness . msct reporting of the paranasal sinuses should be done in a centripetal fashion , thus moving from the centre , i.e. the nasal septum and the respective drainage pathways , to the periphery represented by all sinuses . five patterns of rhinosinusitis , namely the infundibular , sphenoethmoidal , ostiomeatal and sporadic patterns as well as nasal polyposis are described [ 11 , 12 ] . the infundibular pattern relates to the obstruction of the ethmoid infundibulum by soft - tissue thickening , which results in impaired drainage of the maxillary sinus ; this sinus is opacified by thickened mucosa , submucosal oedema and/or entrapped fluid secretions ( fig . similarly , obstruction of the sphenoethmoid recess will impair the drainage of the posterior ethmoid and sphenoid sinus , producing what is referred to as a sphenoethmoid pattern ( fig . the ostiomeatal pattern is a more complex one , reflecting the higher complexity of this anatomical subunit , which , when blocked , simultaneously impairs mucus drainage from the anterior ethmoid cells and the maxillary and frontal sinus ( fig . the mucosa investing the middle turbinate , the uncinate process and the ethmoid infundibulum are the most usual sites of origin of polyps . the hypertrophied , polypoid soft - tissue thickening tends to distort the adjacent bone structures and enlarge the ethmoid infundibulum , quite often invading the maxillary sinus . such a growth pattern almost invariably produces ostiomeatal pattern rhinosinusitis ; pansinusitis due to extension to the posterior ethmoid ( and thus impairment of the sphenoethmoid recess ) is similarly frequent ( fig . 11sinusitis with infundibular pattern : the most proximal part of the ethmoid infundibulum ( asterisks ) is obstructed by thickened mucosa , the maxillary sinus is completely opacified by inflammatory material . although large , the infraorbital cell ( ioc)does not narrow the drainage pathwayfig . 12sinusitis with a sphenoethmoid pattern : the sphenoethmoid recess is obstructed by thickened mucosa ( asterisks ) , both the small left sphenoid sinus and the posterior ethmoid cells are opacified . note the basal lamella ( arrows ) clearly demarcating the anterior ethmoid from the inflamed posterior ethmoid cells . secretions are also retained within the pneumatised vertical lamella of the middle turbinate ( arrowhead)fig . 13ostiomeatal complex pattern : the middle meatus ( asterisk ) and anterior ethmoid cells are occupied by tissue , the medial orbital wall appears slightly displaced ( arrows ) . mucus drainage from the ethmoid infundibulum and frontal recess is simultaneously hindered producing frontal and maxillary sinusitis ( io infraorbital cell)fig . 14nasal polyposis : diffuse bilateral thickening of the mucosa investing the middle meati , the middle turbinate , and the anterior and posterior ethmoid cells . retained secretion can be seen within the blocked maxillary and sphenoid sinuses sinusitis with infundibular pattern : the most proximal part of the ethmoid infundibulum ( asterisks ) is obstructed by thickened mucosa , the maxillary sinus is completely opacified by inflammatory material . although large , the infraorbital cell ( ioc)does not narrow the drainage pathway sinusitis with a sphenoethmoid pattern : the sphenoethmoid recess is obstructed by thickened mucosa ( asterisks ) , both the small left sphenoid sinus and the posterior ethmoid cells are opacified . note the basal lamella ( arrows ) clearly demarcating the anterior ethmoid from the inflamed posterior ethmoid cells . secretions are also retained within the pneumatised vertical lamella of the middle turbinate ( arrowhead ) ostiomeatal complex pattern : the middle meatus ( asterisk ) and anterior ethmoid cells are occupied by tissue , the medial orbital wall appears slightly displaced ( arrows ) . mucus drainage from the ethmoid infundibulum and frontal recess is simultaneously hindered producing frontal and maxillary sinusitis ( io infraorbital cell ) nasal polyposis : diffuse bilateral thickening of the mucosa investing the middle meati , the middle turbinate , and the anterior and posterior ethmoid cells . retained secretion can be seen within the blocked maxillary and sphenoid sinuses the sporadic pattern is a large box in which several different conditions are stored , such as isolated mucosal thickenings , retention cysts , antrochoanal polyp , silent sinus syndrome , odontogenic sinusitis and mucocele . isolated mucosal thickenings are incidentally found in a large number of msct performed for non - sinonasal pathological conditions . as a general rule , such mucosal thickening is unrelated to clinical symptoms , and when seen in the maxillary sinus and ethmoid cells , it does not need to be reported unless the mucosal thickness exceeds 4 mm and 2 mm , respectively . on the other hand , the mucosal lining of the frontal and sphenoid sinuses should not be visible on msct under normal conditions . retention cysts are submucosal ( pseudo-)cystic lesions that may have serous ( fluid accumulation in the submucosa ) or may be of a mucous nature ( blockage of a submucosal mucus gland ) . at imaging , they display a convex shape and sharply demarcated borders , and are attached to a sinus wall ; thus , they do not change with the patient s decubitus . they have little clinical relevance unless large enough to hinder mucus drainage ( fig . 15a retention cyst , displaying typical smooth and convex borders , partially fills the maxillary sinus cavity a retention cyst , displaying typical smooth and convex borders , partially fills the maxillary sinus cavity antrochoanal polyp is a relatively easy diagnosis , based on the pattern of growth ( from the maxillary sinus to the nasal fossa and farther to the choana ) and nature ( cystic appearance ) of this generally isolated polypoid lesion . though much rarer , spheno- and ethmoidochoanal variants are also reported [ 14 , 15 ] ( fig . 16antrochoanal polyp : the polypoid lesion arises from the right maxillary sinus , and extends through an accessory ostium ( arrows ) into the nasal fossa , reaching posteriorly the nasopharynx through the choana antrochoanal polyp : the polypoid lesion arises from the right maxillary sinus , and extends through an accessory ostium ( arrows ) into the nasal fossa , reaching posteriorly the nasopharynx through the choana silent sinus syndrome refers to an obstructed maxillary sinus , occupied by inflamed mucosa and secretions , which is shrunken with depression of the orbital floor causing exophthalmia . it is called silent sinus syndrome when clinically there is no clue as to the maxillary sinus disease , only being discovered on an imaging study . interestingly , antrochoanal polyp and silent sinus syndrome share the same pathophysiological mechanism , i.e. maxillary sinus blockage . in antrochoanal polyp , an increased intrasinusal pressure ( due to partial ostial obstruction with unidirectional flow of air through the ostium into the sinus ) is the trigger to force the expulsion of an intramural cyst through an accessory ostium . in silent sinus syndrome , conversely , complete sinus blockage and progressive resorption of air within the cavity leads to a pressure drop , orbital floor depression and sinus contraction ( fig . 17 ) , is heralded by a combination of signs , namely isolated maxillary sinusitis , detection of periradicular inflammatory lesions , possibly with surrounding bone destruction , and sometimes retention within the sinus cavity of calcifications , or even hyperdense material , with a density similar to dental filling material . the differential diagnosis between dentogenic sinusitis and fungal infection ( see below ) may be difficult on imaging studies . a large area of bone resorption is seen in the alveolar ridge , around the root of a molar tooth ; the floor of the maxillary sinus is interrupted , the sinus filled by inflammatory material . the small calcifications found along the ethmoid infundibulum and in the anterior ethmoid ( arrows ) were proven to be bone fragments , possibly transported by mucociliary clearance odontogenic sinusitis . a large area of bone resorption is seen in the alveolar ridge , around the root of a molar tooth ; the floor of the maxillary sinus is interrupted , the sinus filled by inflammatory material . the small calcifications found along the ethmoid infundibulum and in the anterior ethmoid ( arrows ) were proven to be bone fragments , possibly transported by mucociliary clearance finally , chronic sinus drainage obstruction may end up in mucocele formation : a mucocele is an epithelium - lined mucus collection completely filling a sinus cavity ; while the secretions accumulate , pressure within the cavity increases , inducing sinus expansion and bone remodelling . 18 ) are more frequent in the fronto - ethmoid region but probably more dangerous in the sphenoid sinus , where sinus expansion may result in severe damage to the optic nerve .
fig . 18the right middle turbinate is swollen by a sharply defined lesion exhibiting spontaneously high density on msct , and showing different fluid components on mri . absence of solid , enhancing areas confirms mucocele within a pneumatised middle turbinate the right middle turbinate is swollen by a sharply defined lesion exhibiting spontaneously high density on msct , and showing different fluid components on mri . absence of solid , enhancing areas confirms mucocele within a pneumatised middle turbinate bone remodelling is a broad definition that encompasses the whole spectrum of alterations that may accompany the aforementioned mucosal changes in chronic rhinosinusitis and nasal polyposis . as described by giacchi et al . , bone remodelling is an adaptive process , in which osteoblasts and osteoclasts can be variably involved , that can be triggered by mechanical factors ( pressure , in nasal polyposis and mucocele ) as well as inflammatory mediators or infectious agents ( bacterial invasion of periosteum and bone ) . the prevalence of osteoclastic activity provokes bone resorption and demineralisation ; this is mostly seen in thin bone structures subject to chronic pressure . thinning and displacement of ethmoid septa or the medial wall , as well as truncation of the middle turbinate are , for example , frequent findings in nasal polyposis ; similarly , the expanded sinus walls of mucoceles are often thinned if not totally demineralised . on the other hand , osteoblastic activity manifests itself as thickening and sclerosis of bone ( osteitis ) , more typically encountered in thicker bone structures , such as the walls of the maxillary sinus and sphenoid sinus . as expected , all the described bone changes can be present simultaneously in the same patient , particularly if affected by nasal polyposis and fungal infections ( see below ) ( fig . 19demineralisation and distortion of thin bony laminae ( middle turbinates and ethmoid labyrinth ) and sclerosis of thicker bone structures ( sphenoid sinus ) are present simultaneously in a patient with diffuse nasal polyposis demineralisation and distortion of thin bony laminae ( middle turbinates and ethmoid labyrinth ) and sclerosis of thicker bone structures ( sphenoid sinus ) are present simultaneously in a patient with diffuse nasal polyposis some of the imaging patterns of chronic rhinosinusitis already described ( such as the ostiomeatal pattern and mucocele ) can also be encountered as indirect signs of the presence of a neoplasm either benign or malignant . furthermore , extensive nasal polyposis may completely obstruct sinonasal cavities , severely distorting the normal anatomy . patients examined for rhinosinusitis undergo imaging applying a standard msct protocol , without contrast agent injection , and using bone algorithm reconstructions alone . as a consequence , the major concern for radiologists ( particularly those not routinely involved in sinonasal imaging ) is how to safely rule out a neoplasm harboured amid inflammatory changes . some landmarks may be of help : chronic rhinosinusitis ( and nasal polyposis ) are much more commonly bilateral . whenever a ostiomeatal pattern is seen as unilateral disease , the presence of a neoplasm in the middle meatus should be considered , and endoscopic assessment of the region should be prompted . neoplastic lesions , even when benign , tend to behave more aggressively towards bone , causing more pronounced displacement , erosion and destruction . in chronic rhinosinutis and nasal polyposis bone is remodelled , distorted , demineralised but , as a general rule , not fragmented . mucocele is emblematic : frequently the line of demarcation between demineralised and normal bone is represented by a blunt but sharp border , whereas in neoplasms the border is ill - defined and spiculated . density measurements are not of great help , neither for the differentiation between tumour and polyp ( both are within the solid range of densities ) nor to discriminate tumour from mucocele ( which may exhibit high density if secretions are desiccated ) . in some cases , mri may add information ( polyps are generally more t2 hyperintense than most malignant tumours ; inverted papilloma , the most common benign nasosinusal tumour , may display the almost pathognomonic cerebriform pattern ) . however , the bottom line is that in any unclear case an endoscopic examination should be performed , and a biopsy obtained ( fig . 20the left ostiomeatal complex is occupied by solid tissue , the anterior ethmoid cells , the maxillary and frontal sinus are completely opacified . the absence of inflammatory changes on the right side as well as the displacement and destruction of the anterior ( arrowheadss ) and the posterior ( arrows ) maxillary sinus wall suggests the presence of a neoplasm . actually , mri shows a mass arising from the maxillary sinus and protruding into the nasal fossa , histologically proven as ameloblastoma the left ostiomeatal complex is occupied by solid tissue , the anterior ethmoid cells , the maxillary and frontal sinus are completely opacified . the absence of inflammatory changes on the right side as well as the displacement and destruction of the anterior ( arrowheadss ) and the posterior ( arrows ) maxillary sinus wall suggests the presence of a neoplasm . actually , mri shows a mass arising from the maxillary sinus and protruding into the nasal fossa , histologically proven as ameloblastoma fungal infection is rather common , given the ubiquitous presence of fungal agents in the environment and the high frequency of colonisation of sinonasal mucosa ; the role of fungi in promoting chronic rhinosinusitis has been suggested . fungal infections may be classified as either non - invasive or invasive forms , according to the absence or presence of invasion of mucosa , submucosa , bone and vessels by hyphae . the non - invasive form occurs in immunocompetent subjects and may further be classified as fungus ball and eosinophilic rhinosinusitis ; these forms are seen in patients with chronic rhinosinusitis , and are non - responsive to medical and/or surgical treatment . on msct , non - invasive fungal infection is suspected whenever spontaneously hyperdense material is detected within the sinus cavities . in the case of a fungus ball , such hyperdensity is due to a high content of heavy metals in the hyphae , but small calcifications can also be seen . maxillary fungal sinusitis may also be caused by the presence of intrasinusal dental filling material , which appears extremely hyperdense . in 80% of cases , 21 ) . in eosinophilic rhinosinusitis , besides fungal hyphae , the accumulation of dense eosinophilic mucin may account for the detection of sinus hyperdensities , which are scattered in multiple sinus cavities , uni- or bilaterally . mri can be misleading in both conditions , because heavy metal and calcium , on the one hand , and the tight packing of fungal material within sinuses , on the other hand , produce very hypointense signal which , particularly on t2 sequences , can be as low as the signal void of air ( fig . a , b the maxillary sinus is filled , at the periphery , by hypodense thickened mucosa and contains , in the centre , a calcification ; dense sclerosis of the posterolateral wall secondary to chronic inflammation ( arrows ) . c , d chronic rhinosinusitis with polypoid thickening of the mucosa and hyperdense material scattered in all sinus cavities : this pattern suggests allergic fungal rhinosinusitis . e , f on mri eosinophilic mucin displays t2 hypointense signal comparable with air : the t1 intermediate signal along with bone remodelling and sinus expansion ( arrowheads ) help to make the diagnosis of allergic fungal rhinosinusitis non - invasive fungal rhinosinusitis . a , b the maxillary sinus is filled , at the periphery , by hypodense thickened mucosa and contains , in the centre , a calcification ; dense sclerosis of the posterolateral wall secondary to chronic inflammation ( arrows ) . c , d chronic rhinosinusitis with polypoid thickening of the mucosa and hyperdense material scattered in all sinus cavities : this pattern suggests allergic fungal rhinosinusitis . e , f on mri eosinophilic mucin displays t2 hypointense signal comparable with air : the t1 intermediate signal along with bone remodelling and sinus expansion ( arrowheads ) help to make the diagnosis of allergic fungal rhinosinusitis invasive fungal infection can be divided into an acute invasive form and a chronic invasive form , and both usually occur in immunocompromised subjects ( diabetes mellitus with or without ketoacidosis , organ transplantation , chemotherapy , haematological diseases , hiv infection ) ; the chronic invasive form has also been reported in immunocompetent hosts . sinus hyperdensities are not common in invasive mycoses , bone destruction with invasion of adjacent anatomical structures ( orbit , skull base , cavernous sinus , pterygopalatine fossa , periantral fat tissue ) are the most significant imaging findings in both forms . whilst the differential diagnosis between invasive and non - invasive forms is rather straightforward , the discrimination between acute invasive and chronic invasive is very difficult at imaging ; this differentiation is mainly based on the clinical setting , as the acute invasive form is rapidly progressive and bears a dismal short - term prognosis ( up to 50 - 80% mortality ) . although msct equipment is more readily accessible in nearly every institution , invasive mycoses highly benefit from the high contrast resolution of mri to provide thorough assessment of intracranial and cavernous sinus involvement ( fig . chronic invasive mycosis invading the maxillary sinus mucosa , destroying the pterygoid plates and posterior maxillary sinus wall ( arrows ) , and invading the infratemporal fossa mri se t1 before ( left ) and after ( right ) contrast application . chronic invasive mycosis invading the maxillary sinus mucosa , destroying the pterygoid plates and posterior maxillary sinus wall ( arrows ) , and invading the infratemporal fossa although the paranasal sinuses are a complex framework of air cavities , widely variable in number and size , their anatomy may be simplified using a schematic approach based on drainage pathways . in this perspective , two functionally distinct compartments can be identified : first , the anterior compartment including the anterior ethmoid cells , the frontal and the maxillary sinus , draining into the middle meatus . second , the posterior compartment which is composed of the posterior ethmoid cells and the sphenoid sinus , draining into the superior meatus . the line of demarcation between the two compartments is the basal lamella of the middle turbinate , i.e. the attachment of the turbinate to the lateral nasal wall or medial orbital wall . although the basal lamella can be seen on axial and coronal images , sagittal reconstructions , easily available on multislice computed tomography ( msct ) examinations , are best suited to precisely delineate its obliquely oriented course ( fig . 1 ) . arrows outline the course of the basal lamella of the middle turbinate , which marks the border between anterior and posterior ethmoid air cells ( a agger nasi cell ) sagittal mpr reconstruction . arrows outline the course of the basal lamella of the middle turbinate , which marks the border between anterior and posterior ethmoid air cells ( a agger nasi cell ) for each of the aforementioned compartments some key anatomical landmarks can be defined . in the anterior one , all drainage pathways of the anterior ethmoid cells , and the frontal and maxillary sinus converge in an anatomical area located in the middle meatus ; this is referred to as the ostiomeatal complex ( fig . 2 ) . the ostiomeatal complex is composed of several structures : the uncinate process , a sickle - shaped bone structure projecting from the medial maxillary sinus wall up to the skull base ; the ethmoid bulla , generally the most prominent ethmoid air cell located above the uncinate process ; the ethmoid infundibulum , the thin channel in between the uncinate process and the ethmoid bulla ; the frontal recess , which is the obliquely orientated , funnel - shaped drainage pathway of the frontal sinus . the anatomy of the frontal recess is closely related to that of the uncinate process : the medial and lateral borders of the frontal recess depend upon the superior attachment of the uncinate process . as described by landsberg and friedman and lee et al . , a cranial insertion of the uncinate process on the ethmoid fovea ( the roof of the ethmoid ) or on the middle turbinate will result in a frontal recess coursing lateral to the uncinate process and emptying into the ethmoid infundibulum ; otherwise , if the superior insertion of the uncinate process is on the medial orbital wall , the frontal recess will run medial to the process , closer to the middle turbinate ( fig . 3 ) . the sphenoethmoid recess is the drainage pathway for the sphenoid sinus and posterior ethmoid cells , and is easily seen on axial images as a short and thin channel arising from the anterior sphenoid sinus wall and coursing just medial to the nasal septum . on coronal and sagittal images , the sphenoethmoid recess is less clearly seen ; on msct examinations , correct identification is improved by interactive navigation on 3d workstations ( fig . 4 ) . the ostiomeatal complex is the functional area in which the anterior ethmoid cells , and the maxillary and frontal sinus drain . it is composed of the middle turbinate ( pneumatised in this case , c ) , the uncinate process ( arrowhead ) , the ethmoid bulla ( b ) , the ethmoid infundibulum ( arrow ) , and the frontal recess ( asterisks)fig . 3coronal oblique mpr reconstruction showing the variable cranial attachment of the uncinate process . on the left side , the uncinate process attaches laterally to the medial orbital wall ( arrowheads ) , thus the frontal recess ( asterisks ) courses close to the middle turbinate ( mt ) . the ethmoid infundibulum is obstructed resulting in sinusitis with an infundibular pattern . on the right side , the uncinate process inserts on both the medial orbital wall and the skull base ( arrows ) ; the frontal recess ( not visible ) will drain into the middle meatusfig . 4the sphenoethmoid recess ( asterisks ) is generally well seen on the axial plane along the medial aspect of the anterior sphenoid sinus wall . identification on the coronal and sagittal planes can be simplified navigating through the volume as shown in b , c and d coronal msct . the ostiomeatal complex is the functional area in which the anterior ethmoid cells , and the maxillary and frontal sinus drain . it is composed of the middle turbinate ( pneumatised in this case , c ) , the uncinate process ( arrowhead ) , the ethmoid bulla ( b ) , the ethmoid infundibulum ( arrow ) , and the frontal recess ( asterisks ) coronal oblique mpr reconstruction showing the variable cranial attachment of the uncinate process . on the left side , the uncinate process attaches laterally to the medial orbital wall ( arrowheads ) , thus the frontal recess ( asterisks ) courses close to the middle turbinate ( mt ) . the ethmoid infundibulum is obstructed resulting in sinusitis with an infundibular pattern . on the right side , the uncinate process inserts on both the medial orbital wall and the skull base ( arrows ) ; the frontal recess ( not visible ) will drain into the middle meatus the sphenoethmoid recess ( asterisks ) is generally well seen on the axial plane along the medial aspect of the anterior sphenoid sinus wall . identification on the coronal and sagittal planes can be simplified navigating through the volume as shown in b , c and d overall , the number and frequency of anatomical variants is so high that at least one is to be expected in up to 60% of patients undergoing imaging . some are clinically irrelevant , some may be regarded as cofactors of chronic rhinosinusitis , as they narrow the drainage pathways for the paranasal sinuses ; others need to be reported as a potential source of iatrogenic damage during endoscopic sinus surgery . in the anterior compartment , agger nasi cells , for example , are the result of ethmoidal pneumatisation of the lacrimal bone ; they are the most anterior ethmoidal cells found on standard coronal images , visible anterior to the ethmoid fovea and the crista galli . when large , these cells may narrow the frontal recess , as best displayed by sagittal reformations ( fig . 5 ) . infraorbital and supraorbital cells are the extension of ethmoid pneumatisation to the orbital floor or roof , respectively . the former , more commonly called haller cells , may narrow the ethmoid infundibulum , whereas the relevance of the latter is more strictly related to the correct planning of endoscopic surgery . being located just posterior to the frontal sinus , these supraorbital cells may be easily overlooked on the coronal plane ; the axial and sagittal planes are far more accurate in the detection of these cells , which drain through an ostium located inferior and lateral to the frontal recess ( fig . 6 ) . concha bullosa , i.e. pneumatisation of the middle turbinate , is very frequently associated with contralateral septal deviation , although the developmental correlation between the two is unclear . both the variants may restrict the ostiomeatal complex , however no statistically significant correlation was found between the presence of these variants and any sinus disease . bulla frontalis is a group of variants basically indicating air cells located cranial to agger nasi cells and extending towards the frontal sinus : they can be isolated and protruding ( type iii ) or not ( type i ) ( fig . 7 ) in the frontal sinus , or they can be a chain of air cells ( type ii ) ; finally , type iv relates to isolated cells into the frontal sinus . the uncinate process itself may exhibit variants such as pneumatisation or medial bending of the tip . the most relevant variant of the uncinate process is hypoplasia , which results in occlusion of the ethmoid infundibulum ; this is associated with hypoplasia of the maxillary sinus , and commonly also chronic maxillary inflammation ( fig . the onodi cell is by far the most dreaded variant for the surgeon ; this is generally a rather huge posterior ethmoid cell , which expands on top or lateral to the sphenoid sinus . if overlooked on a presurgical image , it can increase the hazard of middle cranial fossa penetration during endoscopic surgery ( fig . similarly , the risk of iatrogenic damage may be amplified by other variants such as dehiscences and protrusions ( fig . dehiscences are more crucial when seen at the level of the ethmoid roof and medial orbital wall ; their classification as either of post - traumatic or of congenital origin is debated as the apparently congenital ones might also be the result of a neglected trauma . protrusions relate to bulging of the carotid canal , foramen rotundum , vidian canal , optic canal within an hyperpneumatised sphenoid sinus . 5msct coronal ( a , b ) and sagittal reformations obtained on the right ( c ) and left ( d ) sides . bilateral agger nasi cells ( asterisks ) are visible on the coronal images , but sagittal reformations better display their relationships with the frontal recesses ( arrows ) . a type i bulla frontalis ( arrowhead in b ) can be seen on top of the left agger nasi cell . inflammatory material with focal calcifications is retained within the right sphenoid sinus whose walls are densely sclerotic ( arrowheads in c ) : chronic inflammation , possibly a fungus ball ( see below)fig . 6anterior ( a ) and posterior ( p ) infraorbital cells seen in two different patients ; the former is strictly adjacent to the ethmoid infundibulum , the latter minimally displaces the orbital wall . the anterior one is also called a haller cellfig . 7coronal and sagittal msct reformations showing type i bulla frontalis ( bf ) , on top of an agger nasali cell ( asterisk ) , and its relationships with the frontal recess ( arrows ) , posteriorlyfig . the left uncinate process ( u ) is hypoplastic and tightly attached to the medial orbital wall ( compare with the contralateral side ) , hindering maxillary sinus drainage . the sinus cavity is shrunk and the orbital floor is slightly depressed because of chronic hypopressurefig . 9a huge onodi cell extends above and lateral to the right sphenoid sinus ( arrowhead points to the sphenoethmoid recess ) , the arrow points to the protrusion of the optic canal within the air cellfig . 10two focal bone dehiscences are seen along the medial orbital wall , through which a small amount of orbital fat tissue herniates . this condition increases the risks of endoscopic sinus surgery , particularly if extrinsic ocular muscles are entrapped in the bone gap msct coronal ( a , b ) and sagittal reformations obtained on the right ( c ) and left ( d ) sides . bilateral agger nasi cells ( asterisks ) are visible on the coronal images , but sagittal reformations better display their relationships with the frontal recesses ( arrows ) . a type i bulla frontalis ( arrowhead in b ) can be seen on top of the left agger nasi cell . inflammatory material with focal calcifications is retained within the right sphenoid sinus whose walls are densely sclerotic ( arrowheads in c ) : chronic inflammation , possibly a fungus ball ( see below ) anterior ( a ) and posterior ( p ) infraorbital cells seen in two different patients ; the former is strictly adjacent to the ethmoid infundibulum , the latter minimally displaces the orbital wall . the anterior one is also called a haller cell coronal and sagittal msct reformations showing type i bulla frontalis ( bf ) , on top of an agger nasali cell ( asterisk ) , and its relationships with the frontal recess ( arrows ) , posteriorly axial msct and coronal reconstructions of a patient suffering silent sinus syndrome . the left uncinate process ( u ) is hypoplastic and tightly attached to the medial orbital wall ( compare with the contralateral side ) , hindering maxillary sinus drainage . the sinus cavity is shrunk and the orbital floor is slightly depressed because of chronic hypopressure a huge onodi cell extends above and lateral to the right sphenoid sinus ( arrowhead points to the sphenoethmoid recess ) , the arrow points to the protrusion of the optic canal within the air cell two focal bone dehiscences are seen along the medial orbital wall , through which a small amount of orbital fat tissue herniates . this condition increases the risks of endoscopic sinus surgery , particularly if extrinsic ocular muscles are entrapped in the bone gap the rationale for imaging of patients affected with chronic rhinosinusitis , simplified to the extreme , consists in demonstrating any impairment of mucus clearance through the aforementioned pathways , along with any anatomical variant that may either incite rhinosinusitis or raise the risks of endoscopic surgery . given its superb detail in depicting the thinnest bone structures , msct is the most suitable technique for the task ; the inherent contrast displayed on bone algorithm reconstructions among bone , air and soft tissues allows the radiation dose to be decreased to a minimum , thus allowing all information to be obtained with limited biological invasiveness . msct reporting of the paranasal sinuses should be done in a centripetal fashion , thus moving from the centre , i.e. the nasal septum and the respective drainage pathways , to the periphery represented by all sinuses . five patterns of rhinosinusitis , namely the infundibular , sphenoethmoidal , ostiomeatal and sporadic patterns as well as nasal polyposis are described [ 11 , 12 ] . the infundibular pattern relates to the obstruction of the ethmoid infundibulum by soft - tissue thickening , which results in impaired drainage of the maxillary sinus ; this sinus is opacified by thickened mucosa , submucosal oedema and/or entrapped fluid secretions ( fig . similarly , obstruction of the sphenoethmoid recess will impair the drainage of the posterior ethmoid and sphenoid sinus , producing what is referred to as a sphenoethmoid pattern ( fig . the ostiomeatal pattern is a more complex one , reflecting the higher complexity of this anatomical subunit , which , when blocked , simultaneously impairs mucus drainage from the anterior ethmoid cells and the maxillary and frontal sinus ( fig . 13 ) . the mucosa investing the middle turbinate , the uncinate process and the ethmoid infundibulum are the most usual sites of origin of polyps . the hypertrophied , polypoid soft - tissue thickening tends to distort the adjacent bone structures and enlarge the ethmoid infundibulum , quite often invading the maxillary sinus . such a growth pattern almost invariably produces ostiomeatal pattern rhinosinusitis ; pansinusitis due to extension to the posterior ethmoid ( and thus impairment of the sphenoethmoid recess ) is similarly frequent ( fig . 11sinusitis with infundibular pattern : the most proximal part of the ethmoid infundibulum ( asterisks ) is obstructed by thickened mucosa , the maxillary sinus is completely opacified by inflammatory material . although large , the infraorbital cell ( ioc)does not narrow the drainage pathwayfig . 12sinusitis with a sphenoethmoid pattern : the sphenoethmoid recess is obstructed by thickened mucosa ( asterisks ) , both the small left sphenoid sinus and the posterior ethmoid cells are opacified . note the basal lamella ( arrows ) clearly demarcating the anterior ethmoid from the inflamed posterior ethmoid cells . secretions are also retained within the pneumatised vertical lamella of the middle turbinate ( arrowhead)fig . 13ostiomeatal complex pattern : the middle meatus ( asterisk ) and anterior ethmoid cells are occupied by tissue , the medial orbital wall appears slightly displaced ( arrows ) . mucus drainage from the ethmoid infundibulum and frontal recess is simultaneously hindered producing frontal and maxillary sinusitis ( io infraorbital cell)fig . 14nasal polyposis : diffuse bilateral thickening of the mucosa investing the middle meati , the middle turbinate , and the anterior and posterior ethmoid cells . retained secretion can be seen within the blocked maxillary and sphenoid sinuses sinusitis with infundibular pattern : the most proximal part of the ethmoid infundibulum ( asterisks ) is obstructed by thickened mucosa , the maxillary sinus is completely opacified by inflammatory material . although large , the infraorbital cell ( ioc)does not narrow the drainage pathway sinusitis with a sphenoethmoid pattern : the sphenoethmoid recess is obstructed by thickened mucosa ( asterisks ) , both the small left sphenoid sinus and the posterior ethmoid cells are opacified . note the basal lamella ( arrows ) clearly demarcating the anterior ethmoid from the inflamed posterior ethmoid cells . secretions are also retained within the pneumatised vertical lamella of the middle turbinate ( arrowhead ) ostiomeatal complex pattern : the middle meatus ( asterisk ) and anterior ethmoid cells are occupied by tissue , the medial orbital wall appears slightly displaced ( arrows ) . mucus drainage from the ethmoid infundibulum and frontal recess is simultaneously hindered producing frontal and maxillary sinusitis ( io infraorbital cell ) nasal polyposis : diffuse bilateral thickening of the mucosa investing the middle meati , the middle turbinate , and the anterior and posterior ethmoid cells . retained secretion can be seen within the blocked maxillary and sphenoid sinuses the sporadic pattern is a large box in which several different conditions are stored , such as isolated mucosal thickenings , retention cysts , antrochoanal polyp , silent sinus syndrome , odontogenic sinusitis and mucocele . isolated mucosal thickenings are incidentally found in a large number of msct performed for non - sinonasal pathological conditions . as a general rule , such mucosal thickening is unrelated to clinical symptoms , and when seen in the maxillary sinus and ethmoid cells , it does not need to be reported unless the mucosal thickness exceeds 4 mm and 2 mm , respectively . on the other hand , the mucosal lining of the frontal and sphenoid sinuses should not be visible on msct under normal conditions . retention cysts are submucosal ( pseudo-)cystic lesions that may have serous ( fluid accumulation in the submucosa ) or may be of a mucous nature ( blockage of a submucosal mucus gland ) . at imaging , they display a convex shape and sharply demarcated borders , and are attached to a sinus wall ; thus , they do not change with the patient s decubitus . they have little clinical relevance unless large enough to hinder mucus drainage ( fig . 15a retention cyst , displaying typical smooth and convex borders , partially fills the maxillary sinus cavity a retention cyst , displaying typical smooth and convex borders , partially fills the maxillary sinus cavity antrochoanal polyp is a relatively easy diagnosis , based on the pattern of growth ( from the maxillary sinus to the nasal fossa and farther to the choana ) and nature ( cystic appearance ) of this generally isolated polypoid lesion . though much rarer , spheno- and ethmoidochoanal variants are also reported [ 14 , 15 ] ( fig . 16antrochoanal polyp : the polypoid lesion arises from the right maxillary sinus , and extends through an accessory ostium ( arrows ) into the nasal fossa , reaching posteriorly the nasopharynx through the choana antrochoanal polyp : the polypoid lesion arises from the right maxillary sinus , and extends through an accessory ostium ( arrows ) into the nasal fossa , reaching posteriorly the nasopharynx through the choana silent sinus syndrome refers to an obstructed maxillary sinus , occupied by inflamed mucosa and secretions , which is shrunken with depression of the orbital floor causing exophthalmia . it is called silent sinus syndrome when clinically there is no clue as to the maxillary sinus disease , only being discovered on an imaging study . interestingly , antrochoanal polyp and silent sinus syndrome share the same pathophysiological mechanism , i.e. maxillary sinus blockage . in antrochoanal polyp , an increased intrasinusal pressure ( due to partial ostial obstruction with unidirectional flow of air through the ostium into the sinus ) is the trigger to force the expulsion of an intramural cyst through an accessory ostium . in silent sinus syndrome , conversely , complete sinus blockage and progressive resorption of air within the cavity leads to a pressure drop , orbital floor depression and sinus contraction ( fig . 8) . 17 ) , is heralded by a combination of signs , namely isolated maxillary sinusitis , detection of periradicular inflammatory lesions , possibly with surrounding bone destruction , and sometimes retention within the sinus cavity of calcifications , or even hyperdense material , with a density similar to dental filling material . the differential diagnosis between dentogenic sinusitis and fungal infection ( see below ) may be difficult on imaging studies . 17odontogenic sinusitis . a large area of bone resorption is seen in the alveolar ridge , around the root of a molar tooth ; the floor of the maxillary sinus is interrupted , the sinus filled by inflammatory material . the small calcifications found along the ethmoid infundibulum and in the anterior ethmoid ( arrows ) were proven to be bone fragments , possibly transported by mucociliary clearance odontogenic sinusitis . a large area of bone resorption is seen in the alveolar ridge , around the root of a molar tooth ; the floor of the maxillary sinus is interrupted , the sinus filled by inflammatory material . the small calcifications found along the ethmoid infundibulum and in the anterior ethmoid ( arrows ) were proven to be bone fragments , possibly transported by mucociliary clearance finally , chronic sinus drainage obstruction may end up in mucocele formation : a mucocele is an epithelium - lined mucus collection completely filling a sinus cavity ; while the secretions accumulate , pressure within the cavity increases , inducing sinus expansion and bone remodelling . 18 ) are more frequent in the fronto - ethmoid region but probably more dangerous in the sphenoid sinus , where sinus expansion may result in severe damage to the optic nerve .
fig . 18the right middle turbinate is swollen by a sharply defined lesion exhibiting spontaneously high density on msct , and showing different fluid components on mri . absence of solid , enhancing areas confirms mucocele within a pneumatised middle turbinate the right middle turbinate is swollen by a sharply defined lesion exhibiting spontaneously high density on msct , and showing different fluid components on mri . bone remodelling is a broad definition that encompasses the whole spectrum of alterations that may accompany the aforementioned mucosal changes in chronic rhinosinusitis and nasal polyposis . as described by giacchi et al . , bone remodelling is an adaptive process , in which osteoblasts and osteoclasts can be variably involved , that can be triggered by mechanical factors ( pressure , in nasal polyposis and mucocele ) as well as inflammatory mediators or infectious agents ( bacterial invasion of periosteum and bone ) . the prevalence of osteoclastic activity provokes bone resorption and demineralisation ; this is mostly seen in thin bone structures subject to chronic pressure . thinning and displacement of ethmoid septa or the medial wall , as well as truncation of the middle turbinate are , for example , frequent findings in nasal polyposis ; similarly , the expanded sinus walls of mucoceles are often thinned if not totally demineralised . on the other hand , osteoblastic activity manifests itself as thickening and sclerosis of bone ( osteitis ) , more typically encountered in thicker bone structures , such as the walls of the maxillary sinus and sphenoid sinus . as expected , all the described bone changes can be present simultaneously in the same patient , particularly if affected by nasal polyposis and fungal infections ( see below ) ( fig . 19demineralisation and distortion of thin bony laminae ( middle turbinates and ethmoid labyrinth ) and sclerosis of thicker bone structures ( sphenoid sinus ) are present simultaneously in a patient with diffuse nasal polyposis demineralisation and distortion of thin bony laminae ( middle turbinates and ethmoid labyrinth ) and sclerosis of thicker bone structures ( sphenoid sinus ) are present simultaneously in a patient with diffuse nasal polyposis some of the imaging patterns of chronic rhinosinusitis already described ( such as the ostiomeatal pattern and mucocele ) can also be encountered as indirect signs of the presence of a neoplasm either benign or malignant . furthermore , extensive nasal polyposis may completely obstruct sinonasal cavities , severely distorting the normal anatomy . patients examined for rhinosinusitis undergo imaging applying a standard msct protocol , without contrast agent injection , and using bone algorithm reconstructions alone . as a consequence , the major concern for radiologists ( particularly those not routinely involved in sinonasal imaging ) is how to safely rule out a neoplasm harboured amid inflammatory changes . some landmarks may be of help : chronic rhinosinusitis ( and nasal polyposis ) are much more commonly bilateral . whenever a ostiomeatal pattern is seen as unilateral disease , the presence of a neoplasm in the middle meatus should be considered , and endoscopic assessment of the region should be prompted . neoplastic lesions , even when benign , tend to behave more aggressively towards bone , causing more pronounced displacement , erosion and destruction . in chronic rhinosinutis and nasal polyposis bone is remodelled , distorted , demineralised but , as a general rule , not fragmented . mucocele is emblematic : frequently the line of demarcation between demineralised and normal bone is represented by a blunt but sharp border , whereas in neoplasms the border is ill - defined and spiculated . density measurements are not of great help , neither for the differentiation between tumour and polyp ( both are within the solid range of densities ) nor to discriminate tumour from mucocele ( which may exhibit high density if secretions are desiccated ) . in some cases , mri may add information ( polyps are generally more t2 hyperintense than most malignant tumours ; inverted papilloma , the most common benign nasosinusal tumour , may display the almost pathognomonic cerebriform pattern ) . however , the bottom line is that in any unclear case an endoscopic examination should be performed , and a biopsy obtained ( fig . 20the left ostiomeatal complex is occupied by solid tissue , the anterior ethmoid cells , the maxillary and frontal sinus are completely opacified . the absence of inflammatory changes on the right side as well as the displacement and destruction of the anterior ( arrowheadss ) and the posterior ( arrows ) maxillary sinus wall suggests the presence of a neoplasm . actually , mri shows a mass arising from the maxillary sinus and protruding into the nasal fossa , histologically proven as ameloblastoma the left ostiomeatal complex is occupied by solid tissue , the anterior ethmoid cells , the maxillary and frontal sinus are completely opacified . the absence of inflammatory changes on the right side as well as the displacement and destruction of the anterior ( arrowheadss ) and the posterior ( arrows ) maxillary sinus wall suggests the presence of a neoplasm . actually , mri shows a mass arising from the maxillary sinus and protruding into the nasal fossa , histologically proven as ameloblastoma fungal infection is rather common , given the ubiquitous presence of fungal agents in the environment and the high frequency of colonisation of sinonasal mucosa ; the role of fungi in promoting chronic rhinosinusitis has been suggested . fungal infections may be classified as either non - invasive or invasive forms , according to the absence or presence of invasion of mucosa , submucosa , bone and vessels by hyphae . the non - invasive form occurs in immunocompetent subjects and may further be classified as fungus ball and eosinophilic rhinosinusitis ; these forms are seen in patients with chronic rhinosinusitis , and are non - responsive to medical and/or surgical treatment . on msct , non - invasive fungal infection is suspected whenever spontaneously hyperdense material is detected within the sinus cavities . in the case of a fungus ball , such hyperdensity is due to a high content of heavy metals in the hyphae , but small calcifications can also be seen . maxillary fungal sinusitis may also be caused by the presence of intrasinusal dental filling material , which appears extremely hyperdense . in 80% of cases , 21 ) . in eosinophilic rhinosinusitis , besides fungal hyphae , the accumulation of dense eosinophilic mucin may account for the detection of sinus hyperdensities , which are scattered in multiple sinus cavities , uni- or bilaterally . mri can be misleading in both conditions , because heavy metal and calcium , on the one hand , and the tight packing of fungal material within sinuses , on the other hand , produce very hypointense signal which , particularly on t2 sequences , can be as low as the signal void of air ( fig . a , b the maxillary sinus is filled , at the periphery , by hypodense thickened mucosa and contains , in the centre , a calcification ; dense sclerosis of the posterolateral wall secondary to chronic inflammation ( arrows ) . c , d chronic rhinosinusitis with polypoid thickening of the mucosa and hyperdense material scattered in all sinus cavities : this pattern suggests allergic fungal rhinosinusitis . e , f on mri eosinophilic mucin displays t2 hypointense signal comparable with air : the t1 intermediate signal along with bone remodelling and sinus expansion ( arrowheads ) help to make the diagnosis of allergic fungal rhinosinusitis non - invasive fungal rhinosinusitis . a , b the maxillary sinus is filled , at the periphery , by hypodense thickened mucosa and contains , in the centre , a calcification ; dense sclerosis of the posterolateral wall secondary to chronic inflammation ( arrows ) . c , d chronic rhinosinusitis with polypoid thickening of the mucosa and hyperdense material scattered in all sinus cavities : this pattern suggests allergic fungal rhinosinusitis . e , f on mri eosinophilic mucin displays t2 hypointense signal comparable with air : the t1 intermediate signal along with bone remodelling and sinus expansion ( arrowheads ) help to make the diagnosis of allergic fungal rhinosinusitis invasive fungal infection can be divided into an acute invasive form and a chronic invasive form , and both usually occur in immunocompromised subjects ( diabetes mellitus with or without ketoacidosis , organ transplantation , chemotherapy , haematological diseases , hiv infection ) ; the chronic invasive form has also been reported in immunocompetent hosts . sinus hyperdensities are not common in invasive mycoses , bone destruction with invasion of adjacent anatomical structures ( orbit , skull base , cavernous sinus , pterygopalatine fossa , periantral fat tissue ) are the most significant imaging findings in both forms . whilst the differential diagnosis between invasive and non - invasive forms is rather straightforward , the discrimination between acute invasive and chronic invasive is very difficult at imaging ; this differentiation is mainly based on the clinical setting , as the acute invasive form is rapidly progressive and bears a dismal short - term prognosis ( up to 50 - 80% mortality ) . although msct equipment is more readily accessible in nearly every institution , invasive mycoses highly benefit from the high contrast resolution of mri to provide thorough assessment of intracranial and cavernous sinus involvement ( fig . 22 ) . chronic invasive mycosis invading the maxillary sinus mucosa , destroying the pterygoid plates and posterior maxillary sinus wall ( arrows ) , and invading the infratemporal fossa mri se t1 before ( left ) and after ( right ) contrast application . chronic invasive mycosis invading the maxillary sinus mucosa , destroying the pterygoid plates and posterior maxillary sinus wall ( arrows ) , and invading the infratemporal fossa interpretation of the msct examinations of patients affected by chronic rhinosinusitis should always be aimed at answering the key questions asked by clinicians . assessment of location and extent of the disease , along with drainage pathway impairment , contributes to selecting candidates for surgical treatment . detailed reporting on anatomical variants allows the best surgical approach to be identified and the risk of iatrogenic complications to be decreased . finally , accurate evaluation of any suspect msct findings helps to identify those patients in whom chronic rhinosinusitis is the secondary effect of a neoplasm obstructing the sinus drainage pathways . | the complex regional anatomy of the nose and paranasal sinuses makes the interpretation of imaging studies of these structures intimidating to many radiologists .
this paper aims to provide a key to interpretation by presenting a simplified approach to the functional anatomy of the paranasal sinuses and their most common ( and most relevant ) variants .
this knowledge is basic for the full understanding of chronic rhinosinusitis and its computed tomography ( ct ) patterns . as fungal infections
may be observed in the setting of chronic rhinosinusitis , these are also discussed .
chronic sinus inflammation produces bone changes , clearly depicted on ct images .
finally , clues to suspecting neoplastic lesions underlying inflammatory sinus conditions are provided . |
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according to the previously conducted studies around the world , musculoskeletal disorders ( msds ) are the most prevalent disorders among computer operators . the prevalence of the neck and shoulders pain is reported to be high , in comparison with the pains in the other limbs that leading to sickness absence and disability , decreasing work efficiency , and extensive costs upon the organizations and societies . as a matter of fact , developing appropriate programs and prevention of work - related msds among workers is a national precedence in several countries . however , predictors of these diseases are relatively weak . in addition , previously conducted studies have indicated that the related factors of persistent neck / shoulder pains can be different from the etiological factors . in a study conducted by grooten et al . , it is reported that merely almost of only one - third of those who suffer from the neck / shoulder pains could release from these pains after 5 - 6 years . although the relationship between msds and both individual and work - related factors have been studied and confirmed through various researches , these recognized risk factors do not clarify unusual progressive modifications that have been detected in chronic pains and disabilities attributed to the prevalent msds . in 1980s , for instance , a disability resulted from arm pain became epidemic in australia , which could not be found in the other countries with similar work conditions . furthermore , rate of disability due to back pain increased during the years of 1953 - 1992 in britain , although the required physical load at work was largely decreasing . these findings have encouraged the hypothesis that the progress and persistence of nonspecific msds and related disabilities are influenced by culturally - determined health beliefs , in addition to mental health and physical activities . furthermore , several observations have been conducted which support the role of somatization tendency ( i.e. , a ( nonmusculoskeletal symptoms ) on msds . in a longitudinal study conducted by solidaki et al . among 518 nurses , office workers and postal clerks in crete , it was found that persistence of msds significantly associated with somatising tendency , physical loading of work , and beliefs about work - causation of pain with odds ratio ( or ) ( 95% confidence interval [ ci ] ) of 2.6 , 2.3 and 1.9 respectively . ryall et al . in their studies on patients , who were receiving primary care and physiotherapy services , found that fear - avoidance beliefs and mental health are prognostic factors for arm pain . in addition , based on the study of warnakulasuriya et al . among 852 computer operators , sewing machinists , nurses and postal workers in sri lanka , low mood and tendency to somatize were identified to be constantly associated with musculoskeletal pains . coggon et al . , indicated that somatizing tendency , health beliefs , and low mood could influence the persistence of msds and related disabilities more than the rate of acute complaints . this study conducted considering above - mentioned findings , points and international differences in the prevalence of msds and related disabilities among occupational groups who are carrying out similar tasks , which is not completely clarified by the individual and psychosocial risk factors . therefore , this study is aimed to assess the relationship of these new factors with persistence of neck / shoulder pains among computer office workers in shahroud , iran . all eligible computer office workers ( n = 182 ) who were employed in four different universities of shahroud university of medical sciences and its subsidiary units and hospitals , shahroud university of technology , shahroud university of quranic sciences and shahroud azad university and its subsidiary hospital were selected and followed - up for 1-year . inclusion criterion was at least 1-year experience of being a computer operator with at least 4 h / day of working with computer . a standardized cultural and psychosocial influences on disability questionnaire completed at the baseline ( from july 2008 to march 2009 ) and 1-year later at follow - up was applied to collect the required data . considering the base - line questionnaire , the first section was about the individual characteristics ( e.g. gender , age , education etc . ) . for the second one , physical and psychosocial work - related factors ( e.g. , using a computer keyboard for longer than 4 h / day , manual lifting of weights 25 kg , repeated movements of the wrist or fingers for longer than 4 h in an average working day , time pressures , control at work , support from colleagues or supervisor , job satisfaction , decision latitude , job security , etc . ) were included . in the third section , 14 questions were included regarding the neck and shoulder pains ( elements of nordic questionnaire ) and their disabilities for getting dressed and doing normal household jobs . furthermore , a question about the expectation of pain ( i.e. , if you expect your neck / shoulders pain would be a problem for you in next 12 months too ? ) was added to this section . the next section , included with four questions about other people 's pain ( e.g. , do you know anyone who has had neck pain in the past 12 months at work or outside ) . in the section five , each worker was faced with five statements taken from fear - avoidance beliefs questionnaire which were about their beliefs regarding the causation and prognosis of neck / shoulder pains . three of the statements applied in this section were as the following : how strongly the participant agreed with statements of the form , your work may cause you to develop musculoskeletal pain , and rest is needed to get better these problems . the answers were categorized as agree in the cases that the one agrees completely with the statement or tended to agree . in the sixth section , seven questions ( components of the brief symptom inventory ( bsi ) questionnaire ) , ( faintness or dizziness , pains in the heart or chest , nausea or upset stomach , trouble getting breath , numbness or tingling in parts of your body that had been distressing during the past 7 days regarding the somatizing tendency ) were applied . mental health by applying the relevant subscale of the short form-36 questionnaire with five questions was the subject of the last section . some of the questions included in that section are as the following : during the past month were you a happy person ? , have you felt calm and peaceful ? and have you been a very nervous person ? . validity and reliability of these questionnaires have confirmed in many studies . in somatization tendency section , each symptom was scored 0 - 4 , depending on the distress it caused . furthermore , the optional answers for mental health section were as the followings : all of the time , most of the time , a good bit of the time , some of the time , a little of the time , none of the time . the scores attributed to these answers ranged 0 - 6 . in somatization tendency and mental health section of questionnaire scores were summed and subjects were categorized into three bands , representing approximate thirds of the distribution ( denoted low , middle , and high ) for the whole sample . at the base - line , the participants were asked about pains in their neck / shoulders lasted for at least a day over the past 12 months . the follow - up questionnaire was shorter and after an interval of 12 months asked about pain in the last 1 month . participants , who had declared their agreement for take part at follow - up , filled this questionnaire . the original questionnaire by the author was translated from english to farsi , and then by bilingual person it was back translated to english , also confirmed by occupational medicine specialists . the pilot study was conducted among 60 workers who were not included in this study in order to test whether the farsi version of the questionnaire could be satisfactorily understood and completed . reliability of the questionnaire was tested with chronbach alphas of 0.76 . in this study , neck / shoulder pains , was defined as the pains in the neck and/or either the shoulders . furthermore , reported neck / shoulder pain lasting for at least a day over the previous 12 months at baseline and continuing presence of pain lasting for at least a day over 1-month at follow - up defined as persistent pain . ethical approval required to conduct this study was applied and received from the research committee of shahroud university of medical sciences . all eligible computer office workers ( n = 182 ) who were employed in four different universities of shahroud university of medical sciences and its subsidiary units and hospitals , shahroud university of technology , shahroud university of quranic sciences and shahroud azad university and its subsidiary hospital were selected and followed - up for 1-year . inclusion criterion was at least 1-year experience of being a computer operator with at least 4 h / day of working with computer . a standardized cultural and psychosocial influences on disability questionnaire completed at the baseline ( from july 2008 to march 2009 ) and 1-year later at follow - up was applied to collect the required data . considering the base - line questionnaire , the first section was about the individual characteristics ( e.g. gender , age , education etc . ) . for the second one , physical and psychosocial work - related factors ( e.g. , using a computer keyboard for longer than 4 h / day , manual lifting of weights 25 kg , repeated movements of the wrist or fingers for longer than 4 h in an average working day , time pressures , control at work , support from colleagues or supervisor , job satisfaction , decision latitude , job security , etc . ) were included . in the third section , 14 questions were included regarding the neck and shoulder pains ( elements of nordic questionnaire ) and their disabilities for getting dressed and doing normal household jobs . furthermore , a question about the expectation of pain ( i.e. , if you expect your neck / shoulders pain would be a problem for you in next 12 months too ? ) was added to this section . the next section , included with four questions about other people 's pain ( e.g. , do you know anyone who has had neck pain in the past 12 months at work or outside ) . in the section five , each worker was faced with five statements taken from fear - avoidance beliefs questionnaire which were about their beliefs regarding the causation and prognosis of neck / shoulder pains . three of the statements applied in this section were as the following : how strongly the participant agreed with statements of the form , your work may cause you to develop musculoskeletal pain , and rest is needed to get better these problems . the answers were categorized as agree in the cases that the one agrees completely with the statement or tended to agree . in the sixth section , seven questions ( components of the brief symptom inventory ( bsi ) questionnaire ) , ( faintness or dizziness , pains in the heart or chest , nausea or upset stomach , trouble getting breath , numbness or tingling in parts of your body that had been distressing during the past 7 days regarding the somatizing tendency ) were applied . mental health by applying the relevant subscale of the short form-36 questionnaire with five questions was the subject of the last section . some of the questions included in that section are as the following : during the past month were you a happy person ? , validity and reliability of these questionnaires have confirmed in many studies . in somatization tendency section , each symptom was scored 0 - 4 , depending on the distress it caused . furthermore , the optional answers for mental health section were as the followings : all of the time , most of the time , a good bit of the time , some of the time , a little of the time , none of the time . the scores attributed to these answers ranged 0 - 6 . in somatization tendency and mental health section of questionnaire scores were summed and subjects were categorized into three bands , representing approximate thirds of the distribution ( denoted low , middle , and high ) for the whole sample . at the base - line , the participants were asked about pains in their neck / shoulders lasted for at least a day over the past 12 months . the follow - up questionnaire was shorter and after an interval of 12 months asked about pain in the last 1 month . participants , who had declared their agreement for take part at follow - up , filled this questionnaire . the original questionnaire by the author was translated from english to farsi , and then by bilingual person it was back translated to english , also confirmed by occupational medicine specialists . the pilot study was conducted among 60 workers who were not included in this study in order to test whether the farsi version of the questionnaire could be satisfactorily understood and completed . reliability of the questionnaire was tested with chronbach alphas of 0.76 . in this study , neck / shoulder pains , was defined as the pains in the neck and/or either the shoulders . furthermore , reported neck / shoulder pain lasting for at least a day over the previous 12 months at baseline and continuing presence of pain lasting for at least a day over 1-month at follow - up defined as persistent pain . ethical approval required to conduct this study was applied and received from the research committee of shahroud university of medical sciences . in order to investigate the associations between persistence of neck / shoulder pains and all of the variables , initially univariate logistic regression was applied . then in order to investigate the simultaneous relationships between these variables and the persistence of neck / shoulder pains ( i.e. , with the aim of effects of likely confounding factors can be controlled ) , all of the variables in the former process with age , job experience and educational level were entered into the multiple logistic regression analysis model . and , they were summarized as ors with associated 95% cis . the level of significance was set up at p < 0.05 . for data analysis , from 208 computer office workers employed in shahroud universities , 182 ( response rate 88% ) completed the questionnaire at the baseline and 91.2% ( 166 ) of them participated at follow - up in 1-year later . characteristics of the selected participants at the base - line and follow - up are summarized in table 1 . characteristics of study population at baseline and follow - up mean age of the participants and weekly work hours were respectively 32.1 6.7 ( mean standard deviation [ sd ] ) years and 47.4 8.2 ( mean sd ) . 50% ( n = 81 ) of the study population had longer than 5 years experience of work . most of the participants ( 64.8% ) were females and 68.3% reported working under time pressure . in comparison with the females , the males significantly had more second jobs ( p < 0.001 ) . at the baseline of the study , 54.9% ( 100 ) of computer operators reported neck / shoulder pains at previous 12 months . prevalence of neck / shoulder pains among the females ( 63.6% ) significantly ( p < 0.002 ) was more than the males ( 39.1% ) . 57 ( 34.3% ) of the participants who reported pains at the baseline , reported the pains 1-year later at follow - up . persistence of neck / shoulder pains also among females was greater than males ( 40.4% vs. 22.8% ) . however , based on the results derived from the follow - up , disability due to neck / shoulder pains was found to be higher among the females than the males . disability in getting dressed was 57.7% among the females , in comparison with 28.6% found among the males . furthermore , disability in performing the chores was 92.2% and 42.9% in the female and male participants . referring to physician was 19 ( 57.6% ) and the number of males who were visited by physicians was higher than the females ( 57.1% vs. 44.4% ) . sickness absence due to neck / shoulder pains was just reported among the female participants at follow - up ( 25% ) . based on the results from surveying the related risk factors for persistent neck / shoulder pains which was conducted with using univariate logistic regression , significant relationships between persistent neck / shoulder pain and somatization tendency ( p = 0.0 ) , mental health ( p = 0.002 ) and negative expectation of pain ( p = 0.003 ) were found . the or of this outcome was 5.9 times more in those categorized as the middle and high of somatization tendency , in comparison with the low category of somatization tendency and 3.2 times more in those with low and middle mental health scores versus high . furthermore , persistent of neck / shoulder pains among participants with negative beliefs about expectation of pain was 4 times more than others havent this belief . all variables in the former process with age , job experience and educational level were entered into the logistic regression analysis model . finally , associated risk factors of persistent neck / shoulder pains among computer office workers were found to be somatization tendency ( p = 0.01 ) , and negative expectation of pain ( p = 0.002 ) [ table 2 ] . thus , after adjustment for confounding variables the or for persistent neck / shoulder pain was 6.5 ( 1.6 - 27.4 ) in the middle and high versus the low category of somatization tendency score and 8.3 ( 2.1 - 32.9 ) in those with negative beliefs about expectation of pain . it means persistent of neck / shoulder pain among participants with middle and high score of somatization tendency is 6.5 times more than others with low score . participants with negative beliefs about expectation of pain 8.3 times more exposed to these persistent pains . in our study , although no associations were found between persistent neck / shoulder pain and belief about work - relatedness of neck / shoulder pain and avoidance from physical activity in these pains , result showed these beliefs 1.8 times increase risk of persistent pain . physical , organizational , beliefs , somatization tendency and mental health , risk factors for neck / shoulder disorders adjusted for age , gender , and education this study did nt show significant association between individual factors includes work experience and work hours per week with persistent neck / shoulder pain , although work experience increases these pains ( or = 1.5 ) . furthermore , physical and psychosocial risk factors includes , lifting weights of 25 kg or more by hand , working with hands above shoulder height , working under pressure to complete tasks by a fixed time , deciding at work , support from colleagues / supervisor , security at work did not indicate significant association with persistent neck / shoulder pain . on basis of the results , knowing someone at work and outside with pain in the neck , arm , shoulder and mental health did not indicate significant association with persistent neck / shoulder pain , but theses pains among workers with low and middle score of mental health were 2.3 times more than high score . in this study , prevalence of neck or shoulder pains among computer operators was found to be 54.9% , which is < 63% reported in a similar study conducted in usa , and more than the results of studies conducted in nicaragua ( 30% upper extremely pain ) and the netherlands ( neck / shoulder , arm 54% ) . in a cross - sectional study performed by choobineh et al . , among computer operators working in banks of shiraz , iran , neck and shoulder pains with 59.6% and 58.2% , respectively , in previous 12 months of frequency were the most prevalent reported pains , which are higher than the results of the present study . some of the other studies have indicated a range of 10 - 62% of neck / shoulder pains among computer users . based on the results , 34.3% of the subjects which reported neck / shoulder pains at base - line showed also persistence of these pains at follow - up , which is less than some studies . in a study conducted in germany , 41% of the office workers reported persistence of shoulder pains after 1-year follow - up . juul - kristensen and jensen , indicated in their studies that 61% of office workers in 11 danish companies showed the symptoms of persistence of neck / shoulder symptoms in days after 1-year follow - up while persistence of low back or elbow / hand region , were 53% and 41% , respectively . these differences in prevalence and persistence of neck / shoulder pains could be related to different cultures , different compensation and insurance systems . in this study , prevalence and persistence of neck / shoulder pains among female computer operators were found to be higher than the males which are consistent with the results of several previously conducted studies . repetitive work with not as much of rest , increased contact with etiological causes and gender imbalance in domestic work are interlinking factors that explain gender differences in neck / shoulder pains . our results showed that the persistence of neck / shoulder pains at follow - up was strongly influenced by somatization tendency and negative expectation of pain at the baseline , raising risks some 6.5- and 8-fold . however , in a study conducted in australia on three groups of workers including office workers , it was indicated that the relations between msds and somatization tendency was weak . at the present , cognitive behavior therapy is suggested for somatizing tendency and negative belief about pain in many studies . in a study in england among 1798 participants in working age , persistent arm pain was significantly prevalent in persons who believed that arm pain would be a problem in future 12 months which is consistent with our findings . however , nocebo effects create completely from psychological bases and in many studies are related to anxiety ; it can be either psychological or physiological . studies have shown brain processes of pain and the central nervous system , especially nociceptive portions of insula , cingulate and thalamus may be influenced by expectations as psychological factor . although in this study mental health was found to have significant relation with neck / shoulder pains , persistent of neck / shoulders pains among persons with low and medium score of mental health were 2.3 times more than others with high score . in study by palmer et al . , persistent arm pain in persons with low score of mental health were significantly prevalent . , among workers who were performing monotonous and repetitive work ; it was shown that neck / shoulder disorders are strongly associated with decreasing of health - related quality of life . however , in this study , beliefs about causation and prognosis of neck / shoulder pains were not found to have significant relationships ; it was greater among participant with these beliefs . in studies conducted by gimeno et al . health beliefs about prognosis of pain were associated with the change from nondisabling to disabling msds ( or 3.7 ) . furthermore , the results of this study showed that refereeing to physician for neck / shoulder pains was 57.6% and sickness absence due to neck / shoulder pain was 25% which both are higher than some previously conducted studies ( 21 - 38% ) and ( 13 - 21% ) , respectively . it may be related to sickness absence compensation , work conditions , and belief about recovery of pain . this study introduces several potential psychological risk factors , which could be helpful for healthcare in assessing the prognosis of patient with neck / shoulder pain . our study confirms the importance of somatizing tendency and negative expectation about pain as risk factors for persistence of neck / shoulder pain among computer office workers . this finding is novel in our country and needs to be independently replicated in other occupations . however , it suggests that interventions with the aim of reducing the persistence of neck / shoulder pain should focus on recognizing somatization tendency by clinicians and modifying negative expectation about neck shoulders pain and generally ergonomic intervention should be account not only for prevention of injury but also for pleasing workplaces and reducing psychological risk factors . this study was self - reported questionnaire based survey and this is the limitation of this work . however , standardized questionnaire of many important risk factors , including physical , psychological , social and cultural , with follow - up design and high response rate are the points of strength in this study . future studies should consider the importance of all the risk factors during longer follow - up interval and workplace interventions about worker 's health beliefs and somatization tendency and cognitive behavior therapy . this study showed that neck / shoulder pains still pose a major problem among computer office workers and somatization tendency and negative expected pain at the baseline of the study are risk factors for persistent neck / shoulder pain after 1-year follow - up . this confirms similar studies in europe countries and importance of psychological factors on work related msd in recent years . | background : neck and shoulder pains are the prevalent complaints among computer office workers .
the purpose of this study was to assess the relationship of somatization tendency , expectation of pain , mental health and beliefs about causation of pain with persistence of neck / shoulder pains among computer office workers.methods:this research is a kind of prospective cohort study with 1-year follow - up .
it has done among all eligible computer office workers of shahroud universities ( n = 182 ) in 2008 - 2009 and 1-year later .
data were collected using the cultural and psychosocial influences on disability questionnaire .
multiple logistic regression analysis was used to analyze the data through spss ( p < 0.05).results : at the baseline 100 ( 54.9% ) of participants reported neck / shoulder pains and at follow - up 34.3% of them reported persistence pains .
significant relationships were found between persistence of neck / shoulder pains and negative expectation about pain in next 1-year p = 0.002 , ( odds ratio [ or ] = 8.3 , 95% confidence interval [ ci ] : 2.1 - 32.9 ) and somatization tendency p = 0.01 , ( or = 6.5 , 95% ci : 1.6 - 27.4).conclusions : pain expectation and somatization tendency recognized as associated risk factors of persistent neck / shoulder pain among computer operators .
this confirmed some other similar studies on work - related musculoskeletal disorders in europe countries in recent years . |
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airway remodeling is characterized by the accumulation of extracellular matrix ( ecm ) , such as collagen , and thickening of smooth muscle . fibrogenic cytokine transforming growth factor ( tgf- ) and asthma - related il-13 are crucial cytokines for synergistic airway remodeling . matrix metallopeptidase 9 ( mmp-9 ) , one of the extracellular proteases family members , mediates the degradation of the extracellular matrix during tissue remodeling . granulocyte basophils have been demonstrated to bind ige and perform essential roles of th2 cytokine - dependent immunity and allergic inflammation . basophils are rarely found in normal tissues . however , their number increases markedly at allergic inflammatory sites in the airways of asthmatic patients , especially during asthma exacerbation in response to allergen inhalation [ 46 ] . allergic asthma is characterized by the accumulation and infiltration of eosinophils in tissues mediated by the specific eosinophil chemokine eotaxin and vascular cell adhesion molecule ( vcam)-1 and intercellular adhesion molecule ( icam)-1 on epithelial cells , with subsequent release of granular toxic proteins such as eosinophilic cationic protein from eoisnophils . light ( lymphotoxin - related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells ) , also known as tumor necrosis factor superfamily ( tnfsf)14/cd258 , is one of the tnf family members . it is a homotrimer on the surface of several immune cells such as activated t and b cells . many members including tnf- , cd40 ligand ( cd40l ) , fas ligand ( fasl ) , tnf - related activation - induced cytokine ( trance ) , and light can be cleaved from cell surfaces , and their soluble forms have been reported to be involved in various physiological processes with broad biological functions [ 811 ] . since light is a membrane - expressed protein related to the membrane form of lymphotoxin ( lt) , it binds the herpesvirus entry mediator ( hvem ; tnfrsf14 ) and is also a shared ligand with membrane lymphotoxin for ltr [ 12 , 13 ] . light directly induces airway remodeling which is dependent on the induction of the fibrogenic cytokine transforming growth factor ( tgf)-. in mouse models of chronic asthma , pharmacological inhibition of light using a fusion protein between the igg fc domain and ltr can reduce lung fibrosis , smooth muscle / epithelial hyperplasia , and airway hyperresponsiveness via the suppression of the production of lung tgf- and il-13 , which are key cytokines in airway remodeling in humans . on the other hand in line with this , sputum light levels in asthmatic patients were found to correlate with decreased lung function . apart from light , anti - human b- and t - lymphocyte attenuator ( btla ) , an inhibitory receptor on t lymphocytes with similar t - cell inhibitory functions to cytotoxic t lymphocyte - associated antigen 4 ( ctla-4 ) and programmed death 1 ( pd-1 ) , is also the ligand of hvem [ 18 , 19 ] . although btla - hvem complexes have been shown to negatively regulate t - cell immune responses , the expression of btla on asthma - related basophils , eosinophils , and bronchial epithelial cells have not been investigated . recent mechanistic studies have shown that light can induce il-13 and tgf- release from esoinophils and alveolar macrophages , respectively [ 15 , 20 ] . eosinophils express hvem but not ltr , while light can induce mmp-9 release from macrophages via ltr . we have recently demonstrated the crucial roles of the interaction of basophils and eosinophils with bronchial epithelial cells in allergic asthma [ 22 , 23 ] . however , the precise role played by the elevated light in airway hyperresponsiveness is still unresolved , and the intracellular mechanisms by which light can activate bronchial epithelial cells interacting with basophils and eosinophils to release airway remodeling related molecules are not certain . since we hypothesize that light may play an immunological role in airway remodeling through the activation of the intercellular interaction between the granulocyte and airway epithelium , the aim of the present study was to investigate the effects of light on bronchial epithelial cells interacting with basophils / eosinophils and the underlying intracellular mechanisms . the recombinant human light / tnfsf14 was purchased from r&d systems ( minneapolis , mn , usa ) . ib phosphorylation inhibitor bay11 - 7082 , p38 mapk inhibitor sb203580 , c - jun n - terminal protein kinase ( jnk ) inhibitor sp600125 , extracellular signal - regulated kinase ( erk ) inhibitor u0126 , and pi3k inhibitor ly294002 were purchased from calbiochem corporation ( san diego , ca , usa ) . bay11 - 7082 , sb203580 , sp600125 , u0126 , and ly294002 were dissolved in 0.1% ( v / v ) dimethylsulphoxide ( dmso ) . purification of human basophils and eosinophils was performed according to our previous publications [ 2224 ] . fresh human buffy coat obtained from healthy volunteers of the hong kong red cross blood transfusion service was diluted with pbs and centrifuged using ficoll - paque plus solution ( ge healthcare corp . , piscataway , nj , usa ) and isotonic percoll solution ( density 1.082 g / ml ; ge healthcare ) for the purification of basophils and eosinophils , respectively . basophil - rich peripheral blood mononuclear cell ( pbmc ) fraction or eosinophil - rich granulocyte fraction was collected and washed twice with cold pbs containing 2% fetal bovine serum ( fbs ) ( invitrogen corp . , basophils and eosinophils were purified by negative selection using basophil isolation kit and anti - cd16 magnetic beads ( miltenyi biotec , bergisch gladbach , germany ) , respectively , using an ls+ column ( miltenyi ) within a magnetic field . with this preparation , the drop - through fraction contained purified basophils or eosinophils with a purity of at least 99% as assessed by giemsa staining solution ( sigma - aldrich corp . , st . louis , mo , usa ) together with specific basophil cell surface marker cd203c staining or hemacolor rapid blood smear stain ( e merck diagnostica , darmstadt , germany ) , respectively . the isolated basophils / eosinophils were cultured in rpmi1640 medium ( invitrogen ) supplemented with 10% fbs ( invitrogen ) . the above protocol using human basophils / eosinophils purified from human buffy coat was approved by the clinical research ethics committee of the chinese university of hong kong - new territories east cluster hospitals with written consent from all healthy volunteers of hong kong red cross blood transfusion service in accordance with the declaration of helsinki . the human bronchial epithelial cell line ( beas-2b ) was obtained from the american type culture collection ( atcc , manassas , va , usa ) . this cell line has been transformed by adenovirus 12-sv40 virus hybrid ( ad12sv40 ) and used widely as an in vitro bronchial epithelial cell model . beas-2b cells were grown in dulbecco 's modified eagle 's medium nutrient mixture f12 ( invitrogen ) with 10% fbs at 37c in a humidified 5% co2 atmosphere until confluence to cell monolayer . in coculture , the medium of beas-2b cells was replaced with rpmi-1640 medium containing 10% fbs ( invitrogen ) with or without basophils / eosinophils . for inhibition experiments , basophils / eosinophils and beas-2b cells were pretreated with signaling molecule inhibitors for 1 h before coculture and treatment by light . to prevent direct interaction between basophils / eosinophils and beas-2b cells in the coculture , transwell inserts ( pore size : 0.4 mm ) ( bd biosciences corp . , san jose , ca , usa ) confluent beas-2b cells and basophils / eosinophils were cultured together in the presence of transwell inserts , in which basophils / eosinophils and beas-2b cells were placed in the upper and lower compartment , respectively . concentrations of cytokines il-5 , il-6 , il-9 , il-13 , epidermal growth factor ( egf ) , vascular endothelial growth factor ( vegf ) , tgf- , and chemokine cxcl8 in the culture supernatants were measured using the human milliplex map kit assay reagent ( merck millipore corp . , billerica , ma , usa ) with bio - plex 200 suspension array system ( bio - rad laboratories , inc . , hercules , ca , usa ) . concentration of human mmp-9 in the culture supernatants was measured using the milliplex human mmp magnetic panel assay reagent ( merck millipore ) with bio - plex 200 suspension array system ( bio - rad ) . coculture of beas-2b cells ( 1 10 cells ) and basophils / eosinophils ( 3 10 cells ) or beas-2b cells alone ( 3 10 cells ) were maintained in a 24-well plate with transwell inserts ( pore size : 0.4 mm ) ( bd biosciences ) and stimulated with light ( 0100 ng / ml ) for 24 h. the basophils / eosinophils were removed and another batch of freshly isolated basophils / eosinophils ( 3 10 cells ) was then added into the adherent beas-2b cells for the adhesion analysis . cells were cultured in rpmi1640 medium containing 10% fbs and incubated at 37c in a humidified 5% co2 atmosphere for 1 h and digested with trypsin and resuspended in sheath fluid . basophils / eosinophils and beas-2b cells were analyzed separately based on the expression of specific basophilic cell surface marker cd203c in histograms and distinct forward light scatters ( fsc ) together with side light scatters ( ssc ) of eosinophils in dot plots using flow cytometry ( facscalibur flow cytometer , bd biosciences ) . the ratio of the measured number of adherent eosinophils / basophils onto beas-2b cells was calculated [ 2224 ] . to determine the expression of hvem , ltr , and intercellular adhesion molecule ( icam)-1 on the cell surface , adherent bronchial epithelial cells were harvested using cell dissociation solution . after blocking with 2% human pooled serum for 20 min at 4c and washing with pbs supplemented with 0.5% bovine serum albumin , cells were incubated with phycoerythrin ( pe)-conjugated mouse anti - human hvem antibody , pe - conjugated mouse anti - human ltr antibody , apc - conjugated mouse anti - human btla / cd272 antibody ( biolegend , inc . , san diego , ca , usa ) , pe - conjugated mouse igg1 isotype , fluorescein isothiocyanate ( fitc)-conjugated mouse anti - human icam-1 antibody , or fitc - conjugated mouse igg2a , isotype ( biolegend ) for 30 min at 4c in the dark . after washing , cells were subjected to flow cytometric analysis . to determine the intracellular expression of phosphorylated signaling molecules , after centrifugation , cells were permeabilized in ice - cold bd phosflow perm buffer for 30 min and then stained with mouse anti - human phosphorylated ( p ) p38 mapk , perk1/2 , pib , or mouse igg1 antibodies ( bd pharmingen corp . , san diego , ca , usa ) for 60 min followed by fitc - conjugated goat anti - mouse secondary antibody ( life technologies , carlsbad , ca , usa ) for another 45 min at 4c in the dark . cells were then washed , resuspended , and subjected to flow cytometric analysis . expression of surface molecules and intracellular phosphorylated signaling molecules of 5,000 viable cells was analyzed using flow cytometry ( bd facscalibur flow cytometer ) and presented as mean fluorescence intensity ( mfi ) . for the differential analysis of intracellular mapk and nuclear factor ( nf)-b activity of beas-2b cells , nonadherent basophils / eosinophils were separated from the adherent beas-2b cells by washing with pbs after different treatments . adherent beas-2b cells were then harvested using cell dissociation solution for the flow cytometric analysis of intracellular signaling molecules ( sigma aldrich corp . basophils / eosinophils and beas-2b cells were analyzed separately based on the expression of specific basophilic cell surface marker cd203c in histograms and distinct forward light scatters ( fsc ) together with side light scatters ( ssc ) of eosinophils in dot plots using flow cytometry ( facscalibur flow cytometer , bd biosciences ) [ 2224 ] . the statistical significance difference was determined by one - way analysis of variance ( anova ) or unpaired t - test . data were expressed as mean plus standard error of the mean ( sem ) from three independent experiments . any differences with p value < 0.05 were considered significant . when anova indicated a significant difference , bonferroni 's post hoc test was then used to assess the difference between groups . all analyses were performed using spss statistical software for windows ( version 16.0 ; spss inc . , chicago , il , usa ) . as shown in figures 1(a ) , 1(d ) , 1(g ) , 1(h ) , and 1(i ) , the proteins hvem , ltr , and btla were constitutively expressed on the cell surface of bronchial epithelial beas-2b cells but only hvem was observed to be expressed on basophils and eosinophils . results were similar to a previous report that eosinophils expressed cell surface hvem but not ltr ( figures 1(d ) and 1(e ) ) . consistent with a previous publication , a slight decrease in hvem expression on these cells was detected after light stimulation ( data not shown ) . since we observed that plasma light concentration of asthmatic patients using elisa could be up to 100 pg / ml , the local inflammatory concentration could be 101000 fold higher than the circulating levels . in order to mimic the inflammatory condition , we chose 1100 ng / ml concentration for the following in vitro studies that is also comparable to that adopted in a previous publication . figure 2(a ) shows that light ( 100 ng / ml ) could upregulate the cell surface expression of icam-1 on beas-2b cells alone . as shown in figure 2(b ) , the cell surface expression of icam-1 on beas-2b cells alone was significantly enhanced upon stimulation by light at high concentration ( 100 ng / ml ) ( p < 0.05 ) but not with low concentration ( 1 or 10 ng / ml ) . upon interaction with basophils , icam-1 level on beas-2b cells was also upregulated by light at 100 ng / ml only ( figure 2(b ) ) . however , light ( up to 100 ng / ml ) did not show any significant effect on the expression of icam-1 on basophils in the coculture with beas-2b cells ( figure 2(b ) , all p > 0.05 ) . in the coculture of eosinophils and beas-2b cells ( figure 2(c ) ) , light could significantly induce the expression of icam-1 on beas-2b cells ( light , 10 and 100 ng / ml ) and eosinophils ( light , 100 ng / ml ) ( all p < 0.05 ) . as shown in figures 2(d ) and 2(e ) , the increased cell number of basophils or eosinophils ( 0.3 103 10 cells ) could enhance the expression of icam-1 on beas-2b cells in coculture . moreover , the transwell insert could significantly downregulate the icam-1 expression on beas-2b cells in coculture ( all p < 0.05 ) . we did not observe any significant changes in cell surface expression of icam-1 on basophils or eosinophils alone upon treatment with light up to 100 ng / ml ( p > 0.05 ) ( data not shown ) . to further address the light - induced activation in cocultured beas-2b and basophils / eosinophils , we estimated the ability of activated beas-2b in adhesion with basophils / eosinophils ( figure 3 ) . an increased number of adherent basophils / eosinophils could only be observed in coculture upon treatment by 100 ng / ml light ( figures 3(a ) and 3(b ) , both p < 0.05 ) . figure 3(b ) shows that there was a moderate upregulation of eosinophils adhesion onto beas-2b cells when beas-2b cells alone were stimulated with light ( 100 ng / ml ) . the above results therefore were in concordance with the upregulated expression of adhesion molecule on beas-2b cocultured with basophils / eosinophils in the presence of light ( figure 2 ) . figures 3(c ) and 3(d ) showed that basophils / eosinophils and beas-2b cells could be analyzed separately based on the expression of specific basophilic cell surface marker cd203c on basophils ( figure 3(c ) ) and distinct forward light scatters ( fsc ) together with side light scatters ( ssc ) of eosinophils in dot plots ( figure 3(d ) ) using flow cytometry . the milliplex human cytokine / chemokine magnetic panel assay was used to measure the airway remodeling - related cytokines and chemokines , including il-5 , il-6 , il-9 , il-13 , egf , tgf- , vegf , and cxcl8 . as shown in figures 4(a ) and 4(b ) , light ( 100 ng / ml ) upon coculture with basophils , the induction of il-6 and cxcl8 by light ( 100 ng / ml ) was found to be significantly higher than those of beas-2b cells alone ( both p < 0.05 ) . however , light did not show any significant effect on the release of il-6 or cxcl8 from basophils alone , even at high concentration ( 100 ng / ml , figures 4(a ) and 4(b ) ) . figure 4(d ) shows that light ( 100 ng / ml ) could significantly promote the release of cxcl8 from eosinophils alone . light ( 10 ng / ml ) could further induce the release of il-6 and cxcl8 from coculture of eosinophils and beas-2b cells compared to eosinophils or beas-2b cells alone ( all p < 0.05 , figures 4(c ) and 4(d ) ) . in addition , the coculture of beas-2b cells with eosinophils , together with light stimulation ( 100 ng / ml ) , exhibited a synergistic effect on il-6 and cxcl8 production ( all p < 0.05 , figures 4(c ) and 4(d ) ) . the levels of il-5 , il-9 , il-13 , egf , vegf , and periostin were all undetectable using the same experimental conditions ( data not shown ) . moreover , there was induction of il-6 and cxcl8 production from the coculture beas-2b cells and eosinophils without light stimulation ( figure 4 ) . we also found no significant induction of tgf- in the coculture of beas-2b cells and basophils / eosinophils with or without light stimulation ( all p > 0.05 , data not shown ) . as shown in figure 5 , light ( 100 ng / ml ) could significantly induce the release of mmp-9 from beas-2b cells ( p < 0.001 ) . upon coculture with basophils , the induction of mmp-9 by light ( 100 ng / ml ) was significantly higher than control without light treatment ( p < 0.05 ) . however , light did not show any significant effect on the release of mmp-9 from basophils alone , even at high concentration ( 100 ng / ml , p > 0.05 ) . there was no significant induction of mmp-9 in eosinophils alone or the coculture of eosinophils with beas-2b cells with or without light stimulation ( data not shown ) . as shown in figures 6(a ) and 6(b ) , light ( 100 ng / ml ) could significantly activate erk and nf-b in eosinophils ( all p < 0.05 ) but not in basophils ( all p > 0.05 ) . figures 6(c ) , 6(d ) , and 6(e ) show that with the treatment of light ( 100 ng / ml ) , phosphorylation of p38 mapk , erk1/2 , and ib was significantly enhanced in beas-2b cells upon their coculture with eosinophils at 30 and 60 min , and erk1/2 was significantly phosphorylated even at 15 min . the p38 mapk and erk1/2 were significantly phosphorylated in beas-2b cells of the coculture of basophils and beas-2b cells at 30 and 60 min upon light ( 100 ng / ml ) stimulation ( figures 6(c ) and 6(d ) ) . the p38 mapk inhibitor sb203580 ( 7.5 m ) and erk inhibitor u0126 ( 10 m ) could significantly suppress the light - induced expression of icam-1 on beas-2b cells in their coculture with basophils ( figure 7(a ) ) and the release of il-6 , cxcl8 , and mmp-9 from the coculture ( figures 7(b ) , 7(c ) , and 7(d ) ) . the p38 mapk inhibitor sb203580 ( 7.5 m ) , erk inhibitor u0126 ( 10 m ) , and nf-b inhibitor bay11 - 7082 ( 1 m ) could significantly suppress the light - induced expression of icam-1 on beas-2b cells ( figure 7(e ) ) and the production of il-6 ( figure 7(f ) ) from the coculture of eosinophils and beas-2b cells . structural remodeling of the airway involves the accumulation of extracellular matrix proteins and thickening of smooth muscle . we found that allergic asthma - related basophils and eosinophils constitutively expressed hvem ( figure 1 ) , a ligand of airway remodeling cytokine light . bronchial epithelial beas-2b cells also expressed hvem , ltr , and another hvem ligand , btla . therefore , light can play immunomodulatory roles for the activation of basophils / eopsinophils interacting with bronchial epithelial cells in airway remodeling . the present in vitro study has shown that the tnf family member light could significantly promote the cell surface expression of adhesion molecule icam-1 , the release of airway - remodeling cytokine il-6 , chemokine cxcl8 , and extracellular protease mmp-9 from human bronchial epithelial cells upon the interaction with asthma - related basophils or eosinophils . since the increased cell number of basophils / eosinophils could enhance the expression of icam-1 on beas-2b cells , and the disruption of intercellular interaction using transwell inserts could significantly downregulate the icam-1 expression on beas-2b cells ( figures 2(d ) and 2(e ) ) , upregulated icam-1 expression on beas-2b cells was dependent on the direct interaction between basophils / eosinophils and beas-2b cells . as shown in figure 2(e ) , there was a remarkable increase in the icam-1 expression on beas-2b interacted with eosinophils after stimulated with light in the presence of the transwell insert , suggesting that the soluble factors were also associated with the regulation of icam-1 . since the direct interaction of icam-1 and cd18 has been shown to mediate eosinophil adhesion onto bronchial epithelial cells , the increased ability of beas-2b in adhesion with eosinophils could be partially explained by the upregulated icam-1 on beas-2b upon interaction with eosinophils with light stimulation . however , the elevated adhesion between beas-2b and basophils could also be observed , even the icam-1 expression was similar between ( i ) beas-2b coculturing with basophils using transwell inserts ( figure 2(d ) ) and ( ii ) beas-2b cells alone were stimulated by light ( figure 2(b ) ) , thereby suggesting that other factors in coculture , apart from the adhesion molecule icam-1 , were partly contributing to the intercellular adhesion . further study may be required to fully elucidate the biochemical mechanisms for the adhesion of basophils / eosinophils onto beas-2b cells . il-6 has been shown to enhance collagen synthesis , production of tissue inhibitors of metalloproteinases ( timps ) , and airway hyperresponsiveness [ 2830 ] . expression of il-6 in fibroblasts is correlated with fibrosis . by causing airway smooth muscle cell proliferation and migration , cxcl8 released from human bronchial epithelial cells upon leukotriene d4 stimulation is involved in epithelial - mediated asthmatic airway remodeling via the activation of egf receptor . mmp-9 is significantly increased in bronchoalveolar lavage fluid ( balf ) and sputum from patients with allergic asthma [ 33 , 34 ] . an elevated circulation level of mmp-9 is also found in patients suffering from asthma exacerbation . mmp-9-deficient mice challenged with ovalbumin show less peribronchial fibrosis and total lung collagen compared to ovalbumin - challenged wild type . together , the induced il-6 , cxcl8 , and mmp-9 suggested that light may play an important role in airway remolding via the activation of basophils and eosinophils interacting with bronchial epithelial cells . there are three isoforms of tgf- in the normal human lung , and tgf-1 is associated with bronchial epithelial cells , smooth muscle cells , fibroblast - like cells , and the airway extracellular matrix ( ecm ) [ 3841 ] . tgf-1 level is increased in balf of asthmatic patients , and asthmatic animal models also show increased levels of tgf-1 in balf and tissue [ 43 , 44 ] . mice treated with anti - tgf- antibody significantly reduce the deposition of peribronchial ecm , proliferation of airway smooth muscle cell , and mucus production in lung . tgf-1 plays an important role in the regulation of airway remodeling . in the present study , beas-2b cells were found to release tgf-1 ; however , the levels of tgf-1 seemed not to be influenced by light or the interaction with basophils or eosinophils ( data not shown ) . it has been shown that the matricellular protein periostin can interact with cell surface integrin molecules and can be involved in tissue development and remodeling [ 48 , 49 ] . however , in the present study , the level of periostin is undetectable by elisa ( data not shown ) . moreover , other airway remodeling - related cytokines and growth factors including il-5 , il-9 , il-13 , egf , and vegf could not be detected in the present study . the expression of these mediators should be further investigated in the future in vivo study using murine model to better elucidate the detailed effect of light on the interaction of basophils / eosinophils and bronchial epithelial cells in airway remodeling and inflammation . our previous study of airway inflammation has demonstrated that the induction of il-6 and ccl2 upon the interaction of basophils and bronchial epithelial cells under il-17a stimulation was differentially regulated by erk , jnk , p38 mapk , and nf-b pathways . in the present study to investigate the signaling pathways involved in the interaction of basophils / eosinophils and human bronchial epithelial cells upon light stimulation nf-b inhibitor bay11 - 7082 , erk inhibitor u0126 , and p38 mapk inhibitor sb203580 could differentially suppress light - induced icam-1 , il-6 , cxcl8 , and mmp-9 in the coculture of basophils / eosinophils and beas-2b cells ( figure 7 ) . together with the results in figure 6 regarding the light - mediated activation of erk , p38 mapk , and nf-b in eosinophils alone and beas-2b cells in coculture , the results indicated that the induction of icam-1 and release of airway remodeling cytokine il-6 , chemokine cxcl8 , and extracellular protease mmp-9 in light - activated coculture of basophils / eosinophils and beas-2b cells were differentially regulated by intracellular nf-b , erk , and p38 mapk pathways , probably via the regulation of downstream transcription factors and/or microrna [ 22 , 50 ] . these in vitro mechanistic results are actually in concordance with our previous published results that the expressions of cytokines / chemokines and adhesion molecules in the coculture of eosinophils / basophils and bronchial epithelial cells are differentially regulated by distinct activation profiles of signaling molecules [ 2224 ] . since targeting signaling molecules can be a novel strategy for the treatment of asthma , the potential cross - talk between different signaling pathways and the downstream molecular regulatory mechanisms awaits further studies . in summary , the expression of airway remodeling adhesion molecule icam-1 and the release of airway remodeling - related cytokine il-6 , chemokine cxcl8 , and extracellular protease mmp-9 and intercellular adhesion were significantly enhanced in the coculture of basophils / eosinophils and bronchial epithelial cells via the regulation of a distinct intracellular signal transduction mechanism . our previous studies have also shown that the interaction of bronchial epithelial cells and basophils / eosinophils can induce the release of a variety of inflammatory mediators involved in allergic asthma via the upregulated expression of adhesion molecules and the modulation of signaling pathways [ 22 , 23 ] . the release of mediators such as mmp-9 , il-6 , and cxcl8 upon the activation by light should contribute to airway remodeling . however , whether light signals through hvem on basophils / eosinophils and/or ltr on epithelial cells are the direct or indirect consequences involving other receptor - mediated mechanisms requires further investigation . nevertheless , the present cellular mechanistic results may somehow shed light on the potential therapeutic target for airway remodeling . | allergic asthma can cause airway structural remodeling , involving the accumulation of extracellular matrix and thickening of smooth muscle .
tumor necrosis factor ( tnf ) family ligand light ( tnfsf14 ) is a cytokine that binds herpesvirus entry mediator ( hvem)/tnfrsf14 and lymphotoxin receptor ( ltr ) .
light induces asthmatic cytokine il-13 and fibrogenic cytokine transforming growth factor- release from allergic asthma - related eosinophils expressing hvem and alveolar macrophages expressing ltr , respectively , thereby playing crucial roles in asthmatic airway remodeling . in this study , we investigated the effects of light on the coculture of human basophils / eosinophils and bronchial epithelial beas-2b cells .
the expression of adhesion molecules , cytokines / chemokines , and matrix metalloproteinases ( mmp ) was measured by flow cytometry , multiplex , assay or elisa .
results showed that light could significantly promote intercellular adhesion , cell surface expression of intercellular adhesion molecule-1 , release of airway remodeling - related il-6 , cxcl8 , and mmp-9 from beas-2b cells upon interaction with basophils / eosinophils , probably via the intercellular interaction , cell surface receptors hvem and ltr on beas-2b cells , and extracellular signal - regulated kinase , p38 mitogen activated protein kinase , and nf-b signaling pathways . the above results , therefore , enhance our understanding of the immunopathological roles of light in allergic asthma and shed light on the potential therapeutic targets for airway remodeling . |
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for the year 2012 , in brazil , it is an expected 6,110 new cases of laryngeal cancer are expected , with an estimated risk of 6 cases per 100 000 individuals . the laryngeal cancer is the sixth most frequent in the northeast region ( 4/100 000 ) . ) , seventh in the south region ( 9/100 000 ) and north regions ( 2/100 000 ) , occupies the seventh position ; eighth in the southeast region ( 8/100 000 ) , eighth , and ninth in the midwest region ( 5/100 000 ) , the ninth1 position . the therapeutic procedures indicated for patients suffering from laryngeal cancer in advanced stages is the total laryngectomy . despite his statement efforts to be very careful , that is , and operate only when it has there is a high degree of infiltration and involvement ( stages iii and iv)2
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4 , changes in chewing , deglutition , breathing , and speech5 presents serious complications as in the patient 's life , particularly in interaction with their peers . the self - image of laryngectomiyzed patients may be find themselves compromised , further affecting the quality of life in this population6
7 , and may also result in pain , postural changes , difficulty in performing daily tasks , and sleep disorders2
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12 . the communication is put effected into the society through various codes used by men individuals to express their views , and to represent things , beings , and ideas ; , and among them these codes , the most important is the language . many authors describe the impact of communication and adaptation after laryngectomy in surviving patients with survival13
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17 . the ability to communicate has a very strong association with improved quality of life13
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17 . however , other studies do not have point to report the existence of a correlation between quality of life and speech18 . while there is no consensus on the concept of quality of life , three 3 fundamental aspects related to its construct nature were obtained through the work of a group of scholars from different cultures . they are ( 1 ) subjectivity , ( 2 ) multidimensionality , and ( 3 ) presence of positive dimensions ( e.g. : mobility ) and negative ( e.g. : pain ) . the development of these elements led to the definition of quality of life as an individual 's perception of their position in life in the context of ( the ) culture and value system in which they live and in relation to their goals , expectations , standards and concerns , the impairment of quality of life of people affected by cancer can cause devastating damage to entire families . , this is especially true when the head of the family who is the provider of the only source of income gets sick , being the provider of the only source of income , as well as when one 1 parent is affected by the disease and children come to exercise have to care activities of the family , failing to take live their lives within according to the standards expected for their age1 . to this end , we applied the protocol for the evaluation of communication satisfaction of patients after total laryngectomy adapted from bertoncello20 ( table 1 ) , a validated instrument that measures the degree of satisfaction of the patient with his / her 's communication after total laryngectomy , and presents objective questions that facilitate the participant 's response . in order to post scenario , communication difficulties permeate family , professional , and social relationships of a family , professional and social , uniquely affecting uniquely the quality of life of these subject 's patients20 . this article aims to evaluate the satisfaction of communication and its relationship to quality of life in patients after total laryngectomyzed . this study is part of the research entitled quality of life and its relations with the use of diagnostic technologies in human communication disorders in rural workers who underwent total laryngectomy , , call mct / cnpq / health - ct / ms / sctie / decit n. 67/2009 - rebrats , with approval by the ethics committee in research of pernambuco society for fighting cancer / cancer hospital of pernambuco , n. 67/2010 . we adopted the following profile of volunteers to participate in the study : ( a ) inclusion criteria were as follows : an aged between 40 and 75 years ; both sexes ; who underwent surgery for total laryngectomy with or without neck dissection ; at least 45 days ; adjuvant radiotherapy ; being serviced at the speech pathology ( sefon ) department of head and neck ( dccp ) from the present hospital ; and signature on the instrument of consent ( tcle ) . ; ( b ) exclusion criteria criteria were as follows : previous head and neck surgery ; hold another type of laryngectomy , or tracheotomy prior to total laryngectomy ; alternative route of feeding way prior to total laryngectomy or present at the time of application of the protocol , ; patients who underwent history of postoperative complications ( pharyngocutaneous phystula , dehiscence , and tissue necrosis ) ; previous frame of difficulty in understanding simple commands or neurological / neuromuscular / neurodegenerative disease , ; and individuals who refuse to participate on in the study . data were collected and applied on a chip containing participant personal data items relating name , age , gender ( male / female ) , schooling , and occupational status functional ( active and retired ) . on the basis of the schooling , topic the participants were organized into four 4 groups : ( illiterate ; 1 to 4 years of schooling ; 5 to 8 years of schooling ; and 9 to 11 years of schooling ) . here goes for clarification regarding the adoption of the schooling classification criteria . from the federal resolution 4 of 07.13.201021 , which stablisheds the national general curriculum guidelines for basic education , and made 9 years of primary education it became compulsory primary education with nine years . however , we chose to keep the schooling calculations given on eight years of compulsory education , considering that the our research sample was composed of adults and elderly , who would have been schooled so when the previous regime statute still in force with eight years of schooling was still in force . all patients were in had adapted of an esophageal speech process , since because the service of this hospital does not offer the traqueosophagic or electronic larynx prosthesis . the research population belonged to low- income groups and generally therefore also has had no own spare resources for acquiring them prostheses on their own . subsequently , we applied the protocol for the evaluation of communication satisfaction of patients after total laryngectomy adapted from bertoncello20 ( table 1 ) . the adapted instrument for this study consists of fifty five55 specific items for functional communication evaluation with highly satisfactory reliability indices . has each has 5five possible answers : ( 1 ) not at all true , ( 2 ) somewhat true , ( 3 ) more true , ( 4 ) very true , and ( 5 ) very much true . for analysis purposes , we chose to organize the 55 questions from the bertoncello protocol20 in five 5 communication domains ( table 2 ) : ( 1 ) family relationship , ( 2 ) social relationship , ( 3 ) staff analysis , ( 4 ) morphofunctional appearance , and ( 5 ) writing use . the original numbering of the protocol was maintained . the protocol used has the peculiarity to of providing responses that may indicate positive and negative characteristics traits from the five 5 possible answers . to optimize the responses , analysis in each domain all participants were treated in the sefon of the hospital , a reference institution center for oncology treatment in the north and northeast of brazil . in sefon , the patients receive speech therapy before and after surgery to and orientation and rehabilitation of chewing , deglutition , and voice functions . , the study population included two 2 females women ( 13.3% ) and thirteen 13 males men ( 86.6% ) , aged between 47 and 73 years with a mean age of 62.8 years . considering the profile for research participants who were illiterate or poorly schooling , we chose to read the protocol by the researcher , where and the participant would only choose one answer for each question presented ; giving also the patients the option of reading the protocol by themselves . to obtain more reliable answers produced by the participant , and considering that the public profile of the research presents difficulties in speech production due to laryngectomy , we designed a feature assistive technology : the visual response scale . depending on the technical assistance committee , at the ata 's seventh meeting of the committee on technical assistance , special secretariat for human rights22 stated as follows : assistive technology is an area of knowledge , of interdisciplinary character , which includes products , resources , methodologies , strategies , practices , and services that aim to promote the functionality , related to the activity and participation of people with disabilities , incapacities or reduced mobility , seeking autonomy independence , quality of life and social inclusion . the instrument contained in staggered columns with a single color ( blue ) , where each column had a lighter tone subsequent number of responses related to the protocol . the participant indicates the desired response , relating the column size and color depth to the order of their responses . please note that appeal was presented to the participant , however , used only with the concurrence of the same . all caregivers were instructed to wait for the response of the participant , allowing the subject patient himself to answer autonomously themselves . the analysis was performed using descriptive statistics , using a simple frequency and average due to the qualitative profile of the study . the scenario presented in building the profile of participants can be seen in table 3 , relating gender , age , schooling , and job function . contains the following data among the participants , 86.7% are were male and 13.3% were female , with a mean age of 62.8 years . there is was prevalence of low schooling , totaling ( 93.3% ) , considering the illiterate subjects and from those with 1 to 4 years of schooling . of the respondents , 3 ( 20% ) are in were working activity and 12 ( 80% ) are were retired . obs : * these data refer to the mean age of participants . of the possible 825 events , the responses collected from the fifteen 15 participants are were organized from three perspectives ( table 4 ) : ( a ) the five types of responses ( not at all true , little true , more true , very true , and very much true ) , ( b ) the communication domain ( family relationship , social relationship , personnel analysis , appearance morphofunctional appearance , and writing use ) , and ( c ) the positive and negative aspects of each domains .
subtitles : npv , not at all true ; pv , a little true ; mv , true ; bv , very true ; mtv , very much true . ( + ) positive aspect , ( ) negative aspect . the overall picture ( table 5 ) reflects the responses of participants considering which would point to a good communication quality of life and which would point to a poor quality of life in communication , from the number of occurrences in each of the communication domains , categorizing them into two groups : the more negative responses , like , not at all true ( npv ) and somewhat true ( pv ) ; and , the most positive , like , very true ( bv ) and very much true ( mtv ) . ninety - three responses such as very much true ( mv ) had a total of 93 occurrences and were discarded for this analysis to position themselves in a intermediary categorization proposed by the protocol bertoncello ( 2004 ) , aiming for greater objectivity . subtitles : qol , quality of life ; an , negative aspect ; ap , positive aspect ; npv , not at all true ; pv , a little true ; bv , very true ; mtv , very much true . of the 705 events ( table 5 ) present at the end of the protocol application adapted from bertoncello20 , there were 97 cases for the family relationship domain , 147 to for the social relationship domain , 283 instances for the personal analysis item , 141 cases for the appearance morphofunctional item , and 40 occurrences for the writing use item . the results of each items of communication domains in relation to responses that point to indicate a good quality of life in communication ( bgqvc ) and those that who suggest poor quality of life in communication ( mpqvc ) were as follows : family relationship item : - bqvc gqvc , with 30 occurrences ( 31.9% ) and mqvc pqvc , with 64 occurrences ( 68.1% ) ; social relationship item : - bgqvc , with 54 occurrences ( 36.7% ) and mpqvc , with 93 occurrences ( 63.3% ) ; staff analysis item : - bgqvc , with 101 occurrences ( 35.7% ) and mpqvc , with 182 ( 64.3% ) ; morphofunctional appearance item : - bgqvc , with 32 occurrences ( 22.7% ) and mpqvc , with 109 ( 77.3% ) ; writing use item : - bqvcgqvc , with 25 occurrences ( 62.5% ) and mqvcpqvc , with 15 ( 37.5% ) . computed responses suggest show that our findings is of bqvcgqvc of have 242 occurrences ( 34.3% ) and those that suggest of mqvcpqvc , have 463 occurrences ( 65.7% ) . the protocol adopted20 consists of direct questions and easily understood by participants ; , however , we analyzed the need for greater autonomy in the production of responses by subjects . although not shown , it is common to caregivers or companions of people after who have undergone total laryngectomy generally take for them the word of speak for the patients , by because of the own nature of the communication disorder . in while analyzing the profiles of respondents ( table 3 ) , even considering the relatively small sample size ( fifteen 15 members ) , total laryngectomy there were was more frequent in males ( 86.7% ) than in females ( 13.3% ) . this finding agrees with the literature on laryngeal cancer , which occurrs predominantly in males , as well as does the age of the study sample , which shows variation between 50 and 70 years3
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24 . according to the inca in 20121 , it is not recommended to estimate the cancer in women in 2012 is not recommended due to its low magnitude incidence . the schooling of the sample was characterized as low ( 93.3% ) , adding to consider the illiterate ( 20% ) and those who attended between 1 and 4 years of formal education ( 73.3% ) . this is a common fact finding among low - income people in the brazilian reality . only one 1 participant reported having schooling between 5 and 8 years ( 6.7% ) and none between 9 and 11 years . the profile of laryngectomized participants with a low schooling level of this research sample was also found in other studies20
25
26 . thus , the schooling of the participants could interfere in the production provision of answers as this type of activity , because these participants are not familiar with the questionnaire is not very familiar in their daily lives . however , a questionnaires answered by the patients themselves as is a resource commonly used tool for assessing the quality of life24 . when we proposed the use of an assistive technology with the visual response scale was used , we observed that this instrument helped to safety in facilitated the responses of participants who opted for it . sometimes , the verbal response was associated with the use of the instrument , reflecting the need to of the respondent 's to ensure understanding of by the evaluator , since it was someone outside of their immediate communication . this behavior suggests the uncertainty of the respondents in the use of verbal communication in new situations , on the part of respondents . however , it should not fail to consider the artificiality of communication involved in the use of a questionnaire implementation of as an instrument of evaluation should also be considered . even if the evaluator seeks to perform the activity with the greatest possible comfort for the participant , the circumstances of impartiality can not be disregarded . importantly , to point out that all volunteers have demonstrated desire and devotion in the active participation of in the research . of the three subjects patients who that met the functional occupancy ( 20% ) , all are were male and are aged between 58 and 68 years . the total number of retirees consisteds of twelve subjects participants ( 80% ) with aged between 47 and 73 years ; , seven men and two women ( 60% ) have had a lower age for retirement , age being a total of 60% . thus , it is anticipated since the time of retirement is due to the onset of cancer . of respondents who were working , all claimed that their voice can could be understood , even partially , in question 53 . in the discussion in relation to regarding gender , the two women in the study have had undergone schooling between 1 and 4 years , are were retired , and their responses indicated greater impairment in communication , corroborating the findings by gomes17 . for developing a closer look , the table 5 shows the occurrences on the communications domains organized into two groups for the two responses that the said dispute , or type responses not at all true ( npv ) and little true ( pv ) and two 2 more to confirm the saying , and they very true ( bv ) and very much true ( mtv ) . the intermediate response more true ( mv ) was discarded to had provide balance in relation to nature of the responses . with this table , we seek to sharpen the look at more clearly outline the relationship that formed between the quality of responses and their relationship to the quality of life of this research population . writing use is presented as an alternative communication method , used but not necessarily desired , as soon as there is the implication of failure case of verbal communication failed . in question 37 , which talks about discussed not using writing as a means to make himself oneself understood , a balanced was a result of the response was obtained , where with six subjects said they disagreeing , six agreeing , with said and three opting for an intermediate response , more true ( mv ) . these results , an apparently positive writing use item , reflected in 62.5% , still must be balanced , taking into account the low schooling level , so either do not make use of writing , or they use it of restricted mode . the family relationship item was the second of more negative ( 69% ) item , after the morphofunctional aspect item ( 77.3% ) , according to protocol data . patients may have had some difficulties while answering issues related to the family relationship item may have had some degree of difficulty to be answered , since family members often play the role of accompanying the patient . still , the high rate of responses that indicate difficulties of family interaction can be illustrated by the question 03 , which states that the respondent 's way of speaking interferes with family relationships ; where eight subjects ( 53.3% ) confirmed the said with the two more affirmative responses and three did so with an intermediate responses , totaling eleven replies indicating full or partial agreement ( 73.3% ) . this is a group composed consisted of 80% of retirees , so the family life becomes more intense and in view of the participants , the communication aspect is strongly marked in a negative way . although the profile of the study population to provided users of esophageal speech , bertoncello ( 2004 , p.86 ) describes research , as in carr et al , where the subjects , even varying the type of oral rehabilitation , present commitments in relation to quality of life in communication . some data that follow illustrate and may serve as a tipping point for both the speech therapist , as and for related areas . it can be seen that 66.7% of patients responded positively to the items of discomfort and greater effort ( question 15 and 16);. although not being the majority , 33.3% of patients had pain when speaking , because it and this could be controlled . there was a greater negative impact on all questions relating to the act of singing ( question 42 , - 80% ) , change the in voice tone ( question 43 , to 86.6% ) , and send producing some speech sounds ( question 22 , - 80% ) . faced with this panorama this wide variety of conditions , it may be possible to suggest treated by multidisciplinary interventions that will minimize it . talking on the phone was characterized as an indicative outline of the difficulties presented in the communication interaction of people post - who had undergone laryngectomyzed , with family , friends , or strangers ( 80% of respondents as very true more than fixed telephony , mobile telephony is more prevalent than fixed telephon brazil is among the technologies available today , getting to have with more lines of cell phone lines , than the number of people in over twenty brazilian states27 . while talking on the phone is an activity common to most of the brazilians , for patients post - who have undergone laryngectomy people this activity involves great difficulty practice presents itself as an element of great disorder . regarding the social relationship domain , the question 17 portrays the negative impact on dealing with the others , where fourteen ( 93.3% , adding to mtv and bv ) of respondents said that most people have difficulty in understanding them . in this study , the following limits can be highlighted as relevant to communication quality of life in patients surviving after laryngectomy survival : great difficulty in verbal interaction with family , friends , and strangers ; friends move away become distant ; the phone is a feature that worsens the communicative situation ; annoyance when it is not understood ; physical discomfort when speaking ; and finally , failure to express their ideas . for people who have left are able still to express their ideas , participation in this study may had have represented a listening moment . professionals and families to reflect on this data , can show how they are emotional and social interaction committed , and the urgency of changes in a difficult reality experienced by this group . perhaps , the participation of post - laryngectomy patients has provided its own subject reflection on condition to a right of communication . the adapted protocol assessment of communication satisfaction of patients after total laryngectomy21 was relevant to the objective of this research objectives , allowing it to have providing an overview of the communication quality of life of those who have undergone went total laryngectomy and presenting , as a consequence , changes in your previous standards of communicative functioning . the visual response scale , such provided as assistive technology , proved to be a facilitator , contributing to an inclusive stance against the persons subjected to undergoing total laryngectomy . the role of speech therapy interventions pervades these areas of speech communication . from among the techniques used , taking into account various factors , however , none of them allowed a satisfactory level of excellence . it will consider wish the individual , since there is need to develop new learning and skills to be involved by the same , which require flexibility and adaptation , and they can not always reach them28 . concern about the quality of life refers to a movement in the human and biological sciences in appreciating broader parameters that controls symptoms , decreased mortality , and increased life expectancy25 . quality of life can be a very subjective concept , but subjectivity can not be an impediment to be for enhanced techniques , since this could lead to stagnation of this type of research28
29 . language permeates all these domains that make constitute the quality of life , and through it the human relations are established through language26 . after laryngectomy , where once there was a guy person with fluent and independent discourse , is to present a scenario of experience linguistic isolation and silence ; imposed on himself in the person also faces of negative attitudes , subjective or explicit , their from social peers . social relations from a non - standard communication results in changes in the behavior of some patients . the norm centrism imposed by the different models of truth , implies paradigms breaking in the context of their own health professionals . therefore , the stigma of normality reported by larrosa and skliar30 expelled discursive spaces of various groups , among them laryngectomyzed people . knowledge with more properly the questions about of these changes that bring them in produce social limitations , could be a force arm the people submitted to individuals who have undergone total laryngectomy , compared to deal with managers of public politics , allowing more focused on promoting health communication actions . the expansion of this sector of studies favors the demystification of a non - standard human communication , from a multiprophessional intervention , promoting rehabilitation , recovery , or even resetting establishment of new goals near to this for post - laryngectomyzed , with quality of life . to rebrants / cnpq , the electromyography laboratory of the graduate program in pathology , ufpe , and the cancer hospital of pernambuco . | summary
introduction : among people affected by cancer , the impairment of quality of life of people affected by cancer can cause have devastating effects . the self - image of patients after
post - laryngectomyzed patients may be find themselves compromised , affecting the quality of life in this population .
objective : to characterize quality of life in related to communication in people who have undergone went total laryngectomy surgery .
methods : this is an observational study , with a cross - sectional and descriptive series .
design of series study .
the sample were comprised 15 patients interviewed the period from january to february of 2011 .
we used the quality protocol for life communication in post - laryngectomy adapted from bertocello ( 2004 ) ; which this questionnaire contains 55 questions .
the protocol was organized from the nature of using responses classified as positive and negative aspects , proposals in with respect to five 5 communication domains : family relationships , social relationships , personal analysis ; morphofunctional aspect , and use of writing . to promote and guarantee the autonomy of the respondents ,
was examiners made use of used assistive technology with the visual response scale .
results : the responses that total laryngectomy compromises the quality of life in communication amounted to 463 occurrences ( 65.7% ) , and that who responses suggesting good quality of life were represented with amounted to 242 occurrences ( 34.3% ) , from a total of 705 occurrencesresponses . from among the five 5 communication domains , four 4 had percentages of above 63% for occurrences of negative content for impact on communication .
appearance morphofunctional appearance gave the had the highest percentage of negative content , amounting to 77.3% of cases .
conclusions : the results showed important limitations of a personal and social nature due to poor communication with their peers . thus , there is a need for multidisciplinary interventions that aim to minimize the entrapment of negative impact on these people communication among these patients . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a decade after the dawn of the internet age , are people who seek health information better off than they used to be ? the current study by arora and colleagues examines a small slice of the massive health information national trends survey dataset and attempts to understand the experiences of those us adults who have sought cancer information at any point in their lives from any source . one third reported that the information they encountered was hard to understand , and one half questioned the quality of the information they found . several research questions regarding how individuals successfully find information on the internet remain to be answered . a decade after the dawn of the internet age , are people who seek health information better off than they used to be ? early predictions around the turn of the millennium suggested that electronic access to information would revolutionize medical practice , creating legions of download - happy medical consumers armed with the latest information and prepared to tackle their own health care . newfound access to information was predicted to be a great equalizer for patients and to pose a threat to the traditional authority of doctors.13 but has anything changed for the better ? the national cancer institute ( nci ) recognized that the radical shift in the way patients could obtain information from online sources raised new questions about health behaviors and health care , and realized that an understanding of information - seeking behaviors in this new age was necessary to its mission of decreasing the burden of cancer on the health of americans . as a result , the nci sponsored the cross - sectional study known as the health information national trends survey ( hints ) in 20022003 . this study properly recognized that online information - seeking behavior was better understood in the context of health information seeking in general . the current study by arora and colleagues examines a small slice of the massive hints dataset and attempts to understand the experiences of those us adults who have sought cancer information at any point in their lives from any source.4 their findings suggest that more than 90 million adults in the us have sought cancer information , three quarters within the past year . the authors also found that , among the sources consulted first for cancer information , respondents sought information from health care providers only a minority of the time ( 10.7% ) . the internet was used by us adults as the first source of cancer information nearly half of the time , by far the most popular source . what adult information seekers found once they went looking for cancer information was dissatisfying in many cases : one third reported that the information they encountered was hard to understand , and one half questioned the quality of the information they found . individuals with more years of formal education had better experiences , as did those with health insurance . those individuals who went online for information were slightly more satisfied than those who first sought information from health care providers or other sources , as evidenced by the smaller proportion of those who consulted the internet first , who agreed that the information was hard to understand . first , the individuals who responded to the hints survey were mostly not cancer patients themselves , although nearly 11% had a personal history of cancer , and many more reported a family history of cancer . individuals with a personal history of cancer are likely to have encountered specific cancer information in the context of their own health care and treatment , and may , in general , have a different view of cancer information sources . thus , the hints dataset is not , primarily , a study of information seeking by cancer patients . second , the sampled cohort may not be educationally representative of us adults in general , as the 2006 us census data suggests that more than 80% of us adults over the age of 20 are high school graduates,5 whereas only 32% of the hints survey cohort reported having graduated from high school . fewer years of formal education was associated with lower information - seeking experience ( isee ) scores in the current study . finally , about one half of the hints cohort had never sought cancer information , and their views are not the subject of the present analysis . the authors do not indicate whether respondents who had never sought cancer information differed in any substantial ways from those who had sought cancer information at any time in their lives . importantly , respondent characteristics accounted for only a small fraction of the variance found in isee scores , suggesting that important features of the information environment are more likely to influence information - seeking experiences than any of the individual socio - demographic characteristics examined in this study . a decade of research and scholarship on information seeking in the age of the internet has suggested that individuals who are younger , richer , and better educated are more likely to seek health information online . the present study s findings conform substantially to these precepts . evidence that the digital divide still exists may be found in the results of this study , although evidence from other sources suggests that it may be narrowing in significant ways.6 most disturbingly , half of those who had sought cancer information from any source agreed that almost everything causes cancer , and three - quarters agreed that there were too many cancer prevention recommendations and that it was hard to know which to follow . is it a positive or negative finding that so few individuals who sought cancer information did so first from sources other than their health care provider ? what factors contributed to the substantially negative experiences encountered by those who sought cancer information ? imagine for a moment an average us adult who considers seeking cancer information . perhaps , he or she gets a new pain and wonders if it could be caused by cancer or hears a friend say that she has just started a new anti - cancer diet and wonders if food can prevent cancer . perhaps this individual learns that an uncle has just been diagnosed with prostate cancer and wants to learn something about the disease . one can imagine a thousand scenarios in which a person might be driven to seek information about cancer . although a new , personal diagnosis of cancer might mean that a patient is able to see a cancer clinician within 1 to 2 weeks ( thereby gaining access to information quickly ) , a patient who was worried about his uncle s prostate cancer and wants to learn more may not have the option of turning immediately to his or her health care provider . the internet , in such circumstances , provides instant access to such information , at least for those who have access to the internet and the skills necessary for seeking information online . thus , having access to several more - immediately available forms of information ( internet , books ) allows individuals to begin to seek answers to their burning cancer questions much sooner than they would be able to discuss it with a provider . the study s finding that adults turn more frequently to the internet and other sources first before turning to health care providers is easily explained in this way and does not seem to me to be a negative result when seen in this light . however , if people come away from their searches frustrated and confused , then they may not be any better off . the authors of the present study found that individual respondent characteristics had very little relative influence on cancer information seeking experiences . this finding suggests that elements of the information - seeking environment or perhaps , other unmeasured factors are more likely to be responsible for the negative experiences . we know that individuals who seek information online ( the most popular single first information source for individuals in this cohort ) do so often by entering general search terms into a search engine such as google.7 what they encounter next is a large number of links , most of which lead to information that is substantially commercial in nature.8 much of it is of questionable quality.9 some individuals may be able to sort out the good from the bad , but exactly how best to do this is an unanswered research question . we know also that about half of the us adult population has either rudimentary or limited reading skills10 and that online materials are frequently written at about the 12th or 13th grade level , above the reading abilities of the majority of the us population.1113 finally , and perhaps most important , although for a decade , researchers have identified the quality of internet information as being the most important problem associated with open and easy access to online health information , my own experience has been that patients are more commonly frustrated by their ability to find individualized information . they frequently recognize when the information they encounter is of questionable quality , and they certainly know when someone is trying to sell them something . but they have great trouble sorting out what applies to them and what does not . drinking from the fire hose when all you want is a drink of water.14 it is easy to find information about prostate - specific antigen testing ; it is much harder to find online information that helps an individual ( with all his unique attributes ) to weigh the risks and benefits of testing in meaningful ways . the authors of the present study found that respondents with the lowest levels of formal educational attainment had the lowest isee scores . these individuals were more likely to have negative cancer related beliefs , such as agreeing that almost everything causes cancer . if we grant that it is not , in fact , the case that almost everything causes cancer , then why did so many information seekers agree that this is the case ? two possible explanations come to mind : the first is that individuals with less formal education are more likely to hold this type of inaccurate beliefs in the first place and that their information - seeking experiences did not disabuse them of the beliefs . the second is that such individuals seek and encounter information that persuades them to hold such inaccurate beliefs , perhaps , because they are unable to distinguish higher quality from lower quality information . in any case , what we learn from arora s study is that the information - seeking experiences examined in the hints study do not appear to have improved the information - seeking environment for all groups equally . a potentially fruitful area of research that has not received substantial attention to date is the examination of socio - demographic and educational factors that predict successful use of online sources for health information . some individuals may know how to do it in ways that lead to positive outcomes for them , and others may suffer harms as a result of web - based information seeking . understanding the predictors of successful and unsuccessful information - seeking behaviors would undergird future efforts to create means of assisting patients to find high - quality , individualized information helpful to decision making and to efficient and effective medical care . | abstracta decade after the dawn of the internet age , are people who seek health information better off than they used to be ?
the current study by arora and colleagues examines a small slice of the massive health information national trends survey dataset and attempts to understand the experiences of those us adults who have sought cancer information at any point in their lives from any source .
one third reported that the information they encountered was hard to understand , and one half questioned the quality of the information they found .
several research questions regarding how individuals successfully find information on the internet remain to be answered . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
glucocorticoids have been extensively characterized as being anti - inflammatory and/or immunosuppressive in therapeutic settings and are generally discussed as opposed to the development of inflammation and limiting of production or maturation of some effector immune cell types , in a physiological context . nevertheless , there is a well - established association between stress and allergic diseases , including asthma [ 4 , 5 ] . glucocorticoids , which are an essential part of systemic stress responses , play coadjuvant roles in promoting inflammation and may promote th2-type immunity through differential effects on th1 th2 cytokine production [ 5 , 6 ] . chronically stimulated eosinophil production ( eosinopoiesis ) is an important feature of human asthma and of murine allergic asthma models . in both cases , allergen challenge of sensitized subjects increases eosinopoiesis in the bone - marrow [ 7 , 8 ] . this effect is antigen - specific and can be abolished by inducing oral tolerance to the allergen , which affects both eosinophils and neutrophils in bone - marrow . the effects of oral tolerization in bone - marrow neutrophil and eosinophil granulocytes can be duplicated by transfer of splenic t lymphocyte subpopulations from tolerized / sensitized / challenged donors to histocompatible naive recipients . these observations highlight the importance of acquired cellular immunity in regulating the hematological response to allergen sensitization and challenge . they further suggest the possibility that granulopoiesis , encompassing both eosinophil and neutrophil production , might be regulated by lymphocyte populations in nonsensitized subjects as well . in an allergic asthma model , we demonstrated a critical role for endogenous glucocorticoids in the hematological response to allergen challenge : challenge induces a corticosterone surge that is paralleled by increased eosinophilia of bone - marrow in vivo and by increased responsiveness to il-5 , the major eosinopoietic cytokine , ex vivo ; the bone - marrow response to challenge is abolished by blockade of glucocorticoid signaling or glucocorticoid production . on the other hand , in the absence of allergen sensitization and challenge , we have also obtained evidence of a link between the corticosterone surge induced by mild surgical trauma and short - term bone - marrow eosinophilia ; again , blockade of endogenous glucocorticoid production or action abolished the bone - marrow eosinophilia induced by trauma . these in vivo effects of corticosterone , an endogenous glucocorticoid released by the adrenal glands , are paralleled by those of exogenously provided corticosterone or dexamethasone on murine bone - marrow [ 12 , 13 ] and of other glucocorticoids on human hemopoietic cells . in balb / c mice , dexamethasone increases eosinophil production in murine bone - marrow culture [ 12 , 13 ] and primes bone - marrow cells in vivo for increased ex vivo responses to il-5 . during further screening of inbred mouse strains for differences in the granulopoietic responses to dexamethasone , we observed bone - marrow eosinophilia in mice of the c57bl/6 ( b6 ) background injected with dexamethasone , which was undetectable in perforin - deficient b6 mutants submitted to the same treatment . perforin is a major mediator of cellular immunity [ 1519 ] , expressed in lymphocyte populations which fight viral and bacterial pathogens [ 16 , 17 ] as well as malignant cells in the context of both innate and acquired immune responses . perforin is also expressed by murine bone - marrow neutrophils , which have a critical regulatory role in t cell - mediated contact hypersensitivity . perforin deficiency is known to induce complex changes in leukocyte populations in humans and mice [ 16 , 17 , 20 ] , including a classical presentation of familial hemophagocytic lymphohistiocytosis , characterized by early life onset , high mortality , and multiple immunological defects , including uncontrolled activation and proliferation of cd4 + and cd8 + t cells , cytokine storm , macrophage activation and proliferation , pancytopenia , and anemia . here we report that perforin deficiency also presents a selective defect in granulocyte production , which can be corrected by wild - type lymphocyte transfer . sh30088.03 ) , rpmi1640 ( sh30011.01 ) , and imdm ( sh30228.01 ) were from hyclone ( logan , ut , eua ) ; l - glutamine ( g7513 ) , penicillin - streptomycin solution ( p4333 ) , essential amino acids solution ( 50x ) ( m5550 ) , methylcellulose ( m0387 ) , dexamethasone ( 21-phosphate , disodium salt , d1159 ) , mifepristone ( ru486 , m8046 ) , and histopaque-1083 ( 10831 ) were from sigma - aldrich corporation ( st . louis , mo , eua ) ; agar noble ( 0142 - 15/21422 ) was from difco ( detroit , mi , eua ) ; nonessential amino acids solution ( 100x ) , ( 11140 - 050 ) and mem vitamin solution ( 100x ) ( 11120 - 052 ) were from gibco life technologies ( carlsbad , ca , usa ) ; recombinant murine il-5 ( 405-ml-025 ) was from r&d systems ( minneapolis , mn , usa ) ; recombinant murine gm - csf ( 315 - 03 ) was from preprotech ( rocky hill , nj , usa ) ; rat antimouse cd8b ( clone : ebioh35 - 17.2 , 12 - 0083 - 82 , 0.2 mg / ml ) was from ebioscience ( san diego , ca , usa ) ; magnetic microspheres conjugated to antimouse cd4 ( l3t4 , 130 - 049 - 20 ) and to goat antirat igg ( 130 - 048 - 501 , as secondary antibody to primary rat antimouse cd8b ) were from miltenyi biotec ( ambriex , sp , brazil ) . wild - type c57bl/6 , perforin - deficient ( pfp ) mutants of the b6 background , and wild - type b6.129 mice were bred by cecal - fiocruz , rio de janeiro , brazil . the pfp stock was derived from the original b6.129s6-pfp stock by backcrossing to c57bl/6 at fiocruz . unless otherwise indicated , the wild - type controls for the experiments shown were c57bl/6 ; in selected experiments , b6.129 wild - type controls were used and yielded the same results as c57bl/6 ( not shown ) . the animals were housed and handled following institutionally approved guidelines under license l-00209 from ceua - fiocruz . routinely , female mice were used for the experiments , since male mice characteristically fight for dominance in the same cage , and the stress associated with fighting may confound the interpretation of the results . we have no evidence , however , that the granulopoietic responses described here , including the strain differences , are restricted to females . dexamethasone in saline solution was injected i.p . ( 200 l , i.p . , amounting to 5 mg / kg ; ) . ru486 in 0.1% methylcellulose was given intragastrically 2 h before dexamethasone with a gavage needle ( 200 l , amounting to 100 mg / kg ) . for lymphocyte transfers , 10 nylon - wool purified cells from naive c57bl/6 donors in a 100 l volume of sterile saline were injected into the tail vein of pfp recipients , once . controls received an equal number of cells from pfp donors . where lymphocytes depleted of cd4 + or cd8 + cells were used , the amount of cells injected was that recovered from 10 initial unseparated lymphocytes , in 100 l sterile saline , i.v . dexamethasone was administered to the recipients , in both cases , 48 h after lymphocyte transfer . where indicated , bone - marrow , peripheral blood from the abdominal vena cava , spleens , and whole thymuses were collected , for enumeration of cells or determination of relative weight of the thymus ( mg / g body weight ) [ 21 , 23 ] . bone - marrow collected from both femurs of each mouse in 5 ml rpmi1640 medium/1% fbs with a 22-gauge needle and kept on ice was used for total and differential counts and cell culture ( see below ) . spleen mononuclear cells were the source for isolation of lymphocytes ( see below ) [ 24 , 25 ] . differential counts were carried out in cytocentrifugates after fixation in pbs-10% formaldehyde and staining for eosinophil peroxidase ( epo [ 26 , 27 ] ) followed by counterstaining with harris ' hematoxylin . liquid bone - marrow cultures were established at 37c , with 10 bone - marrow cells in 1 ml rpmi1640/10% fbs , in 5% co2/95% air , plated in 24-well plates ( cat . n : 142475 , nunc brand products ) with il-5 ( 1 ng / ml ; 7 days ) . where indicated , dexamethasone was added ( 10 l / well , to 10 m final concentration ) . absolute numbers were calculated from total cell counts in turk 's solution multiplied by the % of epo+ cells ( eosinophil - lineage cells , both mature and immature ) in cytocentrifuge smears . triplicate semisolid ( clonal ) bone - marrow cultures were established for 7 days with 2 10 cells adjusted to 1 ml of 1 : 1 imdm / agar mix , in 35 mm tissue culture plates ( nunc ) , in the presence of gm - csf ( 2 ng / ml final ) , with or without dexamethasone ( 10 m ) , at final 20% fbs and 0.3% agar concentrations . total colonies ( defined as aggregates > 50 cells derived from a single progenitor cell ) [ 8 , 12 ] were scored at day 7 under an inverted microscope ( 400x , phase contrast ) . for lymphocyte isolation , spleens were collected and minced in rpmi1640/1% fbs on tissue culture plates . spleen mononuclear cells ( 2 10 in 10 ml rpmi1640/1% sfb ) were isolated by centrifugation on a 1.083-density ficoll - hypaque cushion ( 3 ml , at 400 g , for 30 minutes , at room temperature , following manufacturer 's instructions ) [ 24 , 25 ] . cells recovered from the medium / ficoll - hypaque interface were collected , washed in serum - free medium , resuspended , counted , and further separated on nylon - wool columns [ 24 , 25 ] at 4 10 cells/2 ml / g nylon wool . cells eluted in a total 25 ml warm medium , dropwise , were washed and counted before cytocentrifugation / staining or incubation with antibodies . depletion of cd4 + cells was done with 10 l l3t4/10 nylon - wool purified lymphocytes in 100 l serum - free rpmi1640 , on ice , following manufacturers ' instructions . depletion of cd8 + cells was in two steps , with rat antimouse cd8b conjugated with pe , followed by goat antirat igg ( 20 l/10 lymphocytes in 80 l medium , on ice ) . columns were eluted with 9 ml medium , dropwise , over a 5-minute period . the numbers of experiments ( n ) are indicated in the caption of the figures , to avoid overcharging the figures and captions . for comparisons of two groups ( figures 1 and 2 ) , we used the two - tailed t - test with separate variances ( systat for windows , version 5 , systat inc . , evanston , il ) . for multiple comparisons ( figures 3 , 4 , 5 , and 6 ) , we used anova , with the tukey hsd correction for groups of equal size ( systat for windows ) or with bonferroni 's correction for groups of unequal size ( using prisma 5 for windows , graph pad , la jolla , ca ) , unless otherwise indicated in section 3 . following preliminary experiments ( not shown ) that evidenced a positive response to dexamethasone in b6 wild - type mice , as well as the absence of any response , positive or negative , in pfp mutants of the b6 background , we reviewed data on freshly harvested bone - marrow from a large number of mice of both strains ( n = 44 and n = 48 , resp . ) , to look for evidence of strain differences in bone - marrow steady - state parameters , in the absence of dexamethasone exposure ( figure 1 ) . a significant difference was observed in this large series , with pfp mice having lower bone - marrow cellularity than wild - type controls ( figure 1(a ) ; p < 0.001 ) . the data available for epo+ cells ( figure 1(b ) ; p = 0.033 ) and neutrophils ( figure 1(c ) ; p = 0.010 ) of the mice in the large series showed significant differences as well , with lower counts in pfp mutants . as the two groups were defined only on the basis of genetic differences ( presence or absence of functional perforin genes ) , these differences could still be accounted for , in principle , by variance due to nongenetic factors within these groups , especially those which influence growth and development . because bone - marrow cellularity is roughly proportional to the size of the animal , we next evaluated whether these differences would disappear in the comparison between groups of control and mutant mice matched by weight ( figures 1(d)1(f ) ) . significant differences were still observed for total cells ( figure 1(d ) ; p = 0.001 ) , eosinophils ( figure 1(e ) ; p = 0.004 ) , and neutrophils ( figure 1(f ) ; p = 0.011 ) , all three parameters being lower in the mutant mice . because age might affect bone - marrow function through mechanisms unrelated to body weight gain ( as senescence may have an earlier onset in some strains ; furthermore , age is a major determinant of incidence of many pathological processes , such as malignancies ) , we next examined whether matching by age ( all animals at 12 weeks ) would eliminate the differences . significant differences were still found for all three parameters ( for figures 1(g)1(i ) , resp . , p = 0.001 , p = 0.006 , p = 0.001 ) , which were lower in the mutant mice . even matching by both weight and age ( for figures 1(j)1(l ) , resp . , p = 0.022 , p = 0.032 , p = 0.001 ) failed to eliminate these significant differences between controls and mutants for any of the three parameters , which were all lower in the mutant mice . overall , this suggests that bone - marrow cellularity , as well as bone - marrow eosinophil and neutrophil counts , is significantly lower in pfp mutants than in b6 mice and that this difference can not be dismissed as created by undue comparisons of two groups differing in body weight , age , or both . we further examined ( figure 2 ) the counts of total cells , lymphocytes , neutrophils , and eosinophils in peripheral blood of weight - matched ( median 21 g , range 1923 g ) b6 and pfp mice . unlike bone - marrow , peripheral blood total nucleated cell counts did not differ significantly between these strains ( figure 2(a ) , p = 0.795 ) , nor did lymphocyte counts ( figure 2(b ) , p = 0.417 ) . by contrast , both neutrophil ( figure 2(c ) , p = 0.025 ) and eosinophil ( figure 2(d ) ; p = 0.030 ) counts were significantly different , and , like in bone - marrow , lower in pfp mice . together , the data in figures 1 and 2 suggest that , even in the absence of dexamethasone , pfp mice have reduced granulocyte numbers both inside and outside of bone - marrow , relative to wild - type controls of comparable body weight . our original observation of this strain difference was increased % eosinophils in cultured bone - marrow from b6 , but not pfp , mice when both il-5 and dexamethasone were present ( jones and cardoso de mendona , unpublished observations ) . this is , however , insufficient to characterize a strain difference in response to dexamethasone , because the % of a given cell type in a bone - marrow sample , which has a highly heterogeneous composition , can be increased artifactually by a corresponding decrease in another cell type , rather than by a positive effect of dexamethasone on the cell type of interest . to confirm a positive effect of il-5 and dexamethasone in stimulation of wild - type and mutant eosinopoiesis , liquid cultures were established from the same number of bone - marrow cells ( 10 ) from b6 or pfp donors , in the presence of il-5 , alone or associated with dexamethasone , and the absolute numbers of eosinophils in the culture were determined . as shown in figure 3 , eosinophils were produced in cultures of il-5-stimulated wild - type and mutant bone - marrow ( figure 3(a ) ) . control cultures lacking il-5 do not contain eosinophils at the end of the culture period ( not shown ; ) . counts of epo+ cells recovered at the end of the culture were increased in the presence of dexamethasone ( 10 m ) , relative to il-5 controls , in cultures from b6 controls , but not from pfp mutants ( figure 3(a ) , p < 0.001 ) . nevertheless , the il-5 present was sufficient to sustain eosinopoiesis in the absence of dexamethasone by pfp bone - marrow to the same level observed in b6 control cultures . on the other hand , data from semisolid cultures , which allowed us to examine the effects of dexamethasone on several classes of progenitors ( colony - forming cells ) , including the granulocyte ( g ) and granulocyte - macrophage ( gm ) progenitors , which produce neutrophils , are also shown in figure 3 . the total counts of colonies formed by gm - csf - stimulated pfp bone - marrow progenitors , in the absence of dexamethasone , also differed significantly from those of b6 controls ( p 0.001 ) : even though identical numbers of bone - marrow cells were plated , less colonies were made by perforin - deficient bone - marrow ( figure 3(b ) ) . furthermore , dexamethasone ( 10 m ) significantly stimulated colony formation by b6 bone - marrow ( p = 0.002 ) but failed to stimulate ( p = 0.558 ) pfp bone - marrow in the same conditions ( figure 3(b ) ) . these observations show that bone - marrow progenitors from pfp mice differ significantly from those of b6 controls , because they form less colonies and do not respond to dexamethasone , which significantly enhances colony formation in the wild - type cultures . we next examined the effect of dexamethasone ( 5 mg / kg injection ) on bone - marrow eosinophils and neutrophils , as well as on the relative weight of the thymus , which is significantly reduced by glucocorticoids . as shown in figure 4 , dexamethasone injection increased the numbers of bone - marrow eosinophils ( figure 4(a ) , p = 0.003 ) and neutrophils ( figure 4(b ) , p = 0.019 ) in vivo , relative to saline - injected controls , in wild - type b6 mice , but not in pfp mice . the effect of dexamethasone on eosinophil numbers in wild - type b6 mice was abolished by ru486 pretreatment , while ru486 had no effect of its own in the absence of dexamethasone , in either b6 or pfp mice ( not shown ) . the relative weight of the thymus was significantly different ( p = 0.010 ) between body weight - matched pfp mice and wild - type controls , suggesting a decreased thymic cellularity in mutant thymus , even without exogenous glucocorticoid administration ( figure 4(c ) ) . importantly , dexamethasone injection did reduce significantly the relative weight of the thymus in both wild - type ( p = 0.007 ) and mutant ( p = 0.001 ) mice ( figure 4(c ) ) . again , the effect of dexamethasone on the thymus was abolished in both cases by ru486 pretreatment ( not shown ) . together , these observations show that pfp bone - marrow is unresponsive to dexamethasone stimulation in vivo , although dexamethasone significantly increases eosinophil and neutrophil numbers in the bone - marrow of wild - type controls . they also show that lack of responsiveness to dexamethasone in pfp bone - marrow is not due to a general lack of response to glucocorticoids , because the thymus of pfp mice responds to dexamethasone injection as expected . because perforin is mainly expressed in various effector / regulatory lymphocyte subsets , it was important to test whether the perforin - related defect in bone - marrow response to dexamethasone could be corrected by introducing b6 lymphocytes in pfp mice . we have done so with splenic lymphocytes from naive b6 donors , because both the baseline granulopoiesis defect and the defective granulopoietic response to dexamethasone were observed in the absence of allergic sensitization . the number ( 10 ) of lymphocytes for transfer into individual mice was defined on the basis of similar reconstitution studies . to define whether elimination of a particular lymphocyte subpopulation defined by standard surface markers ( cd4/cd8 ) prevented reconstitution of the dexamethasone response , 10 total lymphocytes purified from spleen were submitted to alternative depletion protocols with marker - specific microbeads , and the depleted cells in the column effluent , corresponding to the number of cells negative for the selection marker present in the original lymphocyte sample , were injected . as shown in figure 5 , pfp recipients of wild - type lymphocytes ( unseparated ) show a significant in vivo response to dexamethasone injection , by increased total cell ( figure 5(a ) ; p = 0.032 , t - test ) , eosinophil ( figure 5(b ) ; p = 0.04 ) , and neutrophil ( figure 5(c ) ; p < 0.001 ) counts in the bone - marrow , sharply contrasting with observations in pfp mice in the absence of wild - type cell transfer ( compare figure 4 ) . on the other hand , depletion of cd4 + or cd8 + cells in the wild - type lymphocyte preparation gave distinct results in the reconstitution assay , depending on the granulocyte population examined : while depletion of either subset abolished the ability of splenic lymphocytes to reconstitute the eosinopoietic response to dexamethasone ( figure 5(b ) ) , depletion of cd4 + cells did not prevent reconstitution of the neutropoietic response ( figure 5(c ) ; p = 0.013 ) , while depletion of cd8 + cells prevented reconstitution . as a further control , we performed transfer of pfp lymphocytes into pfp recipients and obtained no reconstitution of dexamethasone responses , judged by any of these three parameters ( figure 6 ) . in these controls , dexamethasone reduced significantly the relative weight of the thymus , showing that dexamethasone was able to reach systemically active levels even if bone - marrow showed no evidence of responding to it . overall , these observations suggest that lymphocytes from the spleen of naive wild - type mice , but not pfp mice , are able to reconstitute in the short term the granulopoietic responses to dexamethasone , but different cells may be involved in reconstitution of lineage - specific ( eosinophilic versus neutrophilic ) responses . this is , to our knowledge [ 1520 ] , the first description of a selective defect in granulopoiesis associated with perforin deficiency in mice and of its correction by the intravenous transfer of wild - type lymphocytes . as such , it extends the range of manifestations associated with perforin deficiency and raises the issue of how perforin contributes to regulation of granulocyte lineages in vivo . because the wild - type lymphocyte populations interact with a drug ( dexamethasone ) which initiates signaling through the ru486-inhibitable glucocorticoid receptor , to stimulate in vivo eosinophil and neutrophil production , these observations may be relevant to processes in which bone - marrow is stimulated by immune responses or trauma with involvement of the same receptor and of endogenous glucocorticoids released by the adrenal glands . perforin is a well - characterized effector protein , mainly ( but not exclusively ) expressed in lymphocytes which share the ability to induce cell - mediated cytotoxicity [ 1520 ] . perforin deficiency was initially characterized by the loss of important cytotoxic lymphocyte functions [ 1520 ] . however , it was soon realized that perforin deficiency entails other , more complex , functional consequences , leading to the development of type ii familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 ] , which shares pathophysiological features with the macrophage activation syndrome , including interferon- ( ifn- ) overproduction . hence , perforin deficiency has broader consequences in addition to impairment of cytotoxic lymphocyte function , and ifn- , alone or in association with other cytokine storm components , may transduce its effects on other leukocyte lineages . one of the issues raised by our findings is whether the lower granulocyte numbers in blood and bone - marrow of pfp mice are due to the progression of the murine equivalent of the human familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 , 28 ] . it should be noted that the human familial hemophagocytic lymphohistiocytosis is a severe condition , presenting early in life in most cases and associated with important mortality [ 20 , 28 ] . also , phenotypically distinct forms correspond to different mutations with perforin expressed at lower levels and/or with altered properties , a situation that is not applicable to the present study , which employed perforin null mice . the perforin - deficient mice in the original walsh et al . study ( from which the mice used in this study descend ) were described as healthy for at least 5 months . in our study , mice were certified spf and maintained in microisolator units , with no evidence of mortality , stunting , or signs of infection during the entire observation period of up to 3 months ( 12 weeks ) . on the other hand , pfp defect in response to dexamethasone could be corrected by transfer of as few as 10 lymphocytes 48 h before the stimulus . the prompt reconstitution of this defect argues against the lack of dexamethasone response in pfp animals being caused by a chronic inflammatory disease of the bone - marrow , such as familial hemophagocytic lymphohistiocytosis , which in humans is challenging for therapy . because correction was achieved by simple transfer of lymphocytes from naive donors , we think it is more likely that the transferred lymphocytes provided something that was lacking in the recipient . neutrophils were reduced in bone - marrow and blood of pfp mice relative to b6 controls . this not only shows that the neutrophil deficiency has peripheral expression but further argues against reduced numbers in bone - marrow being due to an increased export of neutrophils to the periphery . the evidence from colony - forming assays suggests , instead , that production is greatly reduced in pfp relative to b6 mice , as there were less gm - csf - responsive progenitors in an identical number of bone - marrow cells . reduced production would explain the decrease in granulocyte counts both outside and inside of bone - marrow . on the other hand , normal numbers of lymphocytes and total leukocyte counts show that the defect is selective for granulocytes , which are not the major circulating leukocyte subpopulation in mice , so that reduction in their numbers does not have a major impact on total circulating leukocyte counts . neutropenia is associated with increased susceptibility to bacterial and fungal infection , provided it is severe enough . this was not observed in our study , so presumably the pfp mice were capable of coping with the microorganisms present in a rather clean environment ( spf conditions , microisolator housing ) . it remains to be seen , however , whether following a more severe infectious exposure , such as that associated with sepsis induction , pfp mice would prove more vulnerable to bacterial dissemination . this will be addressed in future studies , since emergency granulopoiesis , as opposed to baseline granulopoiesis , is driven by gm - csf and related hemopoietic cytokines . the lack of appropriate response to gm - csf , both in the absence and in the presence of dexamethasone , would predict that emergency granulopoiesis would be defective in pfp mice and might therefore negatively influence the outcome of sepsis . the hypothalamus - pituitary - adrenal axis is activated in sepsis , and therefore it is possible that emergency granulopoiesis is driven by endogenous glucocorticoids , in a way consistent with our observations . the prediction , in this case , is that perforin would be required for normal host response to bacterial sepsis through an effect on glucocorticoid - mediated signaling in the bone - marrow . the ability of lymphocyte preparations to reconstitute responses to dexamethasone was abolished by depletion of cd4 + and/or cd8 + cells before transfer , depending on the granulocyte population . for neutrophils , cd8 while this suggests that different mechanisms are involved for eosinophils and neutrophils , it certainly raises the issue of the lymphocyte subpopulation involved . since naive lymphocytes are sufficient , it is not likely that conventional t cells , cd4 + or cd8 + , are responsible , for they would not be activated in this isolation protocol [ 24 , 25 , 31 ] . conventional nk cells would not be a strong candidate , since cd4 + or cd8 + lymphocyte depletion would probably not eliminate them [ 32 , 33 ] . innate lymphocytes other than conventional nk cells , however , would be expected to be present in naive mouse spleens [ 32 , 33 ] . natural killer t cells , which include subsets expressing cd4 and cd8 , are found in significant numbers in b6 spleen , present an activated memory phenotype without known activating exposure , and exert multiple regulatory functions through rapid secretion of large amounts of cytokines [ 32 , 33 ] , might perhaps account for some of our observations . regardless of innate or adaptive lymphocyte population involved , two questions which require further study are as follows : ( a ) is perforin the missing component that is brought by the transferred lymphocytes ? and ( b ) do these lymphocytes act by homing to the recipients ' bone - marrow and interacting locally with granulocyte progenitors / precursors ? both questions , however , require a more complex experimental design and resources to allow us to detect and modify perforin expression inside living lymphocytes , as well as monitor their homing to the bone - marrow compartment . for this reason , such experiments fall outside the scope of this paper , which is limited to the demonstration of the defect and of its correction . finally , it should be noted that lymphocyte transfer restored the short - term response to dexamethasone but did not bring the baseline counts of eosinophils and neutrophils to the same level of the b6 controls . this might be interpreted as evidence that steady - state granulopoiesis and response to dexamethasone are unrelated processes ( i.e. , their perforin deficiency entails two distinct defects in granulopoiesis , rather than one defect with distinct manifestations ) . we do not support this interpretation , however , because the duration of the experiment might be insufficient for an effect of lymphocyte transfer alone on steady - state granulocyte numbers to become noticeable . it is also possible that demonstration of a durable effect on steady - state granulocyte numbers would require not only an extended observation period but also an increased or repeated input of wild - type lymphocytes and an interaction of exogenous lymphocytes with endogenous adrenal glucocorticoids as well . | exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone - marrow .
a hematological response dependent on endogenous glucocorticoids underlies bone - marrow eosinophilia induced by trauma or allergic sensitization / challenge .
we detected a defect in granulopoiesis in nonsensitized , perforin - deficient mice .
in steady - state conditions , perforin- ( pfp- ) deficient mice showed significantly decreased bone - marrow and blood eosinophil and neutrophil counts , and colony formation in response to gm - csf , relative to wild - type controls of comparable age and/or weight .
by contrast , peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency .
dexamethasone enhanced colony formation by gm - csf - stimulated progenitors from wild - type controls , but not pfp mice .
dexamethasone injection increased bone - marrow eosinophil and neutrophil counts in wild - type controls , but not pfp mice .
because perforin is expressed in effector lymphocytes , we examined whether this defect would be corrected by transferring wild - type lymphocytes into perforin - deficient recipients .
short - term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild - type donors .
transfer of the same amount of splenic lymphocytes from perforin - deficient donors was ineffective .
this demonstrates that the perforin - dependent , granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
primary biliary cirrhosis ( pbc ) is an autoimmune hepatic disease characterized by chronic cholestasis that may gradually destruct the intrahepatic duct and cause portal venous inflammation and fibrosis . this condition frequently occurs in middle - aged women , with a prevalence of 2.7 to 40 per 100,000 persons.1 ) pbc is characterized by the presence of antimitochondrial antibodies ( amas ) and high immunoglobulin ( ig ) m level in the blood.2 ) it may remain asymptomatic for an extended period of time or develop rapidly in a short period of time . igm level can increase because of pbc . however , correlation between pbc and igm antibody against hepatitis b core antigen ( igm anti - hbc antibody ) has not been reported . along with a literature review , we report a case of a pbc patient , who was initially suspected to be in the window period of hepatitis b in a local clinic because immunoserological test revealed isolated igm anti - hbc antibodies ( + ) . a 35-year - old asian man was referred to good gang - an hospital mainly because of intermittent pruritus and fatigue that lasted for two months . the patient was suspected to be in the window period of hepatitis b based on the detection of igm anti - hbc antibody ( + ) with increased aspartate transaminase ( ast ) and alanine transaminase ( alt ) levels in his blood about a month ago . he had been drinking approximately a bottle of beer once a month but does not smoke . his family history was not significant . at the time of his visit to this hospital , his blood pressure was 120/80 mm hg ; pulse rate , 76/min ; respiration rate , 20/min ; and temperature , 36.5. moreover , the patient was conscious and had no specific symptom but fatigue . his breath sound was normal during chest auscultation ; neither whole body jaundice nor icteric sclera was observed . no tactile mass was noted in the abdominal region ; neither abdominal pain nor abdominal fluid was found . the peripheral blood test showed that his hemoglobin was 14.6 g / dl ( reference range , 1116 g / dl ) ; white blood cell count , 4,880 cells / mcl ( reference range , 3,6009,500 cells / mcl ) ; and platelet , 181,000 cells / mcl ( reference range , 150,000450,000 cells / mcl ) . the biochemical test revealed the following : total protein , 9.2 g / dl ( reference range , 6.48.3 g / dl ) ; albumin , 4.4 g / dl ( reference range , 3.85.3 g / dl ) ; ast , 107 iu / l ( reference range , 838 iu / l ) ; alt , 104 iu / l ( reference range , 543 iu / l ) ; alkaline phosphatase ( alp ) , 1,431 iu / l ( reference range , 35105 iu / l ) ; gamma - glutamyl transpeptidase ( ggt ) , 813 iu / l ( reference range , 1575 iu / l ) ; total bilirubin , 0.4 mg / dl ( reference range , 0.21.2 mg / dl ) ; direct bilirubin , 0.1 mg / dl ( reference range , 00.4 mg / dl ) ; blood urea nitrogen , 13.9 mg / dl ( reference range , 720 mg / dl ) ; creatinine , 1.0 mg / dl ( reference range , 0.71.3 mg / dl ) ; and lactate dehydrogenase , 518 iu / l ( reference range , 263450 iu / l ) . the immunoserological test found hepatitis b surface ( hbs ) antigen / antibody ( / ) , hepatitis b e ( hbe ) antigen / antibody ( / ) , hepatitis c virus antibody ( ) , anti - hepatitis a virus ( hav ) igm ( ) , anti - hav igg ( ) , igm anti - hbc antibody ( + ) , and hepatitis b virus ( hbv ) dna quantification ( ) ( table 1 ) . he was diagnosed with pbc because of his increased alp and ggt levels in the blood test and increased igm / igg ( 518/1,740 mg / dl ) in the additional blood test ; he was also both ama and antinuclear antibody ( ana ) positive . moreover , no abnormal findings were observed ; however , an approximately 2 cm hepatic cyst was found through abdominal computed tomography ( figure 1 ) . liver biopsy found intrahepatic bile duct injury and epithelial cell infiltration in the lymphocytes , both of which characterize pbc , but did not indicate severe fibrosis or any condition that suggested autoimmune hepatitis ( figure 2a , b ) . based on ama and ana ( + ) and the blood test results , which suggested a cholestatic disease , he was diagnosed with pbc . the patient was administered with a high dose of ursodesoxycholic acid ( udca , 900 mg / d ) for three months . after the treatment , both fatigue and pruritus decreased , and the ast , alt , alp , and ggt were 34 , 18 , 350 , and 107 iu / l , respectively . six months later , the patient exhibited lower alp and ggt levels and negative igm anti - hbc antibody in the follow - up test ( table 2 ) . the most useful index in diagnosing acute hepatitis b is igm anti - hbc antibody , which increased for the first two to four weeks after the hbv infection and then began to decrease gradually . this antibody was detected in the blood for about six months after the acute hbv infection and was the only serological index that could determine the recent hbv infection during the window period between the disappearance of hbs antigen and the appearance of hbs antibody . if igm anti - hbc antibody is separately detected , the patient can be suspected to have anti - hbc false - positive , to be in the window period , to be in the convalescent stage after hbv infection , or to have latent hbv injection . this case was considered anti - hbc antibody false - positive because hbs antigen / antibody , hbe antigen , and anti - hbe negative were maintained and hbv dna was not detected during the six months of follow - up . ninety percent of the pbc patients are female , and this condition frequently occurs in those in their fifties and sixties . pruritus and fatigue are two of the most common initial symptoms , and pbc has recently been detected more frequently in its early stages during medical check - up , with 50% to 60% of all the cases detected by chance in liver function test.3 ) pbc is a cholestatic autoimmune disease , with 90% to 95% of all the cases being ama ( + ) 45 ) and approximately 50% being ana ( + ) . it is accompanied by the increase in alp and ggt levels , which characterize cholestatic liver diseases , and the increase in igm level in the blood in most cases . this phenomenon is attributed to the lymphocytes that are deposited around damaged bile canaliculi , which usually generate igm in pbc patients.6 ) udca is the only medicine for pbc and has been officially approved by the food and drug administration . this medicine may be administered at a dose of 1315 mg / kg / d . its therapeutic effects may be determined through the results from biochemical tests ( alp , bilirubin , etc . ) in six to nine months in most cases and those cases with therapeutic effects may have good long - term prognoses . udca may reduce the need of liver transplantation , improve the survival rate , and control the development of varicose veins.78 ) liver transplantation may be considered when pbc is accompanied by pruritus , which can not be controlled by medication , has severe osteoporosis , suffers hepatic failure because of the condition , or feels badly fatigued.9 ) the patient in this case was suspected to be in the window period of acute hepatitis b because he had non - specific symptoms , such as continuous fatigue and intermittent pruritus , and compromised liver functions . moreover , the patient was igm anti - hbc antibody ( + ) . however , he underwent ama and ana examination and liver biopsy for cholestatic hepatitis because his blood test showed an increase in the alp and ggt levels . he was diagnosed with pbc based on the ama and ana ( + ) finding and the liver biopsy results . the anti - hbc false - positive finding is probably due to the increased igm level in blood , which leads to antibodies with cross reactivity in hbc antigen and their non - specific binding . the patient in this case was igm anti - hbc antibody ( + ) in the immunoserological test when he visited the hospital . his alp and ggt levels then improved with udca administration . after six months , his immunoserological test result showed igm anti - hbc antibody ( ) . this result is probably due to the decreased igm level in the blood with the favorable turn of the condition after the treatment . isolated igm anti - hbc antibody ( + ) is generally found in immunoserological test . the condition may be treated in relation to viral hepatitis : the hbv window period , the convalescent stage after hbv infection , or latent hbv infection . in this case , the patient was also initially strongly suspected to be in the window period of acute hepatitis b because he was a young man with igm anti - hbc antibody ( + ) in two consecutive tests . then , he was suspected of cholestatic hepatitis because hbv dna was undetected and the alp and ggt levels increased remarkably and was diagnosed with pbc in the additional test . the increased igm level in the blood can likely generate antibodies with cross reactivity in hbc antigen , which may be false - positive . however , correlation between pbc and anti hbc - igm antibody or the frequency of being false - positive has not been reported . igm anti - hbc antibody is the most useful index in diagnosing acute hepatitis b. this antibody increases for the first two to four weeks after the hbv infection . however , it is necessary to consider the possibility of pbc when an acute hepatitis patient has liver dysfunction , isolated igm anti - hbc antibody ( + ) , and increased alp and ggt levels . if a patient with abnormal findings from immunoserological test sees the alp and ggt levels raised , it is helpful in seeing a favorable turn of the condition and better prognoses to perform additional blood test for cholestatic hepatitis along with liver biopsy as needed to start proper treatment on the basis of correct diagnosis . | primary biliary cirrhosis ( pbc ) is a slowly progressive cholestatic autoimmune liver disease characterized by progressive bile duct injury .
the most common symptoms of this disease include fatigue and pruritus .
the diagnosis of pbc is based on cholestatic biochemical liver tests , presence of antimitochondrial antibodies , and characteristic histological biopsy findings .
we report a case of a patient with pbs , who was initially suspected to be in the window period of hepatitis b by a private doctor in a local clinic based on the detection of isolated immunoglobulin m antibody against hepatitis b core antigen .
the presence of this antibody is the most useful index in diagnosing acute hepatitis b ( + ) by immunoserological test .
the final diagnosis of the patient in good gang - an hospital was pbc through additional tests .
the patient is receiving outpatient treatment . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
alkhumra haemorrhagic fever virus ( ahfv ) , a tick - borne flavivirus , was firstly discovered in 1995 from a butcher who had lately slaughtered a sheep in the city of alkhumra in makkah province . ahfv is now an emerging virus causing febrile reactions with fatal haemorrhagic and neurological manifestations ( alzahrani et al . ahfv was misnamed as alkhurma virus for many times in scientific papers and publications but has been corrected recently by the international committee on taxonomy of viruses ( ictv ) ( madani 2005 , king et al . ahfv is the first tick - borne haemorrhagic virus whose whole genome has been sequenced and based on the sequence analysis , it has 89 % homology with kyasanur forest disease virus ( kfdv ) which belongs to the genus flavivirus of the flaviviridae family ( mohabatkar 2011 ) . this genus includes nearly over 70 viruses , mostly arthropode - borne , which infect humans and animals ( pastorino et al . kfdv and its genetic variants , ahfv and seven other species including langat , louping ill , omsk haemorrhagic fever , powassan , royal farm , gadgets gully and tick - borne encephalitis are grouped in the same subclassification ( memish et al . however , ahfv among all these viruses makes a major concern in the local health authorities as it can be exported to other countries by the muslims returning from yearly hajj rites . since it is suggested to be one of the most deadly flavivirus infections , with case fatality rates > 30 % ( charrel et al . 2005 ) , it can be one of our major concerns in iran as a muslim country . generally , incidence of many life - threatening viruses in humans has increased during the last two decades ( mohabatkar 2011 ) and there are many others which threaten to increase in the near future . these urgent situations greatly force us to accelerate our investigations and studies . undoubtedly in vitro and in vivo studies are of major importance in discovering the therapeutic and prophylactic solutions , but the fact is that in silico studies and simulations can accelerate these experiments due to the time - saving manner they have . high - accuracy and knowledge - based in silico predictions will result in a better and faster handling of the disease being studied since it can highlights the most important parts to be studied ( mohabatkar et al . , useful data have been computationally produced for many viruses such as human papillomavirus type 16 ( hpv-16 ) ( mohabatkar 2007 ) , influenza a h3n2 virus ( a / hong kong/1/68 ) ( liang et al . 2012 ) , isfahan virus ( isfv ) ( mohabatkar and mohsenzadeh 2009 ) and many others parallel to that of in vivo studies , showing the complementary and necessary role of computational investigations . a comprehensive in silico study on medically important structural properties of protein e of ahfv was done by mohabatkar 2011 , including the prediction of its secondary structure , glycosylation sites , tetraspan membrane protein of hair cell stereocilia ( tmh ) , disulfide connectivity , subcellular localization , evolutionary distance and t - cell epitope prediction , but was not including conformational epitope prediction while lacking a good 3d model ( mohabatkar 2011 ) . it should be noted that , protein e plays a central role in the biology of flaviviruses . it is the dominant antigen in eliciting neutralizing antibodies and plays a main role in inducing immunologic responses in the infected host ( wu et al . the aim of this study was to generate in silico 3d - structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model . however , better models can be generated in the future due to the fact that bioinformatics servers and tools are progressing rapidly . the amino acid sequence of the envelope protein of ahfv was obtained from the protein database of ncbi ( accession number np_775470.1 ) . after ensuring that there is no 3d - structure of the protein in protein data bank ( pdb ) ( http://www.rcsb.org/pdb/ ) , the present work was held . modeling was done using two homology modeling programs , m4 t ( fernandez - fuentes et al . 2007 ) and modweb ( http://salilab.org/modweb ) which are both automated web - servers . since unfavorable distortions are exerted on the side chains of amino acids during homology modeling process , energy minimization was performed on both models to move them to their optimal condition where the protein is in its most stable state . here energy minimization process was performed using gromos96 force - field implementation in swiss pdbviewer software ( version 4.0.1 ) ( kaplan et al . the most common method is root - mean - square deviation ( rmsd ) metric which shows the mean distance between the corresponding atoms in the two structures ( maiorov 1994 ) . rmsds of the modeled structures from the selected template were calculated using rmsd calculator tool in swiss - pdbviewer ( version 4.0.1 ) based on the backbone atoms ( n , c and c ) . the other method to evaluate the generated protein models used here was based on the use of modfold4 web - based server . the server allows user to make quantitative judgments about the credibility of models using different quantitative parameters like global score and p - value ( mcguffin et al . finally , the overall stereochemical property of the more accurate model was further assessed by ramachandran plot analysis using rampage server . the best selected model was subjected to predict its possible conformational epitopes with discotope 2.0 server ( haste andersen et al . 2006 ) , which uses a combination of amino acid statistics , spatial information , and surface exposure . the server is trained on a compiled data set of discontinuous epitopes from 76 x - ray structures of antibody - antigen protein complexes ( haste andersen et al . 2006 ) and is one of the most known and reliable epitope prediction servers . according to the server constructors study , discotope detects 15.5 % of residues located in discontinuous epitopes with a specificity of 95 % . this level of specificity is higher than several similar servers ( haste andersen et al . the amino acid sequence of the envelope protein of ahfv was obtained from the protein database of ncbi ( accession number np_775470.1 ) . after ensuring that there is no 3d - structure of the protein in protein data bank ( pdb ) ( http://www.rcsb.org/pdb/ ) , the present work was held . modeling was done using two homology modeling programs , m4 t ( fernandez - fuentes et al . 2007 ) and modweb ( http://salilab.org/modweb ) which are both automated web - servers . since unfavorable distortions are exerted on the side chains of amino acids during homology modeling process , energy minimization was performed on both models to move them to their optimal condition where the protein is in its most stable state . here energy minimization process was performed using gromos96 force - field implementation in swiss pdbviewer software ( version 4.0.1 ) ( kaplan et al . the most common method is root - mean - square deviation ( rmsd ) metric which shows the mean distance between the corresponding atoms in the two structures ( maiorov 1994 ) . rmsds of the modeled structures from the selected template were calculated using rmsd calculator tool in swiss - pdbviewer ( version 4.0.1 ) based on the backbone atoms ( n , c and c ) . the other method to evaluate the generated protein models used here was based on the use of modfold4 web - based server . the server allows user to make quantitative judgments about the credibility of models using different quantitative parameters like global score and p - value ( mcguffin et al . finally , the overall stereochemical property of the more accurate model was further assessed by ramachandran plot analysis using rampage server . the best selected model was subjected to predict its possible conformational epitopes with discotope 2.0 server ( haste andersen et al . 2006 ) , which uses a combination of amino acid statistics , spatial information , and surface exposure . the server is trained on a compiled data set of discontinuous epitopes from 76 x - ray structures of antibody - antigen protein complexes ( haste andersen et al . 2006 ) and is one of the most known and reliable epitope prediction servers . according to the server constructors study , discotope detects 15.5 % of residues located in discontinuous epitopes with a specificity of 95 % . this level of specificity is higher than several similar servers ( haste andersen et al . homology modeling was performed using two programs : m4 t and modweb which are servers for automated comparative protein structure modeling that calculate their models based on the best available template structures from pdb without the need of manually identifying a template and doing pairwise alignment . however , results of the alignment and the choice of template can be observed in the servers output ( eswar et al . 2003 ) . here , the template by which the two modeling programs generated their models was the crystal structure of chain a of envelope glycoprotein from tick - borne encephalitis virus ( pdb : 1svb_a ) at 2 resolution ( rey et al . alignment results performed automatically by the server revealed that the selected template has almost 82 % identity with the query sequence . although the accuracy of the generated model is highly dependent on its sequence identity with the template sequence , by having the results of rmsd metric and modfold4 server the quality of the model can be reliably assessed . the results of rmsd and modfold4 for the final energy - minimized models are provided in table 1 . during energy minimization process which was perfomed for 5 runs , the final calculated energy of m4 t and modweb generated models were reduced to 15335.445 kj / mol and 14327.418 kj / mol respectively . before energy minimization process , these values were 4808.651 kj / mol and 3147.817 kj / mol for m4 t and modweb generated models respectively . quality assessment of models using different parameters in case of rmsd , m4 t generated model showed 0.46 rmsd with the template and is therefore more accurate than the model generated by modweb with 3.45 rmsd . using modfold 4 , the global model quality score is calculated and from this feature a p - value is calculated which represents the probability that a model is incorrect . the global model quality scores range between 0 and 1 and scores less than 0.2 indicates an incorrectly modeled proteins while scores greater than 0.4 are models with higher quality and indicate that the models are highly similar to the native structures . the other output parameter of the server is p - value . in general , p - values more than 0.05 , represent low - quality models . when p - value reaches 0.010.05 , it can be concluded that the model quality is medium . high quality models show 0.0010.1 p - values and if having absolutely certifiable models , the p - values of less than 0.001 are expected . here both the global model quality score and p - value are acceptable especially for the model generated by m4 t , where p - value is less than 0.001 and the global model quality score is near 1 , at about 0.77 . the p - value and global model quality of 2.92e and 0.773 , respectively , shows that the model scores are both in the range required for a high quality model . therefore the model generated by m4 t was then visualized by pymol ( fig . 3d predicted model visualized by pymol the evaluation of m4 t generated model for its stereochemical quality was performed by ramachandran plot analysis using rampage server and the output plot is shown in fig . it showed 98 % of residues in favored region , 1.5 % in allowed region and just one residue ( 0.3 % ) in outlier region which highly indicates a good streochemical quality of the predicted model . the detailed result from rampage ramachandran plot for the model ramachandran plot analaysis results using rampage having an acceptable model , the prediction of envelope protein of ahfv was undertaken using discotope 2.0 . disco tope 2.0 utilizes calculation of surface accessibility -estimated in terms of contact numbers- and a novel epitope propensity amino acid score . the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers . in the most recent version of discotope which is version 2.0 used in this paper , novel definition of the spatial neighborhood is used to sum propensity scores and half - sphere exposure as a surface measure ( kringelum et al . results from discotope 2.0 showed 26 residues out of the whole 395 total residues of the model as epitopes ( table 3 ) . these residues can then interact as the consequence of protein folding and form conformational epitopes . predicted conformational epitiope residues by discotope 2 homology modeling was performed using two programs : m4 t and modweb which are servers for automated comparative protein structure modeling that calculate their models based on the best available template structures from pdb without the need of manually identifying a template and doing pairwise alignment . however , results of the alignment and the choice of template can be observed in the servers output ( eswar et al . 2003 ) . here , the template by which the two modeling programs generated their models was the crystal structure of chain a of envelope glycoprotein from tick - borne encephalitis virus ( pdb : 1svb_a ) at 2 resolution ( rey et al . alignment results performed automatically by the server revealed that the selected template has almost 82 % identity with the query sequence . although the accuracy of the generated model is highly dependent on its sequence identity with the template sequence , by having the results of rmsd metric and modfold4 server the quality of the model can be reliably assessed . the results of rmsd and modfold4 for the final energy - minimized models are provided in table 1 . during energy minimization process which was perfomed for 5 runs , the final calculated energy of m4 t and modweb generated models were reduced to 15335.445 kj / mol and 14327.418 kj / mol respectively . before energy minimization process , these values were 4808.651 kj / mol and 3147.817 kj / mol for m4 t and modweb generated models respectively . quality assessment of models using different parameters in case of rmsd , m4 t generated model showed 0.46 rmsd with the template and is therefore more accurate than the model generated by modweb with 3.45 rmsd . using modfold 4 , the global model quality score is calculated and from this feature a p - value is calculated which represents the probability that a model is incorrect . the global model quality scores range between 0 and 1 and scores less than 0.2 indicates an incorrectly modeled proteins while scores greater than 0.4 are models with higher quality and indicate that the models are highly similar to the native structures . the other output parameter of the server is p - value . in general , p - values more than 0.05 , represent low - quality models . when p - value reaches 0.010.05 , it can be concluded that the model quality is medium . high quality models show 0.0010.1 p - values and if having absolutely certifiable models , the p - values of less than 0.001 are expected . here both the global model quality score and p - value are acceptable especially for the model generated by m4 t , where p - value is less than 0.001 and the global model quality score is near 1 , at about 0.77 . the p - value and global model quality of 2.92e and 0.773 , respectively , shows that the model scores are both in the range required for a high quality model . therefore the model generated by m4 t was then visualized by pymol ( fig . 3d predicted model visualized by pymol the evaluation of m4 t generated model for its stereochemical quality was performed by ramachandran plot analysis using rampage server and the output plot is shown in fig . it showed 98 % of residues in favored region , 1.5 % in allowed region and just one residue ( 0.3 % ) in outlier region which highly indicates a good streochemical quality of the predicted model . the detailed result from rampage having an acceptable model , the prediction of envelope protein of ahfv was undertaken using discotope 2.0 . disco tope 2.0 utilizes calculation of surface accessibility -estimated in terms of contact numbers- and a novel epitope propensity amino acid score . the final scores are calculated by combining the propensity scores of residues in spatial proximity and the contact numbers . in the most recent version of discotope which is version 2.0 used in this paper , novel definition of the spatial neighborhood is used to sum propensity scores and half - sphere exposure as a surface measure ( kringelum et al . results from discotope 2.0 showed 26 residues out of the whole 395 total residues of the model as epitopes ( table 3 ) . these residues can then interact as the consequence of protein folding and form conformational epitopes . predicted conformational epitiope residues by discotope 2 interaction between b - cell receptors or soluble antibodies and protein antigens is one of the major mechanisms of immune system to overcome infectious pathogens . identification of the epitopes which antigens can bind to antibodies is undoubtedly essential in many biomedical applications including vaccine design , immunotherapeutics studies and also for developing and designing of diagnostic kits . experimental epitope mapping is difficult and expensive in term of time and cost , making in silico methods a good complementary accelerant ( kringelum et al . although most b - cell epitopes are conformational , computational methods and studies often concentrate on mapping linear epitopes ( yasser and honavar 2010 ) . in recent years interests are increasing for mapping conformational epitopes and this assertion can be confirmed by the fact that different tools potential for predicting conformational epitopes are increasingly developing , these are including ellipro ( ponomarenko et al . 2008 ) , pepito ( sweredoski and baldi 2008 ) , seppa ( sun et al . 2009 ) , epsvr ( liang et al . 2010 ) , discotope 1.0 and 2.0 and many others . however due to the computational complexity and lack of sufficient knowledge about antibody - antigen complex structures , there are still a limited number of high accuracy web - based servers and tools . linear b - cell and t - cell epitopes of envelope protein of ahfv have already been predicted and both have essential roles in the immune response to pathogens ( mohabatkar 2011 ) . in this paper , predicting the conformational epitope of the envelope protein of ahfv using a structure - based prediction method was held after generating a certifiable 3d model . there are sequence - based prediction methods available for conformational epitope mapping which does not require the 3d structure of the protein being studied . however , the structure - based prediction can be more accurate if equipping with strong algorithms and therefore , the latter was used in the present study . although we can more conveniently gain information about protein 3d structures in silico , x - ray crystallography and nmr are undoubtedly the first and best choices to get such information . homology modeling of proteins is an alternative way when there is no information about their experimentally - prepared 3d structures . the critical step in homology modeling is the identification of a 3d template chosen by virtue of having the highest sequence identity with the target sequence , if indeed any are available . sometimes there is no suitable template structure available or there are templates with medium - low sequence identities ( less than 5070 % identity ) . when there are template structures available but with low sequence identity to our target protein sequence , it is not recommended to perform homology modeling . here , the selected template structure ( 1svb ) showed 82 % sequence identity to our target sequence . having 82 % sequence identity , it was ensured that homology modeling is an appropriate method to predict the 3d structure of our target protein . furthermore , model accuracy was described based on its rmsd with the template structure and also by modfold4 . according to related literatures and to provide a frame of reference for rmsd values , up to 0.5 rmsd occurs in independent determinations of relatively the same protein , meaning that this range of rmsd value for any modeled protein shows high modeling accuracy ( chothia and lesk 1986 ) . rmsds of m4 t and modweb generated models were 0.46 and 3.45 respectively and it is obvious that m4t - generated model is more accurate . according to previous studies of homology modeling cases , highly accurate models were reported to have 0.41.5 rmsd ( lindin et al . thus , our predicted protein structure has excellent backbone rmsd and can be referred to as highly accurate . it is important to note that , there is a defect in the use of rmsd as a quality assessment method : rmsd metric is sensitive to outlier regions created by poor modeling of individual loop regions in a structure that its core is accurately modeled and for this reason there are cases when as many as one in ten highly accurate homology models ( 60%95% identity ) , have an rmsd value of more than 5 vs. the empirical structure ( guex et al . modfold4 quality assessment server was also used along with rmsd - based accuracy assessment . using the scores given by modfold4 , the similarity between the template and target structures which are identical in amino acid sequences but different in tertiary structures can be described . both models were regarded as accurate due to the results of modfold4 , however , again these scores were better for the m4 t generated model ( 0.7730 and 2.92e-4 for global model quality score and p - value , respectively ) . using this server , the model is ranked by global model quality score , and a p - value is calculated for it , which relates to the likelihood that the global model is incorrect ( mcguffin et al . our m4t - generated model has gained a high global model quality score along with a very low p - value , meaning that the model is highly certifiable . , the choice of epitope prediction server was relied on a study showed that discotope 2.0 server has the highest predictive performance among some other similar servers ( kringelum et al . 2012b ) . results from discotope 2.0 showed 26 residues as conformational epitopes . according to ramachandran plot analysis , alanine 54 which was one of the predicted epitope residues , therefore , it seems to be unacceptable to name this residue as an epitope compartment . however , it should be also noted that it is not bizzare when a residue or more adopt a disallowed stereochemical angle . it can be explained by the fact that they do so to maintain the stability of the whole protein structure . due to this reason , alanine can adopt disallowed conformations to stabilize the whole protein tertiary structure with high rate of occurrence ( pal and chakrabarti 2002 ) . thus , this residue can be also accepted as an epitope compartment regardless of being in the outlier region of ramachandran plot . since no therapeutic or prophylactic vaccine is available for ahflv , such data on its potential conformational epitopes might be helpful for scientists in future drug and vaccine development . | background : the aim of this study was to generate in silico 3d - structure of the envelope protein of ahfv using homology modeling method to further predict its conformational epitopes and help other studies to investigate its structural features using the model.methods:a 3d - structure prediction was developed for the envelope protein of alkhumra haemorrhagic fever virus ( ahfv ) , an emerging tick - borne flavivirus , based on a homology modeling method using m4 t and modweb servers , as the 3d - structure of the protein is not available yet .
modeled proteins were validated using modfold 4 server and their accuracies were calculated based on their rsmds . having the 3d predicted model with high quality , conformational epitopes were predicted using discotope 2.0.results:model generated by m4 t was more acceptable than the modweb - generated model .
the global score and p - value calculated by modfold 4 ensured that a certifiable model was generated by m4 t , since its global score was almost near 1 which is the score for a high resolution x - ray crystallography structure .
furthermore , itsthe p - value was much lower than 0.001 which means that the model is completely acceptable . having 0.46 rmsd ,
this model was shown to be highly accurate .
results from discotope 2.0 showed 26 residues as epitopes , forming conformational epitopes of the modeled protein.conclusion:the predicted model and epitopes for envelope protein of ahfv can be used in several therapeutic and diagnostic approaches including peptide vaccine development , structure based drug design or diagnostic kit development in order to facilitate the time consuming experimental epitope mapping process . |
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\begin{document}$$\omega _ \vargamma ( \mathfrak o ) = \omega ( \mathfrak o ) $ $ \end{document } , where \documentclass[12pt]{minimal }
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\begin{document}$$\omega ( \mathfrak o ) $ $ \end{document } is the unit sum height defined in . with those notations let \documentclass[12pt]{minimal }
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\begin{document}$$f\subset \mathbb c $ $ \end{document } be a field and \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma $ $ \end{document } a finitely generated subgroup of \documentclass[12pt]{minimal }
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\begin{document}$$f^*$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$-1\in \vargamma $ $ \end{document}. let \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document } be a subring of \documentclass[12pt]{minimal }
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\begin{document}$$f$$\end{document } that is generated as a \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z $ $ \end{document}-module by a finite set \documentclass[12pt]{minimal }
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\begin{document}$$\varepsilon \subset \vargamma \cap r$$\end{document}. assume that for given integers \documentclass[12pt]{minimal } \usepackage{amsmath }
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\begin{document}$$n\ge i \ge 2$$\end{document } the equation1.2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } u_1+\cdots + u_i = n , \qquad u_1,\ldots , u_i \in \vargamma \cap r \end{aligned}$$\end{document}has a solution \documentclass[12pt]{minimal }
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\begin{document}$$(u_1,\ldots , u_i)\ne ( 1,\ldots , 1)$$\end{document}. then we have \documentclass[12pt]{minimal }
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\begin{document}$$\omega _ \vargamma ( r ) \le n-1$$\end{document}. the following section , sect . 2 , is devoted to the proof of theorem 1.2 . in the third section we apply our main theorem , theorem 1.2 , to some special orders of shanks simplest cubic fields . a special case of that theorem yields applications to double - base expansions . there we choose \documentclass[12pt]{minimal }
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\begin{document}$$f=\mathbb q $ $ \end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$r=\mathbb z $ $ \end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma = \langle -1,p , q\rangle $ $ \end{document } , where \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } are coprime integers . we discuss that in sect . we start this section by giving a short plan of the proof . let \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } be arbitrary . our goal is to find a representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } of the form ( 1.1 ) in which the coefficients \documentclass[12pt]{minimal }
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\begin{document}$$a_1,\ldots , a_\ell $ $ \end{document } are all bounded by \documentclass[12pt]{minimal }
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\begin{document}$$n-1$$\end{document}. we first show that \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } can be represented as a linear combination of the form ( 1.1 ) with \documentclass[12pt]{minimal }
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\begin{document}$$\nu _ 1,\ldots , \nu _ \ell $ $ \end{document } chosen in a particular way . the idea of the proof is rather simple and is based on induction over the total weight of this representation ( this is the sum of all of its coefficients , see definition 2.2 ) . start with a representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } as above and choose a coefficient which is greater than or equal to \documentclass[12pt]{minimal }
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\begin{document}$$n$$\end{document } ( if such a coefficient does not exist , we are finished ) . this leads to a new representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } of the form ( 1.1 ) whose total weight does not increase ( and actually remains the same after excluding some trivial cases ) . this process is now repeated until we either have a representation in which all coefficients are bounded by \documentclass[12pt]{minimal }
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\begin{document}$$n-1$$\end{document } , or the support of the representation contains big gaps . in the first case we are finished . in the second case we can split the representation into two parts which are separated by a large gap . the total weight of each part is less than the total weight of the original representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document}. we thus use the induction hypothesis on both of them , so we get a new representation of each part with coefficients bounded by \documentclass[12pt]{minimal }
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\begin{document}$$n-1$$\end{document}. now , since the gap between the supports of these two parts is large , they do not overlap after we apply ( 1.2 ) to them in the appropriate way and we can put them together to find a representation as desired also in this case . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } now we start with the proof of theorem 1.2 . \usepackage{amsmath }
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\begin{document}$$a$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document } we write\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned}{}[\ ! [ { a},{b}]\ ! ] : = { \left\ { { a , a+1,\ldots , b}\right\ } } \end{aligned}$$\end{document}for the integers in the interval from \documentclass[12pt]{minimal }
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\begin{document}$$a$$\end{document } to \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document}. for tuples \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf{x}={\left({x_1,\ldots , x_m}\right)}$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\varvec{\varepsilon } = { \left({{\varepsilon } _ 1,\ldots , { \varepsilon } _ m}\right)}$$\end{document } we set\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \varvec{\varepsilon } ^\mathbf{x}:= { \varepsilon } _ 1^{x_1 } \cdots { \varepsilon } _ m^{x_m}. \end{aligned}$$\end{document}observe first that each element of r has at least one representation of the form ( 1.1 ) . the coefficients of that representation are integers , but not necessarily smaller than \documentclass[12pt]{minimal }
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\begin{document}$$n$$\end{document}. there exists a \documentclass[12pt]{minimal }
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\begin{document}$$k$$\end{document}-th root of unity \documentclass[12pt]{minimal }
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\begin{document}$$\zeta $ $ \end{document } , elements \documentclass[12pt]{minimal }
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\begin{document}$$\eta _ 1,\ldots , \eta _ l \in \mathcal{e } $ $ \end{document } , and multiplicatively independent elements \documentclass[12pt]{minimal }
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\begin{document}$${\varepsilon } _ \vargamma \cap r$$\end{document } , abbreviated as \documentclass[12pt]{minimal }
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\begin{document}$$\varvec{\varepsilon } = { \left({{\varepsilon } _ 1,\ldots , { \varepsilon } _ m}\right)}$$\end{document } , such that\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } u_i = \zeta ^{k_i } \varvec{\varepsilon } ^\mathbf{r ^{(i ) } } , \qquad i\in [ \ ! [ { 1},{i}]\ ! ] , \end{aligned}$$\end{document}for some \documentclass[12pt]{minimal }
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\begin{document}$$k_1,\ldots , k_i\in [ \ ! [ { 0},{k-1}]\!]$$\end{document } and some \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf r ^{(1)},\ldots , \mathbf r ^{(i ) } \in \mathbb z ^m$$\end{document } , each \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } can be written as\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k\in [ \ ! [ { 0},{k-1}]\ ! ] } \sum _ { \ell \in [ \ ! [ { 1},{l}]\ ! ] } \sum _ { \mathbf{x}\in \mathbb z ^m } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } , and such that no relation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \zeta ^{k } \eta _ i \varvec{\varepsilon } ^\mathbf{x}= \eta _ j \varvec{\varepsilon } ^\mathbf y , \quad i \ne j \end{aligned}$$\end{document}with integer exponents and \documentclass[12pt]{minimal }
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\begin{document}$$k\in \mathbb z $ $ \end{document } holds . let \documentclass[12pt]{minimal }
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\begin{document}$$u_1,\ldots , u_i$$\end{document } be as in ( 1.2 ) . choose a \documentclass[12pt]{minimal }
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\begin{document}$$k$$\end{document}-th root of unity \documentclass[12pt]{minimal }
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\begin{document}$$\zeta \in \vargamma \cap r$$\end{document } ( note that the torsion group of \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma $ $ \end{document } is finite and cyclic ) and multiplicatively independent \documentclass[12pt]{minimal }
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\begin{document}$${\varepsilon } _ 1,\ldots , { \varepsilon } _ m \in \vargamma \cap r$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$m \le i$$\end{document } , such that\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } u_i = \zeta ^{k_i } { \varepsilon } _ m^{r_m^{(i ) } } = \zeta ^{k_i } \varvec{\varepsilon } ^\mathbf{r ^{(i ) } } \qquad ( i\in [ \ ! [ { 1},{i}]\ ! ] ) \end{aligned}$$\end{document}holds for some \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf r ^{(1)},\ldots , \mathbf r ^{(i ) } \in \mathbb z ^m$$\end{document}. we set2.1\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } r:= \max \{r_m^{(i)}:\quad { i\in [ \ ! [ { 1},{i}]\!],m\in [ \ ! [ { 1},{m}]\!]}\ } \end{aligned}$$\end{document}and want to mention that we reference to that \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document } later in this section . let us consider a finite subset \documentclass[12pt]{minimal }
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\begin{document}$${\left\ { { \eta _ 1,\ldots , \eta _ l}\right\ } } \subset \mathcal{e } $ $ \end{document } such that all \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } can be written as a linear combination\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k\in [ \ ! [ { 0},{k-1}]\ ! ] } \sum _ { \ell \in [ \ ! [ { 1},{l}]\ ! ] } \sum _ { \mathbf{x}\in \mathbb z ^m } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}\in \mathbb z $ $ \end{document } ( which is possible since \documentclass[12pt]{minimal }
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\begin{document}$$\mathcal{e } $ $ \end{document } finitely generates \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document } as \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z $ $ \end{document}-module ) . we can ( and do ) choose that finite subset such that no relation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \zeta ^k \eta _ i \varvec{\varepsilon } ^\mathbf{x}= \eta _ j \varvec{\varepsilon } ^\mathbf y , \quad i \ne j \end{aligned}$$\end{document}with integer exponents \documentclass[12pt]{minimal }
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\begin{document}$$x , y \in \mathbb z ^m$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$k\in \mathbb z $ $ \end{document } holds . note that \documentclass[12pt]{minimal }
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\begin{document}$$\zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\in \vargamma \cap r$$\end{document}. furthermore , we can choose the coefficients \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } to be non - negative , since , by assumption , we have \documentclass[12pt]{minimal }
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\begin{document}$$-1\in \vargamma $ $ \end{document } , which allows us to choose the signs in our representation . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } from now on we suppose that \documentclass[12pt]{minimal }
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\begin{document}$$\zeta $ $ \end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$\eta _ 1,\ldots , \eta _ l$$\end{document } , and \documentclass[12pt]{minimal }
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\begin{document}$$\varvec{\varepsilon } $ $ \end{document } are fixed and given as in a. we use the following convention on representations . let \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } and suppose we have a representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } where the coefficients are denoted by \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } ( small latin letter with some index ) , i.e. , \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } is written as\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k\in [ \ ! [ { 0},{k-1}]\ ! ] } \sum _ { \ell \in [ \ ! [ { 1},{l}]\ ! ] } \sum _ { \mathbf{x}\in \mathbb z ^m } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}we denote by \documentclass[12pt]{minimal }
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\begin{document}$$a \subset \mathbb z ^m$$\end{document } ( capital latin letter corresponding to the letter used for the coefficients ) the minimal \documentclass[12pt]{minimal }
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\begin{document}$$m$$\end{document}-dimensional interval including all \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf{x}$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x } } \ne 0$$\end{document}. we write\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } a = [ \ ! [ { \underline{a}_1},{\overline{a}_1}]\ ! ] \times \ldots \times [ \ ! [ { \underline{a}_m},{\overline{a}_m}]\ ! ] . \end{aligned}$$\end{document}we omit the range of the indices \documentclass[12pt]{minimal }
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\begin{document}$$k$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\ell $ $ \end{document } since they are always the same . thus \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } will be written as\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document } an important quantity is the weight of a representation . let \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } and suppose we have a representation as in a , i.e.,\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document}with non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document}. we call the minimum of all\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \end{aligned}$$\end{document}among all possible representations ( as above ) of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } the total weight of\documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } and write \documentclass[12pt]{minimal }
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\begin{document}$$w_\alpha $ $ \end{document } for it . as mentioned in the plan of the proof of theorem 1.2 , we apply eq . ( 1.2 ) to an existing representation to get another one . in the following paragraph , we define that replacement step , which will then always be denoted by \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document}. \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } ( replacement step ) . suppose we have a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x } , \end{aligned}$$\end{document}where at least one coefficient \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x } } \ge n$$\end{document}. we get a new representation by applying\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } u_1 + \cdots + u_i = n. \end{aligned}$$\end{document}more precisely , if \documentclass[12pt]{minimal }
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\begin{document}$$u_i = \zeta ^{k_i } \varvec{\varepsilon } ^\mathbf{r ^{(i)}}$$\end{document } , then the coefficient \documentclass[12pt]{minimal }
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\begin{document}$$a_{k+k_i,\ell , \mathbf{x}+\mathbf r ^{(i)}}$$\end{document } is increased by \documentclass[12pt]{minimal }
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\begin{document}$$1$$\end{document } for each \documentclass[12pt]{minimal }
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\begin{document}$$i \in [ \ ! [ { 1},{i}]\!]$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } is replaced by \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}-n$$\end{document}. the following statements b and c deal with two special cases . if \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$w_\alpha < i$$\end{document } , then theorem 1.2 holds . we use that statement as the basis of our induction on the total weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document}. since \documentclass[12pt]{minimal }
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\begin{document}$$i \le n$$\end{document } we have \documentclass[12pt]{minimal }
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\begin{document}$$w_\alpha < n$$\end{document}. so the sum of all ( non - negative ) coefficients is smaller than \documentclass[12pt]{minimal }
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\begin{document}$$n$$\end{document}. therefore all coefficients themselves are in \documentclass[12pt]{minimal }
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\begin{document}$$[\ ! [ { 0},{n-1}]\!]$$\end{document } , which proves the theorem in that special case.\documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } from now on suppose we have an \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } with a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x } , \end{aligned}$$\end{document}which has minimal weight . that means , we have \documentclass[12pt]{minimal }
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\begin{document}$$w : = w_\alpha $ $ \end{document}. if \documentclass[12pt]{minimal }
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\begin{document}$$i < n$$\end{document } , then theorem 1.2 holds . assume that there is a coefficient \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x } } \ge n$$\end{document } in the representation of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document}. we apply \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } to obtain a new representation . but because \documentclass[12pt]{minimal }
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\begin{document}$$i < n$$\end{document } , the new one has smaller total weight , which is a contradiction to the fact that \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } was chosen minimal . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } because of b and c we suppose from now that \documentclass[12pt]{minimal }
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\begin{document}$$w \ge i$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$i = n$$\end{document}. as indicated above , we prove theorem 1.2 by induction on the total weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document}. more precisely we want to prove the following claim by induction . assume that \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in r$$\end{document } has a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } and with minimal total weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document}. then \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } has also a representation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in g } g_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document}with integers \documentclass[12pt]{minimal }
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\begin{document}$$g_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document } and where\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } g = [ \ ! [ { \underline{a}_1-f(w)},{\overline{a}_1+f(w)}]\ ! ] \times \cdots \times [ \ ! [ { \underline{a}_m - f(w)},{\overline{a}_m+f(w)}]\ ! ] \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$f(1)=0$$\end{document } and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } f(w ) = t(w ) r+f(w-1)\qquad ( w\in \mathbb n ) , \end{aligned}$$\end{document}where\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } t(w ) = \left(w+2(w-1)f(w-1)\right)^{mw } k^w l^w . \end{aligned}$$\end{document } in order to prove theorem 1.2 it is sufficient to prove claim 2.3 . as already mentioned , we use induction on the total weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document}. note that the induction basis has been shown above in b. let us start by looking what happens if one applies \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document}. repeatedly applying \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } yields pairwise essentially different representations of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document}. more precisely , by repeatedly applying \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } , it is not possible to get two representations\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}= \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell { \varvec{\varepsilon } ^{\mathbf{x}+\mathbf l } } \end{aligned}$$\end{document}with some \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf l \in \mathbb z ^m \setminus { \left\ { \mathbf{0 } \right\ } } $ $ \end{document}. remember that we assumed \documentclass[12pt]{minimal }
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\begin{document}$$i = n$$\end{document}. first , let us note that we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } n \le \sum _ { i\in [ \ ! [ { 1},{n}]\ ! ] } \left|{u_i}\right| \end{aligned}$$\end{document}because of eq . schwarz inequality yields\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } n^2\le \left(\sum _ { i\in [ \ ! [ { 1},{n}]\ ! ] } 1\cdot \left|{u_i}\right|\right)^2 \le n\sum _ { i\in [ \ ! [ { 1},{n}]\ ! ] } \left|{u_i}\right|^2 . \end{aligned}$$\end{document}hence,\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } n<\sum _ { i\in [ \ ! [ { 1},{n}]\ ! ] } \left|{u_i}\right|^2 , \end{aligned}$$\end{document}unless \documentclass[12pt]{minimal }
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\begin{document}$$ \left|{u_1}\right|=\cdots = \left|{u_n}\right|=1$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\sum _ i u_i = n$$\end{document } , i.e. , \documentclass[12pt]{minimal }
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\begin{document}$$u_1=\cdots = u_n=1$$\end{document}. since the trivial solution has been excluded , we see that every application of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } makes the quantity2.2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \left ( \left|{{\varepsilon } _ 1}\right|^{x_1 } \cdots \left|{{\varepsilon } _ m}\right|^{x_m}\right)^2 \end{aligned}$$\end{document}larger , i.e. , the quantity ( 2.2 ) coming from coefficients \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}^{\prime } $ $ \end{document } is larger than ( 2.2 ) from \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document } , where the \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}^{\prime } $ $ \end{document } are the coefficients after an application of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } on a representation with coefficients \documentclass[12pt]{minimal }
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\begin{document}$$a_{k,\ell , \mathbf{x}}$$\end{document}. note that the \documentclass[12pt]{minimal }
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\begin{document}$${\varepsilon } _ statement a. hence , repeatedly applying \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } produces pairwise disjoint representations . moreover , we can not get the same representation up to linear translation in the exponents twice , i.e. , we can not get representations\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}= \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^{\mathbf{x}+\mathbf l } \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$\mathbf l \in \mathbb z ^m \setminus { \left\ { \mathbf{0 } \right\ } } $ $ \end{document}. such a relation would imply that \documentclass[12pt]{minimal }
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\begin{document}$$\varvec{\varepsilon } ^\mathbf l = 1$$\end{document } , which is a contradiction to the assumption that the \documentclass[12pt]{minimal }
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\begin{document}$${\varepsilon } _ \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } now we look what happens after sufficiently many applications of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document}. set\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } t(w ) : = \left ( w+2(w-1)f(w-1 ) \right)^{mw } k^w l^w \end{aligned}$$\end{document}and suppose we have a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in a } a_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document}after at most \documentclass[12pt]{minimal }
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\begin{document}$$t(w)$$\end{document } applications of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } we get a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in b } b_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x } , \end{aligned}$$\end{document}such that one of the following assertions is true : each coefficient satisfies \documentclass[12pt]{minimal }
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\begin{document}$$b_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m \le w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document } holds for all \documentclass[12pt]{minimal }
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\begin{document}$$m \in [ \ ! [ { 1},{m}]\!]$$\end{document}.there exists an index \documentclass[12pt]{minimal }
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\begin{document}$$m$$\end{document } such that \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m > w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document } holds . each coefficient satisfies \documentclass[12pt]{minimal }
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\begin{document}$$b_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m \le w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document } holds for all \documentclass[12pt]{minimal }
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\begin{document}$$m \in [ \ ! [ { 1},{m}]\!]$$\end{document}. there exists an index \documentclass[12pt]{minimal }
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\begin{document}$$m$$\end{document } such that \documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m > w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document } holds . each replacement step \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } yields an essentially different representation , see d , and there are at most \documentclass[12pt]{minimal }
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\begin{document}$$t(w)$$\end{document } possibilities to distribute our new coefficients in an interval \documentclass[12pt]{minimal }
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\begin{document}$$[\ ! [ { 0},{k-1}]\!]\times [ \ ! [ { 1},{l}]\!]\times b$$\end{document } with\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m \le w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document}for each \documentclass[12pt]{minimal }
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\begin{document}$$m$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$1\le m\le m$$\end{document}. therefore after at most \documentclass[12pt]{minimal }
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\begin{document}$$t(w)$$\end{document } replacement steps we are either in case 1 or in case 2 of e. \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } with the setup and notations of e , a possible translation of the indices stays small . more precisely , we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \max \left\ { \left|{\underline{a}_m-\underline{b}_m}\right|:\quad { m\in [ \ ! [ { 1},{m}]\!]}\right\ } \le t(w ) r , \end{aligned}$$\end{document}and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \max \left\ { \left|{\overline{a}_m-\overline{b}_m}\right|:\quad { m\in [ \ ! [ { 1},{m}]\!]}\right\ } \le t(w ) r , \end{aligned}$$\end{document}where \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document } is as defined as in ( 2.1 ) . the quantity \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document } is the maximum of all exponents in the representation of the \documentclass[12pt]{minimal }
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\begin{document}$$u_i$$\end{document } as powers of the \documentclass[12pt]{minimal }
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\begin{document}$${\varepsilon } _ m$$\end{document}. thus , an application of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } can change the exponents , and therefore the upper and lower bounds , respectively , by at most \documentclass[12pt]{minimal }
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\begin{document}$$r$$\end{document}. we have at most \documentclass[12pt]{minimal }
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\begin{document}$$t(w)$$\end{document } applications of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } , so the statement follows . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } now we look at the two different cases of e. the first one leads to a result directly , whereas in the second one we have to use the induction hypothesis to get a representation as desired . if we are in case ( 1 ) of e , then we are finished . since\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left|\overline{a}_m - \overline{b}_m\right|\le t(w ) r < t(w)r+f(w-1)=f(w ) \end{aligned}$$\end{document}and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \left|\underline{a}_m - \underline{b}_m\right|\le t(w ) r < t(w)r+f(w-1)=f(w ) \end{aligned}$$\end{document}hold for each \documentclass[12pt]{minimal }
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\begin{document}$$m\in \mathbb n $ $ \end{document } we have found a representation as desired in claim 2.3 . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } if we are in case ( 2 ) of e , then we can split the representation into two parts and between them there is a large gap . more precisely , there is a constant \documentclass[12pt]{minimal }
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\begin{document}$$c$$\end{document } such that we can write \documentclass[12pt]{minimal }
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\begin{document}$$\alpha = \gamma + \delta $ $ \end{document } with\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \gamma = \sum _ { k,\ell } { \mathop { \mathop { \sum } \limits _ { \mathbf{x}\in b}}\limits _ { x_m < _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta = \sum _ { k,\ell } { \mathop { \sum \limits _ { \mathbf{x}\in b}}\limits _ { x_m > c + 2f(w-1 ) } } b_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document } in case 2 of e we have an index \documentclass[12pt]{minimal }
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\begin{document}$$m \in [ \ ! [ { 1},{m}]\!]$$\end{document } with\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \overline{b}_m - \underline{b}_m \ge w + 2(w-1)f(w-1 ) \end{aligned}$$\end{document}the total weight of \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } is \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } , so the representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in b } b_{k,\ell , \mathbf{x } } \zeta ^k \eta _ \ell \varvec{\varepsilon } ^\mathbf{x } , \end{aligned}$$\end{document}has at most \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } non - zero coefficients . therefore , by the pigeon hole principle we can find an interval \documentclass[12pt]{minimal }
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\begin{document}$$j$$\end{document } of length at least \documentclass[12pt]{minimal }
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\begin{document}$$2f(w-1)$$\end{document } and with the property that all coefficients \documentclass[12pt]{minimal }
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\begin{document}$$a_{\mathbf{x } , i}$$\end{document } fulfilling \documentclass[12pt]{minimal }
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\begin{document}$$x_m \in j$$\end{document } are zero . therefore we can split up \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } as mentioned . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } if we have the splitting described in h , then claim 2.3 follows for weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document}. after renaming the intervals and coefficients , we have \documentclass[12pt]{minimal }
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\begin{document}$$\alpha = \gamma + \delta $ $ \end{document } with\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \gamma = \sum _ { k,\ell } \sum _ { \mathbf{x}\in c } c_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta = \sum _ { k,\ell } \sum _ { \mathbf{x}\in d } d_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}. \end{aligned}$$\end{document}both total weights \documentclass[12pt]{minimal }
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\begin{document}$$w_\gamma $ $ \end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$w_\delta $ $ \end{document } , respectively , are smaller than \documentclass[12pt]{minimal }
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\begin{document}$$w = w_\alpha $ $ \end{document } , so we can use induction hypothesis : we get representations2.3\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \gamma = \sum _ { k,\ell } \sum _ { \mathbf{x}\in e } e_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$e_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document } and2.4\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \delta = \sum _ { k,\ell } \sum _ { \mathbf{x}\in f } f_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$f_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document}. the upper and lower bounds of the intervals in \documentclass[12pt]{minimal }
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\begin{document}$$c$$\end{document } to \documentclass[12pt]{minimal }
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\begin{document}$$e$$\end{document } differ by at most \documentclass[12pt]{minimal }
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\begin{document}$$f(w_\gamma ) \le f(w-1)$$\end{document } in each coordinate . the same is valid for the intervals of \documentclass[12pt]{minimal }
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\begin{document}$$d$$\end{document } to \documentclass[12pt]{minimal }
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\begin{document}$$f$$\end{document}. since the intervals in \documentclass[12pt]{minimal }
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\begin{document}$$c$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$d$$\end{document } were separated by intervals of length at least \documentclass[12pt]{minimal }
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\begin{document}$$2f(w-1)$$\end{document } , therefore the intervals in \documentclass[12pt]{minimal }
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\begin{document}$$e$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$f$$\end{document } are disjoint . in other words , so we can add these two representations and obtain\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { k,\ell } \sum _ { \mathbf{x}\in g } g_{k,\ell , \mathbf{x } } \zeta ^k\eta _ \ell \varvec{\varepsilon } ^\mathbf{x}\end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$g_{k,\ell , \mathbf{x } } \in [ \ ! [ { 0},{n-1}]\!]$$\end{document}. we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \max \left\ { \left|{\overline{g}_m-\overline{a}_m } \right|:\quad { m\in [ \ ! [ { 1},{m}]\!]}\right\ } \quad \le t(w ) r+f(w-1 ) = f(w ) \end{aligned}$$\end{document}and\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \max \left\ { \left|{\underline{g}_m-\underline{a}_m}\right|:\quad { m\in [ \ ! [ { 1},{m}]\!]}\right\ } \le t(w ) r+f(w-1 ) = f(w ) , \end{aligned}$$\end{document}which finishes the proof . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } let \documentclass[12pt]{minimal }
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\begin{document}$$a$$\end{document } be an integer and let \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } be a root of the polynomial\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } x^3-(a-1)x^2-(a+2)x-1 . \end{aligned}$$\end{document}then the family of real cubic fields \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb q ( \alpha ) $ $ \end{document } is called the family of shanks simplest cubic fields . these fields and the orders \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ \alpha ] $ $ \end{document } have been investigated by several authors . in particular , in a recent paper of the second and third author it was shown that the unit sum height of the orders \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ \alpha ] $ $ \end{document } is \documentclass[12pt]{minimal }
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\begin{document}$$1$$\end{document } in case of \documentclass[12pt]{minimal }
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\begin{document}$$a=0,1,2,3,4,6,13,55$$\end{document } and the unit sum height \documentclass[12pt]{minimal }
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\begin{document}$$a=5$$\end{document}. moreover , it was conjectured that \documentclass[12pt]{minimal }
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\begin{document}$$\omega ( \mathbb z [ \alpha ] ) = 1$$\end{document } for all \documentclass[12pt]{minimal }
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\begin{document}$$a\in \mathbb z $ $ \end{document}. using our main theorem we are able to prove the following result . we have \documentclass[12pt]{minimal }
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\begin{document}$$\omega ( \mathbb z [ \alpha ] ) \le 2$$\end{document } for all \documentclass[12pt]{minimal }
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\begin{document}$$a\in \mathbb z $ $ \end{document}. first let us note some important facts on \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb q ( \alpha ) $ $ \end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ \alpha ] $ $ \end{document } , see for example shanks original paper . we know that \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb q ( \alpha ) $ $ \end{document } is galois over \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb q $ $ \end{document } with galois group \documentclass[12pt]{minimal }
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\begin{document}$$g=\{\text { i}d , \sigma , \sigma ^2\}$$\end{document } and with \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ 2:=\sigma ( \alpha ) = -1-\frac{1}{\alpha } $ $ \end{document}. if we set \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ 1:= \alpha $ $ \end{document } , then \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ 1$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\alpha _ 2$$\end{document } are a fundamental system of units . now we know enough about the structure of \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ \alpha ] $ $ \end{document } to apply theorem 1.2 . if we can find three units \documentclass[12pt]{minimal }
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\begin{document}$$u_1,u_2,u_3\in \mathbb z [ \alpha ] ^*$$\end{document } such that \documentclass[12pt]{minimal }
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\begin{document}$$u_1+u_2+u_3=3$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$u_i \ne 1$$\end{document } , then the theorem is a direct consequence of theorem 1.2 . indeed we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 3&= \overbrace{(\alpha _ 1 ^ 2+(-a+2)\alpha _ 1-a)}^{=u_1 } \\&+ \overbrace{(-2\alpha _ 1 ^ 2+(2a-1)\alpha _ 1 + a+4)}^{=u_2 } \\&+ \overbrace{(\alpha _ 1 ^ 2+(-a-1)\alpha _ 1 - 1)}^{=u_3 } \\&= \alpha _ 1\alpha _ 2 ^ 2+\alpha _ 1^{-2}\alpha _ 2^{-1}+\alpha _ 1\alpha _ 2^{-1}. \end{aligned}$$\end{document}\documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } we start with the definition of a signed double - base expansion of an integer . ( signed double - base expansion ) let \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be different integers . let \documentclass[12pt]{minimal }
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\begin{document}$$n$$\end{document } be an integer with\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } n = \sum _ { i\in \mathbb n _ 0 , j\in \mathbb n _ 0 } d_{ij } p^i q^j , \end{aligned}$$\end{document}where \documentclass[12pt]{minimal }
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\begin{document}$$d_{ij } \in { \left\ { { -1,0,1}\right\ } } $ $ \end{document } and only finitely many \documentclass[12pt]{minimal }
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\begin{document}$$d_{ij}$$\end{document } are non - zero . then such a sum is called a signed\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion of\documentclass[12pt]{minimal }
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\begin{document}$$n$$\end{document}. the pair \documentclass[12pt]{minimal }
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\begin{document}$$(p , q)$$\end{document } is called base pair . a natural first question is , whether each integer has a signed double - base expansion for a fixed base pair . if one of the bases \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } is either \documentclass[12pt]{minimal }
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\begin{document}$$2$$\end{document } or \documentclass[12pt]{minimal }
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\begin{document}$$3$$\end{document } , then existence follows since every integer has a binary representation ( base \documentclass[12pt]{minimal }
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\begin{document}$$2$$\end{document } with digit set \documentclass[12pt]{minimal }
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\begin{document}$${\left\ { { 0,1}\right\ } } $ $ \end{document } ) and a balanced ternary representation ( base \documentclass[12pt]{minimal }
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\begin{document}$$3$$\end{document } with digit set \documentclass[12pt]{minimal }
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\begin{document}$${\left\ { { -1,0,1}\right\ } } $ $ \end{document } ) , respectively . to get the existence results for general base pairs , we use the following theorem , cf . ( birch ) let \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be coprime integers . then there is a positive integer \documentclass[12pt]{minimal }
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\begin{document}$$n(p , q)$$\end{document } such that every integer larger than \documentclass[12pt]{minimal }
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\begin{document}$$n(p , q)$$\end{document } may be expressed as a sum of distinct numbers of the form \documentclass[12pt]{minimal }
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\begin{document}$$p^iq^j$$\end{document } all with non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$i$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$j$$\end{document}. let \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be coprime integers . then each integer has a signed \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion . next we want to give an efficient algorithm that allows to calculate a signed double base expansion of a given integer . birch s theorem , or more precisely the proof in , does not provide an efficient way to do that . however , using our main result , there is a way to compute such expansions efficiently at least for certain base pairs . let \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be coprime integers with absolute value at least \documentclass[12pt]{minimal }
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\begin{document}$$3$$\end{document}. if there are non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$x$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$y$$\end{document } such that4.1\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2 = \left|{p^x - q^y}\right|\ ! , \end{aligned}$$\end{document}then each integer has a signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion which can be computed efficiently ( there exists a polynomial time algorithm ) . in particular given a \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-adic expansion of an integer \documentclass[12pt]{minimal }
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\begin{document}$$\alpha $ $ \end{document } , one has to apply ( 4.1 ) at most \documentclass[12pt]{minimal }
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\begin{document}$$o(\log ( \alpha ) ^2)$$\end{document } times . we start to prove the first part of the corollary and therefore apply theorem 1.2 with \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb f = \mathbb q $ $ \end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$r=\mathbb z $ $ \end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma $ $ \end{document } is the multiplicative group generated by \documentclass[12pt]{minimal }
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\begin{document}$$-1,p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}. since by assumption \documentclass[12pt]{minimal }
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\begin{document}$$2=\pm ( p^x - q^y)$$\end{document } we have a solution to ( 1.2 ) and theorem 1.2 yields that \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansions exist . \usepackage{amsmath }
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\begin{document}$$\alpha $ $ \end{document } the \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-adic expansion\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = a_{0}+a_{1}p+\cdots + a_{k } p^{k } \end{aligned}$$\end{document}is given , with \documentclass[12pt]{minimal }
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\begin{document}$$a_{0},\ldots , a_{k}\in [ \ ! [ { 0},{p-1}]\!]$$\end{document}. let us note that the weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document } of this representation is at most \documentclass[12pt]{minimal }
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\begin{document}$$o(\log \alpha ) $ $ \end{document}. now the following claim yields the corollary . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } assume\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { i\in [ \ ! [ { 0},{i}]\ ! ] } a_i p^i \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$a_i\in \mathbb z $ $ \end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$i\in \mathbb n _ 0$$\end{document } , and set \documentclass[12pt]{minimal }
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\begin{document}$$w = \sum _ { i\in [ \ ! [ { 0},{i}]\ ! ] } \left|{a_i}\right|$$\end{document}. then , after at most \documentclass[12pt]{minimal }
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\begin{document}$$\frac{w^2-w}{2}$$\end{document } replacement steps \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } we arrive at a representation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { j\in [ \ ! [ { 0},{j}]\ ! ] } q^{jy } \sum _ { k\in [ \ ! [ { 0},{k}]\ ! ] } b_{k , j}p^k , \end{aligned}$$\end{document}where the \documentclass[12pt]{minimal }
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\begin{document}$$b_{k , j}$$\end{document } are integers with \documentclass[12pt]{minimal }
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\begin{document}$$ \left|{b_{k , j}}\right| \le 1$$\end{document } , and \documentclass[12pt]{minimal }
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\begin{document}$$j , k\in \mathbb n _ 0$$\end{document}. we prove the claim by induction on \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document}. if \documentclass[12pt]{minimal }
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\begin{document}$$w\le 1$$\end{document } the statement of the claim is obvious . further , if all the \documentclass[12pt]{minimal }
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\begin{document}$$a_i$$\end{document } are in \documentclass[12pt]{minimal }
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\begin{document}$${\left\ { { -1,0,1}\right\ } } $ $ \end{document } we are done . therefore we assume that there is at least one index \documentclass[12pt]{minimal }
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\begin{document}$$i$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$ \left|{a_i}\right|>1$$\end{document}. we now apply the replacement step \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } in the following way : if \documentclass[12pt]{minimal }
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\begin{document}$$a_i>1$$\end{document } , then \documentclass[12pt]{minimal }
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\begin{document}$$a_i$$\end{document } is replaced by \documentclass[12pt]{minimal }
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\begin{document}$$a_i-2$$\end{document } , if \documentclass[12pt]{minimal }
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\begin{document}$$a_i<-1$$\end{document } , then \documentclass[12pt]{minimal }
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\begin{document}$$a_i$$\end{document } is replaced by \documentclass[12pt]{minimal }
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\begin{document}$$a_i+2$$\end{document}. after at most \documentclass[12pt]{minimal }
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\begin{document}$$w-1$$\end{document } such steps , we get a new representation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \sum _ { i\in [ \ ! [ { 0},{i_c}]\ ! ] } c_ip^i + q^y \sum _ { i\in [ \ ! [ { 0},{i_d}]\ ! ] } d_ip^i , \end{aligned}$$\end{document}\documentclass[12pt]{minimal }
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\begin{document}$$i_c , i_d\in \mathbb n _ 0$$\end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$c_i , d_i\in \mathbb z $ $ \end{document } , such that all \documentclass[12pt]{minimal }
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\begin{document}$$c_i$$\end{document } fulfil \documentclass[12pt]{minimal }
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\begin{document}$$ \left|{c_i}\right|\le 1$$\end{document}. note that no replacement step \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } increases the weight \documentclass[12pt]{minimal }
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\begin{document}$$w$$\end{document}. now consider\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \beta = \sum _ { i\in [ \ ! [ { 0},{i_d}]\ ! ] } d_ip^i . \end{aligned}$$\end{document}the weight of \documentclass[12pt]{minimal }
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\begin{document}$$\beta $ $ \end{document } fulfils\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } w_\beta = \sum _ { i\in [ \ ! [ { 0},{i_d}]\ ! ] } \left|{d_i}\right| \le w-1 , \end{aligned}$$\end{document}since in each replacement step it is increased exactly by \documentclass[12pt]{minimal }
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\begin{document}$$1$$\end{document}. now , by induction , hypothesis we obtain a representation\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \beta = \sum _ { j\in [ \ ! [ { 0},{j_e}]\ ! ] } q^{jy } \sum _ { k\in [ \ ! [ { 0},{k_e}]\ ! ] } e_{k , j}p^k , \end{aligned}$$\end{document}where the \documentclass[12pt]{minimal }
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\begin{document}$$e_{k , j}$$\end{document } are integers with \documentclass[12pt]{minimal }
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\begin{document}$$ \left|{e_{k , j}}\right| \le 1$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$j_e , k_e\in \mathbb n _ 0$$\end{document}. further , this can be done in \documentclass[12pt]{minimal }
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\begin{document}$$\frac{w_\beta ^2-w_\beta } { 2}$$\end{document } steps . setting \documentclass[12pt]{minimal }
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\begin{document}$$b_{i,0 } = c_i$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$b_{i , k } = e_{i , k-1}$$\end{document } for \documentclass[12pt]{minimal }
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\begin{document}$$k>0$$\end{document } yields the desired representation . moreover , this can be done with at most\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \frac{w_\beta ^2-w_\beta } { 2 } + w-1 \le \frac{(w-1)(w-2)}{2 } + w-1 = \frac{w(w-1)}{2 } \end{aligned}$$\end{document}applications of \documentclass[12pt]{minimal }
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\begin{document}$$*$$\end{document } , which finishes the proof of the claim . \documentclass[12pt]{minimal }
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\begin{document}$$\square $ $ \end{document } now we want to give some examples for base pairs , where the corollary can be used . let \documentclass[12pt]{minimal }
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\begin{document}$$(p , q)$$\end{document } be a twin prime pair , i.e. , we have \documentclass[12pt]{minimal }
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\begin{document}$$q = p+2$$\end{document } and both \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } are primes . then clearly\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2 = q - p , \end{aligned}$$\end{document}so , by corollary 4.4 , every integer has a signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion , which can be calculated efficiently . let \documentclass[12pt]{minimal }
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\begin{document}$$p=5$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q=23$$\end{document}. we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2 = 5 ^ 2 - 23 , \end{aligned}$$\end{document}therefore every integer has a signed \documentclass[12pt]{minimal }
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\begin{document}$$5$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$23$$\end{document}-double - base expansion , which can be calculated efficiently . , we calculated the following:\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 995&= - 5^{5 } + 5^{4 } + 5^{3 } \cdot 23 - 5^{2 } + 5 \cdot 23 + 23^{2 } + 1 \\ 996&= - 5^{3 } + 5^{2 } \cdot 23 + 23^{2 } - 5 + 23 - 1 \\ 997&= - 5^{3 } + 5^{2 } \cdot 23 + 23^{2 } - 5 + 23 \\ 998&= 5^{4 } - 5^{3 } - 5^{2 } + 23^{2 } - 5 - 1 \\ 999&= 5^{4 } - 5^{3 } - 5^{2 } + 23^{2 } - 5 \\ 1000&= 5^{4 } - 5^{3 } - 5^{2 } + 23^{2 } - 5 + 1 \\ 1001&= - 5^{3 } + 5^{2 } \cdot 23 + 23^{2 } + 23 - 1 \\ 1002&= - 5^{3 } + 5^{2 } \cdot 23 + 23^{2 } + 23 \\ 1003&= 5^{4 } - 5^{3 } - 5^{2 } + 23^{2 } - 1 \end{aligned}$$\end{document}in each case we started with an initial expansion , which is obtained by a greedy algorithm : for a \documentclass[12pt]{minimal }
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\begin{document}$$v\in \mathbb z $ $ \end{document } find the closest \documentclass[12pt]{minimal }
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\begin{document}$$5^i \cdot 23^j$$\end{document } , change the coefficient for that base , and continue with \documentclass[12pt]{minimal }
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\begin{document}$$v - 5^i \cdot 23^j$$\end{document}. then we calculated the expansion by applying the equation \documentclass[12pt]{minimal }
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\begin{document}$$2 = 5 ^ 2 - 23$$\end{document } as in the proof of theorem 1.2 . one can find pairs \documentclass[12pt]{minimal }
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\begin{document}$$(p , q)$$\end{document } where corollary 4.4 does not work . consider the equation4.2\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2 = \left|{p^x - q^y}\right| \end{aligned}$$\end{document}with non - negative integers \documentclass[12pt]{minimal }
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\begin{document}$$x$$\end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$y$$\end{document}. a first example , where the corollary fails , is \documentclass[12pt]{minimal }
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\begin{document}$$p=5$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q=11$$\end{document}. indeed , looking at eq . another example is \documentclass[12pt]{minimal }
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\begin{document}$$p=7$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q=13$$\end{document } , where looking at ( 4.2 ) modulo \documentclass[12pt]{minimal }
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\begin{document}$$7$$\end{document } , yields a contradiction . a third example is \documentclass[12pt]{minimal }
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\begin{document}$$p=7$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q=11$$\end{document}. so in the cases given in the remark above , as well as in a lot of other cases , we can not use the corollary to compute a signed double - base expansion efficiently . is there an efficient ( polynomial time ) algorithm for each base pair \documentclass[12pt]{minimal }
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\begin{document}$$(p , q)$$\end{document } to compute a signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion for all integers ? for some combinations of \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } we can get a weaker result . first , we define an extension of the signed double - base expansion : we allow negative exponents in the \documentclass[12pt]{minimal }
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\begin{document}$$p^i q^j$$\end{document } , too . ( extended signed double - base expansion ) let \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be different integers ( usually coprime ) . let \documentclass[12pt]{minimal }
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\begin{document}$$z\in \mathbb q $ $ \end{document}. if we have\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } z = \sum _ { i\in \mathbb z , j\in \mathbb z } d_{ij } p^i q^j , \end{aligned}$$\end{document}where \documentclass[12pt]{minimal }
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\begin{document}$$d_{ij } \in { \left\ { { -1,0,1}\right\ } } $ $ \end{document } and only finitely many \documentclass[12pt]{minimal }
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\begin{document}$$d_{ij}$$\end{document } are non - zero , then we call the sum an extended signed\documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion of\documentclass[12pt]{minimal }
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\begin{document}$$z$$\end{document}. with that definition , we can prove the following corollary to theorem 1.2 . let \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } be coprime integers . if there are integers \documentclass[12pt]{minimal }
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\begin{document}$$a$$\end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$c$$\end{document } , and \documentclass[12pt]{minimal }
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\begin{document}$$d$$\end{document } with \documentclass[12pt]{minimal }
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\begin{document}$$(a , b , c , d)\ne ( 0,0,0,0)$$\end{document } and such that4.3\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2 = p^a q^b \pm p^c q^d , \end{aligned}$$\end{document}then every element of \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ 1/p,1/q]$$\end{document } has an extended signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion which can be computed efficiently ( polynomial time algorithm ) . if we have a solution to the equation in corollary 4.4 , then corollary 4.11 works , too . but more can be said about the existence and efficient computability of extended double - base expansions for the elements of \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ 1/p,1/q]$$\end{document}. if each integer has an efficient computable signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion , then each element of \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb z [ 1/p,1/q]$$\end{document } has an extended signed \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion which can be computed efficiently . the proof of this corollary runs along the same lines as the proof of corollary 4.4 . we apply theorem 1.2 with \documentclass[12pt]{minimal }
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\begin{document}$$\mathbb f = \mathbb q $ $ \end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$r=\mathbb z [ 1/p,1/q]$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$\vargamma $ $ \end{document } is the multiplicative group generated by \documentclass[12pt]{minimal }
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\begin{document}$$-1$$\end{document } , \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}. since , by assumption , \documentclass[12pt]{minimal }
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\begin{document}$$2=\pm ( p^aq^b - p^cq^d)$$\end{document } we have a solution to ( 1.2 ) , theorem 1.2 yields that \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-\documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansions exist . next , we claim that we may assume \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document } are odd and \documentclass[12pt]{minimal }
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\begin{document}$$p , q>3$$\end{document}. indeed assuming that \documentclass[12pt]{minimal }
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\begin{document}$$p\in { \left\ { { 2,3}\right\ } } $ $ \end{document } , then we can write \documentclass[12pt]{minimal }
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\begin{document}$$\alpha \in \mathbb z [ 1/p,1/q]$$\end{document } in the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \alpha = \frac{\widetilde{\alpha } } { p^{x_p}q^{x_q } } \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$\widetilde{\alpha } \in \mathbb z $ $ \end{document } and appropriate exponents \documentclass[12pt]{minimal }
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\begin{document}$$x_p$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$x_q$$\end{document}. moreover , \documentclass[12pt]{minimal }
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\begin{document}$$\widetilde{\alpha } $ $ \end{document } has a representation of the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } \widetilde{\alpha } = \sum _ { i\in [ \ ! [ { 0},{k}]\!]}a_i p^i \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$a_i\in { \left\ { { -1,0,1}\right\ } } $ $ \end{document}. however the computation of such a represenation can be done efficiently and takes polynomial time in the height \documentclass[12pt]{minimal }
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\begin{document}$$h(\alpha ) $ $ \end{document } , where\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } h(n / m)=\max \{\log \left|{n}\right| , \log \left|{m}\right| , 1\ } \end{aligned}$$\end{document}provided \documentclass[12pt]{minimal }
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\begin{document}$$n , m\in \mathbb z $ $ \end{document } are coprime . since we may assume \documentclass[12pt]{minimal }
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\begin{document}$$p , q>3$$\end{document } , we want to show next that a solution to eq . ( 4.3 ) necessarly takes the form\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2=\pm p^{-a}\pm p^{-a}q^b , \end{aligned}$$\end{document}with \documentclass[12pt]{minimal }
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\begin{document}$$a , b\ge 0$$\end{document}. we observe that a solution to ( 4.3 ) with \documentclass[12pt]{minimal }
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\begin{document}$$a , c>0$$\end{document } or \documentclass[12pt]{minimal }
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\begin{document}$$b , c>0$$\end{document } does not exist , since otherwise \documentclass[12pt]{minimal }
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\begin{document}$$p\vert 2$$\end{document } or \documentclass[12pt]{minimal }
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\begin{document}$$q\vert 2$$\end{document}. next we note that if \documentclass[12pt]{minimal }
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\begin{document}$$a\ne c$$\end{document } ( \documentclass[12pt]{minimal }
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\begin{document}$$b\ne d$$\end{document } respectively ) the \documentclass[12pt]{minimal }
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\begin{document}$$p$$\end{document}-adic valuation ( \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-adic valuation ) on the right hand side of ( 4.3 ) would be the minimum of \documentclass[12pt]{minimal }
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\begin{document}$$a$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$c$$\end{document } ( \documentclass[12pt]{minimal }
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\begin{document}$$b$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$d$$\end{document } respectively ) and in view of the left hand side , this minimum must be \documentclass[12pt]{minimal }
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\begin{document}$$0$$\end{document}. thus any solution to eq . ( 4.3 ) must be of one of the following forms:\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2&= \pm p^aq^b \pm 1 , \\ 2&= \pm p^{-a}q^{-b } \pm p^{-a}q^{-b } , \\ \end{aligned}$$\end{document}or\documentclass[12pt]{minimal }
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\begin{document}$$\begin{aligned } 2&= \pm p^a\pm q^b , \end{aligned}$$\end{document}where \documentclass[12pt]{minimal }
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\begin{document}$$a$$\end{document } and \documentclass[12pt]{minimal }
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\begin{document}$$q$$\end{document}-double - base expansion with minimal weight for a given integer ? a greedy approach for solving this question can be found in berth and imbert , some further results can be found in dimitrov and howe . | let \documentclass[12pt]{minimal }
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\begin{document}$$u , v\in \mathfrak o ^*$$\end{document}. we generalize this result and give applications to signed double - base digit expansions . |
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they stimulate expression of certain proinflammatory cytokines and adsorb plasma proteins , which initiate immune response cascades that stimulate macrophage infiltration and multinuclear giant cell formation . cytokines produced by giant cells and macrophages recruit other inflammatory cells and stimulate fibrous encapsulation of the material by fibroblasts . encapsulation isolates the material from the surrounding tissue , facilitating foreign body elimination and tissue repair . however , failure to phagocytize the material is often associated with release of damaging lysosomal enzymes and reactive oxygen species . this report describes a case in which polymerizing resin was forced into a surgical extraction site , where it remained for ten months . a healthy 45-year - old caucasian female presented with swelling and discomfort in the upper right posterior area , which had persisted since the extraction of her upper right second premolar . immediately after the extraction , the adjacent teeth were prepared for a fixed prosthesis and a temporary bridge was fabricated . periapical radiograph of the maxillary right posterior teeth , taken 53 months prior to extraction of the upper right second premolar the patient returned the following day with pain , swelling , and facial bruising . when symptoms persisted to the eleventh post - operative day , her dentist removed the temporary bridge , curetted the extraction site , and prescribed clindamycin . when she was re - evaluated on the twenty - third postoperative day , there was continued swelling , but wound healing appeared to be progressing . within three months after the extraction , the site was covered by intact keratinized tissue . radiographs revealed a radiopaque object distal to the upper right first premolar and a mesial osteotomy defect [ figure 2 ] . a segmental osteotomy performed thirty years earlier to correct a maxillary midline discrepancy had failed to heal completely . the edentulous site exhibited firm facial swelling , but did not appear granulomatous . after obtaining local anesthesia , a facial full thickness flap was reflected from the distal of the first molar to the distal of the canine . an encapsulated acrylic object and associated granulation tissue since the site was inflamed and access was limited by the bridge , no bone graft was placed prior to wound closure . at a follow - up appointment however , the gingival margin on the distofacial and distal aspects of the premolar had receded 2.5 mm in conjunction with tissue shrinkage at the edentulous site . periapical radiograph of the maxillary right posterior teeth , taken 9 months after extraction of the upper right second premolar longitudinal view of the resin foreign body after surgical removal from the upper right second premolar extraction site final healing was evaluated eight months later . a radiograph suggested that the density of the bone distal to the first premolar had increased [ figure 4 ] . post - operative radiograph of the maxillary right posterior teeth , taken 8 months after removal of the foreign body the decision to prepare teeth adjacent to a fresh surgical extraction site contributed to the complication described in this report . acrylic was presumably forced into the surgical site during fabrication of the temporary bridge , molded by the socket , and later displaced by postoperative curettage . if a connection had persisted between the entrapped acrylic and the temporary bridge , the entrapped mass might have been discovered . from the earliest postoperative stages , the patient experienced swelling and delayed healing that persisted despite treatment with curettage , anti - inflammatory agents , and antibiotics . this resistance to treatment signaled the need for further investigation . a radiograph would have been the single most useful source of information to explain the delayed healing . ultimately , healing progressed to fibrous encapsulation of the acrylic mass . by isolating the foreign body from the bone and the oral environment , encapsulation may have limited bone loss , reduced the magnitude of inflammatory episodes and enhanced the potential for favorable osseous healing . while complications of this nature are rare , this case reinforces the need to account for all materials introduced into the oral cavity , investigate the etiology of persistent swelling and inflammation , and incorporate time for complete postoperative healing in the treatment sequence . in cases of foreign body gingivitis , histological evaluation shows granulomatous foci contained particles of foreign material that are often inconspicuous and easily overlooked . home care recommendations of patients with foreign body gingivitis include avoidance of dentifrices with certain chemical additives and rinses with high alcohol content . | when acrylic resin is inadvertently embedded in oral tissue , it can result in a pronounced chronic inflammatory response .
this report describes a case in which temporary crown and bridge resin was forced into a surgical extraction site after the two adjacent teeth were prepared for a bridge immediately following extraction of a maxillary premolar .
the patient experienced swelling at the extraction site over a ten month period despite treatment with antibiotics and anti - inflammatory drugs .
after detection and removal of the foreign body , the symptoms resolved .
the episode contributed to periodontal bone loss around an adjacent tooth .
while morbidity of this nature is rare , this case reinforces the need to investigate persistent signs of inflammation and account for dental materials that are lost during the course of treatment . |
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a 60-year - old male was referred to the ophthalmology clinic with recurrent uveitis of the right eye and severe corneal edema . he had been diagnosed with anterior uveitis one month previous at a private eye clinic , for which administration of topical 1% prednisolone acetate led to remission . anterior uveitis recurred with severe corneal edema one month after the first episode . despite topical and systemic steroid treatment , the anterior chamber ( ac ) inflammation and corneal edema worsened , leading to the referral of this patient to our clinic . at initial presentation , visual acuity was 0.2 od , and 0.9 os using a snellen visual acuity chart . intraocular pressure ( iop ) was 15 mmhg od and 17 mmhg os as measured with a non - contact tonometer ( nct ) . an od slit lamp examination revealed grades 1 and 2 ac reactions with flare and severe corneal edema . a small whitish fragment , previously diagnosed as hypopion at the private eye clinic , the od vitreous and fundus were not visualized due to severe corneal edema , but b - ultrasonography revealed no remarkable signs . specular pachymetry of the right eye was not possible due to the severe corneal edema and endothelial decompensation . it was concluded that the whitish material in the inferior angle of the ac was a retained lens fragment , because a retained lens fragment was also discovered in the superior capsule . a clear corneal incision was made at the superior cornea , and hyaluronate sodium ( 13.8 mg/0.6 ml / syr , healon 5 inj ; meditip co. , seoul , korea ) was injected into the anterior chamber . the lens fragment was removed with lens forceps , and the remaining hyaluronate sodium was removed with irrigation and aspiration ( stellaris vision enhancement system ; bausch & lomb , rochester , ny , usa ) . on postoperative day 1 , od corneal edema was slightly aggravated , and visual acuity was limited to a finger count at 10 cm . after one month , od corneal edema was much improved , and visual acuity had increased to 0.4 by snelln visual acuity chart . a two - month post - operative od follow - up examination revealed reduced corneal edema and subsequent improvement of visual acuity to 0.7 by snellen visual acuity chart ( fig . although the od cornea was clear , an endothelial cell count showed 458 cells / mm , and specular pachymetry revealed significant loss of hexagonality ( fig . postoperative anterior segment inflammation after cataract surgery can be a difficult diagnostic and management problem . causes of late onset postoperative inflammation include retained lens fragments , reactivated uveitis , and infections , such as endophthalmitis and wound abscess . late onset of corneal edema after a cataract surgery also has been reported to be caused by retained lens fragments [ 7 - 9 ] , intraocular lens fragments , and endothelitis . a retained lens fragment in the anterior segment after phacoemulsification with an intact posterior capsule is an uncommon complication . a retained cortical fragment causes an inflammatory reaction that can generally be treated with topical corticosteroids , resulting in resolution of the inflammation and resorption of the cortical material . retained unencapsulated nuclear material frequently causes an inflammatory reaction in the ac as well as corneal edema of varying severity . bohigian and wexler suggested that direct mechanical trauma to the corneal endothelium played a role in the corneal edema caused by retained ac lens fragments . some researchers have suggested that lens fragments may be trapped in the posterior chamber during phacoemulsification in high myope patients with small pupils and a hard nucleus , especially if significant shattering of fragments occurs during phacoemulsification . together with the lens fragments themselves , lens epithelial cells on those fragments the patient had undergone cataract surgery at another other eye clinic 15 years previous ; it was impossible to determine the preoperative refractive errors and keratometry values . anterior uveitis with corneal edema was refractory despite topical and systemic steroid therapy , so the lens fragment in this patient was thought to be a nuclear fragment . as in prior cases , surgical removal of the nuclear fragment was required in order to improve the ac reaction and corneal edema [ 7 - 9 ] . a unique feature of this case is that the interval between cataract surgery and anterior uveitis was 15 years . in previous cases , the interval was between 6 weeks to 27 months , much shorter than in this case [ 7 - 9,14,15 ] . we hypothesize that the lens fragment in this patient remained sequestered behind the iris or posterior chamber for 15 years after cataract surgery . alternatively , the lens fragment may have been sequestered in the posterior capsule with no inflammation after the cataract operation . it is possible that the lens fragment migrated into the anterior chamber , possibly due to minor trauma or capsule deformation during fibrosis , leading to the acute onset of the ac reaction with severe corneal edema . at the first diagnosis of anterior uveitis one month before the initial visit to our clinic , the lens fragment might have been located in the inferior angle , as in a previously recorded case . the lens fragment might have migrated to the surface of the iris to come into contact with the corneal endothelium , leading to corneal edema and decreased vision . this case highlights that retained lens fragments should be considered as causes for anterior uveitis in a pseudophakic eye , even years after cataract surgery . this case has shown that the time interval between surgery and anterior uveitis can be as long as 15 years . therefore , when a pseudophakic patient presents with anterior uveitis , a possible hidden lens fragment should be considered . in such cases careful examination including gonioscopy should be performed . in a patient with some cortical remnant , especially within the superior lens capsule , a follow - up showed be performed to assess the possibility of lens fragment migration and subsequent chronic uveitis . | a 60-year - old male was referred to the ophthalmologic clinic with aggravated anterior uveitis and corneal edema despite the use of topical and systemic steroids .
he had undergone cataract surgery in both eyes 15 years previous .
slit lamp examinations revealed a retained lens fragment in the inferior angle of the anterior chamber , with severe corneal edema and mild anterior uveitis .
the corneal edema and uveitis subsided following surgical extraction of the lens fragment . that a retained lens fragment caused symptomatic anterior uveitis with corneal edema 15 years after an uneventful cataract surgery is unique .
a retained lens fragment should be considered as one of the causes of anterior uveitis in a pseudophakic patient . |
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we obtained nucleotide sequences of the envelope ( e ) and nonstructural ( ns)3/ns5 protein regions of 6 wnv human strains isolated during the 20082009 outbreaks in italy and analyzed their phylogenetic relationships with other wnv strains isolated in europe and the mediterranean basin . virus isolation on vero e6 cells was performed from human serum samples obtained from patients with neuroinvasive wnv infection : 5 samples were collected during the 2009 outbreak from patients residing in the provinces of ferrara ( 2 patients ) , modena ( 1 ) , and mantua ( 2 ) ; 1 sample was collected during the 2008 outbreak from a patient living in ferrara . cytopathic effects were observed 6 days after the serum was injected into cell cultures ; wnv infection was identified by immunofluorescence staining and confirmed by real - time reverse transcription pcr ( 9 ) . virus isolates were designed as follows : tos-09 ( ferrara ) , nal-09 ( ferrara ) , chi-09 ( mantua ) , bal-09 ( mantua ) , fan-09 ( modena ) , and man-08 ( ferrara ) . after 1 passage , supernatants of infected tissue cultures were processed for viral rna extraction and pcr amplification . nucleotide sequences were obtained for the e gene ( 1,503 nt ) and for a region of 5,759 nt encompassing the ns3 , ns4a , ns4b , and ns5 genes , which are considered to be the most phylogenetic informative regions ( 12,13 ) . multiple alignments of the 6 new wnv sequences of e and ns3/ns5 regions , along with the corresponding sequences from 30 other wnv strains and isolates available in genbank and usutu virus as outgroup virus , were generated by using clustalw software ( www2.ebi.ac.uk/clustalw ) . phylogenetic analyses of the e ( figure 1 ) and ns3/ns5 ( figure 2 ) sequences generated highly comparable topologies and confirmed that strains from italy and all western mediterranean strains belonging to lineage 1 , clade 1a , were highly related . in addition , on the basis of the phylogenetic trees of e and ns3/ns5 sequences , we found that the 6 new sequences clustered with other strains isolated in italy in 2008 and 2009 , constituting a distinct and well - supported group within the western mediterranean cluster ( 100% bootstrap on e and ns3/ns5 regions ) ( figure1 , figure 2 ) . phylogenetic trees of west nile virus strains isolated during outbreaks in italy , 20082009 , based on nucleotide sequences of the complete envelope gene . phylogenetic tree and distance matrices were constructed by using nucleotide alignment , the kimura 2-parameter algorithm , and the neighbor - joining method implemented in mega version 4.1 ( www.megasoftware.net/mega4/mega41.html ) . the percentage of successful bootstrap replicates is indicated at nodes , showing only values > 80 . strains sequenced in this study are tos-09 , fan-09 , bal-09 , chi-09 , nal-09 , and man-08 . envelope nucleotide sequence genbank accession nos : nal-09 , hm991272 ; tos-09 , hm991273 ; bal-09 , hm991274 ; chi-09 , hm991275 ; man-09 , hm991276 ; fan-09 , hm991277 . boldface indicates the 6 newly sequenced strains . genomic region encompassing nonstructural ( ns ) 3 , ns4a , ns4b , and ns5 genes . phylogenetic tree and distance matrices were constructed by using nucleotide alignment , the kimura 2-parameter algorithm , and the neighbor - joining method implemented in mega version 4.1 ( www.megasoftware.net/mega4/mega41.html ) . the percentage of successful bootstrap replicates is indicated at nodes , showing only values > 80 . strains sequenced in this study are tos-09 , fan-09 , bal-09 , chi-09 , nal-09 and 191 man-08 . : nal-09 , hm641230 ; tos-09 , hm641225 ; bal-09 , hm641226 ; chi-09 , hm641227 ; man-09 , hm641229 ; fan-09 , hm641228 . the mean genetic distance within the 6 new isolates from italy was 0.08% at the nucleotide level , whereas the derived amino acid sequences were totally conserved . for the ns3/ns5 region , the mean genetic distance within the 6 new isolates from italy was 0.10% at the nucleotide and amino acid levels . within the 20082009 cluster from italy , the 6 newly sequenced human strains exhibited a 0.14% mean nucleotide distance with the 2 strains isolated in the emilia romagna region in 2008 from magpies ( italy 15803/08 and italy 15217/08 ) and 0.15% with the first human wnv strain isolated in italy in 2009 ( italy/09 ) in the veneto region ( 14 ) , in the e and ns3/ns5 genomic regions . on the basis of ns3/ns5 sequences , all strains from italy isolated during the 20082009 outbreaks showed the highest relatedness to morocco/03 , france/04 , and portugal/04 with 1.08% , 1.28% , and 1.45% mean nucleotide distance , respectively , whereas they show a mean nucleotide distance of 1.56% with italy/98 . the close relatedness to morocco/03 , france/04 , and portugal/04 was also confirmed on e gene sequences ( 1.44% , 1.51% , and 1.17% mean nucleotide distance , respectively ) , compared with a mean nucleotide distance of 2.06% with italy/98 . comparison of the amino acid sequences of the e and ns3/ns5 region showed that all 20082009 strains from italy exhibit a set of amino acid substitutions in the ns4a and ns5 proteins not shared with other western mediterranean strains that could be considered as molecular signatures , e.g. , i2209 and s2224 in the ns4a protein , a2786 and k2950 in the ns5 protein ( table a1 ) . notably , the ns3 thr249pro substitution , suggested to be associated with increased virulence for american crows ( 15 ) , was not uniformly present in all the 20082009 isolates in italy . it was detected in all 2008 isolates , but heterogeneously distributed among the 2009 isolates . in particular , among the newly sequenced strains , p at ns3249 was present in the bal-09 , fan-09 , and man-08 isolates , whereas t was present in the tos-09 , nal-09 , and chi-09 isolates . thus , this substitution could not be considered a hallmark of the well - defined 20082009 cluster in italy . although in the past decades several outbreaks of wnv infection have occurred in europe and in the mediterranean basin ( 6 ) , few viral isolates and nucleotide sequences are available from this geographic area , thus preventing an in - depth analysis of the molecular epidemiology of west nile infection in the region . we provide 6 new nucleotide sequences of the e and ns3/ns5 regions from wnv strains isolated in 20082009 from patients in italy with west nile neuroinvasive disease and describe the isolates phylogenetic relationships with other wnv strains in the western mediterranean cluster . the analysis of the e and ns3/ns5 sequences enabled a new phylogenetic reconstruction of the western mediterranean cluster , suggesting that the 20082009 strains from italy constitute a distinct and well - supported group within the european mediterranean clade , characterized by specific amino acid changes in ns4a and ns5 proteins . the structure of phylogenetic trees , together with the presence of amino acid signatures , is consistent with a common origin of the strains circulating 20082009 , supporting the concept of endemic circulation of wnv in italy ( 6 ) . furthermore , the t249p change in wnv - ns3 helicase has been suggested as increasing the virulence of wnv in birds in north america ( 15 ) , although recent data do not support this hypothesis ( 8) . our results indicate that , at least in 2009 in italy , there was a co - circulation of wnv strains carrying either a p or a t in this position . on the basis of our study , it is not possible to establish whether this was because of different virus introductions in italy or reversion of some originally mutated strains . however , our findings do not support the hypothesis that the recent emergence of this mutation might have contributed to rapid spread of wnv infection with the occurrence of human cases in the mediterranean basin . more accurate studies are required to define the role of the t249p mutation in the pathogenesis and evolutionary history of wnv in recent outbreaks . | to determine the lineage of west nile virus that caused outbreaks in italy in 2008 and 2009 , several west nile virus strains were isolated from human specimens and sequenced .
on the basis of phylogenetic analyses , the strains isolated constitute a distinct group within the western mediterranean cluster . |
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fecundity is reduced in male patients with congenital adrenal hyperplasia ( cah ) due to 21-hydroxylase deficiency . recent studies suggested that development of testicular adrenal rest tumors ( tarts ) , which cause an obstruction of the seminiferous tubules , may play a major role [ 1 , 2 ] . in addition , suppression of the gonadal axis due to adrenal androgen excess might also cause reduced fertility . both pathomechanisms are thought to be a consequence of insufficient hormonal control . besides these somatic causes of impaired fertility in cah males , there might be aspects of psychosocial adaption and sexual well - being which may be additional factors for impaired fertility . however , up to now there are no studies investigating sexual well - being in male cah patients . sexual function is best measured by patient self - report avoiding interviewer bias and only patients can report on issues such as sexual interest and the extent to which sexual dysfunction has an adverse effect on their quality of life . the brief sexual function inventory ( bsfi ) provides an excellent tool to assess a self - reported measure of current sexual functioning . the aims of our two - year prospective study in adult male patients with congenital adrenal hyperplasia wereto investigate changes in hypothalamic - pituitary - testicular regulation by gnrh testing , to evaluate changes in sexual functioning and quality of life . to investigate changes in hypothalamic - pituitary - testicular regulation by gnrh testing , to evaluate changes in sexual functioning and quality of life . the subjects were adult male patients with confirmed classical cah due to 21-hydroxylase deficiency with regular hormonal follow - up at the outpatient clinic of the department of endocrinology of the charit campus mitte hospital , berlin . the study was approved by the ethics committee of the charit campus mitte berlin ( permit no . exclusion criteria were other diseases with impairment of gonadal capacity and other general and psychiatric diseases . all patients were seen at the outpatient clinic by two experienced endocrinologists ( m.q . ; m.v . ) on a regular basis every six months . in each patient physical examination , blood drawings ( between 0800 and 1000 h , 2 h after morning medication ) , questionnaires , and testicular ultrasounds were performed at study start ( baseline ) and two years later ( follow - up ) . the treating physicians tried to optimize treatment during the study period according to the endocrine practice guidelines . the standard medications for the treatment of 21-ohd deficiency are hydrocortisone ( hc ) , prednisolone ( pr ) , and dexamethasone ( dx ) [ 6 , 7 ] . since these glucocorticoids have different biological strengths , dosage for pr and dx were converted into hydrocortisone equivalent ( pr was converted 1 to 5 to hc , dx 1 to 70 to hc ) [ 8 , 9 ] . after conversion to the hydrocortisone equivalent dose , the daily total amount of hydrocortisone equivalent in milligrams was calculated as well as the total daily dose per body surface area ( mg / m ) . grey - scale and color doppler ultrasonography of the testes was obtained in longitudinal and transverse sections . circulating concentrations of 4-androstenedione ( ad ) ( beckmanncoulter , krefeld , germany ) , testosterone , dheas ( dpc biermann gmbh ; bad nauheim , germany ) , lh , fsh , renin concentrations , acth , and 17-hydroxy - progesterone ( 17-ohp ) ( mp biomedicals gmbh ; eschwege , germany ) were measured by commercially available assays . the gnrh stimulation test was performed by administering 100 g gnrh ( aventis pharma gmbh , frankfurt , germany ) as an i.v.bolus . serum fsh and lh levels were measured at 0 and 30 min after gnrh dose . we used the differences between peak and basal lh and fsh concentrations , referred to as max , as response variables to eliminate the additive effect of basal lh or fsh level on the peak . a normal lh response in gnrh testing was assumed if lh levels rise was at least 3-fold ; normal fsh response if fsh rises was at least 50% or above 3 psychometric evaluation of patients was performed using three validated self - assessment subjective health status ( shs ) questionnaires : the sf-36 , the brief form of the giessen complaint list ( gbb-24 ) , and the hospital anxiety and depression scale ( hads ) . in addition , the sexual functioning was assessed by the male brief sexual function inventory ( bsfi ) . all four questionnaires were presented as self - explanatory , multiple - choice self - assessments . the sf-36 questionnaire is the most widely used generic instrument to assess quality of life ( qol ) . it consists of eight multi - item domains representing physical functioning ( pf ) , role functioning physical ( rp ) , bodily pain ( bp ) , general health perception ( gh ) , vitality ( vt ) , social functioning ( sf ) , role functioning emotional ( re ) , and mental health ( mh ) . the domain scores range from 0 to 100 with higher values indicating better qol [ 12 , 13 ] . each item is scored as a number , with a maximum score of 21 for each subscale . higher scores indicate higher levels of anxiety or depression . a cut - off value of 8 is regarded as indicating mild impairment , and a cut - off value of 11 is indicative of severe impairment . the short form of the gbb-24 questionnaire consists of 24 items defining four subscales ( exhaustion tendency , gastric symptoms , pain in the limbs , and heart complaints ) , each including six items with ratings from 0 to 4 . in addition , a global score of discomfort ( gsd ) is calculated by adding the four subscale scores . the maximum value for each subscale is 24 , and for the global score 96 . higher scores indicate greater impairment of well - being . regarding control group data we calculated the z - scores by using reference data for sf-36 scores obtained from the german national health survey ( bundesgesundheits - survey 1998 , robert koch institut , berlin 2000 , public use file bgs 98 ) comprising a representative random sample of 7124 subjects from the german population aged between 18 and 79 yr . reference data for the hads ( n = 4410 ) and the gbb-24 ( n = 2076 ) were obtained from previously performed surveys [ 1517 ] . the brief sexual function inventory ( bsfi ) was used to assess perceived problems associated with sexual drive ( two items ) , erection ( three items ) , problem assessment ( three items ) , ejaculation ( two items ) , or overall satisfaction ( one item ) . each question was scored on a 5-point scale , ranging from 0 to 4 , with lower scores indicating worse sexual function . regarding control group data we calculated the z - scores by using normative data for the bsfi obtained from a representative random sample of 1185 subjects from the norwegian population aged between 20 and 79 yr . results are expressed as mean standard deviation ( sd ) if not stated otherwise . the significance of data was determined by students t test in normally distributed and in not normally distributed data by mann - whitney - wilcoxon test where appropriate . eight patients were excluded due to testicular operations or other exclusion criteria . finally 20 patients were enrolled into the study . three patients did not participate in the 2-year follow - up visit and were not included into the 2-year follow - up analysis . clinical and genetic characteristics are shown in table 1 : 14 patients had salt - wasting cah and 6 patients had simple - virilizing cah . patients with salt - wasting cah were diagnosed within the first week after birth ; patients with simple - virilizing form were diagnosed in the first 7 years after birth . biochemical and hormonal parameters of the 17 patients at baseline and at the 2-year follow - up visit are presented in table 2 . over the study period bmi , systolic blood pressure , lipids , androgens , and androgen precursors did not change significantly in the whole cohort or in the sw and sv subgroups . of these , two were fathers ( 12.5% ) , one of one child and the other of two children . during the study period three adrenal crises occurred resulting in a calculated incidence of 8.8 adrenal crises per 100 patients / year , which is higher than the recently reported frequency in cah patients ( 4.8 crises per 100 patients / year ) and resembles more the frequency in patients with primary adrenal insufficiency ( 6.6 crises per 100 patients / year ) . decreased dheas levels were measured in 15 patients ( 88.2% ) at baseline and in all patients at follow - up ( 100% ) . nmol / l ) was observed in no patients at baseline ( 0% ) and in only one patient ( 5.9% ) at follow - up , whereas elevated levels of 17-ohp in serum ( > 36 nmol / l ) were present in 4 patients ( 23.5% ) at baseline and 2 patients at follow - up ( 11.8% ) . the androstenedione to testosterone ( ad / t ) ratio as indicator of testicular testosterone production was normal ( < 0.2 ) in 11 patients ( 64.7% ) at baseline and follow - up ; three patients ( 17.6% ) had an ad / t ratio > 1 suggesting testosterone from predominantly adrenal origin ( table 2 ) . estradiol levels were within the normal male range ruling out any suppression of the hypothalamus - pituitary - gonadal axis by estradiol . total testosterone levels were decreased in 4 patients ( 23.5% ) at baseline and in 2 patients ( 11.8% ) at follow - up ; calculated free testosterone index was diminished in 5 patients ( 29.4% ) at baseline and in 8 patients ( 47.1% ) at follow - up . iu / l ) in all patients at baseline and in all but one patient at follow - up . basal fsh levels were elevated in three patients at baseline ( 17.6% ) and normal in all patients at follow - up . gnrh stimulation induced an adequate increase in lh in all but one patient at baseline ( 5.9% ) and in all but two patients ( 11.8% ) at follow - up . fsh failed to increase sufficiently by gnrh stimulation in two patients at baseline and at follow - up ( 11.8% ) . three patients ( 18% ) showed tart in testicular ultrasound with a size of 611 mm . in one patient tart regressed and was not detected after 2 years . in a subset of patients ( 6 of the 17 patients ) patients with an ad / t ratio below 0.2 , indicating sufficient adrenal suppression and a testosterone of testicular origin , showed significant lower 17-ohp and ad levels than patients with an ad / t ratio > 0.2 ( table 3 ) . significantly more patients with an ad / t ratio < 0.2 received dexamethasone . basal lh and fsh levels as well as testosterone levels were not different between the groups . however , the max increase in lh in gnrh testing was significantly higher in the patients with an ad / t ratio < 0.2 than those with an ad / t ratio > 0.2 ( table 3 ) . analysis of the qol questionnaires ( gbb-24 , hads , and sf-36 ) revealed no significant changes in z - scores during the 2-year study period in our adult male cah patient cohort ( figure 1 ) . however , all dimensions of the gbb-24 showed a trend to increased z - scores indicating an impairment of qol ( figure 1(a ) ) . similar results were found for the anxiety and depression z - scores of the hads questionnaires ( figure 1(b ) ) . z - scores of the sf-36 questionnaire showed a trend to impairments especially in the dimensions physical functioning , general health perception , and emotional role functioning ( figure 1(c ) ) . the dimensions role physical functioning and the analysis of the participants ' z - scores revealed that male cah patients exhibited a slightly reduced sexual drive . no significant differences in bsfi z - scores were found between patients with ad / t ratio < 0.2 or > 0.2 . further analysis showed that ad levels significantly negatively correlated with z - scores of the dimension sexual drive ( p < 0.05 ; figure 3 ) with higher ad levels associated with lower z - scores ( = impaired sexual drive ) . decreases in qol and sexual well - being were not correlated with the presence of tarts . development of tarts and suppression of the gonadal axis are possible factors that might cause reduced fertility in male cah patients [ 1 , 2 , 21 ] . it is suggested that adrenal - derived androgen excess due to insufficient hormonal control might be the underlying cause [ 2 , 3 ] . a recent study in adult male cah patients revealed a high prevalence of impaired leydig cell function and impaired spermatogenesis . however , the authors found no correlation between semen parameters , hormonal control , and tart prevalence or size . in our current study , the majority of patients showed basal testosterone and lh within the normal range of young healthy men suggesting normal leydig cell function in most of the patients . after two years no significant differences were observed in our patients indicating stable therapeutic regimens . however , lh and fsh showed a more pronounced increase ( max ) after gnrh stimulation than reported in healthy normal males . we further subdivided our cohort into a group with good hormonal control and only testicular testosterone production indicated by an androstenedione / testosterone ( ad / t ) ratio < 0.2 and a group with poorer disease control and mixed adrenal and testicular testosterone production indicated by an ad / t ratio > 0.2 . the group with an ad / t ratio > 0.2 showed a normal lh and fsh response ( max ) to gnrh compared to healthy young men . however , the group with an ad / t ratio < 0.2 presented a significant higher lh response to gnrh testing . this resembled a prepubertal response in gnrh testing but might be also due to a suppressed hypothalamic - pituitary axis with a decreased release of gnrh from the hypothalamus . this might be caused by abundant adrenal androgens , which seems not to be the case in this group with ad / t ratio < 0.2 . interestingly , the percentage of dexamethasone treated patients was significantly higher in the group with an ad / t ratio < 0.2 compared to the group with a ratio > 0.2 , but we did not find a significant difference in total daily glucocorticoid equivalent dose per body surface between the two groups . in addition , the amount of glucocorticoid used was approximately similar to that used in other recent studies with male cah patients [ 1 , 23 ] . we assume that total glucocorticoid doses were not too high because our patients showed still normal and not suppressed lh levels . in summary , this suggests that dexamethasone has a profound effect on the hypothalamic - pituitary feedback regulation . this is in accordance with previous reports that changing glucocorticoid medication from hydrocortisone to dexamethasone resulted in an increased fertility . besides these somatic causes of impaired fertility in cah males , there might be aspects of psychosocial adaption and sexual well - being which might be additional factors for impaired fertility . we performed this in a prospective fashion and used also quality of life questionnaires to detect possible other changes or general influences during the 2-year study period . during the study period our cah patients showed unchanged bmi , unchanged metabolic and hormonal parameters , and unchanged impaired z - scores in qol questionnaires . impaired qol in male cah patients has been shown in previous studies [ 7 , 2527 ] ; however , these had only cross - sectional and not a longitudinal design . we did not find differences in qol z - scores in patients that were on dexamethasone and prednisolone treatment compared to hydrocortisone only as previously reported . sexual drive , erections , and ejaculations were impaired in our cohort . it is important to point out that overall satisfaction should not be confused with the mean score of the functional domains of the bsfi , and additional factors might be involved not covered by the questions . it is known that patients with low scores on functional domains , for example , ejaculatory impairment as a side - effect of an anti - depressant drug , do not necessarily report reduced overall sexual satisfaction . we are the first to describe a clearly impaired sexual well - being in male cah patients by using an established sexual function questionnaire . interestingly , we observed that poor disease control , according to elevated androstenedione levels , was associated with a reduced sexual drive . therefore , we believe that aspects of psychosocial adaption and sexual well - being might be important additional factors for impaired fertility in our male cah patients first , cah patients have had a chronic disease since their childhood , as well as having been exposed to exogenous glucocorticoids also during pubertal development . secondly , male cah patients have still a lower height than the average male population and this might cause problems in psychosocial adaptation . however , a recent hungarian study showed that sexual activity was not clearly related to other anthropometric parameters such as height . ( 1 ) there is no normative data for germany for calculating z - scores for the bsfi , and we had to rely on normative data from norway . however , no significant differences in functional bsfi scores were found between the norwegian data and american data from the olmsted county . ( 2 ) there is increasingly reduced sexual function concerning drive , erection , ejaculation , and problem assessment with age with most of these age - related effects starting at > 50 years old . however , our patients were all below the age of 50 y. ( 3 ) our study is a rather small cohort of male cah patients ; however , this is the first longitudinal study in adult male cah patients . in conclusion , we showed that male cah patients with a normal ad / t ratio showed an increased lh and fsh response in gnrh testing indicating possible decreased hypothalamic gnrh release by glucocorticoid therapy . secondly , we found that male cah patients had impaired sexual well - being , especially regarding erections , ejaculations , and sexual drive . | introduction . men with congenital adrenal hyperplasia ( cah ) due to 21-hydroxylase deficiency show impaired fecundity due to testicular adrenal rest tumors and/or suppression of the gonadal axis .
sexual well - being might be an additional factor ; however , no data exists .
patients and methods .
prospective longitudinal monocentric study included 20 male cah patients ( 14 salt wasting , 6 simple virilizing ; age 1849 yr ) . clinical assessment , testicular ultrasound , biochemical and hormonal parameters , three validated self - assessment questionnaires ( sf-36 , gbb-24 , and hads ) , and male brief sexual function inventory ( bsfi ) were analyzed at baseline and after two years
. results .
basal lh and testosterone levels suggested normal testicular function .
lh and fsh responses to gnrh were more pronounced in patients with a good therapy control according to androstenedione / testosterone ratio < 0.2 .
this group had significant higher percentage of patients on dexamethasone medication .
gbb-24 , hads , and sf-36 showed impaired z - scores and no changes at follow - up .
bsfi revealed impairments in dimensions sexual drive , erections , and ejaculations , whereas problem assessment and overall satisfaction revealed normal z - scores .
androstenedione levels correlated ( p = 0.036 ) inversely with z - scores for sexual drive with higher levels associated with impaired
sexual drive .
conclusion .
male cah patients showed a partly impaired sexual well - being which might be an additional factor for reduced fecundity . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
it leads to prolonged hospital stay , increases the cost of treatment , morbidity and mortality . fn commonly affects gastrointestinal tract ( git ) , where chemotherapy induced mucosal damage causes blood stream invasion by gut flora . invasive devices like central venous catheter ( cvc ) also become a source for blood stream infections . inadvertently , fn leads to decrease in dose intensity and poor outcome of cancer . with the use of empiric antibiotic , the mortality due to fn has gradually reduced in cancer patients from 75% to < 10% . more so , with the availability of broad spectrum antibiotics , highly aggressive chemotherapeutic regimens are being used . while there are innumerable studies from the western countries with respect to fn and the infectious agent , choice of empiric antibiotic and outcome , data from india is relatively less . hence , we thought it is worthwhile to evaluate the clinical profile , pathogenic organisms , pattern of antibiotic sensitivity and the outcome of treatment in high risk hematological malignancies ( hm ) with fn . in this prospective study , 72 patients with hm , diagnosed and treated for 108 high risk febrile neutropenic events from august 2011 to january 2013 at a regional cancer center , in south india were evaluated . fn was diagnosed in the occurrence of a single oral temperature of 38.3c ( 101f ) or 38.0c ( 100.4f ) for more than 1 h along with an absolute neutrophil count ( anc ) 500/l or 1000/l with predicted rapid decline during next 48 h. all febrile patients were evaluated for relevant history , physical examination , complete hemogram , liver function test , renal function test , serum electrolytes and chest x - ray . microbiological cultures of blood from peripheral vein , central line ( if present ) and urine were conducted routinely for all febrile episodes . blood culture ( two sets of blood culture , 10 ml each , one from central line and the other from peripheral line ) was repeated every 48 h if fever persisted . antibiotic susceptibility testing was carried out in the first half of the study using the kirby - bauer disk diffusion method . computed tomography ( ct ) scan of paranasal sinuses was done if patients had paranasal sinus tenderness or if fungal infection of the sinus was suspected . ct scan thorax was done in patients who had pleuritic chest pain ; chest x - ray was suggestive of fungal mass or infiltrate or in clinical suspicion of fungal infection . these patients were categorized as high risk based on infectious diseases society of america guidelines . it includes patients with profound neutropenia 100 cells/l , neutropenia anticipated to extend beyond 7 days , significant comorbid conditions , hemodynamic instability , pneumonia , new onset of abdominal pain or neurologic change . once microbiological culture report was available , antibiotics were modified accordingly . however , in culture negative patients same antibiotic was continued . vancomycin was administered additionally to the patients , who had persistent fever , hypotension , redness or tenderness at central line insertion site and pneumonia . amphotericin b was initiated empirically in patients in whom fever persisted despite antibiotics on day 4 or 5 , in sinusitis with suspected fungal infection , pleuritic chest pain or chest x - ray suggested presence of fungal ball . in culture negative and stable patients intravenous antibiotics were continued for 57 days or until anc recovered to > 500/l . subsequently , the patients were started on oral antibiotics which included amoxicillin / clavulanate , ciprofloxacin or levofloxacin on an outpatient basis . bacterial pathogens in all samples yielding culture positivity were identified and their antibiogram was recorded . diagnosis , clinical features , chest x - ray findings , type of chemotherapy , duration of neutropenia , use of growth factor , culture positivity , antibiotic use and outcome of fn were recorded for all patients . a total of 72 patients with hm were diagnosed and treated for 108 fn events . totally 22 patients had cvc , 21 had recurrent fn ( episodes 14 patients had two episodes , 2 patients had three episodes , 2 had four episodes and 3 patients had five episodes of fn ) . acute myeloid leukemia ( 55 episodes ) was the most common etiology in our study followed by non hodgkins lymphoma ( 31 episodes ) and acute lymphoblastic leukemia ( 16 episodes ) . only in 25% episodes growth factor majority of the patients with fn had neutropenia for more than 7 days ( 72/108 episodes , 76.67% ) . each of these 108 fn events were analyzed for associated clinical symptoms as shown in table 1 . overall the culture positivity was identified in 32 of 108 episodes ( 29.62% ) : ( blood culture 19.44% , sputum 7.41% and pus 2.77% ) . gram - negative bacilli ( gnb ) ( 63.64% ) were isolated more commonly followed by gram - positive cocci ( gpc ) ( 36.36% ) . the details of which are given in table 2 . among gram - negative infections , escherichia coli was the most common isolate ( 32.14% ) followed by klebsiella pneumoniae , acinetobacter baumannii and pseudomonas aeruginosa ( 25% , 21.43% , and 17.86% ) respectively . staphylococcus aureus was the most common isolate amongst gram - positive infections ( three were methicillin - resistant s. aureus [ mrsa ] ) . gpc accounted for 42.86% of blood stream infection with s. aureus being the most common isolate . the most common isolate in sputum was p. aeruginosa and e. coli followed by k. pneumoniae , a. baumannii and s. aureus . fungi were isolated in four sputum samples with two samples yielding aspergillus species and two candida species . bacteriological profile the antibiotic sensitivity among gnb was highest for imipenem ( 100% ) , followed by pipercillin - tazoactum ( 86.95% ) . most of the e. coli isolates were resistant to cefoperazone - sulbactum , piperacillin - tazobactum . gram - positive isolates including mrsa were uniformly sensitive to vancomycin , teicoplanin , linezolid and levofloxacin . with first line antibiotic ( cefoperazone - sulbactum ) , 68.5% fn episodes showed an improvement , 26% episodes improved with second line ( piperacillin - tazobactum with amikacin ) , another 5.5% required third line antibiotics ( imipenem / cilastin or meropenem ) . additional vancomycin was required in 14.5% fn episodes , while 17% episodes required addition of antifungal agent on day 5 due to unresponsive fever . fifteen ( 13.5% ) patients died due to septicemia ( 8 acute myeloid leukemia , 3 diffuse large b - cell lymphoma , 2 peripheral t - cell lymphoma , 1 acute lymphoblastic leukemia and 1 acute promyelocytic leukemia ) . four patients succumbed to blood stream infections . in five patients , the source of infection one patient developed multiorgan dysfunction and one had septicemic shock . in eight patients who have died , a bacterial pathogen was isolated either from blood or sputum ( 6-gnb , 2-gpc ) . this study was undertaken to review the clinical profile , antibiotic sensitivity pattern , severity of infection and outcome of treatment in fn with high risk hm patients . this high incidence of fn in acute myeloid leukemia could be attributed to the use of intensive chemotherapy leading to prolonged and profound neutropenia , which increases the risk of infection . similar to other studies fn more commonly occurred during induction chemotherapy , than during consolidation and maintenance chemotherapy . in this study , majority of the patients had prolonged neutropenia ( > 7 days ) and had more episodes of fn ( both bacterial and fungal ) with associated morbidity and mortality , which was in concordance to other studies . culture positive episodes have been identified in 1538% patients in other studies . in the western countries , while infections with gnb predominated during 1970 and 80 , a predominant shift to gpc infections has been reported in later years . this is usually attributed to frequent use of indwelling cvcs and the use of fluoroquinolone prophylaxis which suppresses the aerobic gnb colonizing the git but fails to suppress the microaerophilic gpc . in our study in contrast to the western studies , studies from india including the present one still find gnb as the predominant infection in hm . overall e. coli was found to be the most common isolate including all cultures and s. aureus was the most common isolate from blood . the reason for low incidence of s. aureus infection in our study is due to infrequent use of cvcs . increased incidence of s. aureus isolates from the blood in our study was due to use of cvcs in all acute leukemia patients . also gram - negative isolates from only two cases produced extended spectrum beta lactamases . we found that gnb were highly sensitive to imipenem , piperacillin / tazobactum and meropenem . similar antibiotic sensitivity pattern of high sensitivity of gnb to carbapenems , piperacillin / tazobactum , cefoperazone / sulbactum and resistance to third generation cephalosporins has been reported in studies . even though , in vitro sensitivity to cefoperazone / sulbactum was low , most patients responded to it clinically , when administered empirically . all s.aureus including mrsa were highly sensitive to amikacin , linezolid , vancomycin , teicoplanin , and levofloxacin . cancer patients on chemotherapy are significantly predisposed to fn due to immune deficiency mediated either by the underlying malignancy and/or anticancer chemotherapy and mucosal breach due to chemotherapy . this is reflected by the fact that nearly 80% of these infections are due to endogenous flora . with the present menace of antibiotic resistant superbugs , judicious use of available antibiotics the strength of our study is that it is a large single institutional study which has taken into account the fn in hm . though observational , it can go a long way in transforming the health policies and antibiotic policies in our tertiary care oncology center . furthermore , the importance of clinical judgment and not chasing laboratory reports is well emphasized through this study . for instance though cefoperazone - sulbactum was only 70% sensitive according to the culture sensitivity , 70% of episodes did respond to the first line antibiotic suggesting an in vivo and in vitro discordance , which has been proven before . since sporadic data exist from this part of the world , this study can be a useful guide for oncologists managing fn in india and building institutional antibiotic policy . this study attempts to highlight the common infectious agents , their antibiotic sensitivity pattern and outcome of high risk fn with cefoperazone - sulbactum . empiric therapy with cefoperazone - sulbactum leads to satisfactory clinical outcome in high risk fn and indiscriminate use of higher antibiotics must be restricted even in this medical emergency and therapy should be tailored to the most appropriate antibiotic according to the bacterial culture . | background and objectives : febrile neutropenia ( fn ) is considered a medical emergency .
patients with hematological malignancies ( hm ) commonly experience fn .
broad spectrum antibiotics have to be started empirically to prevent complications .
this study depicts the clinical profile , microbiological profile , antibiotic sensitivity pattern , and outcome in high risk hm.materials and methods : in this prospective study , 72 patients with hematologic malignancies , diagnosed and treated for 108 high risk febrile neutropenic episodes from august 2011 to january 2013 at a regional cancer center , in south india were analyzed .
cefoperazone - sulbactum was used as a first - line empiric antibiotic.results:majority of the patients with fn episodes had acute myeloid leukemia .
overall culture positivity was 29.62% .
the most common organisms isolated were gram - negative bacilli ( 63.64% ) , with escherichia coli being the most frequent pathogen .
all gram - negative organisms were sensitive to imipenem , whereas sensitivity pattern to other antibiotics were as follows : 85.71% , 78.26% , 69.52% , 63.64% , 41.66% and 47.05% for pipercillin - tazoactum , meropenem , cefoperazone - sulbactum , amikacin , ceftazidime , ciprofloxacin respectively .
overall mortality was 13.5% .
most of the patients responded to empiric antibiotic cefoperazone-sulbactum.conclusions:in the hematologic malignancies particularly in acute leukemia , there is high risk of developing fn .
empiric therapy with cefoperazone - sulbactum as a first line leads to satisfactory outcome in high risk fn and therapy should be tailored to the most appropriate antibiotics according to the bacterial culture results . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
we conducted a literature search through 15 december 2011 using the pubmed and embase databases without restrictions on language or publication date . the search term diabetes was used in combination with fish , seafood , fatty acids , long - chain omega-3 , long - chain n-3 , docosahexaenoic acid , or eicosapentaenoic acid and cohort , prospective , follow - up , or longitudinal . moreover , we reviewed the reference lists from retrieved articles to identify additional relevant studies . studies were included in the meta - analysis if the following criteria were met and reported : 1 ) prospective design ( prospective cohort studies or nested case - control studies ) ; 2 ) the exposure studied was self - reported fish consumption or dietary intake of long - chain n-3 fatty acids ( marine derived n-3 fatty acids such as epa and dha ) ; 3 ) the outcome of interest was incidence of type 2 diabetes ; and 4 ) relative risk ( rr ) estimates with 95% cis . we considered odds ratios , risk ratios , and hazard ratios as estimates of the rr . dose - response meta - analysis requires the distribution of cases and noncases or person - time , and rrs with 95% cis for at least three quantitative exposure categories or for a continuous exposure . for all identified studies that met inclusion criteria but failed to report sufficient information ( 813 ) if data were duplicated in more than one study , we included the study with the largest number of cases . the following data were extracted from each study : the first author s last name , publication year , country where the study was performed , study period , number of cases and cohort size , sex and age of study participants , measure and range of exposure , methods for identifying type 2 diabetes , variables adjusted for in the analysis , and rr estimates with corresponding 95% cis for each category of consumption of fish and/or long - chain n-3 fatty acids . when several risk estimates were presented , we extracted the ones that reflected the greatest degree of control for potential confounders . the study quality was assessed using the newcastle - ottawa quality assessment scale for cohort studies , with which each study is judged based on the selection of study groups , the comparability of the groups , the exposure assessment , and ascertainment of outcome ( 14 ) . study selection , data extraction , and quality assessment were conducted independently by two authors ( a.wa . and d.d.g . we conducted a two - stage dose - response random - effects meta - analysis to combine study - specific rrs ( 15 ) . linear dose - response curves were estimated taking the covariance among risk estimates for different exposure categories into account ( 15 ) . because the included studies used different units to report fish consumption ( i.e. , grams or servings ) , we rescaled consumption into servings per week using 100 g as the approximate average serving size . in addition , we explored how this assumption affected the results by changing the approximated serving size to 80 g and 140 g. the median level of consumption for each category was assigned to the corresponding rr estimate . when the median consumption per category was not presented in the article , the midpoint between the upper and lower boundary was used . if the lowest category was open - ended , the lower boundary was assumed to be zero . open - ended top categories were assumed to be of the same magnitude as the preceding category . we assessed a potential departure from linearity in the dose - response relationship by modeling consumption levels using restricted cubic splines with three knots at fixed percentiles 10 , 50 , and 90% of the distribution . a p value for nonlinearity was calculated by testing the null hypothesis that the coefficient of the second spline is equal to 0 in a multivariable random - effects meta - regression model ( 16 ) . to examine potential sources of heterogeneity , prespecified sensitivity analyses were defined , including stratifications by sex , geographical region , methods for exposure assessment and ascertainment of outcome , and quality score . data were not available in the published articles to examine other potentially influential factors , such as age and bmi categories . further , we conducted sensitivity analyses omitting one study at the time to evaluate whether results were affected markedly by single studies . statistical heterogeneity among studies was evaluated with the cochran q - test and the i
statistic ( 17 ) , which assesses the proportion of total variation that is due to between - study variation . all statistical analyses were performed with stata software , version 10.1 ( stata corp , college station , texas , usa ) . we conducted a literature search through 15 december 2011 using the pubmed and embase databases without restrictions on language or publication date . the search term diabetes was used in combination with fish , seafood , fatty acids , long - chain omega-3 , long - chain n-3 , docosahexaenoic acid , or eicosapentaenoic acid and cohort , prospective , follow - up , or longitudinal . moreover , we reviewed the reference lists from retrieved articles to identify additional relevant studies . studies were included in the meta - analysis if the following criteria were met and reported : 1 ) prospective design ( prospective cohort studies or nested case - control studies ) ; 2 ) the exposure studied was self - reported fish consumption or dietary intake of long - chain n-3 fatty acids ( marine derived n-3 fatty acids such as epa and dha ) ; 3 ) the outcome of interest was incidence of type 2 diabetes ; and 4 ) relative risk ( rr ) estimates with 95% cis . we considered odds ratios , risk ratios , and hazard ratios as estimates of the rr . dose - response meta - analysis requires the distribution of cases and noncases or person - time , and rrs with 95% cis for at least three quantitative exposure categories or for a continuous exposure . for all identified studies that met inclusion criteria but failed to report sufficient information ( 813 ) if data were duplicated in more than one study , we included the study with the largest number of cases . the following data were extracted from each study : the first author s last name , publication year , country where the study was performed , study period , number of cases and cohort size , sex and age of study participants , measure and range of exposure , methods for identifying type 2 diabetes , variables adjusted for in the analysis , and rr estimates with corresponding 95% cis for each category of consumption of fish and/or long - chain n-3 fatty acids . when several risk estimates were presented , we extracted the ones that reflected the greatest degree of control for potential confounders . the study quality was assessed using the newcastle - ottawa quality assessment scale for cohort studies , with which each study is judged based on the selection of study groups , the comparability of the groups , the exposure assessment , and ascertainment of outcome ( 14 ) . study selection , data extraction , and quality assessment were conducted independently by two authors ( a.wa . and d.d.g . we conducted a two - stage dose - response random - effects meta - analysis to combine study - specific rrs ( 15 ) . linear dose - response curves were estimated taking the covariance among risk estimates for different exposure categories into account ( 15 ) . because the included studies used different units to report fish consumption ( i.e. , grams or servings ) , we rescaled consumption into servings per week using 100 g as the approximate average serving size . in addition , we explored how this assumption affected the results by changing the approximated serving size to 80 g and 140 g. the median level of consumption for each category was assigned to the corresponding rr estimate . when the median consumption per category was not presented in the article , if the lowest category was open - ended , the lower boundary was assumed to be zero . open - ended top categories were assumed to be of the same magnitude as the preceding category . we assessed a potential departure from linearity in the dose - response relationship by modeling consumption levels using restricted cubic splines with three knots at fixed percentiles 10 , 50 , and 90% of the distribution . a p value for nonlinearity was calculated by testing the null hypothesis that the coefficient of the second spline is equal to 0 in a multivariable random - effects meta - regression model ( 16 ) . to examine potential sources of heterogeneity , prespecified sensitivity analyses were defined , including stratifications by sex , geographical region , methods for exposure assessment and ascertainment of outcome , and quality score . data were not available in the published articles to examine other potentially influential factors , such as age and bmi categories . further , we conducted sensitivity analyses omitting one study at the time to evaluate whether results were affected markedly by single studies . statistical heterogeneity among studies was evaluated with the cochran q - test and the i
statistic ( 17 ) , which assesses the proportion of total variation that is due to between - study variation . all statistical analyses were performed with stata software , version 10.1 ( stata corp , college station , texas , usa ) . , a total of 2,473 articles were identified by searching the databases , 434 duplicated articles and 2,019 articles that did not meet the selection criteria were excluded after screening of abstract and/or title . the remaining 20 articles and 1 additional article ( 12 ) identified from reference lists , 5 articles were excluded because they assessed only total polyunsaturated fatty acids and/or outcome was defined as impaired glucose tolerance rather than incidence of type 2 diabetes ( 1923 ) . in addition , two duplicate reports from the same study population were excluded ( 24,25 ) . the remaining 14 publications , reporting results from 16 separate cohort studies , were included in the meta - analysis ( 813,2633 ) . systematic review flow diagram . characteristics of the included studies are presented in table 1 . the 14 articles were published between 2001 and 2011 and involved a total of 527,441 participants and 24,082 cases of diabetes . two articles reported results from 2 ( 11 ) or 3 ( 28 ) independent cohorts . three studies that also assessed biomarkers of fatty acid composition , available only in subsets of the study populations , used a nested case - control design within cohorts with prospective collection of dietary data to study the association between intake of long - chain n-3 fatty acids and risk of type 2 diabetes ( 8,9,31 ) . among them , one reported on the association with fish consumption in the whole cohort in a separate article ( 10 ) . among the 16 cohorts , 10 assessed both fish consumption and intake of long - chain n-3 fatty acids ( 911,13,2830,32 ) , whereas 3 reported only on the fatty acids ( 8,26,31 ) and 3 only on fish consumption ( 12,27,33 ) . ( 2628,30,32 ) , 4 in europe ( 9,10,12,29,31 ) , 4 in asia ( 11,13,33 ) , and 1 in australia ( 8) . all studies reported rrs adjusted for age and sex ; all but 2 were further adjusted for total energy intake ( 8,27 ) , smoking ( 8,27 ) and bmi ( 27,29 ) ; and all but 3 were adjusted for physical activity ( 12,27,29 ) and alcohol intake ( 12,26,27 ) . eleven studies used self - administered food frequency questionnaires ( ffq ) to collect dietary data ( 810,2628,3033 ) , whereas 5 used interviewer - administered ffqs ( 1113,29 ) . in 1 study , total fish consumption was assessed by a single question ( 27 ) , whereas it was obtained as the sum of different types of fish in other studies . long - chain n-3 fatty acids was defined as the sum of epa and dha in 11 studies ( 8,9,11,13,28,29,31,32 ) , as the sum of epa , dha , and docosapentenoic acid in 1 study ( 30 ) , and not further explained in 1 study ( 26 ) . six studies updated the dietary information with additional ffqs after baseline ( 11,28,32 ) . incident diabetes cases were identified by self - report in 13 studies , of which 7 followed up the information with supplementary questionnaires or interviews ( 28,30 ) , with physician s confirmation ( 8,31 ) , or with linkage to medical registries ( 9,10 ) . one study used information from general practitioners and pharmacy databases to identify incident cases ( 29 ) , 1 used medication inventory information together with measurements of fasting glucose ( 32 ) , and 1 used linkage to a nationwide register of drug reimbursement ( 12 ) . among studies that included all self - reported cases ( 11,13,26,27,33 ) , 2 stated that validity of self - reports was reasonable ( 13,33 ) and 1 that results did not differ substantially when using a more strict case definition ( 11 ) . the mean range of intake between the highest and the lowest category across studies was about 5.0 servings per week for fish and about 0.38 g per day for long - chain n-3 fatty acids . the study quality was generally good , with all but 3 studies ( 8,27,30 ) reaching criteria for 6 stars on the scale of 9 ( 14 ) . prospective studies on fish consumption , dietary long - chain n-3 fatty acids , and risk of type 2 diabetes the results across all studies were mixed , with positive , inverse , or null associations . for total fish consumption , there was a high degree of heterogeneity between the 13 included studies ( i
= 81.3% ; p < 0.001 ) . for dietary intake of long - chain n-3 fatty acids , substantial heterogeneity was also observed between the 13 included studies ( i
= 78.3% ; p < 0.001 ) . omitting 1 study at a time showed that neither summary risk estimates nor measures of statistical heterogeneity were markedly influenced by single studies ( i
range , fish : 70.282.8% ; long - chain n-3 fatty acids : 73.280.1% ; p < 0.001 ) . further , results of the analysis on fish consumption were not substantially affected by changing the approximated average serving size from 100 g to 80 g ( i
= 82.5% ; p < 0.001 ) or 140 g ( i
= 78.9% ; p < 0.001 ) . excluding the 3 nested case - control studies on long - chain n-3 fatty acid intake ( 8,9,31 ) also had little influence ( i
= 79.7% ; p < 0.001 ) . because of the high degree of heterogeneity , we did not combine results across all studies into an overall summary risk estimate . results of the sensitivity analyses conducted to investigate potential sources of heterogeneity are shown in table 2 , including stratifications by sex , geographical region , and ffq type , and exclusion of studies with a quality score < 6 ( 8,27,30 ) and studies that ascertained type 2 diabetes through self - report only ( 11,13,26,27,33 ) . there were substantial differences according to geographical region , in part explaining the heterogeneity in the overall estimates . summary rrs of type 2 diabetes for an increment of 1 serving / week in total fish consumption and 0.30 g / day in dietary intake of long - chain n-3 fatty acids ( corresponding to approximately 1 serving / week of fatty fish ) the estimated rrs of type 2 diabetes associated with fish consumption and intake of long - chain n-3 fatty acids are shown in fig . 2a and b for individual studies and combined by geographical region . for each serving per week increment in total fish consumption , the summary rrs of type 2 diabetes were 1.05 ( 95% ci 1.021.09 ) , 1.03 ( 0.961.11 ) , and 0.98 ( 0.971.00 ) combining studies from the u.s . , for each 0.30 g per day increment in dietary long - chain n-3 fatty acid intake ( corresponding to approximately one serving per week of fatty fish ) , the corresponding summary rrs were 1.17 ( 1.091.26 ) , 0.98 ( 0.701.37 ) , and 0.90 ( 0.820.98 ) . excluding one australian nested case - control study on long - chain n-3 fatty acid intake ( 8) did not markedly affect the summary estimate of asian / australian studies ( 0.90 [ 0.811.00 ] ) , whereas all six of the u.s . studies were of cohort design , and only a single european cohort study remained after exclusion of two nested case - control studies ( 9,31 ) in this subgroup . a : rr estimates of type 2 diabetes associated with a one serving per week increment in total fish consumption , for individual studies and combined by geographical region . b : rr estimates of type 2 diabetes associated with a 0.3 g / day increment in dietary intake of long - chain n-3 fatty acids ( corresponding to approximately one serving per week of fatty fish ) , for individual studies and combined by geographical region . ( a high - quality color representation of this figure is available in the online issue . ) the egger test showed no evidence of publication bias for total fish consumption ( p = 0.85 ) or dietary intake of long - chain n-3 fatty acids ( p = 0.10 ) . we flexibly modeled the dose - response relationships using restricted cubic splines , and we found no evidence of departure from the simpler linear trends ( p
nonlinearity = 0.81 for fish ; p
nonlinearity = 0.76 for long - chain n-3 fatty acids ) . , a total of 2,473 articles were identified by searching the databases , 434 duplicated articles and 2,019 articles that did not meet the selection criteria were excluded after screening of abstract and/or title . the remaining 20 articles and 1 additional article ( 12 ) identified from reference lists , 5 articles were excluded because they assessed only total polyunsaturated fatty acids and/or outcome was defined as impaired glucose tolerance rather than incidence of type 2 diabetes ( 1923 ) . in addition , two duplicate reports from the same study population were excluded ( 24,25 ) . the remaining 14 publications , reporting results from 16 separate cohort studies , were included in the meta - analysis ( 813,2633 ) . systematic review flow diagram . characteristics of the included studies are presented in table 1 . the 14 articles were published between 2001 and 2011 and involved a total of 527,441 participants and 24,082 cases of diabetes . two articles reported results from 2 ( 11 ) or 3 ( 28 ) independent cohorts . three studies that also assessed biomarkers of fatty acid composition , available only in subsets of the study populations , used a nested case - control design within cohorts with prospective collection of dietary data to study the association between intake of long - chain n-3 fatty acids and risk of type 2 diabetes ( 8,9,31 ) . among them , one reported on the association with fish consumption in the whole cohort in a separate article ( 10 ) . among the 16 cohorts , 10 assessed both fish consumption and intake of long - chain n-3 fatty acids ( 911,13,2830,32 ) , whereas 3 reported only on the fatty acids ( 8,26,31 ) and 3 only on fish consumption ( 12,27,33 ) . ( 2628,30,32 ) , 4 in europe ( 9,10,12,29,31 ) , 4 in asia ( 11,13,33 ) , and 1 in australia ( 8) . all studies reported rrs adjusted for age and sex ; all but 2 were further adjusted for total energy intake ( 8,27 ) , smoking ( 8,27 ) and bmi ( 27,29 ) ; and all but 3 were adjusted for physical activity ( 12,27,29 ) and alcohol intake ( 12,26,27 ) . eleven studies used self - administered food frequency questionnaires ( ffq ) to collect dietary data ( 810,2628,3033 ) , whereas 5 used interviewer - administered ffqs ( 1113,29 ) . in 1 study , total fish consumption was assessed by a single question ( 27 ) , whereas it was obtained as the sum of different types of fish in other studies . long - chain n-3 fatty acids was defined as the sum of epa and dha in 11 studies ( 8,9,11,13,28,29,31,32 ) , as the sum of epa , dha , and docosapentenoic acid in 1 study ( 30 ) , and not further explained in 1 study ( 26 ) . six studies updated the dietary information with additional ffqs after baseline ( 11,28,32 ) . incident diabetes cases were identified by self - report in 13 studies , of which 7 followed up the information with supplementary questionnaires or interviews ( 28,30 ) , with physician s confirmation ( 8,31 ) , or with linkage to medical registries ( 9,10 ) . one study used information from general practitioners and pharmacy databases to identify incident cases ( 29 ) , 1 used medication inventory information together with measurements of fasting glucose ( 32 ) , and 1 used linkage to a nationwide register of drug reimbursement ( 12 ) . among studies that included all self - reported cases ( 11,13,26,27,33 ) , 2 stated that validity of self - reports was reasonable ( 13,33 ) and 1 that results did not differ substantially when using a more strict case definition ( 11 ) . the mean range of intake between the highest and the lowest category across studies was about 5.0 servings per week for fish and about 0.38 g per day for long - chain n-3 fatty acids . the study quality was generally good , with all but 3 studies ( 8,27,30 ) reaching criteria for 6 stars on the scale of 9 ( 14 ) . prospective studies on fish consumption , dietary long - chain n-3 fatty acids , and risk of type 2 diabetes the results across all studies were mixed , with positive , inverse , or null associations . for total fish consumption , there was a high degree of heterogeneity between the 13 included studies ( i
= 81.3% ; p < 0.001 ) . for dietary intake of long - chain n-3 fatty acids , substantial heterogeneity was also observed between the 13 included studies ( i
= 78.3% ; p < 0.001 ) . omitting 1 study at a time showed that neither summary risk estimates nor measures of statistical heterogeneity were markedly influenced by single studies ( i
range , fish : 70.282.8% ; long - chain n-3 fatty acids : 73.280.1% ; p < 0.001 ) . further , results of the analysis on fish consumption were not substantially affected by changing the approximated average serving size from 100 g to 80 g ( i
= 82.5% ; p < 0.001 ) or 140 g ( i
= 78.9% ; p < 0.001 ) . excluding the 3 nested case - control studies on long - chain n-3 fatty acid intake ( 8,9,31 ) also had little influence ( i
= 79.7% ; p < 0.001 ) . because of the high degree of heterogeneity , we did not combine results across all studies into an overall summary risk estimate . results of the sensitivity analyses conducted to investigate potential sources of heterogeneity are shown in table 2 , including stratifications by sex , geographical region , and ffq type , and exclusion of studies with a quality score < 6 ( 8,27,30 ) and studies that ascertained type 2 diabetes through self - report only ( 11,13,26,27,33 ) . there were substantial differences according to geographical region , in part explaining the heterogeneity in the overall estimates . summary rrs of type 2 diabetes for an increment of 1 serving / week in total fish consumption and 0.30 g / day in dietary intake of long - chain n-3 fatty acids ( corresponding to approximately 1 serving / week of fatty fish ) the estimated rrs of type 2 diabetes associated with fish consumption and intake of long - chain n-3 fatty acids are shown in fig . 2a and b for individual studies and combined by geographical region . for each serving per week increment in total fish consumption , the summary rrs of type 2 diabetes were 1.05 ( 95% ci 1.021.09 ) , 1.03 ( 0.961.11 ) , and 0.98 ( 0.971.00 ) combining studies from the u.s . , for each 0.30 g per day increment in dietary long - chain n-3 fatty acid intake ( corresponding to approximately one serving per week of fatty fish ) , the corresponding summary rrs were 1.17 ( 1.091.26 ) , 0.98 ( 0.701.37 ) , and 0.90 ( 0.820.98 ) . excluding one australian nested case - control study on long - chain n-3 fatty acid intake ( 8) did not markedly affect the summary estimate of asian / australian studies ( 0.90 [ 0.811.00 ] ) , whereas all six of the u.s . studies were of cohort design , and only a single european cohort study remained after exclusion of two nested case - control studies ( 9,31 ) in this subgroup .
a : rr estimates of type 2 diabetes associated with a one serving per week increment in total fish consumption , for individual studies and combined by geographical region . b : rr estimates of type 2 diabetes associated with a 0.3 g / day increment in dietary intake of long - chain n-3 fatty acids ( corresponding to approximately one serving per week of fatty fish ) , for individual studies and combined by geographical region . ( a high - quality color representation of this figure is available in the online issue . ) the egger test showed no evidence of publication bias for total fish consumption ( p = 0.85 ) or dietary intake of long - chain n-3 fatty acids ( p = 0.10 ) . we flexibly modeled the dose - response relationships using restricted cubic splines , and we found no evidence of departure from the simpler linear trends ( p
nonlinearity = 0.81 for fish ; p
nonlinearity = 0.76 for long - chain n-3 fatty acids ) . we report the first systematic review and meta - analysis of the prospective association between total fish consumption , dietary intake of long - chain n-3 fatty acids , and risk of incident type 2 diabetes . results from this large analysis including 24,082 cases of diabetes and 527,441 participants show substantial heterogeneity across study results , potentially explained by differences between geographical regions . the summary risk estimates indicate no association among european studies , a direct association among u.s . . however , results are also partly inconsistent within study areas , and the number of studies outside the u.s . is limited . compared with major risk factors for type 2 diabetes , such as overweight and obesity ( associated with rrs of 2.99 [ 95% ci 2.423.72 ] and 7.19 [ 5.749.00 ] in a recent meta - analysis ) , the strength of the summary risk estimates in the current study is modest . a 5% increase in risk for each weekly serving of fish in u.s . studies , however , is comparable to risk estimates reported for other dietary factors in relation to type 2 diabetes , such as red meat ( 1.19 [ 1.041.37 ] per 100 g per day ) ( 35 ) . strengths of this meta - analysis include its large size , and that the assessment was based on data from prospective studies only . this minimizes the possibility that the results were due to recall or selection bias , which are of particular concern in other epidemiological study designs . recall bias may be especially problematic when studying associations between dietary factors and type 2 diabetes , as diagnosed patients commonly get specific dietary advice and thus may change their habits . in addition , the use of a dose - response approach is superior to the conventional methodology of comparing only the extreme categories of intake , which can vary considerably between studies conducted in different populations . from a public health perspective , a dose - response meta - analysis can provide more useful estimates for better quantifying associations between specified amounts of food and disease risk . however , the approach requires a number of assumptions to be made . in terms of associations between absolute intake levels and disease risk , a meta - analysis can only be as good as the individual study instruments that were used . dietary questionnaires such as the ffqs are generally more valuable for ranking study participants according to their consumption than for measuring exact consumption levels . assumptions also had to be made regarding the median level of consumption for each category when this information was missing in the individual studies . finally , since six studies expressed fish consumption in grams rather than in servings , we had to make assumptions about average serving size . there are also potential limitations of our findings that must be taken into consideration when interpreting the results . first , a meta - analysis can not solve inherent problems with confounding in the included studies , which may bias the results toward exaggeration or underestimation of risk estimates . although most studies controlled for total energy intake , smoking , bmi , physical activity , and alcohol , other factors were less consistently included in the multivariate models . to evaluate the impact of these differences on heterogeneity between studies , we separately combined age- and sex - adjusted ( plus race and area where applicable ) rr estimates from the 7 studies on fish consumption providing such data ( 10,27,28,32,33 ) . the statistical heterogeneity in the summary estimate remained ( i
= 79.5% ; p < 0.001 ) , indicating that differences in included covariates is not an important explanation of the differences between studies observed in this meta - analysis . second , most of the included studies assessed diet with a self - administered ffq , and in all but 6 studies ( 11,28,32 ) dietary intake was based on a single questionnaire administered at baseline . therefore , our findings are likely to be affected by some misclassification of exposure . in cohort studies , misclassification is generally nondifferential , which most likely attenuates the association . our meta - analysis included questionnaire data on fatty acid intake , while objectively measured fatty acids ( e.g. , in plasma phospholipid or erythrocyte membrane fractions ) would reduce measurement error . among the 13 studies included in the meta - analysis of long - chain n-3 fatty acids , 4 used biomarker measurements in addition to intake derived from ffqs ( 8,9,31,32 ) . neither epa nor dha in plasma phospholipids ( 8,9,32 ) or erythrocyte membrane phospholipids ( 9,31 ) were significantly associated with risk of type 2 diabetes in individual studies . however , because of the limited number of studies , we did not pool the study - specific risk estimates . measurement error may potentially also have affected the outcome variable ( diabetes incidence ) as the result of underreporting of diagnosis in asymptomatic individuals . it is plausible that rates of screening and surveillance may be higher among participants with a healthier lifestyle associated with higher fish consumption , which could lead to greater case ascertainment among such individuals . further , it is possible that people who are diagnosed with one or several risk factors for diabetes increase their fish consumption as part of a lifestyle change . however , studies that additionally adjusted for hypertension and serum cholesterol ( 12 ) restricted analyses to individuals without hypercholesterolemia or hypertension ( 28 ) , tested for interaction with hypertensive status ( 13 ) , or excluded participants who were diagnosed with diabetes during the first years of follow - up ( 11,13,30,31 ) observed no substantial differences in risk estimates . finally , in a meta - analysis of published studies , publication bias could be of concern because studies with null results or small sample sizes tend not to be published . however , we found no evidence of publication bias in this meta - analysis . intake of long - chain n-3 fatty acids has been hypothesized to have beneficial effects on insulin resistance and type 2 diabetes because of their ability to inhibit inflammatory pathways and suppress expression of genes related to lipid metabolism ( 5 ) . however , most randomized controlled trials that have examined the effects of long - chain n-3 fatty acid supplementation on insulin sensitivity have found no effects ( 6 ) . if an association between fish consumption and risk of type 2 diabetes is attributable to a high intake of epa and dha , the association should be more evident with fatty than with lean fish . however , only three studies assessed types of fish separately ( 10,29,33 ) . the rotterdam study reported positive associations with total and lean fish ( accounting for 81% of total fish consumption ) , but not with fatty fish or with long - chain n-3 fatty acids ( 29 ) . results from the european prospective investigation of cancer ( epic)-norfolk cohort indicated inverse associations with total , white and oily fish , although statistically significant only for total fish ( 10 ) . in a subsample of the same cohort , the association with intake of long - chain n-3 fatty acids was nonsignificantly inverse ( 9 ) . in the japanese study , risk of type 2 diabetes tended to decrease with oily fish consumption , but not with lean fish among men ; no associations were observed with either total or types of fish among women ( 33 ) . it is possible that consumption of fried and deep - fried fish , as well as the use of batter and type of frying media may differ between populations , potentially in part explaining the differences between geographical regions . in addition to long - chain n-3 fatty acids , fish is an important contributor to selenium intake . findings from randomized trials indicate that selenium supplementation may be associated with an increased risk of type 2 diabetes ( 36 ) . in one of the included studies on long - chain n-3 fatty acids , additional adjustment for selenium intake markedly attenuated the risk estimates ( 29 ) . regarding vitamin d , another nutrient abundant in fish , there is accumulating evidence that inadequate vitamin d status may be associated with development of type 2 diabetes , but evidence from longitudinal studies measuring circulating 25-hydroxyvitamin d is sparse and inconsistent ( 37 ) . the available evidence of an association between exposure to persistent organic pollutants and risk of type 2 diabetes is relatively consistent ( 38 ) . however , as it is conceivable that the metabolism of the pollutants could be altered as a consequence of the disease , identification of mechanisms by which these compounds could influence risk is needed to determine if the association reflects a causal relationship ( 38 ) . further , fish consumption is one of the major sources of mercury exposure , which has been shown to affect insulin secretion through oxidative stress a recent large scale cross - sectional study suggested that both blood mercury and serum dioxin levels are associated with increased risk of insulin resistance , and that simultaneous exposure additionally increases the risk ( 40 ) . in conclusion , results from this meta - analysis indicate differences between geographical regions in observed associations of total fish consumption and dietary intake of long - chain n-3 fatty acids with risk of type 2 diabetes , with an increased risk among studies conducted in the u.s . , no association in european populations , and an inverse association in asian / australian populations . in consideration of the heterogeneous results , as well as the cardiovascular benefits associated with the studied exposures , the relationship and its possible mechanisms warrant further investigation . types of fish consumed ( lean , fatty , shellfish ) , dietary factors in fish , as well as preparation methods and levels of contamination , which could vary across different countries , should be comprehensively investigated when the potential role of fish and long - chain n-3 fatty acids in the development of type 2 diabetes is examined in future studies . | objectivethe evidence on the association between fish consumption , dietary long - chain n-3 fatty acids , and risk of type 2 diabetes is inconsistent .
we therefore performed a systematic review and meta - analysis of the available prospective evidence.research design and methodsstudies were identified by searching the pubmed and embase databases through 15 december 2011 and by reviewing the reference lists of retrieved articles .
prospective studies were included if they reported relative risk ( rr ) estimates with 95% cis for the association between fish consumption and/or dietary long - chain n-3 fatty acids and incidence of type 2 diabetes .
a dose - response random - effects model was used to combine study - specific rrs .
potential sources of heterogeneity were explored by prespecified stratifications.resultssixteen studies involving 527,441 participants and 24,082 diabetes cases were included .
considerable statistical heterogeneity in the overall summary estimates was partly explained by geographical differences . for each serving per week increment in fish consumption ,
the rrs ( 95% cis ) of type 2 diabetes were 1.05 ( 1.021.09 ) , 1.03 ( 0.961.11 ) , and 0.98 ( 0.971.00 ) combining u.s . , european , and asian / australian studies , respectively .
for each 0.30 g per day increment in long - chain n-3 fatty acids , the corresponding summary estimates were 1.17 ( 1.091.26 ) , 0.98 ( 0.701.37 ) , and 0.90 ( 0.820.98).conclusionsresults from this meta - analysis indicate differences between geographical regions in observed associations of fish consumption and dietary intake of long - chain n-3 fatty acids with risk of type 2 diabetes .
in consideration of the heterogeneous results , the relationship warrants further investigation . meanwhile , current public health recommendations on fish consumption should be upheld unchanged . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the age - old model of observation followed by attempt ( ie , see one , do one , teach one ) still exists and is still considered common in residency and fellowship training.1 other modalities available to today s medical learner include formal didactic lectures and multimedia learning ( eg , mobile devices , textbooks , and internet).24 traditionally , teaching methods have been introduced with minimal studies of efficacy , if any . today , new methods are often held to a higher standard and are required to demonstrate superiority over some existing comparator . those methods with proven efficacy may be integrated into medical education curricula , either individually or in combination with other methods as appropriate . simulation can be defined as something that is made to look , feel , or behave like something else especially when applied to research or education.5 the use of simulation to mimic real life in the educational setting has arguably many origins but is closely tied to kolb s experiential learning theory.6 the educational experience provided by simulation is often hands - on , practical , provides immediate feedback , and allows for repetition . training with simulation does no harm to patients ; errors can be allowed to occur , can even be scheduled , and can provide realistic experiences managing common and rare situations which differs to training by chance where exposure is limited to real life cases that may or may not occur for every trainee . in the medical education setting , simulation - based interventions are now mature with a wide variety of applications.7 for procedural skills , deliberate practice in a simulated - setting is one example of an effective teaching strategy that has been used for specific skills training in central venous catheter insertion , subarachnoid block placement , and laryngoscopy.8 medical residents trained to place central venous catheters in simulation improve their clinical performance in the intensive care unit.9 the translation of knowledge , skills , and attitudes from the simulation - based classroom to clinical care is important to show effectiveness or efficacy and drive curricular change.10 regional anesthesia inherently requires precise procedural performance due to target nerve locations near vital structures ( eg , blood vessels , pleura , organs , and nerves themselves ) and seems naturally suited for the incorporation of simulation within the training curricula . further , the recent evolution of ultrasound guidance in the practice of regional anesthesia has created great demand for training in this imaging modality for physicians who completed training more than a decade ago and are still in clinical practice.11 the american society of regional anesthesia and pain medicine ( asra ) and european society of regional anesthesia and pain therapy ( esra ) have published joint committee guidelines for training in ultrasound - guided regional anesthesia , and the asra esra guidelines suggest that simulation play an important role.12 although there is widespread use of the modality , the evidence basis for simulation in regional anesthesia training is not completely established.2 in this review , we provide an up - to - date summary of the literature related to the use of simulation in regional anesthesia education and assess its effectiveness . a search of the medline database ( pubmed.gov ; united states national library of medicine , national institutes of health , bethesda , md , usa ) using regional anesthesia , simulation , regional anesthesia simulator , regional anesthesiology simulation , regional anesthesiology simulator , nerve block simulator , and nerve block simulation , was conducted between january and march 2015 , and resulted in 393 citations . three of the authors ( tk , au , and em ) excluded non - english language , veterinary , nerve conduction ( without nerve blockade ) , and magnetic resonance imaging articles and eliminated any duplicate citations based on the search terms and phrases ; 64 articles remained . the reference lists of pertinent articles were also manually searched and revealed 15 additional articles not included in the original medline search . the final sample of 79 articles was critically reviewed for simulation - based educational interventions and their effectiveness . fourteen articles published to date study the effect on learners who underwent a simulation - based educational intervention in regional anesthesia ( table 1).1326 out of the 14 , only studies by niazi et al,15 baranauskas et al,20 and udani et al14 tested a simulation - based teaching strategy against an equivalent control group not receiving simulation ; all three studies enrolled anesthesiology trainees.14,15,20 based on the results of niazi et al , residents in anesthesiology who received 1 hour of simulation training on needling and proper hand eye coordination using ultrasound were more successful than a control group receiving no simulation , as assessed by blocks performed on real patients.15 baranauskas et al studied different durations of simulation training and the potential effect on learners , including 0 hours of simulation.20 students with 2 hours of simulation training in needling with ultrasound performed faster and with fewer technical flaws than students with 1 hour of simulation training . additionally , those with 1 hour of training performed better than those with 0 hours of simulation training . udani et al presented a randomized study in which residents are assigned to receive simulation - based deliberate practice teaching or a base curriculum without simulation to learn subarachnoid blocks.14 in this study , performance scores using a task checklist improved in the participants receiving simulation - based teaching . however , there may not be a translational benefit as there is no difference between groups in the time required for participants to place subarachnoid blocks in actual patients . table 1 includes other studies that on some level assess effectiveness of simulation - based educational interventions , but the ability to discern the impact of simulation alone in these studies is limited by methodology . woodworth et al , gasko et al , friedman et al , and liu et al describe controlled studies.13,17,19,22 however , the interventions under study are more complex than just incremental simulation - based teaching compared to a control group without simulation . for example , woodworth et al include a teaching video in addition to simulation training , which is then compared to a control group.13 gasko et al compare a combination of cd - rom teaching material with simulation versus cd - rom teaching alone versus simulation teaching alone.17 friedman et al compare a high - fidelity simulator versus a low - fidelity epidural simulator without a comparison to no simulator.19 although these studies demonstrate the benefits of enhanced and more rigorous training , the results can not be attributed entirely to the introduction of simulation . liu et al evaluate three different types of simulators for regional anesthesia and conclude that novice practitioners decrease the number of errors in a simulated block with each additional practice attempt in simulation , regardless of the type of simulator used.22 moore et al , garcia - tomas et al , ouanes et al , and bretholz et al describe comprehensive regional anesthesia curricula and demonstrate their effectiveness.16,18,21,25 these interventions all include simulation - based teaching but are combined with other teaching strategies ( eg , web - based tutorials , journal clubs , anatomy labs , etc ) ; thus it is not possible to identify the specific contribution of simulation . kim et al , cheung et al , and brenner et al use a single simulation - based teaching intervention , not in the context of a comprehensive curriculum , but do not include a control group comparison.23,24,26 due to the nature of all participants receiving some type of teaching , the reported effectiveness is mostly positive . participants report feeling more comfortable with proceedures , and post - intervention written test scores increase . the studies by garcia - tomas et al and ouanes et al show post - intervention objective structured clinical examination ( osce ) scores also increase.18,21 brenner et al report that interprofessional debriefings in their crisis management course lead to richer discussions.26 however , moore et al show that although written test scores improve after implementation of their educational curriculum , there is no difference in block accuracy or efficiency as assessed in a simulator.16 bretholz et al also report that the initial increase in comfort after their educational intervention is not sustained one month later.25 to date , 18 articles describe the design of a novel regional anesthesia simulator ( table 2).2744 simulators used for part - task training ( eg , phantoms ) in ultrasound - guided regional anesthesia vary based on the materials used and indications ( ie , tasks to be taught ) . inorganic materials are common in commercially available phantoms but often lack realistic tactile sensation and haptic feedback and do not allow for injection of liquid solutions as is common in regional anesthesia techniques ( figures 1 and 2 ) . these phantoms are useful for teaching procedural steps , dexterity , target identification , and needle guidance . in contrast , organic phantoms ( ie , meat ) arguably produce the most realistic sonoanatomy and tactile sensation and do allow for injection and even catheter insertion , but they are not reusable and must be replaced for subsequent training sessions ( figure 3 ) . since it is not possible to present every model used for regional anesthesia practice , for the purpose of this article we define a novel simulator design as a new , previously undescribed product employing innovative technology to represent the realistic scenario of performing a regional anesthesia procedure . the novel simulator design takes one of the following forms : 1 ) a physical model ( ie , phantom ) ; 2 ) virtual reality model ; 3 ) robot - assisted model ; or 4 ) environmental modification . only niazi et al,32 lim et al,34 lee et al,28 and morse et al27 report an effect on learners attributable to the use of their novel simulators . none of the studies that report an effect on learners include a control group against which simulation - based teaching is compared . rather , these studies focus on before and after test comparisons and show positive results associated with training on the novel simulators described . lim et al demonstrate that after using their virtual reality simulator , participants skills in identifying surface landmarks for block placement improve.34 lee et al demonstrate that participants time performing an epidural block decreases with 20 repetitions on their simulator.28 morse et al use a crossover study design to show that performance is more consistent and that learning is quicker using a robot - assisted regional anesthesia technique in simulation when compared to a traditional , manual technique using practitioner s hands.27 eleven articles present a new medical device or evaluate an established nerve block technique in a simulated environment ( table 3).4555 examples include primarily case reports on devices or techniques such as luer connectors , echogenic needles , needle guides , air test for inferring perineural catheter tip location , and a hand - on - syringe technique.4750,52,53 only kilicaslan et al,49 whittaker et al,45 neal et al,46 johnson et al,47 and gupta et al48 provide effectiveness data . simulation itself is not evaluated for efficacy , and studies that include a control group expose both the control and intervention groups to the simulated environment . reported outcomes include improvements in knowledge , skills , and/or behaviors with the use of the new device or technique . neal et al46 evaluate the use of a treatment checklist for the management of local anesthetic systemic toxicity ( last ) . the authors describe creating a simulated , clinical environment utilizing a mannequin to represent a clinical patient who receives an inadvertent toxic dose of intravascular local anesthetic . the learners , anesthesiology residents , and fellows , are inserted into the simulated crisis with or without a last treatment checklist . in their findings , the authors demonstrate that physician trainees using a treatment checklist make better medical decisions related to the critical management of last than trainees who do not use a checklist . the remaining articles span a broad range of topics , and none test the effectiveness or efficacy of simulation - based teaching in regional anesthesia . some authors collect the needs of training regional anesthesiologists and establish metrics to assess performance , using simulators.5660 others use a simulated environment to calculate procedural learning curves and observe practitioner ergonomics.6167 although these articles touch on the topic of medical education in regional anesthesia , we consider them outside the scope of the present review on simulation - based educational interventions and their effectiveness . although simulation - based medical education has been shown to be effective for specific applications , our review reveals that similar evidence in regional anesthesia training is limited . we especially note a lack of comparative evidence studying the effectiveness of a simulation - based teaching strategy versus a proper control : participants receiving identical teaching as the intervention group less simulation - based instruction . this would mean that control group participants should have an equal amount of time in an educational setting even when they are not receiving simulation - based instruction . medical education research is expected to show efficacy or comparative - effectiveness against an established method , or else educators may presume if you teach [ students ] , they will learn.68 we know that the addition of simulation - based instruction automatically means extra training , but we can not assume that this extra training automatically leads to the acquisition of new knowledge or skills . we encourage the development , implementation , and scientific investigation of comprehensive regional anesthesia training curricula . from the work published to date , we are unable to deduce the incremental effectiveness ( or ineffectiveness ) of simulation - based regional anesthesia education , although we acknowledge that it has face validity . the description of novel simulators and development of new regional anesthesia techniques in a simulated environment represent just the first step to fully assess the role of simulation in teaching the knowledge , skills , and behaviors necessary for regional anesthesia competency . recently , regional anesthesia has been evolving further into the medical subspecialty of regional anesthesiology and acute pain medicine ( raapm ) , and guidelines have been established for fellowship training.69 however , teaching strategies are not clearly recommended or identified . learners are taught using various techniques , most commonly as observers who transition to active participants in the apprenticeship model . a mix of didactic , multimedia , and simulation - based learning would augment this apprenticeship . the effectiveness of the variations in the raapm curricula is largely unknown , and curricula may very well be institution - specific for many reasons ( eg , faculty and resources available).70,71 we see many advantages to simulation - based education in raapm . the key elements of simulation - based education , namely repetitive practice , targeted feedback , self - reflection , and avoiding harm to patients make it a useful teaching strategy . the simulated environment is well - suited for providing learners with the time and means to gain effective feedback and reflect on their performance ; something that is difficult to obtain when taking care of real patients as time in the clinical setting is often limited and rushed . many aspects of raapm are procedure based , and practice in simulation can recreate a realistic experience . we believe raapm may be the ideal specialty to embrace hybrid simulation ( figure 4 ) , a combination of part - task training and mannequin - based simulation to facilitate procedural practice in the context of life - like scenarios ( eg , the anxious patient , a vasovagal response , or even last ) . repetitive practice in a controlled , simulated environment has been shown to lead to better procedural performance in the clinical setting.9 this is one of the tenets of deliberate practice , an effective use of simulation - based medical education.14 as educators , we hope that a student s mistakes will be made in the simulated environment , and corrected prior to actual patient care . another advantage of simulation - based education is that it exemplifies principles of adult learning and kolb s experiential learning theory . in our review of published articles , it is clear that learners of all stages in medical school , residency , fellowship , and clinical practice react and enjoy participating in simulation - based education even though it may initially cause some anxiety.72,73 alternatively , there may be disadvantages to simulation - based education in raapm . first , one may assume that a large capital investment in equipment and time is required.74 in our review , we find that this may not be true as many novel low - cost simulator designs have been described to help keep costs to a minimum while maintaining fidelity and achieving learning objectives.30,33,36,75 time in simulation is time away from clinical care . however , training guidelines established by the accreditation council for graduate medical education for anesthesiology residents now require time in simulation annually , and simulation is included in the requirements for maintenance of board certification in anesthesiology . there is momentum to encourage novice physicians to practice in simulation prior to engaging in clinical care . the same may be said for physicians who have already completed their training and are trying to learn a new technique such as ultrasound - guided peripheral nerve blockade . simulation may provide a more effective alternative to other methods currently available ( eg , continuing medical education or industry - sponsored workshops ) , and self - teaching is still widespread in this population.76 there may be costs involved for practicing physicians to participate in continuing education as well as loss of income . in our opinion , the time spent in simulation may be a solid investment since a complication prevented by practice in simulation may actually save time and decrease complications in clinical practice . the search terms related to regional anesthesiology , regional anesthesia , simulation , and simulation - based education have many pseudonyms that may have resulted in an incomplete literature search and inability to evaluate every study exploring the effectiveness of simulation - based education in regional anesthesia . each of the authors completed his own web - based and manual searches to generate as comprehensive a reference list as was feasible . we define simulation broadly in this review and have included published articles that describe any type of simulated environment or training , including as little as 5 minutes in simulated - training,13 for the sake of completeness . in reality , we believe effective simulation - based education in raapm necessitates stricter criteria including personal feedback , repetitive practice , and student reflection . we as educators see additional benefits of simulation - based education . as anesthesiologists who specialize in raapm , the scope is not limited to just performance of nerve blocks . while achieving mastery in a new technical skill is a measurable and achievable result with simulation , this should not ever be a physician s final goal . studies by neal et al46 investigating the use of emergency checklists during a simulated last crisis and brenner et al26 employing similar crisis management simulations for pain physicians demonstrate the potential role of simulation in teaching professional practice far beyond mere technical skills . in raapm , the frontier for simulation - based education should evolve to include teaching difficult patient interactions ( eg , demented elderly with hip fracture ) , ethical dilemmas ( eg , wrong side blocks ) , interdisciplinary team - based care , safety culture , and more . as technological advances in regional anesthesia and analgesia emerge , such as new robot - assisted procedures , medications , equipment , and techniques , they will require rigorous testing followed by an effective means to update training . the simulated environment is ideal for trialing these innovations , comparing them to current practices , and proving them effective prior to full implementation in clinical care . when there is sufficient evidence to support a change in clinical practice , simulation may even be able to facilitate dissemination and implementation . in summary , the emerging subspecialty of raapm represents an opportunity to critically evaluate the current methods of teaching regional anesthesia techniques and the practice of acute pain medicine . to date , there have been a wide variety of simulation applications in this field , and efficacy has largely been assumed . however , a thorough review of the literature reveals that effective teaching strategies , including simulation , are not yet established completely , in raapm . future research should be directed toward comparative - effectiveness of simulation versus other accepted teaching methods , exploring the combination of procedural training with realistic clinical scenarios , and the application of simulation - based teaching curricula to a wider range of learner from the student to the practicing physician . | the emerging subspecialty of regional anesthesiology and acute pain medicine represents an opportunity to evaluate critically the current methods of teaching regional anesthesia techniques and the practice of acute pain medicine . to date
, there have been a wide variety of simulation applications in this field , and efficacy has largely been assumed .
however , a thorough review of the literature reveals that effective teaching strategies , including simulation , in regional anesthesiology and acute pain medicine are not established completely yet .
future research should be directed toward comparative - effectiveness of simulation versus other accepted teaching methods , exploring the combination of procedural training with realistic clinical scenarios , and the application of simulation - based teaching curricula to a wider range of learner , from the student to the practicing physician . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the major extrarenal complications of adpkd include cerebral aneurysms , hepatic and pancreatic cysts , cardiovascular diseases , colonic diverticula , and malignancies.1)2 ) in cardiovascular diseases , the most common abnormalities include mitral valve prolapse and aortic regurgitation , which occur less frequently in adpkd.3)4 ) in addition , aortic4 ) and coronary aneurysms5 ) and coronary artery dissection6 ) have been described in patients with adpkd . here , we report a case of a patient with adpkd who presented with complex cardiovascular manifestations , as well as a literature review on the cardiovascular complications of adpkd . a 64-year - old man was referred to our hospital because of worsening dyspnea and peripheral edema . he had a 2-week history of shortness of breath on exertion . seven years previously , he visited an outpatient clinic for dyspnea on exertion . at that time , atrial fibrillation on electrocardiography and mild mitral regurgitation and moderate aortic regurgitation on echocardiography were detected . his medical record listed the following values : left ventricular ( lv ) end - diastolic dimension / body surface area ( bsa ) , 33 mm / m ; lv end - systolic dimension / bsa , 28 mm / m ; left atrial ( la ) antero - posterior diameter , 40 mm ; and ejection fraction , 57% by m - mode . abdominal computed tomography he was diagnosed with heart failure caused by atrial fibrillation with aortic regurgitation . his mother and maternal uncle , who both had adpkd , died suddenly at the age of 40 years cardiac auscultation revealed a holosystolic murmur at the apex and diastolic murmur in the right upper parasternal area . laboratory test results revealed elevated levels of brain natriuretic peptide to 272 pg / ml and creatinine to 1.78 mg / dl ( estimated glomerular filtration rate , 23.1 mlmin1.73 m ) . chest radiography revealed massive cardiomegaly with a cardiothoracic ratio of 90% and bilateral pulmonary congestion ( fig . 1 ) . two - dimensional echocardiography showed an extremely dilated la with lv end - diastolic dimension / bsa of 44 mm / m , lv end - systolic dimension / bsa of 30 mm / m , la antero - posterior diameter of 93 mm , and la volume index of 614 ml / m ( fig . 2 ) . the lv ejection fraction calculated by the modified simpson method was 55% . color doppler imaging revealed severe eccentric mitral regurgitation jet toward the lateral side ( fig . moderate regurgitation of the aortic valve was evaluated as a central flow with vena contracta of 6 mm . incomplete coaptation of the aorta with dilatation of the aortic root and annulus resulted in aortic regurgitation ( fig . severe dilatation of the left main coronary artery ( 15 mm ) was noted at the aortic valve level of the parasternal short - axis ( fig . this echocardiographic finding suggested combined coronary aneurysm , although angiographic evaluation for coronary aneurysm was not performed owing to poor renal function . a small amount of pericardial effusion and patent foramen ovale also was observed . multiple variable - sized cystic lesions in the liver and both kidneys observed on abdominal - pelvic computed tomography were compatible with adpkd ( fig . the patient was diagnosed with decompensated heart failure caused by complex cardiovascular abnormalities of adpkd . the patient was discharged with heart failure medications , and his follow - up course has been uneventful . adpkd is characterized by the progressive expansion of numerous cysts , resulting in substantial enlargement of the kidneys.1 ) in addition , patients exhibit a variety of extrarenal complications and concomitant systemic disorders with extrarenal cyst formation in the liver , pancreas , seminal vesicles , and meninges.2 ) noncystic manifestations of adpkd - related extrarenal complications affect mostly the vascular , cardiac , and connective tissues . the cardiovascular complications are the major cause of morbidity and mortality in patients with adpkd.7 ) the most common cardiovascular complication is hypertension.7 ) in structural cardiovascular abnormalities , mitral and aortic valvular prolapse with regurgitation and dilatation of the aortic root and annulus are considered as important extrarenal manifestations of adpkd.2)3)4 ) lumiaho et al.8 ) reported an increased prevalence of valvular abnormalities in patients with adpkd , such as mitral valve prolapse , mitral regurgitation , aortic regurgitation , and tricuspid regurgitation.adpkd also may be associated with an increased incidence of coronary aneurysm and coronary artery dissection.5 ) the mechanism underlying the development of valvulopathies and annuloaortic ectasia in adpkd is uncertain . adpkd is related to mutations in the pkd1 and pkd2 genes , which encode polycystin-1 and polycystin-2.9 ) these proteins are expressed in vascular smooth muscle cells and are associated with increased rates of proliferation and apoptosis . these changes may increase susceptibility to vascular injury , and result in direct aneurysmal changes and rupture of the vasculature.9 ) a few studies have discovered that abnormalities in collagen and extracellular matrix may be responsible for valvular disease in adpkd.4)10 ) on the other hand , polycystin-2 is a nonselective cation channel capable of transporting calcium ions,11 ) which plays an important role in the regulation of intracellular calcium stores and calcium homeostasis . reduced calcium stores in the sarcoplasmic reticulum resulting from a lack of polycystin-2 may be related to cardiac systolic dysfunction.11 ) this mechanism supports the notion of intrinsic myocardial dysfunction in patients with adpkd . previously , choe et al.12 ) reported a case of dilated cardiomyopathy associated with adpkd . in this case , moderate aortic regurgitation , annuloaortic ectasia , severe mitral regurgitation , and coronary aneurysm were observed concurrently . therefore , the main cause of mitral regurgitation progression was believed to be a tethering effect and that of lv dilatation to be aortic regurgitation . because the dilatation of the lv and la was extensive , even considering valvular regurgitation , we could not rule out that this intrinsic myocardial dysfunction may be combined with valvular and vascular diseases . although routine screening of all adpkd patients using echocardiography is not recommended , we suggest careful evaluation and serial follow - up of these patients presenting with symptoms or signs of cardiac disease to enable early decision - making regarding treatment . | autosomal dominant polycystic kidney disease ( adpkd ) is a systemic disorder associated with various extrarenal complications .
the major cardiovascular complications of adpkd include valvulopathies and vascular ectasia .
a 64-year - old man who was diagnosed with adpkd seven years previously was admitted to our hospital for heart failure .
pelvic computed tomography revealed multiple variable - sized cysts in both kidneys .
transthoracic echocardiography showed enlargement of the left ventricle and left atrium .
severe mitral regurgitation and moderate aortic regurgitation with annuloaortic ectasia were observed .
the left main coronary artery was dilated .
the patient had various cardiovascular features associated with adpkd . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
dentin pulp complex injuries are often induced by invasion of microorganisms and their components via dentinal tubules towards the pulp . caries , cracks , fractures , and leakage from restorations provide pathways for microorganisms and their toxins to enter the pulp . the inflammation can spread to the surrounding alveolar bone and cause periapical pathosis . in this process , bacterial lipopolysaccharides ( lpss ) play a potential role in several responses to pulpal infection . lipopolysaccharide ( lps ) can induce the expression of proinflammatory cytokines and chemokines such as tnf- and il-6 and elicit the innate immune response in dental pulp cells ( dpcs ) . signaling pathways initiated by engagement of toll - like receptors ( tlrs ) , such as tlr2 and tlr4 , by bacterial products lead to enhanced transcription of genes responsible for the expression of cytokines , chemokines , adhesion molecules , and other mediators of the inflammatory response associated with bacterial infection . of note , the activation of mitogen - activated protein kinases ( mapks ) is important in the production of inflammatory cytokines by lps stimulation . the mapk family includes extracellular - signal - related protein kinase ( erk ) , c - jun n - terminal kinase / stress - activated protein kinases ( jnk / sap ) and p38mapk . the mapk signaling pathway is involved in various kinds of cellular processes including differentiation , development , proliferation , and survival , as well as cell death , depending on cell type and stimulus [ 5 , 6 ] . pulpal p38mapk signaling is activated by lps stimulation during the induction of local proinflammatory response [ 79 ] . telomeres are specialized structures at the ends of chromosomes that have a role in protecting the chromosome ends from dna repair and degradation . telomerase is a cellular reverse transcriptase ( tert , telomerase reverse transcriptase ) which prevents premature telomere attrition and maintains normal length and function . human reverse transcriptase subunit of telomerase ( htert ) has become an attractive target for cancer vaccines due to it being expressed in 8590% of human cancer tissues , whereas it is almost never expressed in normal tissues . gv1001 peptide , which is a peptide corresponding to amino acids 611626 of htert ( earpalltsrlrfipk ) , has been developed as a vaccine against various cancers and has been reported to have the ability to penetrate into various cells , including cancer cell lines and primary blood cells . gv1001 was found to localize predominantly in the cytoplasm and could successfully deliver macromolecules such as proteins , dna , and sirna into cells . because of this novel pharmaceutical potential and cell - penetrating ability , as well as its own anticancer activity , gv1001 peptide is very promising for use in the medical field . here , we observed that this peptide could also penetrate into human dental pulp stem cells and , furthermore , that it had a self - anti - inflammatory effect without affecting cell viability . the purpose of this study was to evaluate the cell - penetrating function of gv1001 peptide in human dental pulp cells ( hdpc ) and to investigate the anti - inflammatory effect of gv1001 and its related mechanism in p. gingivalis lps - induced inflammation through regression of inflammatory cytokine production . all of the peptides used in this study were synthesized by the fmoc- ( 9-fluorenylmethoxycarbonyl- ) based solid - phase method and characterized by peptron inc . the purities of all peptides used in this study were greater than 95% , as determined by high - performance liquid chromatography . the impacted third molars of human adults were collected from 18- to 22-year - old patients after obtaining informed consent . the isolated dental pulp was cut into small pieces and digested in a solution of 3 mg ml type i collagenase and 4 mg ml dispase for 3060 min at 37c ( sigma aldrich , st . subsequently , the solution was filtered through a 70 mm cell strainer ( becton / dickinson , franklin lakes , nj , usa ) . the single - cell suspensions were seeded in 35 or 60 mm culture dishes and maintained in a culture media consisting of -minimum essential medium ( -mem ; invitrogen , carlsbad , ca , usa ) supplemented with 15% fetal bovine serum ( fbs ; gibco - brl , life technologies inc . , gaithersburg , md , usa ) , 0.292 mg ml glutamine ( invitrogen ) , 100 units ml penicillin g , 100 mg ml streptomycin , and 50 mg ml ascorbic acid ( sigma ) . the cells were incubated in a humidified atmosphere containing 5% co2 at 37c and cells between 3 and 4 passages were used in the following experiments . porphyromonas gingivalis atcc 33277 was purchased from the american type culture collection ( atcc ; manassas , va ) and cultured with brain heart infusion broth ( bhi ; bd bioscience , sparks , md , usa ) supplemented with hemin ( 1 g ml ) and vitamin k ( 0.2 g ml ) in anaerobic conditions at 37c . lipopolysaccharide was extracted from p. gingivalis cultured according to the method described by lee et al . . hdpcs were seeded and cultivated in 2-chamber glass slides ( nunc , roskilde , denmark ) for 12 h. after washing with pbs , cells were incubated in serum - free opti - mem for an hour . fluorescein isothiocyanate- ( fitc- ) labeled 1 , 10 , and 50 m of gv1001 peptides were added to cells and incubated for 2 h. the cells were fixed with 4% paraformaldehyde solution for 20 min at room temperature . cells were stained with to - pro-3 iodide 642/661 nm ( invitrogen , grand island , ny , usa ) to visualize nuclei and were subjected to confocal microscopy . colocalization of the peptides and nuclei was assessed using an fv1000 lager scanning confocal microscope ( olympus ) . after addition of 0.75 m10 m gv1001 to lps - treated cells , the samples were prepared for electrophoresis and were separated using a 10% sodium dodecyl sulfate - polyacrylamide ( sds - page ) gel with a previously established buffer system . after electrophoresis , the proteins were transferred onto a nitrocellulose membrane and blocked by tris - buffered saline ( tbst , 0.05% tween-20 ) containing 5% nonfat dried milk . the membranes were then incubated with anti - p - erk and anti - p - p38 ( cell signaling technology , beverly , ma , usa ) antibodies and subsequently washed with tbst . antigen - antibody complexes were visualized using an enhanced chemiluminescent detection system ( west - zol , intron , seoul , korea ) by incubating membranes with goat anti - rabbit igg or goat anti - mouse igg antibody coupled to horseradish peroxidase ( santa cruz biotechnology , inc . , santa cruz , ca , usa ) , diluted at 1 : 2000 . the blots were stripped and reprobed with an anti--actin polyclonal antibody to ensure that equal amounts of protein were used . after treatment with 0.7520 g ml lps for 2 , 4 , 6 , and 8 h , total rna was isolated from the cells using trizol reagent ( life technologies inc . , gaithersburg , md , usa ) according to the manufacturer 's suggested protocol and treated with dnase i ( rnase - free , rq1 ; promega , madison , wi , usa ) . one microgram of total rna was used as a template to create first - strand cdna with oligo - dt priming using an omniscript rt kit ( qiagen inc . , the quantitative real - time pcr analyses were performed using an abi prism 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) with a sybr premix ex taq ii kit ( takara , otsu , japan ) and 35 cycles of pcr . the denaturing , annealing , and extension conditions of each pcr cycle were 95c for 10 s , 60c for 15 s , and 72c for 10 s , respectively . the relative amount or fold change of the target gene was normalized relative to the level of the control ( untreated cells ) . the following primer sequences were used in the real - time pcr reactions : 5-cga aag tca act cca tct gcc-3 and 5-ggc aac tgg ctg gaa gtc tct-3 for il-6 gene ; 5-cca gga gaa agt cag cct cct-3 and 5-tca tac cag ggc ttg agc tca-3 for tnf- gene ; 5-gtg gtg gac ctg acc tgc-3 and 5-tga gct tga caa agt ggt cg-3 for gapdh gene . to evaluate the cytotoxicity of gv1001 on hdpcs , cells ( 2 10/well ) were treated with several concentrations of gv1001 ( 0 , 1 , 5 , 10 , and 50 m / well ) for 48 hours . mol l of mtt solution for 4 h in a tissue - culture incubator . a 200-ll quantity of dimethyl sulfoxide solution was then added to the cell - culture wells , and the plates were shaken for 10 min at room temperature to dissolve the precipitated formazan crystals . the solution was centrifuged for 10 min , and the optical density of the supernatant was measured at wavelength of 540 nm using an elisa plate reader ( powerwave x 340 ; biotek instruments , vt ) . the data are expressed as the mean standard deviation ( sd ) of at least 3 separate experiments . to confirm the cell - penetrating activity of gv1001 , fitc was conjugated to the c - terminus of 1 , 10 , and 50 m gv1001 peptides ( gv1001-f ) and subjected to an internalization assay using confocal microscopy . as shown in figure 1 , gv1001-f showed mostly cytoplasmic distribution in hdpcs in various levels of peptide concentration . to verify the effects of various doses of lps on inflammatory cytokine expression in hdpcs , the levels of tnf- and il-6 expression were evaluated using real - time pcr after exposing hdpc cells to lps of p. gingivalis or 1 g ml of e. coli at concentrations of 1 , 5 , 10 , or 20 g ml for 10 hrs . the results showed that il-6 transcript was profoundly induced in the presence of 1 , 5 , 10 , or 20 g ml of p. gingivalis lps or 1 g ml of e. coli lps ( p < 0.05 ) , and tnf- transcript was profoundly induced in the presence of 10 or 20 g ml of p. gingivalis or 1 g ml of e. coli lps ( p < 0.05 ) ( figure 2(a ) ) . to evaluate the effects of lps on inflammatory cytokine expression in hdpcs over a period of time , the levels of tnf- and il-6 expression were evaluated using real - time pcr after exposing hdpcs to 20 g ml of p. gingivalis lps or 1 g ml of e. coli lps for 2 , 4 , 6 , or 8 hrs . upregulation was found in hdpcs after 4 to 8 hrs of exposure to 20 g ml of p. gingivalis lps or 1 g ml of e. coli lps ( p < 0.05 ) ( figure 2(b ) ) . the expression of tnf- and il-6 in hdpcs were upregulated by lps in a time- and dose - dependent manner . we measured tnf- and il-6 expression after lps stimulation to determine the anti - inflammatory activity of gv1001 peptide by real - time pcr . lps - stimulated tnf- and il-6 production was significantly inhibited ( p < 0.05 ) ( figures 3(a ) and 4(a ) ) . to investigate whether the anti - inflammatory effect of gv1001 is mediated by the suppression of erk and p38mapk activation , regulation of phosphorylated erk and phosphorylated p38mapk was evaluated by western blot analysis . as shown in figures 3(b ) and 4(b ) , gv1001 decreased lps - induced phosphorylation of erk and p38mapk . incubating gv1001 peptide with hdpcs for 48 h did not affect cell viability in all concentrations tested ( 050 m ) , which showed a pattern similar to that seen with 0.9% nacl ( p < 0.05 ) ( figure 5 ) . although endodontically treated teeth can maintain their function for a prolonged period of time , there are many advantages to maintaining pulp vitality . in immature permanent teeth with incomplete apical and dentinal wall development , reparative dentin formation is critical for further development of the teeth . maintaining the vital pulp also has the benefit of reducing the occurrence of apical periodontitis by blocking bacterial infections . based on these advantages , it is preferable to maintain or renew pulp vitality rather than use current endodontic therapies . recently , successful pulp regeneration and revascularization techniques have been developed and are gaining popularity . p. gingivalis is an obligately anaerobic , gram - negative bacterium that has been positively associated with destructive periodontal disease [ 17 , 18 ] . it is one of the most pathogenic species among black - pigmented gram - negative anaerobes ; it is frequently present in root canal infections and other odontogenic abscesses of patients not suffering from periodontal disease , which confirms its pathogenic role . p. gingivalis may also play a role in symptomatic infections , such as acute apical abscess [ 21 , 22 ] . furthermore , there is a positive association between porphyromonas spp . and pain , mechanical allodynia , swelling , and purulent exudates in root canals [ 22 , 23 ] . pathogens are generally detected by specific receptors , one of which is the toll - like receptor ( tlr ) family . tlr stimulation initiates host defense through the activation of several intracellular signaling pathways , including activation of the mitogen - activated protein ( map ) kinases . exposure of cells to inflammatory stimuli , including lps and proinflammatory cytokines , results in phosphorylation of p38mapk . once the tlr is stimulated by a pathogen , proinflammatory cytokines and chemokines are produced by the odontoblast , resulting in recruitment and stimulation of immune effector cells and also direct bacterial killing . although these biological responses protect the host against invading pathogens , the inflammatory response also leads to host tissue damage . the extra - telomeric functions of htert are suggested regarding cellular proliferation , stem cell mobilization , and antiapoptotic , antiaging , and antioxidant effects through mitochondrial stabilization , transcriptional regulation [ 26 , 27 ] . if htert peptide treatment that mimics the extra - telomeric functions of htert could be developed , it could be used in the treatment of pulpal diseases . in this study , the potential use of a small peptide of human origin to control pulpal inflammation as a therapeutic agent was investigated . the anti - inflammatory effect of gv1001 is achieved by modulating the suppression of the activation of erk and p38 mapk and the subsequent cytokine production induced by p. gingivalis lps stimulation . p. gingivalis and e. coli lpss induced tnf- and il-6 expression in hdpc in a time- and dose - dependent manner , while gv1001 peptide downregulated inflammatory cytokines , il-6 and tnf-. hdpcs expressed tlr2 ( data are not shown ) and upregulated phosphorylated erk and p38 mapk in response to stimulation by lps . from the similar suppression patterns of the two , it could be assumed that the downregulation of inflammatory il-6 and tnf- by gv1001 was dependent on p38 mapk and erk - signaling , respectively , thus indicating that mapk signaling was required in downregulation of inflammatory cytokines in dental pulp stem cells by gv1001 peptide . as shown in figure 3 , higher dose of gv1001 exhibited a lower effect on tnf induced by p. gingivalis lps . one of the possible reasons is that gv1001 can partially aggregate in the high concentration in cell culture medium because cell culture medium does not include carrier protein . thus , the lower concentration of gv1001 except aggregating gv1001 penetrates into the cells and reacts with signaling molecules related with inflammation . in another previous study , gv1001 peptide was reported to have the ability to penetrate into various cells , including cancer cell lines and primary blood cells , without affecting cell viability . gv1001 was predominantly located in the cytoplasm and was used to successfully deliver macromolecules such as proteins , dna , and sirna into cells . these cell - penetrating peptides ( cpps ) have become one of the most popular and efficient tools for delivering various molecules into cells owing to the fact that they have the ability to enter cells independently of a membrane receptor , and they show no cell - type specificity . this study is significant in that it is the first to demonstrate gv1001 peptide as a cpp of human origin with a self - anti - inflammatory effect and without affecting cell viability . the use of gv1001 peptide can be a potential therapeutic approach for treating pulpal inflammation and the peptide can also be used as an intracellular delivery tool for bioactive molecules . using gv1001 peptide as a pulp - capping agent on reversibly inflamed pulp or an alternative to antibiotics in regeneration therapy may be effective . conjugating growth factors such as tgf-s and bmps with gv1001 can facilitate induction of hdpcs to differentiate effectively into odontoblast - like cells . moreover , gene therapy can be implemented using gv1001 to fuse a growth / differentiation factor for application in vital pulp therapy , regenerative endodontic fields and tissue engineering . the results from this study may support further research on the gv1001 peptide and its various clinical applications . the downregulation of lps - induced tnf- and il-6 expression was mediated through erk and p38 map kinase pathways . these findings provide mechanistic insight into the ability of gv1001 peptide to cause anti - inflammatory actions in lps - stimulated pulpal inflammation without significantly affecting cell viability . | porphyromonas gingivalis is considered with inducing pulpal inflammation and has lipopolysaccharide ( lps ) as an inflammatory stimulator .
gv1001 peptide has anticancer and anti - inflammation activity due to inhibiting activation of signaling molecules after penetration into the various types of cells .
therefore , this study examined inhibitory effect of gv1001 on dental pulp cells ( hdpcs ) stimulated by p. gingivalis lps .
the intracellular distribution of gv1001 was analyzed by confocal microscopy .
real - time rt - pcr was performed to determine the expression levels of tnf- and il-6 cytokines .
the role of signaling by map kinases ( erk and p38 ) was explored using western blot analysis .
the effect of gv1001 peptide on hdpcs viability was measured by mtt assay .
gv1001 was predominantly located in hdpc cytoplasm .
the peptide inhibited p. gingivalis lps - induced tnf- and il-6 production in hdpcs without significant cytotoxicity .
furthermore , gv1001 treatment markedly inhibited the phosphorylation of map kinases ( erk and p38 ) in lps - stimulated hdpcs .
gv1001 may prevent p. gingivalis lps - induced inflammation of apical tissue .
also , these findings provide mechanistic insight into how gv1001 peptide causes anti - inflammatory actions in lps - stimulated pulpitis without significantly affecting cell viability . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the ongoing debate over the use of pulmonary artery catheters is focused on their impact on outcome . the value of any diagnostic tool in improving outcome depends , first , on its ability to correctly diagnose the disorders it is meant to diagnose , second , on the relevance of such disorders to the outcome of interest , and third , on the availability of treatment to correct the diagnosed disorders . the rationale for using clinical monitoring tools can be based as well on prevention of disorders that , if allowed to develop , can have relevant effects on outcome . do we have evidence from randomized controlled trials that any diagnostic tool , per se , currently used in intensive care patients improves outcome ? here such diagnostic tools include echocardiography , ct scan , magnetic resonance imaging , angiography , ultrasound and doppler measurements for any purposes , measurement of intracranial pressure , and gastroscopy , just to name a few . does this mean that all such diagnostic tools that have not been proven to improve outcome in randomized controlled trials should be abandoned ? here do we have evidence from randomized controlled trials that any monitoring tool , per se , currently used in intensive care patients improves outcome ? here the answer is also clearly no . such monitoring tools include pulse oximetry , measurement of arterial blood pressure and other intravascular pressures ; monitoring of electrocardiogram ( ecg ) , electroencephalogram ( eeg ) , and concentrations of inspiratory , expiratory , and end - tidal gases ; intracranial pressure monitoring , monitoring of cardiac output by any method , and monitoring of intracardiac and intrathoracic blood volumes or extravascular lung water by any method , just to name a few . does this mean that all such diagnostic tools that have not been proven to improve outcome in randomized controlled trials should be abandoned ? here what is the background of the controversy surrounding the use of the pulmonary artery catheter , a very powerful tool for assessment and monitoring of cardiovascular physiology ? first , there is little doubt that the use of the pulmonary artery catheter in revenue - driven health care systems has been extensive , in part due to the financial incentives involved . hence , it is very likely that this tool has been widely used in patients who have had either no physiological problems that need to be solved , or a low risk of developing such problems . second , there is no doubt that the measurement of complex physiological interactions is , by definition , complex , and requires careful performance and sufficient knowledge of the underlying physiology in order to correctly interpret the results . third , gross misinterpretation of the available measurements has been common , and therapeutic strategies based on such interpretations widely advocated . in comparison with any other diagnostic and monitoring strategy currently used in the intensive care unit , the pulmonary artery catheter has undergone an unusually detailed evaluation several single- or multicentre trials [ 1 - 5 ] , some of them randomized and controlled , have demonstrated that the routine use of the pulmonary artery catheter without any specified therapeutic interventions does not influence the outcome that is , is neither dangerous nor beneficial . with respect to a lack of benefit in routine use , this puts the pulmonary artery catheter in the same position as the other monitoring technologies with the major difference being that it has been assessed in the randomized controlled setting . in terms of not producing harm , the data available for the pulmonary artery catheter are superior to those available for other diagnostic and monitoring technologies practically no other technology has comparable safety data obtained in randomized controlled trials . the ability of the pulmonary artery catheter to measure intravascular pressures , blood flow , mixed venous saturation and , more recently , intracardiac volumes and right ventricular ejection fraction with reasonable accuracy , providing that the measurements have been correctly made , is rarely an issue . the interpretation of these measurements ( diagnostic interpretation ) and application of correct interventions to treat the disorders ( therapeutic or preventive strategy ) are difficult and subject to controversy . despite this , several single - centre , randomized , controlled trials have successfully applied therapeutic strategies based on information obtained from the pulmonary artery catheter , and have used these strategies to improve outcome(s ) in surgical patients [ 6 - 9 ] . all the successful studies have applied strictly controlled therapeutic strategies to affect physiological variables in a pre - emptive fashion , that is , either to treat the disorders early or to prevent their onset . it is reasonable to assume that the success in these trials is based on the success in designing appropriate protocols and selecting the correct groups of patients at risk , and not on the presence of a diagnostic or monitoring technology per se . notably , the largest trial evaluating the use of the pulmonary artery catheter with therapeutic guidelines in surgical patients showed no benefit ( and no harm ) . what is the difference between this landmark multicentre study and the increasing number of smaller , single - centre trials showing controversial results ? first , the multicentre study by sandham and colleagues included patients with a considerably lower mortality than all the successful studies in high - risk patients . it is conceivable that any beneficial effect of physiology - oriented protocol - driven treatments on mortality will be evident in patients at a higher risk of mortality regularly around twice as high in the positive studies compared to the study of sandham and colleagues . second , the sandham trial used guidelines instead of a treatment protocol , and the adherence to these guidelines is scarcely reported . third , the treatment goals in the sandham trial may not have been well selected indeed , they are very similar to those used in a study in young trauma patients , where goal - directed treatment attempting to increase the oxygen delivery beyond the patients ' ability to respond resulted in increased mortality . this underscores that , to improve outcome , a diagnostic or monitoring tool must be coupled with a treatment that improves outcome . at the current stage , routine clinical use of the pulmonary artery catheter has been shown to be safe ( comparable to central venous catheters ) , as long as physiologically reasonable therapeutic goals are used . the pulmonary artery catheter should be used with the same scrutiny as any other diagnostic and monitoring tool used to diagnose disorders and adjust therapy in critically ill patients patients without an actual hemodynamic problem or without a high risk of developing one should not receive a pulmonary artery catheter . the pre - emptive use in high - risk surgery requires definition of patients at high risk , and a treatment strategy proven to work . the information obtained should also be used to find better treatment strategies , and these strategies , instead of the tool itself , should be tested in clinical trials an approach that so far has been almost unique to the pulmonary artery catheter . recently , the ards ( acute respiratory distress syndrome ) clinical trials network published a study in which two treatment strategies were tested simultaneously with two different monitoring approaches in patients with established acute lung injury . one thousand patients were randomized to have their hemodynamic management guided by monitoring using either the pulmonary artery catheter or the central venous catheter . both groups had explicit treatment protocols to guide the hemodynamic support and were simultaneously randomized to two different strictly defined fluid management regimens ( restrictive and liberal fluid administration ) . the rates of death at 60 days before discharge home ( primary outcome variable ) , as well as the number of ventilator - free days and days not spent in intensive care , and various other secondary outcome variables were similar between the two groups with different monitoring strategies . the rates of catheter - related ( either central venous or pulmonary artery catheter ) adverse events were low and similar per catheter inserted , but since the pulmonary artery catheter group had more catheters , they also had significantly more adverse events total , most of which were arrhythmias . notably , arrhythmias were not prospectively recorded by insertion of central venous lines . independent of the monitoring strategy , the restrictive fluid management strategy increased the number of ventilator - free days and days not spent in intensive care in these patients . this study has a very clear message : the use of the pulmonary artery catheter , as defined in the protocol , did not offer any benefit compared to the central venous catheter - guided protocol . while a major strength of the study was the use of strict protocols , this is at the same time a limitation . as in the case of successful studies , the conclusions of unsuccessful studies are limited to the protocol , and the way the monitoring tool guides treatment within the protocol . in this respect , the study of the ards network deserves some scrutiny and comments . in patients with pulmonary artery catheters , cardiac index and pulmonary artery occlusion pressure clinical assessment of circulation was explicitly not used for decision making . in the central venous catheter - guided treatment , clinical assessment of circulation was explicitly included with the central venous pressure , blood pressure and urine output to guide the treatment . although prompt reversal of hypotension , oliguria , and ineffective circulation was a foreseen overriding goal , neither blood lactate levels nor mixed or central venous oxygen saturation were measured . the main uses of the pulmonary artery catheter in the study to evaluate whether cardiac index was higher than 4.5 l / min / min order to avoid fluid administration for normotensive patients with normal urinary output but low filling pressures ; and to measure pulmonary artery occlusion pressure to define when to stop giving furosemide or start giving fluids in normotensive patients with normal urinary output . it is also of interest that 29% of all patients with pulmonary artery catheters had a pulmonary artery occlusion pressure higher than 18 mmhg at protocol start . in order to recruit 1,001 patients , the study screened over 11,000 patients , more than 2,100 of whom were excluded because they already had a pulmonary artery catheter in place these are among the issues that should be considered when applying the results of this important trial in clinical practice . perhaps the most important message from this study is that too much fluid in established acute lung injury is harmful and that the iatrogenic effects of hemodynamic support can be reduced by protocols using clinical judgement and central venous pressure measurement of cardiac output and pulmonary artery occlusion pressures offers no additional benefit in this context . the search for alternative and complementary strategies of hemodynamic monitoring has brought exciting new technologies to clinicians . before repeating many of the pitfalls that have characterized the debate on the value of the pulmonary artery catheter , the search for effective therapeutic interventions should be emphasized . the need for any diagnostic or monitoring tool depends fundamentally on what information is needed to perform such interventions all of the existing and future technologies have a potential value in this context . the clinic of intensive care medicine , university hospital bern , has existing research and/or consultation agreements with the following companies involved in clinical monitoring : edwards lifesciences , general electric , pulsion medical systems ag . | the pulmonary artery catheter ( pac ) is a powerful tool that has been used extensively in the assessment and monitoring of cardiovascular physiology .
gross misinterpretation of data gathered by the pac is common , and its routine use without any specific interventions has not been shown to influence outcome . however , there currently is no evidence from randomized , controlled trials that any diagnostic or monitoring tool used in intensive care patients improves outcome .
studies evaluating the use of the pac have included numerous potential confounding factors , and should be interpreted with caution .
the information obtained with the pac should be used to find better treatment strategies , and these strategies , instead of the tool itself , should be tested in clinical trials . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
polycystic ovarian syndrome ( pcos ) , which is one of the common endocrine disorders , is one of the main causes of ovulatory infertility , affecting 510% of women of reproductive age [ 1 , 2 ] . pcos is characterized by the presence of typical ultrasound features of polycystic ovaries , oligomenorrhea , and clinical and/or biochemical hyperandrogenism and , commonly , by insulin resistance , hyperinsulinemia , morbid obesity , and infertility [ 39 ] . , insulin promotes intraovarian steroidogenesis by interacting with luteinizing hormone ( lh ) leading to inappropriate advancement of granulose cell differentiation and arrest of follicle growth . the outcomes with hyperinsulinemia may directly enhance ovarian secretion and abnormal follicular development , which ultimately lead to ovarian dysfunction [ 11 , 12 ] . insulin resistance or obesity with pcos leads to a significant increase in gestational outcomes and difficulties during labor . during recent years , metformin , which is an effective oral biguanide insulin sensitizer , has been widely used for treating type 2 diabetes mellitus ( t2 dm ) as an antihyperglycemic agent , by improving tissue sensitivity to insulin while inhibiting hepatic glucose production , enhancing peripheral glucose uptake , and decreasing insulin levels [ 14 , 15 ] , and approved by the united states food and drug administration ( fda ) . therefore , metformin has become an ideal first line therapy for individuals with t2 dm . when treated in women with pcos , especially these patients with hyperinsulinemia , metformin corrects hyperinsulinemia and also reduces ovarian androgens , lh and sex hormone binding globulins . metformin has been increasingly regarded to be effective and safe medicine for the metabolic and endocrine abnormalities in pcos [ 17 , 18 ] . its use as a drug for ovulation induction in pcos has been extensively investigated and has been found to increase the likelihood of ovulation and decrease miscarriage rates , particularly in patients who have clomiphene resistance before . as we all know , metformin is a category b drug for use in pregnancy ( absence of teratogenic effects based on animal data ) and its characteristics of effectiveness and safety and , as a result , its use in pregnancy have become increasingly popular worldwide , although there are no guidelines for its continuous use in pregnancy and there is debating on potential adverse effects on both the mother and the fetus because of its crossing the placenta [ 19 , 20 ] . moreover , metformin has been considered as a potentially effective agent during pregnancy to treat gestational diabetes mellitus . on the other hand , several randomized controlled trials ( rcts ) evaluating the reproductive effect of metformin administration and reporting the occurrence of gdm are not available in literatures . experimental and clinical studies seem to suggest that metformin does not have any effect on the incidence of gdm in women with pcos . furthermore , unclear and nonpowered data support the use of metformin for the prevention of gdm , preeclampsia , and other gestational complications in pcos patients . based on these considerations , we conducted a systematic review and meta - analysis of trials for a more objective appraisal of evidence regarding the effect of pregestational metformin administration on gestational complications , especially the gestational diabetes in women with pcos . a systematic review was conducted with all pertinent studies that were found in the electronic databases medline , embase , and the cochrane central register of controlled trials ( central ) that examined metformin and pregnancy outcome on the women with pcos from 1966 to december , 2013 . the search strategy included the terms pregnancy , pregnant complication , gdm , gestational diabetes mellitus , diabetes , metformin , biguanide , insulin - sensitizing drugs , insulin - sensitizers , polycystic ovary syndrome , and pcos . the search was concluded by ( 1 ) the perusal of the reference sections of all relevant studies in all languages and ( 2 ) a manual search of the key journals and abstracts from the major annual meetings in the fields of endocrinology and obstetrics and gynecology . articles were excluded from the analysis if they did not have either adequate disease - matched control groups or data on the outcome of the pregnancy with respect to major gestational diabetes and exposure to metformin throughout pregnancy or in at least the first trimester . the control groups consisted of women with pcos who were not treated with metformin or placebo . inclusion criteria : pcos was exposed to metformin throughout the pregnant period , or at least during first trimester;studies could be a prospective , retrospective , or a case - control study ; however , there had to be a control group;studies were written in any language . pcos was exposed to metformin throughout the pregnant period , or at least during first trimester ; studies could be a prospective , retrospective , or a case - control study ; however , there had to be a control group ; studies were written in any language . exclusion criteria : if type of articles was review or letter to the editor;animal studies;studies with no control group or with an inappropriate control group;women with exposures to other known pathogenic factors or other maternal disorders that might affect the outcome.titles and , especially , abstracts were screened , and the potential relevant researches were identified . two independent reviewers ( zhuo and yu ) , who were not blinded to the names of investigators or sources of publications , identified and selected the articles that met the inclusion and exclusion criteria below . meta - analysis was conducted using review manager 5.2 software ( review manager ( revman ) ( computer program ) ( version 5.2 for windows ) , oxford , england : the cochrane collaboration , 2012 ) . if type of articles was review or letter to the editor ; studies with no control group or with an inappropriate control group ; women with exposures to other known pathogenic factors or other maternal disorders that might affect the outcome . we included trials evaluating the reproductive effects of metformin administration on the gestational complications especially gdm in the women with pcos in the current meta - analysis . we considered all studies in which the diagnosis of pcos was based on the normal criteria such as rotterdam eshre / asrm consensus criteria , nih criteria , or aes criteria eligible . specifically , allocation concealment , blinding , intention - to - treat ( itt ) analysis , and followup were assessed for each trial . allocation concealment was graded as adequate ( a ) , unclear ( b ) , or inadequate ( c ) according to the criteria provided by the cochrane group particularly . blinding was reported as yes , no , or not reported for patients , outcome assessors , investigators , or data analysts . the q - test of cochran revman was used to measure the heterogeneity between articles and was calculated as the weighted sum of squared differences between individual study effects . a cochran q - test p 0.05 represents a statistical homogeneity . for data with statistical homogeneity , the mantel - haenszel method was used to calculate the weighted summary rr under the fixed effects model . on the other hand , the random - effect model of meta - analysis the flowchart of the study selection according to the inclusion and exclusion criteria above is shown in figure 1 . a total of 13 trials potentially resulted for inclusion in the meta - analysis [ 22 , 2536 ] . in particular , we excluded the trials as follows : the data on the occurrence of gdm were not available from papers and could not be obtained from the investigators by e - mail contact ; it was not possible to contact the corresponding investigators and the occurrence rate of gestational diabetes has not been evaluated . the main characteristics of the populations , the interventions received , and the outcomes obtained in the trials are summarized in tables 2 and 4 , respectively . a total of five rcts were also included in the final analysis as table 3 . however , three rcts including fougner et al . , vanky et al . , and salvesen et al . reported an epianalysis of two randomized controlled trials as vanky et al . and salvesen et al . finally , we reviewed two rcts [ 32 , 35 ] in our meta - analysis . no research included in the system review and meta - analysis had occurrence of gestational diabetes as a primary end point , and none was powered to detect differences in gdm incidence . in addition , other potential gestational complications , such as miscarriage , preeclampsia , and preterm delivery , including the infants ' characteristics , were investigated in these studies . the populations studied in the different trials were heterogeneous for several demographic and biochemical / metabolic characteristics . 13 studies met the inclusion criteria , including 5 random control trial studies in the calculation of overall gdm occurrences . exposure to metformin throughout the pregnancy was not associated with a decreased rate of gdm occurrence . the or for gdm in rcts with 289 subjects was 1.07 ( 95% ci 0.601.92 , p = 0.89 , and i = 0% ) ( figure 2 ) ( fougner et al . and selvesen et al . 's study was enrolled because of the same object of three rcts ; meanwhile , vanky et al . 's study was eliminated because of the fact that it is an epianalysis of vanky et al . and vanky et al . ) ; inversely , the or including the studies with disease - matched controls or patient self as control was 0.19 ( 95% confidence interval ( ci ) , 0.130.27 , p = 0.48 , i = 0% ) ( figure 3 ) . subanalysis was conducted to separate studies into those with pcos and those with patients ' selves with pcos subjects as controls ( figures 4 and 5 , resp . ) . with 457 subjects in the treatment group , the or for the nondiabetes pregnancy subjects group was 0.17 ( 95% ci , 0.110.27 , p = 0.40 , i = 2% ) . with 118 subjects in the treatment group for pcos with self as control , the or was 0.24 ( 95% ci , 0.110.54 , p = 0.29 , and i = 11% ) . in our mind , this is the first systematic review and meta - analysis of trials including rcts performed to establish the potential effect of metformin invention during the gestational period on gestational diabetes occurrence risk in patients with polycystic ovary syndrome . gestational diabetes mellitus ( gdm ) represents an important medical and social problem in the general pregnancy population , and it becomes harmful to the health of mother and fetus as well as neonate . investigated that women with pcos demonstrated a significantly higher risk of developing gdm ( or = 2.94 ) , pregnancy - induced hypertension ( or = 3.67 ) , and preeclampsia ( or = 3.47 ) , and their babies had a significantly higher risk of admission to a neonatal intensive care unit ( or = 2.31 ) and a higher perinatal mortality ( or = 3.07 ) . has included that the traditional therapy in pregnancy for gdm includes the use of diet and exercise and/or insulin injection . meanwhile , several researchers have already demonstrated that the administration of metformin throughout pregnancy may decrease miscarriage and improve pregnancy outcomes including gdm , preeclampsia , and preterm delivery [ 34 , 3944 ] . although a population - based cohort research , a system review , and meta - analysis recently identified that the patients with pcos are at higher risk for pregnancy complications [ 45 , 46 ] , moreover , unless metformin was recommended for treatment in pregnancy complications especially dm because of its characteristics of safety , effectiveness , and nonteratogenicity , they could not reach an agreement on its use in prevention or treatment of gdm , hyperinsulinemia , and hyperandrogenemia throughout the pregnancy in women with pcos . in our knowledge , during the past several decades , the retrospective and nonrandomized studies have confirmed beneficial effects of metformin on pregnancy miscarriage and pregnancy complications , in particular gdm in women with pcos , whereas the potential effect of metformin to pregnancy complications in women with pcos has been explored in five randomized , placebo - controlled trials so far [ 26 , 32 ] . the studies suggested no statistical difference in the prevalence of gestational diabetes , preeclampsia , and preterm delivery between the metformin- and the placebo - treated groups . as a result , meta - analytical summaries of existing studies therefore are of importance to gdm in pcos . although it yields a reassuring trend , this analysis exemplifies the need for more research on metformin to dm during pregnancy . it was our initial aim to identify all possible adverse pregnancy outcomes including miscarriage , minor anomalies , major malformations , intrauterine growth retardation , gdm , preterm delivery , live birth , caesarean section rate , or the characteristics of infants in particular the birth - height , birth - weight , apgar in 1 or 5 or 10 minutes , and head circumference . however , we were limited to focusing mainly on the pregnancy complications on gdm because of the paucity of data and the paradoxical effect of metformin on gdm in pcos . notwithstanding our data , metformin administered alone throughout the pregnancy , or at least during first trimester , or in combination with other infertility treatments for inducing ovulation in patients with pcos seems to have significant effect on gdm prevention . in fact , our non - rcts data demonstrated reduction of gdm occurrence risk in patients who received metformin in comparison with those who did not , for an overall relative risk for gdm of 0.19 ( 95% ci , 0.130.27 ) . nevertheless , on the basis of rct studies available now , metformin does not appear to be unavailable for use during pregnancy with respect to diabetes mellitus , for an overall relative risk for gdm of 1.07 ( 95% ci , 0.601.92 ) . as with placebo use in pregnancy , it is challenging to establish the necessity of metformin use in pregnancy considering the background incidence of diabetes in the confounder of the underlying diseases with pcos that have been linked to increased rates of diabetes . however , it is not always true that rcts and non - rcts arrive at the same conclusions and , consequently , it is important to ask what should be done when they do not agree . first of all , the evidence of data derived from multiple randomized clinical trials or meta - analyses is stronger than data derived from a single randomized trial or unrandomized studies . thus , if a rct study differs from several non - rcts , then there is a pressing need to identify the studies much more deeply . we examine the possible reasons why two types of studies arrived at different conclusions and the potential threats to validity of observational outcomes and rcts . our analysis and review indicate that several methodological issues are relevant because they may affect the interpretation of some of the studies published on gdm . most of the studies were retrospective or prospective of cohorts or case - control studies or observation researches and conducted at single center or multiple centers , and many studies included a small number of women , or else , the five rcts , including the three studies of the same research object and one study epianalyzing the other two trials , took place in the same country as norway and the study group of patients mainly recruited from the near clinic center imposed the restrictions on the outcomes , and thus the current situation limited the validity of the results . perhaps the most serious advantages of rct are blindness and randomization because there may be large observed and unobserved differences in patients between the treatment and control groups . these differences may explain why our meta - analysis concludes different results of rcts and non - rcts as rcts were developed for the purpose of eliminating this bias . during pregnancy , extra care must be taken to ensure the safety of the fetus or neonates . our meta - analytical summary and review of inclusive studies therefore have indicated monitoring metformin safety . although all included studies are very heterogeneous for protocols and doses of the drug administered and for characteristics of the studied populations , the detected observation of the results , such as lactic acidosis , birth defects , and maternal or neonatal hypoglycemia , is just a case - report and has no statistical analytical significance . our systematic review and meta - analysis of rcts studies demonstrates no statistically significant benefit of metformin administration on the dm risk in pcos patients who received the drug as monotherapy . however , we examine that non - rcts have a characteristic of a lower gdm occurrence rate in the metformin group compared with the pcos - matched control group . thus , at the moment , there is no clinical evidence to suggest that metformin administration reduces the risk of diabetes in women with pcos . however , the functional consequences of metformin administration throughout pregnancy on gestational diabetes mellitus ( gdm ) have not been assessed . metformin has been increasingly regarded to be effective and safe medicine for the metabolic and endocrine abnormalities in pcos . in our knowledge , this is the first systematic review and meta - analysis of trials including rcts performed to establish the potential effect of metformin intervention during the gestational period on gestational diabetes occurrence risk in patients with polycystic ovary syndrome . we therefore performed a meta - analysis and system review to determine the effect of metformin on gdm in patients with pcos . meta - analysis examined whether metformin could reduce gdm occurrence in pcos with a fixed effect model . the odds ratio ( or ) with 95% confidence interval ( 95% ci ) was calculated to estimate the strength of association . a total of 13 studies including five rcts and eight non - rcts were enrolled . ultimately , effectiveness analysis demonstrated that , in total , there was no significant availability of metformin on gdm in pcos in contrast to placebo ( or = 1.07 , 95% ci 0.601.92 ) in rcts and significant availability of metformin on gdm ( or = 0.19 , 95% ci 0.130.27 ) was indicated in non - rcts . subanalysis was conducted to separate studies into those with pcos and those with patients ' selves with pcos subjects as controls . with 457 subjects in the treatment group , the or for the non - diabetes pregnancy subjects group was 0.17 ( 95% ci , 0.110.28 ) . with 118 subjects in the treatment group for pcos with self as control , , according to the results of our meta - analysis , strictly , metformin did not significantly have effect on gdm with pcos , though more multicenters rcts still need to be investigated . thus , at the moment , there is no clinical evidence to suggest that metformin administration reduces the risk of diabetes in women with pcos . | metformin is an effective insulin sensitizer treating type 2 diabetes mellitus .
however , the functional consequences of metformin administration throughout pregnancy on gestational diabetes mellitus ( gdm ) with polycystic ovary syndrome ( pcos ) have not been assessed .
we therefore performed a meta - analysis and system review to determine the effect of metformin on gdm in pcos .
a meta - analysis was performed on the published studies before december , 2013 .
meta - analysis examined whether metformin could reduce gdm occurrence in pcos with a fixed effect model .
the odds ratio ( or ) with 95% confidence interval ( 95% ci ) was calculated to estimate the strength of association .
a total of 13 studies including 5 rcts and 8 non - rcts were enrolled .
ultimately , effectiveness analysis demonstrated that , in total , there was no significant availability of metformin on gdm in pcos in contrast to placebo ( or = 1.07 , 95% ci 0.601.92 ) in rcts and significant availability of metformin on gdm ( or = 0.19 , 95% ci 0.130.27 ) was indicated in non - rcts . in summary , according to the results of our meta - analysis , strictly , metformin did not significantly effect on gdm with pcos , though more multicenters rcts still need to be investigated . |
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evidence that continuing professional development impacts positively on patient and families outcomes is necessary to sustain an on - going investment in learning activities . in order to optimize the opportunities afforded by emerging web - based technology rural nurses need to develop and maintain their computer competencies . further investigation of the impact of specialist clinical placements on rural nurses palliative care capabilities is also indicated . medication is the main component of nurses function and is a major part of patients care and treatment process . there are annually thousands of medication errors among physicians and para medicine personnel reported in the united state . unfortunately , there is a catastrophic statistic on these errors , which has remained not reported . these errors may be due to function of physicians , pharmacists , medication technicians , nurses or midwives . medication errors may occur in any step of prescription and drug distribution process as prescribing medication is a sophisticated process and needs knowledge , decision making and proper function of the staffs working in various hospital wards . their direct outcomes threaten patients life and increase costs while their indirect outcomes include nurses occupational problems and reduction of trust to them as well as their diminished function . based on research , the burden of medication errors in national health services in england accounts to an annual 500 million dollars in addition to prolonged hospitalization days . nurses spend about 40% of their time on preparation of drugs and medication so , they play a highlighted role in patients medication process . based on research , medication errors originate from physicians in 39% of the cases , from nurses in 38% and from pharmacists in 23% of cases . , in a study on medication errors resulting from drug preparation and administration in internal wards , concluded that most of errors are resulted from nurses acknowledge of medication orders and their method of application . in an analysis of 469 life threatening errors between 1993 and 1998 in usa , human factors were reported to have the most efficient role , mostly due to a defect in practice and knowledge ( 0.44 ) . the their study found out that nurses face problems due to lack of attention to prescribed medications dosages , ordered by a physician , wrong dosage calculation ( exchange ) and not application of their theoretical knowledge in clinical setting . therefore , a comprehensive action is needed to solve this problem . in studies conducted in iran , more attention of authorities and nursing managers it seems that holding educational courses to empower nurses scientific knowledge and motivation and making it more beneficial for the patients , education process modification through encouragement of nurses to report their medication errors and preparation of processes for physicians encouragement to order medication more readably in clinical setting can have a more positive and efficient outcomes . numerous studies , conducted on the effect of continuing education on nurses efficiency , self - confidence , knowledge and skills , all show positive results so that several international studies report the effect of continuing education on improvement of cares quality . the results of research in usa , canada , australia and england have shown the positive effect of education on quality of nursing care as well as the importance of educational programs and their effect on nurses knowledge , attitude and experimental and scientific skills . the researcher had already determined these needs in her study on needs assessment of nurses working in psychiatric wards of hospitals in isfahan in 2011 and her obtained results showed that the most important needs of working nurses in these wards were in association with medical and nonmedical interventions including familiarization with side - effects of drugs , management of side - effects , medication indications and necessary cares at the time of medication . with regard to above - mentioned issues and determination of nurses educational needs in relation with medical and nonmedical interventions , there is not any medical intervention workshop in iran , so we need to assess the effect of this type of program . this study aimed to define the efficiency of an educational workshop on medical and nonmedical interventions to promote the knowledge and attitude of nurses working in psychiatric wards of educational hospitals in isfahan until 3 months after holding the workshop . it has been coordinated to the nursing managers in noor and farabi hospitals for workshop conduction . study population comprised all nurse working in psychiatric wards of two educational hospitals affiliated to isfahan university of medical sciences ( nour and farabi ) in 20122013 . a total of 64 nurses ( as employees , staffs on a contract and casual staffs ) participated in the study . inclusion criteria were at least having a bs degree in nursing and working in a psychiatric ward as an employee , on a contract or a casual staff as well as all nurses who worked part time or did overtime in the ward . data were collected by a researcher made questionnaire which contained three sections of employment and personal characteristics , knowledge ( including 17 multiple choice questions ) and investigation of attitude ( containing 14 likert 's scale question on about psychiatry drugs indication , their side - effects , nursing care and the management of drugs side - effects ) . employment and personal characteristics were recorded and knowledge and attitude were assessed by questionnaire . whose reliability and validity were confirmed before data collection . validity of the questionnaire was made by consideration of scientific references , and was confirmed by some academic members . the educational method employed in the present study was lecture , question and answer and group discussion . it should be noted that the researchers conducted the study with regard to the results obtained from the needs assessment of nurses working in psychiatric wards of hospitals affiliated to isfahan medical sciences university in 2012 . educational protocol and related workshops were designed through formation of a scientific committee ( including some of psychiatry professors and nursing educators of isfahan university of medical sciences as well as nursing managers and educational supervisors ) and discussion about obtained educational needs . the outlines of the educational workshop contained six sections : the generic name of drugs , recognition of dosage and forms , indication and diagnose side effects , electroconvulsive therapy ( ect ) indication and side effects , caring for drugs prescription and the management of drugs side - effects . nurses level of knowledge and attitude was investigated by the questionnaire before beginnings of the workshop , at its end and 3 months after holding the educational workshop . to have the highest possible number of attendants in the workshop , the workshop was held by academic members of the related university in two different times and in two various hospitals ( nour and farabi ) . at the end of workshop , the attendants were given workshop educational materials in form of a cd and the credit of continuing education course . repeated measure anova with control of some baseline and confounding factors as well as bonferroni test were used for data analysis . sixty - four nurses attended the present study of whom 29.7% were male and 70.3% were female . the highest frequency of employment condition was for casual staffs ( 87.5% ) and permanent staffs ( 12.5% ) . the results showed a significant increase in nurses knowledge mean scores in relation with medical and nonmedical immediately after and 3 months after education ( p < 0.001 ) so that it increased from 61.5 14.8 ( before education ) to 81.7 1.8 ( immediately after ) and to 79.9 13.8 ( 3 months after ) [ table 1 ] . mean scores of nurses knowledge and attitude before , immediately after and 3 months after interventions furthermore , there were a significant difference between mean scores of nurses attitude about medical and nonmedical interventions before , immediately after and 3 months after education ( p < 0.009 ) [ table 1 ] . based on table 2 , comparison of nurses knowledge mean scores of three stages ( before education and immediately after ) , ( before education and 3 months after ) and ( immediately after education and 3 months after ) , there were significant association between before education and immediately after it . also , there were significant relation between mean scores nurses knowledge before education and after 3 months . comparison of nurses knowledge concerning medical and nonmedical interventions nurses knowledge and attitude as adjusted variables , before education and immediately after ) , ( before education and 3 months after ) and ( immediately after education and 3 months after ) was evaluated by the bonferroni correction . based on table 3 , the mean of attitude scores showed a significant difference between immediately after education and 3 months after it . comparison of nurses attitude concerning medical and nonmedical interventions based on table 4 , the results showed a significant increase in nurses knowledge about dangerous side - effects of clozapine , side - effects of new antidepressant drugs and monoamine oxidase ( mao ) inhibitors , antidepressant drugs groups , extra pyramidal side effects , side - effects of selective serotonin reuptake inhibitors ( ssris ) and lithium and drug interactions with ect . distribution of nurses correct answers in response to medical and nonmedical interventions question before , immediately after and 3 months after intervention the obtained results showed that 86.3% of nurse were either satisfied with the educational course held for them and believed existence of such a course led to their scientific information improvement . this study aimed to investigate the efficiency of educational intervention on the levels of nurses knowledge and attitude in psychiatric wards about medicational and nonmedicational interventions . the obtained results showed the effect of a medicational and nonmedicational interventional educational workshop on improvement of knowledge level of nurses working in psychiatric wards immediately after and 3 months after education in 15 items . also , only one item ( recognition hypotension orthostatic ) , the knowledge was decreased however it was not statistically significant . these results is consistent with study of pasyar et al . on the effect of continuing education concerning medication indications on promotion of nurses knowledge and attitude . this also confirms the high effect of education on increase of the knowledge in treatment personnel , and consequently , on promotion of patients health and relatively improvement of services quality and nurses job satisfaction . this method seems to have a high effect on preservation of information until 3 months after intervention . in a study by abbaszadeh et al . in kerman , iran , suggests that continuing education program promote knowledge , attitude and performance of nurses on documentation . in a study conducted in northern italian university on effectiveness of a clinical skills workshop for drug - dosage calculation with participation of 77 nursing students , the results showed that clinical skills workshop increased drug - dosage calculation as well as students understanding of mathematical calculation , which is consistent with the present study . in evaluation of the effect of educational workshop on nurses knowledge concerning medicational and nonmedicational interventions , the results showed a significant increase in nurses knowledge about dangerous side effects of clozapine , side effects of new anti - depressant drugs and mao inhibitors , antidepressant drugs groups , extrapyramidal side effects , side effects of ssris and lithium and drug intractions with ect . the results of the present study seemed to have met most of the nurses needs revealed in the nurses needs assessment , conducted several months before holding the educational workshop by the researcher , including familiarization with drugs side effects , management of side effects , and necessary nursing cares in drugs consumption . research showed that nurses staffs education in hospitals about paying a close attention to medication on precise time leads to improvement of medication management and quality of patients care as well as reduction of side - effects , which is consistent with the present study . it seems that current continuing education programs held for nurses have not been adequately successful in increase of nurses knowledge and attitude due to lack of purposefulness of educations , lack of needs assessments and the type of education ( no workshop held ) . research showed that if educational programs are purposefully held based on nurses needs and in form of workshops , they can significantly affect nurses knowledge , attitude and skills , something we observed in the present study . another variable investigated in the present study was determination of nurses attitude for which the results showed the effect of educational intervention on the attitude of nurses working in psychiatric wards immediately after and 3 months after education . dadgari et al . showed that educational workshop leads to improvement of knowledge , attitude and practice of nurses in critical care units . mehdipur showed that educational classes result in an increase in nurses attitude and their more preparation to educate the patients , which is in line with the present study . several studies showed weakness of the nursing students and personnel working in hospitals about psychiatric medications , insufficient skills of correct medication due to brand name and labels similarities as well as wrong dosage calculations . on the other hand , other studies indicated the effect of educational programs on improvement of medicational care quality . therefore , nursing personnel education is going ahead with a rapid pace toward professionalism and employees empowerment parallel to coordination with other medical professions , and curriculum education and just attaining a university certificate can not be a license for work during an employee 's working life any more . in fact , education in medical groups does not end in university but continues during professional activities . brady and hyde believes nurses knowledge and preparation for continuing education is essential . in this direction , detection of educational needs and provision of facilities therefore , the role of continuing education should be considered as a basic need for nurses adaptation with rapid increasing scientific changes , and determination of educational priorities based on needs assessment as the main step in educational planning . nurses are frequently exposed to new advancements during their professional carrier and should update their knowledge about drugs specially the generic names of medications , dosage and medication types , and drugs side effects and contraindications . according to the results of the present study , a proper background for nurses attendance in educational programs based on needs assessment should be prepared for their professional promotion . our obtained results showed that holding an educational workshop was effective on nurses knowledge and attitude toward medical and nonmedical interventions . results of this study confirm that purposeful continuing education is essential for nurses to promote the quality of nursing cares . with regard to the role of nurses in medical interventions and the necessity of their being up - to - date with medical information as one of their necessary skills to reduce medication errors and the most important and life - threatening treatment complications , nurses should be provided with the required conditions for attendance in educational programs based on their professional needs . the researchers faced problems and limitations in conducting this study of which the most important ones were the limited population of participants , and short length of education period and follow - up . the researchers faced problems and limitations in conducting this study of which the most important ones were the limited population of participants , and short length of education period and follow - up . | background : the medical and nonmedical care of patients is the necessary skills in nursing profession . that it needs proper knowledge and attitude .
hence , it is important to promote nurses knowledge and attitude by education based on need assessment .
this study aimed to define the efficacy of a medical and nonmedical intervention educational workshop on nurses knowledge and attitude until 3 months after holding the workshop in psychiatric wards of educational hospitals in isfahan.materials and methods : this is a quasi - experimental study .
the study population comprised all nurses working in psychiatric wards of nour and farabi hospitals ( 64 ) in isfahan in 2012 .
an educational workshop was held through educational sessions in the form of lectures and group discussion in two above - mentioned hospitals .
nurses level of knowledge and attitude were investigated by a researcher made questionnaire before , immediately after and 3 months after intervention .
data were analyzed by descriptive statistical tests of repeated measure anova and bonferroni.results:a significant increase was observed in mean scores of nurses knowledge immediately after and 3 months after education compared to before education ( p < 0.001 ) .
also , the mean of attitude score in 3 phases have significant different ( p < 0.009 ) .
nurses have the high satisfaction ( 86.3% ) of need assessment based education workshop .
moreover , it is effective in the science information revival of nurses.conclusion:educational sessions notably affected the promotion of nurses knowledge and attitude . with regard to nurses satisfaction with the workshop which was held , designing and organizing educational workshops based on constant needs assessment is suggested for the promotion of nursing cares . |
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procedures and experiments described herein were approved by the centers for disease control and prevention ( cdc ) institutional animal care and use committee and conducted in strict accordance with the guide for the care and use of laboratory animals ( 10 ) . cdc is a fully accredited research facility of the association for assessment and accreditation of laboratory animal care international . healthy adult male and female strain 13/n guinea pigs , 1.02.5 years of age , were housed in a biosafety level 4 laboratory in microisolator cage systems filtered with high - efficiency particulate arrestance filters . groups of 5 animals , distributed proportionally by age and sex , were inoculated intraperitoneally with a 50% tissue culture infectious dose ( tcid50 ) at low ( 5 tcid50 ) or high ( 5,000 tcid50 ) levels of gp - ebov - mayinga , or with 5 10 tcid50 of either the wt - ebov - mayinga 1976 variant ( ebola virus / h . sapiens - tc / cod/1976/yambuku - mayinga ) or the wt - ebov - makona 2014 variant ( ebola virus / h . animals were monitored for signs of clinical illness , and body weight and temperature readings were obtained daily . oral swab samples were collected daily for isolation of rna and analyzed by qrt - pcr . postmortem oral swab samples were obtained from 10 animals that were euthanized because of severe clinical illness consistent with ebov . carcasses of the dead animals were kept in an incubator at 30c to simulate conditions in equatorial africa . samples were obtained from 9 of the 10 animals up to 5 days after death and from 1 animal at 2 days after death . in addition to oral swab samples , paired blood samples were collected from the cranial vena cava of anesthetized animals at 3 days postinfection ( dpi ) and by cardiac puncture at the time of death for euthanized animals . low and high doses of gp - ebov - mayinga were uniformly lethal . clinical illness was delayed in 1 animal in the high - dose group ; the animal was euthanized at 12 dpi , but all other animals were euthanized by 9 dpi . one animal infected with nonadapted wt - ebov - mayinga was euthanized at 9 dpi because of clinical illness . no severe clinical illness developed in any of the other animals infected with wt - ebov - mayinga or wt - ebov - makona ( figure , panel a ) . fever developed in all animals infected with low- and high - dose gp - ebov - mayinga , in 20% of animals infected with wt - ebov - makona or wt - ebov - mayinga , and in none of the negative control animals . hypothermia , typical during the terminal phases of many disease processes , was observed in animals with end - stage evd ( figure , panel b ) . substantial weight loss ( > 15% ) was observed in all febrile animals ( figure , panel c ) . the 1 animal infected with wt - ebov - makona that showed clinical sign experienced transient fever and weight loss but started to regain weight by 9 dpi clinical course of guinea pigs infected with guinea pig adapted ebola virus ( gp - ebov ) , wild - type ebov makona , and wild - type ebov mayinga , by number of days postinfection . oral swab samples were analyzed by qrt - pcr targeting the ebov nucleoprotein gene ; 18s ribosomal rna levels were also analyzed to serve as a sampling control . ebov rna abundance was calculated by comparing the cycle threshold values to an in vitro transcribed small - segment rna standard of known copy number . all oral swab samples that were collected 04 dpi were negative for ebov nucleoprotein rna ( table ) . at 3 dpi , blood samples from 7 ( 41% ) of 17 infected animals from which blood samples could be obtained were positive for ebov , but no viral rna was detected in any of the paired oral swab samples . the earliest detection of ebov rna by oral swabbing was at 5 dpi in an animal infected with wt - ebov - mayinga . at 6 dpi , coinciding with the time of overt clinical signs of disease ( i.e. , fever , weakness , anorexia , and ruffled fur ) , qrt - pcr of oral swab samples detected ebov rna in 8 ( 73% ) of 11 animals in which fatal illness developed and in 10 ( 50% ) of 20 infected animals . d , days postinfection ; dmem , dulbecco 's modified eagle 's medium ; ebov , ebola virus ; gp , guinea pig adapted ; i d , identification number ; neg , negative ; ns , not sampled ; qrt - pcr , quantitative reverse transcription pcr ; tcid50 , 50% tissue culture infectious dose ; wt , wild - type . ebov rna copies / ml : , negative ; + , 1010 ; + + , 10 ; + + + ; 10 ; + + + + , 10 ; + + + + + , 1010 . our data suggest that oral swab samples obtained early in the course of infection , before death , are not a reliable method for diagnosing infection with ebov . paired oral swab and blood samples collected at 3 dpi and at time of euthanasia showed that sensitivity of oral swab samples was low compared with the sensitivity of traditional blood samples . testing of oral swab samples did not indicate infection until 3 days after ebov rna was detectable in blood samples , with the exception of 1 animal in which oral swab samples revealed viral rna 2 days after the blood sample . at the time of overt clinical disease , the utility of oral swab samples for diagnostics improved but was not completely consistent with infection until postmortem analysis . our studies also enabled us to investigate whether the virulence of the wt - ebov - makona variant in guinea pigs was as low as that of the prototypic wt - ebov - mayinga variant . as shown in previous studies ( 3,5,6 ) , wt - ebov is less pathogenic than gp - ebov , regardless of variant , in this animal model . investigating the utility of oral swab samples for diagnosing evd in humans is challenging because paired blood and oral swab samples are rarely available and because the timing of sample collection relative to onset of disease and course of infection is often estimated . although evd in the nonhuman primate model mimics many aspects of the disease in humans , sampling from nonhuman primates in an experimental setting is problematic because of the species temperament , which requires anesthesia during specimen collection and venipuncture . the guinea pig model of evd ( 3,5,6 ) offers the convenience of daily oral swab sampling without the need for anesthesia . although suggestive , as with any animal model system , when extrapolating these data to human diagnostics , the effect of potential differences in oral milieus ( e.g. , saliva composition and oral cavity tissue structure ) must be considered . in the future , additional studies that use paired oral swab and blood samples from humans would provide information for continued discussion of antemortem swab sampling as a useful diagnostic modality of evd in humans . our data support the use of oral swab samples as a sensitive modality for postmortem diagnostics ; however , the utility of oral swab samples under field conditions , especially those collected before death , may decrease because of inherent problems with sampling techniques and specimen handling conditions ( i.e. , delays in transport and storage at typically high ambient temperatures ) . despite these considerations , oral swab sample collection could be a useful sampling strategy for humans and animals with unknown causes of death when evd is suspected and when other types of samples are more prohibitive to obtain . | to determine the utility of oral swabs for diagnosing infection with ebola virus , we used a guinea pig model and obtained daily antemortem and postmortem swab samples . according to quantitative reverse transcription pcr analysis ,
the diagnostic value was poor for antemortem swab samples but excellent for postmortem samples . |
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guillain - barre syndrome ( gbs ) is one of the acute flaccid paralysis syndromes . different subtypes producing the clinical picture of gbs have been described including acute inflammatory demyelinating polyradiculoneuropathy ( aidp ) , acute motor axonal neuropathy ( aman ) , acute motor and sensory axonal neuropathy ( amsan ) , acute sensory neuropathy , acute pandysautonomia and the fisher syndrome . presentations with central features such as depressed level of consciousness , and hyper - reflexia were reported in bickerstaff brainstem encephalitis ( bbe ) . in bickerstaff ophthalmoplegia and ataxia were common among patients diagnosed with bickerstaff encephalitis and fisher syndrome ( fs ) . it was always thought that both bbe and fs were distinct disorders from gbs , but considered as a spectrum disorder with an autoimmune mechanism targeting primarily the central nervous system . the discovery of anti - gq1b antibody was a turning point in explaining the fact that certain patients share similar clinical features suggestive of gbs , bbe or fs . the gq1b antigen is highly expressed in the paranodes and the neuromuscular junctions of the oculomotor , trochlear and abducens nerves , muscle spindles in the limbs , and probably reticular formation in the brainstem . the neurological effects of anti - gq1b antibodies are induced by complement - mediated destruction of both perisynaptic schwann cells ( by inhibiting ca influx ) and axonal terminals resulting in neuromuscular junction blockade and ultimately muscle weakness . herein , we report a case of atypical anti - gq1b syndrome in a patient presented with both central and peripheral nervous system involvements . a fifteen years - old right - handed boy presented to the emergency department with progressive weakness of upper and lower extremities for 3 days . he was unable to walk and was having difficulty elevating his arms above the shoulder . although he did not describe any bulbar symptoms , he was complaining of significant binocular diplopia . in addition , he described abdominal pain associated with urinary retention , and a foley s catheter was inserted draining over 500 ml of urine . there was no associated history of headache , fever , recent immunization or travel , or any changes in level of consciousness . on initial exam , he was hemodynamically stable with temperature 36.7c and o2 saturation of 98% on room air . cranial nerve examination revealed normal pupillary reaction , upward and gaze - evoked horizontal nystagmus . motor exam assessment according to medical research council ( mrc ) scale revealed flaccid tone with significant symmetrical proximal more than distal weakness as follows : deltoid 3/5 , biceps 2/5 , triceps 3/5 , wrist and finger extensors 3/5 , wrist and fingers flexors 2/5 , hip flexors 1/5 , knee flexors 2/5 , knee extensors 3/5 , dorsiflexors 4/5 , planterflexors 4-/5 . deep tendon reflexes were reduced ( 1 + ) in triceps and biceps and absent in brachioradialis , knees , and ankles . cerebellar and gait exam were deferred due to his significant weakness . his basic laboratory investigation including hematological , liver and renal function testes were within normal limits . erythrocyte sedimentation rate was elevated at 32 . magnetic resonance imaging ( mri ) of brain / spine with gadolinium was unremarkable . cerebral spine fluid ( csf ) examination revealed 20 white cells ( 100% lymphocytic ) while protein and glucose were within normal limits . the gramstain was unremarkable . given that he had a prodromal illness along with the presentation of symmetrical proximal more than distal weakness with areflexia , gbs was suspected . however , given the presence of brainstem features ( nystagmus ) , pyramidal signs ( extensor plantars ) along with acute urinary retention , other diagnoses such as bickerstaff encephalitis and acute disseminated encephalomyelitis ( adem)-like syndrome were considered despite the preservation of consciousness and normal mri . forced vital capacity was 2.2 l on second day of admission and remained between 2.0 - 2.6 l in the first week . an improvement in the power of upper limbs and neck flexor weakness were evident on day 3 ( deltoid 4/5 , biceps 3/5 , triceps 4/5 , wrist and finger extensors 4/5 , wrist and finger flexors 3/5 ) . his lower limb power was essentially the same apart from improvement of knee extensors 4+/5 , knee flexors 3/5 . nerve conduction study was performed on day 4 of admission , and revealed normal sensory nerve action potentials ( snaps ) but absent compound muscle action potentials ( cpams ) in the lower limbs and reduced cmaps in the upper extremities without conduction block . on day 4 , he developed bilateral lateral abduction paresis and mild facial weakness indicative of the involvement of both cn vi and vii . the decision was made to start iv immunoglobulin 2 g / kg course over 3 days given his clinical progression . he continued to have supportive measures by means of physical therapy , dvt prophylaxis along with urological follow - up in order to train his bladder muscles . a follow - up mri brain and spine , which was performed on day 10 , was unremarkable . on day 14 , he started to regain the function of upper extremities and was able to lift his arms above his shoulders with good support of neck muscles . on day 30 , the power of hip flexors was 3/5 and he was able to stand with 2-person assist . on day 42 , he started to walk unassisted but needed support in rising from a chair . on day 50 , the urinary catheter was removed after bladder training and he was able to urinate without any difficulty . on day 62 , his neurological examination revealed mild limitation of both lateral recti with no evidence of nystagmus . in terms of power testing , his mrc grade for muscles were as follows : deltoid 4 + , biceps 5 , triceps 5 , intrinsic hand muscles 4 + , hip flexor 4- , knee flexors 4 + , knee extensor 4 + , dorsiflexors 5 , planterflexors 5 . he was referred to an outpatient rehabilitation center . at one - year follow - up , our case illustrated atypical presentations and findings , which pointed toward a central involvement since gaze - evoked horizontal nystagmus is usually localized to the cerebellum while upbeat nystagmus is commonly associated with lesions in the rostral pons . in addition , the presence of extensor plantars was indicative of corticospinal tract involvement , which in association with acute urinary retention , was pointing towards spinal cord localization . on the other hand , certain clues in the examination ( distribution of weakness being proximal more than distal , neck flexor weakness and areflexia ) were indicative of polyradiculitis , raising the suspicion of a gbs - variant . although few cases were reported in the literature with central features in gbs patients , it was proposed that corticospinal tract might be functionally involved in patients with anti - ganglioside antibody associated syndromes . kariyama et al . described a patient who had asymmetrical weakness , which was confused for a stroke initially but later was diagnosed with gbs . the central weakness was confirmed by mean of abnormal central motor conduction time ( cmct ) on a motor evoked potential study . the autonomic dysfunction manifested by the acute urinary retention in our patient added to the dilemma of the co - existence of both upper and lower motor signs . in gbs , bladder or bowl disturbance at presentation watson et al . studied 26 children with gbs , of whom 7 had bladder or bowl disturbance at presentation . one patient presented with , and three subsequently developed urinary retention necessitating catheterization for a median of 7.5 days . dysautonomia is usually associated with aidp and it is rare in other types such as aman , and the nerve conduction study in our patient revealed features supportive of the latter . on the other hand , it is not unusual to have urinary retention with areflexia in a spinal shock stage resulting from an acute myelitis . in our case , the persistent urinary retention makes it different from gbs where urinary retention is a transient occurrence . the presence of anti - gq1b antibody in our patient reflects a unique clinical spectrum , which represents an existing continuum between several conditions ( fisher , bickerstaff and guillain - barre syndromes ) presenting with variable central and peripheral nervous system involvements . fisher syndrome presents when the binding of anti - gq1b antibodies to gq1b antigens expressed on the relevant cranial nerves and muscle spindles whereas bickerstaff brainstem encephalitis is induced when the anti - gq1b antibodies enter the brainstem and bind to gq1b antigens . a more inclusive nomenclature such as anti - gq1b antibody syndrome could be used to include the common serological profile when referring to the clinical syndromes described by both bickerstaff and fisher . however , nystagmus ( observed in our patient ) was never reported in the literature to be associated with anti - gb1b antibody syndrome despite the occasional presence of ophthalmoplegia and other brainstem features odaka et al . evaluated 194 anti - gq1b seropositive patients , among whom 110 had fs , 31 had an overlap of fs with gbs , 12 had bbe , and 11 had an overlap of bbe with gbs . in another study of 62 patients with bbe , the authors described positive anti - gq1b antibody in 66% of patients and approximately 60% of the overall cohort associated flaccid symmetrical tetraparesis . maier et al . studied 13 autopsy cases of gbs patients and they revealed evidence of pathological mononuclear cell infiltrates composed of macrophages and t - lymphocytes within the spinal cord in 8 out of 13 cases , within the medulla oblongata in 8 out of 12 cases , within the pons in 5 out of nine cases , and in one out of four midbrains . the authors did not find any evidence of primary demyelination in the cns but rather axonal with secondary myelin impairment , microglial activation and inflammatory infiltration . given the challenging diagnostic nature of our case , and the predominant central features , we elected to treat the patient with a combination of iv methylprednisone ( ivmp ) and iv immunoglobulin ( ivig ) . the rationale was based on the faster effect of ivmp in view of myelitis - like picture , which tends to respond to high dose parental steroids . on the hand , given that the pathogenesis of anti - gq1b antibody syndrome is similar to gbs , ivig was subsequently added as it has been shown to be efficacious in improving outcomes in gbs . although there have been reports of clinical improvement with steroid administration , its role in the anti - gq1b antibody syndrome is less clear . the presence of nystagmus along with other central features may pose diagnostic dilemma in patients suspected to have gbs variants . our case highlights the importance of broad - based approach in dealing with atypical presentations involving both peripheral and central nervous system where the clinical spectrum of anti - gq1b syndrome might be considered . | variants of guillain - barre syndrome such as bickerstaff encephalitis and miller - fisher syndrome have been reported .
we report a 15-year - old boy who presented , after a prodromal illness , with 3-day progressive limb weakness , diplopia , and acute urinary retention .
clinically , he had horizontal gaze - evoked and upbeat nystagmus , bilateral extensor plantars in addition to quadriparesis and areflexia .
magnetic resonance imaging of the brain and spine was unremarkable and cerebral spinal fluid analysis showed lymphocytic pleocytosis .
nerve conduction study revealed symmetrical axonal neuropathy .
anti - gq1b antibody was positive .
a combination of iv methylprednisone followed by ivig was instituted which led to remarkable clinical recovery . this case underpins the importance of recognizing atypical presentations of acute autonomic dysfunction and central nervous system features such as nystagmus , which may be associated with anti - gq1b antibody syndrome
. features mimicking myelitis and brainstem encephalitis may pose diagnostic and therapeutic dilemma among the treating physicians . |
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population structure was assessed by examining the principal components ( pc ) of the variance of the genotype profiles , using eigenstrat software6 . initial examination revealed several clusters of siblings and other close relatives ( cousins or similar ) whose similarity skewed the axes ; where data were available , these identities were in agreement with participant records . after removal of these relatives , analysis of 163 unrelated individuals revealed seven significant eigenvectors . none of these explain more than 5% of the variance , and pc3 through pc7 are heavily weighted by large clusters of snps on one or a few chromosomes . as described by others , such axes are commonly observed and do not provide reliable genome - wide estimates of population structure7,8 , but it is interesting to note that pc3 distinguishes ighrem from the other locations ( supplementary fig . pc1 separates just a dozen individuals , and we inferred that this axis represents a sub - saharan african contribution , consistent with expected levels of admixture in morocco , by performing an analysis including 21 yoruban individuals ( supplementary fig . pc2 is highly correlated with both location and self - reported ethnicity , so is inferred to capture the major component of arab - amazigh ancestry . a surprising aspect of this analysis is the positioning of ighrem arabs between boutroch amazigh and half of the agadir arabs along pc2 . this was confirmed by structure analysis9 of 16,000 randomly chosen autosomal snps assuming admixture of two ancestral populations ( fig . 2b ) , which indicates that ighrem residents tend to be a mixture , while most amazigh are derived from one population , and only a handful of agadir arabs represent the other . there has thus likely been considerable admixture between these two groups over an extended period of time , possibly with movement of arabs from other locations into agadir recently . a slight shift of ighrem arabs toward the amazigh pole of pc2 , relative to agadir arabs , would also be consistent with some genetic exchange between the villages over 50 generations . further sampling of villages in the region may reveal subtle population structure across southern morocco1013 . next we asked whether region , location and ethnicity impact gene expression profiles , and if they do so in a gender - specific manner . since location and ethnicity are confounded in the villages , several parallel analyses were undertaken to tease apart these influences . transcript abundance data was transformed by median centering on the log base 2 scale ( supplementary fig . 2 online ) , which results in maximal overlap of profiles without altering their variance . gene specific analysis of variance14 with expression as a function of region , gender , and their interaction discovered 1,521 probes significant at a 1% false discovery rate ( fdr ; p < 0.0007 ) . region , namely the rural ( boutroch plus ighrem ) versus city ( anza plus dchiera ) comparison is by far the main effect in this joint analysis . approaching 7% of all expressed genes differentiate these individuals by this conservative criterion , whereas considerably fewer than 1% of the probes show gender differences . a full list of genes is provided in supplementary table 1 online . among several classes of over - represented genes for this lifestyle comparison , small nucleolar rna genes stand out : 5 of the top 8 overall and 15 of 29 members of the snord family are in the highly significant list , compared with just 1 of 10 snora genes . there is little in the literature to indicate why this is the case , or what the physiological consequences may be , but it is interesting to note that epigenetic modification has been observed for many small nucleolar rna genes15 . even more differentiation was observed when we fit analysis of variance models including location , gender , and their interaction . since exploratory analyses indicated that the anza and dchiera samples are indistinguishable either for gene expression or genotypes , these were combined into a single location , agadir , in all subsequent analyses . in the three - way comparison , 8,459 probes ( 38% ) were significant at the 1% fdr threshold for location ( supplementary table 2 online ) . boutroch differs from both ighrem and agadir at over seven thousand probes each , with a high degree of overlap ( fig . ighrem and agadir are much more similar to one another , in part because there is considerably more diversity within the ighrem sample that reduces the significance of the location contrast . we also noted that women are much more differentiated among locations than men ( table 1 ) . these results confirm our previous report2 of substantial differentiation between bedouin nomads , urban anza , and another remote amazigh village , sebt nabor . in order to evaluate the possible independent contribution of ethnicity more carefully , variance component analysis of the expression variation was performed . within agadir alone , neither ethnicity ( modeled as the second eigenvector of the genotype data , gpc2 ) nor gender have a noteworthy impact on the principal components of the expression variation , as shown by the bar charts in figure 3b . however , in the total dataset there is some evidence for a contribution : figure 3c shows that when fit jointly with location , the ethnicity , ethnicity - and gender - by - location interaction terms make a substantial contribution to the expression profiles . although gender and ethnicity affect the expression of fewer genes than location , the plot of expression pc1 by pc2 for the most differentially expressed 1,500 genes in figure 4 indicates that for many genes the interaction between these three factors is quite complex this can also be seen in the expression profiles of characteristic individual genes ( supplementary fig . boutroch and ighrem villagers in general separate along pc1 , while high values of pc2 are obtained for all boutroch residents ( cluster 1 ) and for arab women in ighrem ( cluster 2 ) . amazigh women from ighrem ( cluster 3 ) and the ighrem men ( cluster 4 ) have lower values of pc2 similar to those observed for all agadir residents . the simplest interpretation is that cultural or behavioral differences , likely including time spent outside the village , contribute strongly to the observed gender and ethnicity effects . deeper sampling would be required to firmly establish whether intrinsic biological differences between the sexes and/or populations also make significant contributions to expression divergence in lymphocytes , as they appear to do for lymphoblast cell lines grown in culture1619 . these are ribosomal proteins of both the small and large subunits as well as the cytoplasmic and mitochondrial compartments , and proteins involved in oxidative phosphorylation , which are highly up - regulated in half of the agadir residents ( supplementary fig . all of the transcripts encoding these proteins form a module of co - regulated genes , but as shown in supplementary figure 4b online , it is noteworthy that this module is not co - expressed with the snords , which tend to be relatively down - regulated in agadir but are particularly high in the arab women from ighrem . regulation of ribosomal biosynthesis may be related to response to viral infection , and it also seems to be involved in tumorigenesis in conjunction with mitochondrial activity20,21 . oxidative phosphorylation is correlated with renal health and the production or disposal of free radicals22 , so our data suggests that deeper evaluation of health risks associated with lifestyle transitions may be revealing . the genetic contribution to expression variation was evaluated by genome - wide association with expression of all 22,300 probes . starting with a simple test of the correlation between each transcript abundance and each genotype , and filtering to retain only esnps with a minor allele frequency greater than 0.05 further filtering of esnps to retain only autosomal associations with annotated genes , and imposing the additional stringency of p < 10 for putative trans associations between an esnp on one chromosome and a probe on another chromosome , reduced this to 1,636 associations . 1,569 ( 96% ) of these are intra - chromosomal linkages , the vast majority within 50 kb and hence cis - acting(supplementary fig . 5 online ) , and only 3 clearly in different chromosomal intervals . facsimile associations were observed for 39 of the target genes represented by a second probe ( 37 cis , 2 trans ) . reducing the dataset further to exclude linked associations within haplotype blocks leaves 346unique cis and 10 unique trans associations at the stringent genome - wide 5% significance level . these proportions are in good agreement with most other gwas expression studies on blood or lymphocyte cell lines16,17,2326 , and a 30-fold or greater excess of cis over trans associations is also supported by 1% fdr estimates of 600 and 20 genes respectively . complete lists of peak cis and trans associations are provided in supplementary table 3 online . given the high degree of population structure for gene expression , we addressed the possibility that differentiation of esnp allele frequencies may contribute to the observed associations by calculating fst estimates for each pair - wise comparison of location for the 516,972 snps and 16,500 of the genes . no fixed differences were observed and plots of the fst comparisons ( supplementary fig . 6a online ) indicate only moderate overall genetic differentiation , with occasional snps having fst values between 0.12 and 0.3 . there was no tendency for these outliers to have elevated expression differentiation and in fact almost all of the top 10% most differentially expressed genes are among the least genetically differentiated . nor was there any correlation between fst and significance of gene expression divergence ( supplementary fig . 6b online ) , confirming that the observed expression differences between locations are for the most part not attributable to gene - specific allelic frequency differences between locations . the robustness of the 3,430 associations to environmental sources of variance and population structure was further evaluated by fitting two additional linear trend models to the data . the second included two measures of ethnicity ( the first three genotype eigenvectors and a four - way categorical ethnicity cluster : see methods ) , a matrix of relatedness based on an identity by descent measure27 , as well as gender interactions with ethnicity cluster and genotype . figures 5a and 5b show the manhattan plot of associations by chromosomal location for the second of these models , and the cis - trans plot of target against esnp location , respectively . online show that the logarithm of the genotype significance term is highly correlated ( r > 0.95 ) between both of these models and the original correlation test . furthermore , figure 5d shows that there is no evidence for significant genotype - by - location interactions in any of the association trend tests . neither the ethnicity nor the relatedness variance components explain an appreciable amount of the expression variation for any of the transcripts ( supplementary fig . 8 online ) . the absence of interaction effects is readily visualized by plotting expression as a function of genotype with color coding of each location , for each association . an example of a trans association in supplementary figure 9 online shows the clear trend of increased expression of amy1a ( chromosome 1 ) in homozygotes for the a allele of actg1 gamma actin ( chromosome 17 ) , consistently across the three locations despite slight overall location effects . expression of amy1a is highly correlated with that of amy1b ( r > 0.8 ) as well as of dozens of other genes in a co - expression module , but the esnp only regulates amy1a , because it increases expression of the gene two - fold in an additive manner . a similar plot for a representative gene that shows highly significant location and genotype effects in cis , c21orf57 , is provided in figure 6a and further discussed below , and further examples can be seen in supplementary figure 9c online . gwas - expression associations detected in one tissue can identify regulatory variants that may be active in other tissues that are directly engaged in the etiology of disease23,25,26 . one example is the cis - linkages in peripheral blood with the t1d susceptibility locus at chromosome 12q13 . the strongest expression association is with transcription of the rps26 ribosomal protein gene , and network analyses have been employed to argue that this is the more likely diabetes candidate gene than the initially reported erbb328 . however , the strongest t1d association involves a different snp than that associated with expression and/or splicing24 of rps26 . we further find that the same linkage group of esnps , centered on the rs10876864 in the suox gene 35 kb from rps26 , is also associated in trans with half a dozen other rp26 paralogs ( probably due to cross - hybridization ) , and with ccdc4 on chromosome 4 , albeit at the suggestive significance level ofp = 3.510 . intriguingly , expression of rps26 is only weakly correlated with that of the module of ribosomal proteins that differentiate locations ( supplementary fig . 4b online ) , so this association does not contribute to the environmental effect on transcription of ribosomal protein genes . another trans - association of interest involves rs11987927 in myom2 at 8p23 with znf71 at 19q13 , but also with its own myom2 transcript . logic suggests that the cis - association likely affects the abundance of the myom2 myomesin protein , which in turn regulates znf71 , but the trans association is actually significantly stronger and conditional dependence analysis29,30 points in the opposite direction , namely that the myom2 regulatory site influences znf71 , which then feeds back on the myom2 transcript ( supplementary fig . it is worth mentioning that four of the seven strongest trans associations involve regulation by loci that include genes that encode structural proteins , the others being the lama5 laminin ( 20q13 ) with osbpl2 , and the plekhm1 plekstrin homology domain protein ( 17q21 ) with mapk8ip1 . we found association of two probes that detect both hbg1 and hbg2 transcripts from 11p15 with rs766432 in the second intron of the bcl11a zinc finger proto - oncogene at 2p16 . this same snp has previously been associated with the fraction of erythrocytes that contain measurable fetal hemoglobin31 , and alteration of bcl11a activity was recently shown drive differences in globin switching between mice and humans32 . another snp in bcl11a , rs4671393 has been associated with abundance of two bcl11a transcript isoforms in ceu and yri hapmap lymphoblast cell lines33 , but is not associated with bcl11a transcript abundance in our leukocyte data , suggesting that regulation of bcl11a translation or protein activity is more likely to be affecting hbg expression in our sample . we scanned the gwas association database for overlap between our study and established disease associations at p<10 . of 1,628 entries , 10 involve cis associations observed in our dataset that explain between 15 and 55% of the transcript variance ( supplementary table 4 online ) . five of the associations are with disease conditions ( rheumatoid arthritis , celiac disease , t1d , ulcerative colitis , and sle ) and five are with endophenotypes ( pafah1b2 and icam-1 protein levels , triglycerides , ldl cholesterol , and hip bone mineral density ) . the two serum protein associations34,35 are with the same snps as we detect and hence suggest that protein abundance is largely regulated at the transcriptional level . population structure was assessed by examining the principal components ( pc ) of the variance of the genotype profiles , using eigenstrat software6 . initial examination revealed several clusters of siblings and other close relatives ( cousins or similar ) whose similarity skewed the axes ; where data were available , these identities were in agreement with participant records . after removal of these relatives , analysis of 163 unrelated individuals revealed seven significant eigenvectors . none of these explain more than 5% of the variance , and pc3 through pc7 are heavily weighted by large clusters of snps on one or a few chromosomes . as described by others , such axes are commonly observed and do not provide reliable genome - wide estimates of population structure7,8 , but it is interesting to note that pc3 distinguishes ighrem from the other locations ( supplementary fig . pc1 separates just a dozen individuals , and we inferred that this axis represents a sub - saharan african contribution , consistent with expected levels of admixture in morocco , by performing an analysis including 21 yoruban individuals ( supplementary fig . pc2 is highly correlated with both location and self - reported ethnicity , so is inferred to capture the major component of arab - amazigh ancestry . a surprising aspect of this analysis is the positioning of ighrem arabs between boutroch amazigh and half of the agadir arabs along pc2 . this was confirmed by structure analysis9 of 16,000 randomly chosen autosomal snps assuming admixture of two ancestral populations ( fig . 2b ) , which indicates that ighrem residents tend to be a mixture , while most amazigh are derived from one population , and only a handful of agadir arabs represent the other . there has thus likely been considerable admixture between these two groups over an extended period of time , possibly with movement of arabs from other locations into agadir recently . a slight shift of ighrem arabs toward the amazigh pole of pc2 , relative to agadir arabs , would also be consistent with some genetic exchange between the villages over 50 generations . further sampling of villages in the region may reveal subtle population structure across southern morocco1013 . next we asked whether region , location and ethnicity impact gene expression profiles , and if they do so in a gender - specific manner . since location and ethnicity are confounded in the villages , several parallel analyses were undertaken to tease apart these influences . transcript abundance data was transformed by median centering on the log base 2 scale ( supplementary fig . 2 online ) , which results in maximal overlap of profiles without altering their variance . gene specific analysis of variance14 with expression as a function of region , gender , and their interaction discovered 1,521 probes significant at a 1% false discovery rate ( fdr ; p < 0.0007 ) . region , namely the rural ( boutroch plus ighrem ) versus city ( anza plus dchiera ) comparison is by far the main effect in this joint analysis . approaching 7% of all expressed genes differentiate these individuals by this conservative criterion , whereas considerably fewer than 1% of the probes show gender differences . a full list of genes is provided in supplementary table 1 online . among several classes of over - represented genes for this lifestyle comparison , small nucleolar rna genes stand out : 5 of the top 8 overall and 15 of 29 members of the snord family are in the highly significant list , compared with just 1 of 10 snora genes . there is little in the literature to indicate why this is the case , or what the physiological consequences may be , but it is interesting to note that epigenetic modification has been observed for many small nucleolar rna genes15 . even more differentiation was observed when we fit analysis of variance models including location , gender , and their interaction . since exploratory analyses indicated that the anza and dchiera samples are indistinguishable either for gene expression or genotypes , these were combined into a single location , agadir , in all subsequent analyses . in the three - way comparison , 8,459 probes ( 38% ) were significant at the 1% fdr threshold for location ( supplementary table 2 online ) . boutroch differs from both ighrem and agadir at over seven thousand probes each , with a high degree of overlap ( fig . ighrem and agadir are much more similar to one another , in part because there is considerably more diversity within the ighrem sample that reduces the significance of the location contrast . we also noted that women are much more differentiated among locations than men ( table 1 ) . these results confirm our previous report2 of substantial differentiation between bedouin nomads , urban anza , and another remote amazigh village , sebt nabor . in order to evaluate the possible independent contribution of ethnicity more carefully , variance component analysis of the expression variation was performed . within agadir alone , neither ethnicity ( modeled as the second eigenvector of the genotype data , gpc2 ) nor gender have a noteworthy impact on the principal components of the expression variation , as shown by the bar charts in figure 3b . however , in the total dataset there is some evidence for a contribution : figure 3c shows that when fit jointly with location , the ethnicity , ethnicity - and gender - by - location interaction terms make a substantial contribution to the expression profiles . although gender and ethnicity affect the expression of fewer genes than location , the plot of expression pc1 by pc2 for the most differentially expressed 1,500 genes in figure 4 indicates that for many genes the interaction between these three factors is quite complex . this can also be seen in the expression profiles of characteristic individual genes ( supplementary fig . boutroch and ighrem villagers in general separate along pc1 , while high values of pc2 are obtained for all boutroch residents ( cluster 1 ) and for arab women in ighrem ( cluster 2 ) . amazigh women from ighrem ( cluster 3 ) and the ighrem men ( cluster 4 ) have lower values of pc2 similar to those observed for all agadir residents . the simplest interpretation is that cultural or behavioral differences , likely including time spent outside the village , contribute strongly to the observed gender and ethnicity effects . deeper sampling would be required to firmly establish whether intrinsic biological differences between the sexes and/or populations also make significant contributions to expression divergence in lymphocytes , as they appear to do for lymphoblast cell lines grown in culture1619 . these are ribosomal proteins of both the small and large subunits as well as the cytoplasmic and mitochondrial compartments , and proteins involved in oxidative phosphorylation , which are highly up - regulated in half of the agadir residents ( supplementary fig . all of the transcripts encoding these proteins form a module of co - regulated genes , but as shown in supplementary figure 4b online , it is noteworthy that this module is not co - expressed with the snords , which tend to be relatively down - regulated in agadir but are particularly high in the arab women from ighrem . regulation of ribosomal biosynthesis may be related to response to viral infection , and it also seems to be involved in tumorigenesis in conjunction with mitochondrial activity20,21 . oxidative phosphorylation is correlated with renal health and the production or disposal of free radicals22 , so our data suggests that deeper evaluation of health risks associated with lifestyle transitions may be revealing . the genetic contribution to expression variation was evaluated by genome - wide association with expression of all 22,300 probes . starting with a simple test of the correlation between each transcript abundance and each genotype , and filtering to retain only esnps with a minor allele frequency greater than 0.05 further filtering of esnps to retain only autosomal associations with annotated genes , and imposing the additional stringency of p < 10 for putative trans associations between an esnp on one chromosome and a probe on another chromosome , reduced this to 1,636 associations . 1,569 ( 96% ) of these are intra - chromosomal linkages , the vast majority within 50 kb and hence cis - acting(supplementary fig . 5 online ) , and only 3 clearly in different chromosomal intervals . facsimile associations were observed for 39 of the target genes represented by a second probe ( 37 cis , 2 trans ) . reducing the dataset further to exclude linked associations within haplotype blocks leaves 346unique cis and 10 unique trans associations at the stringent genome - wide 5% significance level . these proportions are in good agreement with most other gwas expression studies on blood or lymphocyte cell lines16,17,2326 , and a 30-fold or greater excess of cis over trans associations is also supported by 1% fdr estimates of 600 and 20 genes respectively . complete lists of peak cis and trans associations are provided in supplementary table 3 online . given the high degree of population structure for gene expression , we addressed the possibility that differentiation of esnp allele frequencies may contribute to the observed associations by calculating fst estimates for each pair - wise comparison of location for the 516,972 snps and 16,500 of the genes . no fixed differences were observed and plots of the fst comparisons ( supplementary fig . 6a online ) indicate only moderate overall genetic differentiation , with occasional snps having fst values between 0.12 and 0.3 . there was no tendency for these outliers to have elevated expression differentiation and in fact almost all of the top 10% most differentially expressed genes are among the least genetically differentiated . nor was there any correlation between fst and significance of gene expression divergence ( supplementary fig . 6b online ) , confirming that the observed expression differences between locations are for the most part not attributable to gene - specific allelic frequency differences between locations . the robustness of the 3,430 associations to environmental sources of variance and population structure was further evaluated by fitting two additional linear trend models to the data . the first included location , gender and the interaction between them . the second included two measures of ethnicity ( the first three genotype eigenvectors and a four - way categorical ethnicity cluster : see methods ) , a matrix of relatedness based on an identity by descent measure27 , as well as gender interactions with ethnicity cluster and genotype . figures 5a and 5b show the manhattan plot of associations by chromosomal location for the second of these models , and the cis - trans plot of target against esnp location , respectively . 7 online show that the logarithm of the genotype significance term is highly correlated ( r > 0.95 ) between both of these models and the original correlation test . furthermore , figure 5d shows that there is no evidence for significant genotype - by - location interactions in any of the association trend tests . neither the ethnicity nor the relatedness variance components explain an appreciable amount of the expression variation for any of the transcripts ( supplementary fig . 8 online ) . the absence of interaction effects is readily visualized by plotting expression as a function of genotype with color coding of each location , for each association . an example of a trans association in supplementary figure 9 online shows the clear trend of increased expression of amy1a ( chromosome 1 ) in homozygotes for the a allele of actg1 gamma actin ( chromosome 17 ) , consistently across the three locations despite slight overall location effects . expression of amy1a is highly correlated with that of amy1b ( r > 0.8 ) as well as of dozens of other genes in a co - expression module , but the esnp only regulates amy1a , because it increases expression of the gene two - fold in an additive manner . a similar plot for a representative gene that shows highly significant location and genotype effects in cis , c21orf57 , is provided in figure 6a and further discussed below , and further examples can be seen in supplementary figure 9c online . gwas - expression associations detected in one tissue can identify regulatory variants that may be active in other tissues that are directly engaged in the etiology of disease23,25,26 . one example is the cis - linkages in peripheral blood with the t1d susceptibility locus at chromosome 12q13 . the strongest expression association is with transcription of the rps26 ribosomal protein gene , and network analyses have been employed to argue that this is the more likely diabetes candidate gene than the initially reported erbb328 . however , the strongest t1d association involves a different snp than that associated with expression and/or splicing24 of rps26 . we further find that the same linkage group of esnps , centered on the rs10876864 in the suox gene 35 kb from rps26 , is also associated in trans with half a dozen other rp26 paralogs ( probably due to cross - hybridization ) , and with ccdc4 on chromosome 4 , albeit at the suggestive significance level ofp = 3.510 . intriguingly , expression of rps26 is only weakly correlated with that of the module of ribosomal proteins that differentiate locations ( supplementary fig . 4b online ) , so this association does not contribute to the environmental effect on transcription of ribosomal protein genes . another trans - association of interest involves rs11987927 in myom2 at 8p23 with znf71 at 19q13 , but also with its own myom2 transcript . logic suggests that the cis - association likely affects the abundance of the myom2 myomesin protein , which in turn regulates znf71 , but the trans association is actually significantly stronger and conditional dependence analysis29,30 points in the opposite direction , namely that the myom2 regulatory site influences znf71 , which then feeds back on the myom2 transcript ( supplementary fig . it is worth mentioning that four of the seven strongest trans associations involve regulation by loci that include genes that encode structural proteins , the others being the lama5 laminin ( 20q13 ) with osbpl2 , and the plekhm1 plekstrin homology domain protein ( 17q21 ) with mapk8ip1 . we found association of two probes that detect both hbg1 and hbg2 transcripts from 11p15 with rs766432 in the second intron of the bcl11a zinc finger proto - oncogene at 2p16 . this same snp has previously been associated with the fraction of erythrocytes that contain measurable fetal hemoglobin31 , and alteration of bcl11a activity was recently shown drive differences in globin switching between mice and humans32 . another snp in bcl11a , rs4671393 has been associated with abundance of two bcl11a transcript isoforms in ceu and yri hapmap lymphoblast cell lines33 , but is not associated with bcl11a transcript abundance in our leukocyte data , suggesting that regulation of bcl11a translation or protein activity is more likely to be affecting hbg expression in our sample . we scanned the gwas association database for overlap between our study and established disease associations at p<10 . of 1,628 entries , 10 involve cis associations observed in our dataset that explain between 15 and 55% of the transcript variance ( supplementary table 4 online ) . five of the associations are with disease conditions ( rheumatoid arthritis , celiac disease , t1d , ulcerative colitis , and sle ) and five are with endophenotypes ( pafah1b2 and icam-1 protein levels , triglycerides , ldl cholesterol , and hip bone mineral density ) . the two serum protein associations34,35 are with the same snps as we detect and hence suggest that protein abundance is largely regulated at the transcriptional level . our geographical genomic survey of gene expression variation in southern morocco has highlighted two parallel and for the most part non - overlapping insights . on the one hand , it is evident that as much as half of the transcriptome is influenced by the environment in a highly coordinated manner such that where a person lives explains up to a quarter of the variation for a substantial fraction of the transcripts . the environmental influences are likely a combination of biotic and abiotic factors , as well as cultural and behavioral ones , while genetic differences between the two north african ethnicities are relatively minor . on the other hand , the genome is littered with strong genetic associations , mainly in cis , that explain between 15 and 60 percent of the variance of 5% of the transcripts . impressive as these associations are , particularly since they are discovered in a sample of just under 200 individuals , they have essentially no bearing on the vast majority of the transcriptional variation , and are not informative of the genetic basis of the environmental response . the robustness of the observed associations to the environmental effect raises the issue of whether genotype - by - environment interactions influence the peripheral blood transcriptome at all . genome - wide significant interaction effects are generally unlikely to occur in the absence of significant main genotype effects36 . the only circumstances in which they will are if the genotype effect is in the opposite direction in two locations , and if the genetic effect in these locations is at least the same magnitude as the main effects detected in this gwas , namely explaining over 30% of the variance of a particular transcript . while a few such interactions may exist , it would take a study comparing several thousand individuals from each location to reveal weaker genotype - by - environment interactions . if the genetic architecture of transcription is generally similar to that of visible phenotypes like height and body mass37,38 , even such a study will be underpowered to explain the vast majority of transcriptional variance . a related question is whether or not genotype - by - environment interactions at the level of transcription are necessary to explain genotype - by - environment interactions for disease . it is possible the small interactions beneath the level of detection of gwas are prevalent , or alternatively that disease arises primarily as a result of rare alleles of major effect , whose penetrance may be modulated in an environment - specific manner . however , transcriptional interactions are not required to explain the increased incidence of chronic disease . it is not difficult to imagine that individuals that fall into the major categories of transcriptome profiles ( such as those implicated in fig . 4 and supplementary fig . 4 online ) have different distributions of disease susceptibility that alter the genotype - disease association matrix genome - wide , thereby inducing environment - by - genotype interactions for disease . transcription of some genes that contribute to this expression component may also correlate with disease directly , effectively uncovering cryptic variation and resulting in environment - specific esnp disease associations without any interaction effect at the level of transcription ( fig . a corollary of this is that gene expression profiling might be used to stratify individuals at elevated risk for disease , thereby increasing the resolution of genome - wide association studies by focusing attention on the subset of individuals where genetic effects on disease are most pronounced . our geographical genomic survey of gene expression variation in southern morocco has highlighted two parallel and for the most part non - overlapping insights . on the one hand , it is evident that as much as half of the transcriptome is influenced by the environment in a highly coordinated manner such that where a person lives explains up to a quarter of the variation for a substantial fraction of the transcripts . the environmental influences are likely a combination of biotic and abiotic factors , as well as cultural and behavioral ones , while genetic differences between the two north african ethnicities are relatively minor . on the other hand , the genome is littered with strong genetic associations , mainly in cis , that explain between 15 and 60 percent of the variance of 5% of the transcripts . impressive as these associations are , particularly since they are discovered in a sample of just under 200 individuals , they have essentially no bearing on the vast majority of the transcriptional variation , and are not informative of the genetic basis of the environmental response . the robustness of the observed associations to the environmental effect raises the issue of whether genotype - by - environment interactions influence the peripheral blood transcriptome at all . genome - wide significant interaction effects are generally unlikely to occur in the absence of significant main genotype effects36 . the only circumstances in which they will are if the genotype effect is in the opposite direction in two locations , and if the genetic effect in these locations is at least the same magnitude as the main effects detected in this gwas , namely explaining over 30% of the variance of a particular transcript . while a few such interactions may exist , it would take a study comparing several thousand individuals from each location to reveal weaker genotype - by - environment interactions . if the genetic architecture of transcription is generally similar to that of visible phenotypes like height and body mass37,38 , even such a study will be underpowered to explain the vast majority of transcriptional variance . a related question is whether or not genotype - by - environment interactions at the level of transcription are necessary to explain genotype - by - environment interactions for disease . it is possible the small interactions beneath the level of detection of gwas are prevalent , or alternatively that disease arises primarily as a result of rare alleles of major effect , whose penetrance may be modulated in an environment - specific manner . however , transcriptional interactions are not required to explain the increased incidence of chronic disease . it is not difficult to imagine that individuals that fall into the major categories of transcriptome profiles ( such as those implicated in fig . 4 and supplementary fig . 4 online ) have different distributions of disease susceptibility that alter the genotype - disease association matrix genome - wide , thereby inducing environment - by - genotype interactions for disease . transcription of some genes that contribute to this expression component may also correlate with disease directly , effectively uncovering cryptic variation and resulting in environment - specific esnp disease associations without any interaction effect at the level of transcription ( fig . a corollary of this is that gene expression profiling might be used to stratify individuals at elevated risk for disease , thereby increasing the resolution of genome - wide association studies by focusing attention on the subset of individuals where genetic effects on disease are most pronounced . | studies of the genetics of gene expression reveal expression snps that explain variation in transcript abundance . here
we address the robustness of esnp associations to environmental geography and population structure in a comparison of 194 arab and amazigh individuals from a city and two villages in southern morocco .
gene expression differed between pairs of locations for up to a third of all transcripts , with notable enrichment for ribosomal biosynthesis and oxidative phosphorylation .
robust associations were observed in the leukocyte samples with cis - esnps ( p < 1008 ) for 346 genes , and trans - esnps ( p < 1011 ) with 10 genes .
all of these were consistent across the three sample locations and after controlling for ethnicity and relatedness .
no evidence for large - effect trans - acting mediators of the pervasive environmental influence was found and instead genetic and environmental factors acted in a largely additive manner . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
a critical step in regulating many transcriptional responses is the import of transcription factors from the cytosol to the nucleus . many transcription factors are held outside the nucleus in a complex with cytosolic proteins or with membrane receptors , and translocate to the nucleus in response to various stimuli . alternatively , transcription factors may be inserted directly into the membrane , thereby preventing their access to the nucleus . a handful of such proteins has been shown to be released from membranes by a process known as regulated intramembrane proteolysis ( rip ) . this process is best understood for srebp-1 and srebp-2 , two basic leucine zipper ( bzip ) transcription factors that normally reside in the membrane of the endoplasmic reticulum and golgi apparatus . when cellular sterol levels dip , srebps are liberated from the membrane in a two - step mechanism involving the action of site-1 protease , a site - specific protease that cleaves the protein within the golgi lumen , followed by site-2 protease , an integral membrane protease , that cleaves a membrane - spanning helix . once liberated from the membrane , transport to the nucleus enables these transcription factors to initiate expression of genes involved in cholesterol uptake and biosynthesis . several more examples of membrane - tethered transcriptional regulators have recently been identified by biochemical means , notably atf6 , g13 , cadc , toxr , lzip ( luman ) , notch and spt23 . all appear to undergo proteolytic cleavages to release a fragment that is targeted to dna or the nucleus , but may use different proteases . for example , atf6 uses the same proteolytic machinery , as do srebps , whereas notch is cleaved by different proteases . tumor necrosis factor ( tnf)-converting enzyme catalyzes the cleavage of the extracellular domain of notch , followed by presenilin / gamma - secretase - like activity to liberate the intracellular fragment . thus , the release of some membrane - bound nuclear proteins involves regulated cleavages in the lumenal or extracellular space , followed by a cleavage by an integral membrane protease to release an active fragment . using conventional biochemistry , the identification of transmembrane transcription factors ( tmtfs ) can be easily overlooked . for example , transcription factors are generally assumed to be soluble proteins and , consequently , membrane fractions are often discarded during purification . moreover , the nuclear form of the protein may be rapidly degraded and thus difficult to detect , as is the case for srebps . cell - fractionation studies of other transcription factors show smaller - molecular - weight forms of these proteins enriched in the nucleus , suggestive of a cleavage event . we thus investigated the prevalence of transmembrane transcription factors using computational tools to search for membrane - spanning proteins that contain conserved dna - binding domains . we used pfam hidden markov models for 53 dna - binding domains ( see materials and methods ) to search all proteins in swiss - prot / trembl and swisspfam protein databases . the 9,261 proteins identified by our search are presumed members of dna - binding protein families , and most are expected to be transcription factors . these proteins were then scored for the presence of one or more transmembrane segments using prediction programs phdhtm [ l8 ] , tmhmm , hmmtop and psortii . only those proteins containing membrane - spanning helices predicted by at least three of the four programs were deemed significant in our analysis . by these stringent criteria , 76 proteins from 20 organisms and our analysis predicted a surprisingly large and diverse set of membrane - tethered dna - binding proteins . seventeen of the 53 dna - binding domains chosen for this analysis were represented in the final set of tmtfs . of these , the most abundant is the zf - c4 ( zinc - finger type c4 ) nuclear hormone receptor dna - binding domain , found in 14 proteins in caenorhabditis elegans and avian erythroblastosis virus . tmtfs in arabidopsis were the most diverse , and were associated with eight different dna - binding domains . all but two proteins have dna - binding domains that could be separated from the rest of the protein by a single hypothetical cleavage event , if singly predicted transmembrane segments are discounted ( figure 1 ) . dna - binding domains were also frequently juxtaposed to bipartite nuclear localization signals , suggesting that transmembrane and dna - binding domains in tmtfs are modular . thus , the overall topology of these proteins is consistent with other known tmtfs . c. elegans has an impressive 25 predicted tmtfs , suggesting that rip may be particularly important in the regulation of transcriptional responses in the worm . we deliberately used a stringent method to increase the likelihood of identifying only bona fide tmtfs and , as expected , most experimentally known tmtfs were detected by our analysis , including cadc , lzip , toxr and all swiss - prot / trembl orthologs of srebp - l and srebp-2 . also found were several well - characterized proteins whose predicted membrane insertion had not been recognized . for example , the human doublesex - related protein dmrt2 , drosophila b - h2 ( barh2 ) protein , c. elegans unc-86 , and mouse oasis protein are predicted tmtfs . the transmembrane helix of atf6 was predicted by only two programs : psort and hmmtop . the immunoglobulin dna - binding domain ( tig ) of spt23 is found in both cell - surface proteins as well as transcription factors and was therefore excluded from the set of dna - binding domains . these results indicate that reducing the stringency of our prediction method will expand the number of predicted tmtfs . in some cases we found data in the literature to support our computational predictions . for example , cell - fractionation studies using antibodies directed at the carboxyl terminus of the chaperonin mtj1 showed that the full - length ( 62 kda ) protein exists in microsomes , whereas a smaller 42 kda form of the protein is found in the nucleus . the 42 kda species was hypothesized to represent a product of internal translation . because mtj1 contains putative myb dna - binding domains within the carboxy - terminal half of the protein , we re - examined the subcellular localization of carboxy - terminal - tagged mtj1 in cos-7 cells ( figure 2a ) . our results show clearly that full - length mtj1 is normally associated with the endoplasmic reticulum . in contrast , a truncated form mtj1 ( approximately 40 kda in size ) lacking the transmembrane segment accumulates in the nucleus . therefore , we propose that the 42 kda nuclear form of mtj1 observed in cells arises by cleavage of mtj1 from the membrane , rather than from aberrant translation of the mrna . dmrt2 , a human homolog of c. elegans mab-3 , was identified in our analysis as having a carboxy - terminal transmembrane segment ( figure 1 ) . dmrt2 has gained recent attention as a candidate gene for sex - reversal phenotypes in humans . to verify our prediction that dmrt2 is a membrane - tethered transcription factor , we examined the subcellular localization of full - length and truncated forms of dmrt2 in cos-7 cells ( figure 2b ) . full - length dmrt2 is localized primarily , but not exclusively , to vesicles outside the nucleus . a carboxy - terminal truncation containing the dna - binding domain is , however , concentrated almost entirely in the nucleus . these results are consistent with the idea that dmrt2 is cleaved from the membrane to produce a nuclear fragment . interestingly , transformer protein tra-2a , an indirect activator of mab-3 , has been identified recently as a membrane - tethered nuclear protein . thus , rip maybe a conserved mechanism common to sex determination in humans and worms . we used pfam hidden markov models for 53 dna - binding domains ( see materials and methods ) to search all proteins in swiss - prot / trembl and swisspfam protein databases . the 9,261 proteins identified by our search are presumed members of dna - binding protein families , and most are expected to be transcription factors . these proteins were then scored for the presence of one or more transmembrane segments using prediction programs phdhtm [ l8 ] , tmhmm , hmmtop and psortii . only those proteins containing membrane - spanning helices predicted by at least three of the four programs were deemed significant in our analysis . by these stringent criteria , 76 proteins from 20 organisms and our analysis predicted a surprisingly large and diverse set of membrane - tethered dna - binding proteins . seventeen of the 53 dna - binding domains chosen for this analysis were represented in the final set of tmtfs . of these , the most abundant is the zf - c4 ( zinc - finger type c4 ) nuclear hormone receptor dna - binding domain , found in 14 proteins in caenorhabditis elegans and avian erythroblastosis virus . tmtfs in arabidopsis were the most diverse , and were associated with eight different dna - binding domains . all but two proteins have dna - binding domains that could be separated from the rest of the protein by a single hypothetical cleavage event , if singly predicted transmembrane segments are discounted ( figure 1 ) . dna - binding domains were also frequently juxtaposed to bipartite nuclear localization signals , suggesting that transmembrane and dna - binding domains in tmtfs are modular . thus , the overall topology of these proteins is consistent with other known tmtfs . c. elegans has an impressive 25 predicted tmtfs , suggesting that rip may be particularly important in the regulation of transcriptional responses in the worm . we deliberately used a stringent method to increase the likelihood of identifying only bona fide tmtfs and , as expected , most experimentally known tmtfs were detected by our analysis , including cadc , lzip , toxr and all swiss - prot / trembl orthologs of srebp - l and srebp-2 . also found were several well - characterized proteins whose predicted membrane insertion had not been recognized . for example , the human doublesex - related protein dmrt2 , drosophila b - h2 ( barh2 ) protein , c. elegans unc-86 , and mouse oasis protein are predicted tmtfs . the transmembrane helix of atf6 was predicted by only two programs : psort and hmmtop . the immunoglobulin dna - binding domain ( tig ) of spt23 is found in both cell - surface proteins as well as transcription factors and was therefore excluded from the set of dna - binding domains . these results indicate that reducing the stringency of our prediction method will expand the number of predicted tmtfs . in some cases we found data in the literature to support our computational predictions . for example , cell - fractionation studies using antibodies directed at the carboxyl terminus of the chaperonin mtj1 showed that the full - length ( 62 kda ) protein exists in microsomes , whereas a smaller 42 kda form of the protein is found in the nucleus . the 42 kda species was hypothesized to represent a product of internal translation . because mtj1 contains putative myb dna - binding domains within the carboxy - terminal half of the protein , we re - examined the subcellular localization of carboxy - terminal - tagged mtj1 in cos-7 cells ( figure 2a ) . our results show clearly that full - length mtj1 is normally associated with the endoplasmic reticulum . in contrast , a truncated form mtj1 ( approximately 40 kda in size ) lacking the transmembrane segment accumulates in the nucleus . therefore , we propose that the 42 kda nuclear form of mtj1 observed in cells arises by cleavage of mtj1 from the membrane , rather than from aberrant translation of the mrna . dmrt2 , a human homolog of c. elegans mab-3 , was identified in our analysis as having a carboxy - terminal transmembrane segment ( figure 1 ) . dmrt2 has gained recent attention as a candidate gene for sex - reversal phenotypes in humans . to verify our prediction that dmrt2 is a membrane - tethered transcription factor , we examined the subcellular localization of full - length and truncated forms of dmrt2 in cos-7 cells ( figure 2b ) . full - length dmrt2 is localized primarily , but not exclusively , to vesicles outside the nucleus . a carboxy - terminal truncation containing the dna - binding domain is , however , concentrated almost entirely in the nucleus . these results are consistent with the idea that dmrt2 is cleaved from the membrane to produce a nuclear fragment . interestingly , transformer protein tra-2a , an indirect activator of mab-3 , has been identified recently as a membrane - tethered nuclear protein . thus , rip maybe a conserved mechanism common to sex determination in humans and worms . we have used computational methods to investigate the prevalence of membrane - tethered transcription factors . the identification of 76 predicted tmtfs by our method , and the supporting cell biology , indicate that membrane - tethering may be a common mechanism for regulating transcriptional responses . as stringent criteria were used to identify transmembrane segments and dna - binding domains , we believe that the actual number of tmtfs is likely to be much larger . compared to other signal transduction mechanisms , tethering transcription factors in the membrane provides an expeditious route to the nucleus in response to stimuli that must be communicated across a membrane . our understanding of this process will be enhanced as more tmtfs are studied and the signals for membrane cleavage and their proteases are discovered . pfam hidden markov models for 53 dna - binding domains ( see dna - binding domains below ) were used to search proteins in swiss - prot / trembl ( october 2000 release ; 388,909 proteins ) with p - value < 0.0019 ( 0.01/53 ) . swisspfam proteins identified as having any of the 53 domains were also included in our analysis . default parameters were used for hmmtop , phdhtm [ l8 ] , and tmhmm ( version 2 ) . a higher stringency ( -5.0 ) than default was used for psort ii ( alom2 ) . transmembrane segments predicted by individual programs were considered overlapping if ten or more amino acids were shared by each segment . proteins containing transmembrane helices predicted by at least three of the four programs were included in the final set . three predicted tmtfs were discounted as false - positives on the basis of partial or complete overlap of transmembrane helices with other pfam domains ( o01612 , o23045 and q13771 ) . the following pfam models for dna - binding domains were used ( abbreviated as in pfam ) : 7 kda dna - binding ; ap2-domain ; arid ; asnc trans reg ; at hook ; arg represser ; b3 ; bah ; bro ; bac dna - binding ; basic ; bzip ; cbfb nfya ; csd ; cut ; copper - fist ; dm - domain ; e2f tdp ; fork head ; gata ; halz ; hlh ; homeobox ; hsf dna - binding ; hth 3 ; hth 4 ; hth 5 ; irf ; lexa dna - binding ; mbd ; metj ; myb dna - binding ; muts n ; myc - lz ; phd ; rfx dna - binding ; rhd ; runt ; sap ; sigma70 ; srf - tf ; stat ; sigma54 factors ; sigma70 ecf ; t - box ; tbp ; yeast dna - binding ; trans reg c ; zf ( zinc finger)-c2h2 ; zf - c2hc ; zf - c4 ; zf - nf - x1 ; zn - clus . full - length dmrt2 and mtj1 were generated by pcr using pfu polymerase ( stratagene ) and cloned directionally into bamhi / xbai sites of pcdna3 ( invitrogen ) . truncated mtj1 , in which an atg ( methionine ) was added immediately before amino acid 171 ( q61712 ) , was amplified from expressed sequence tag ( est ) ai790297 ( incyte genomics ) and a myc tag was added at the carboxyl terminus . full - length mtj1 was prepared by digesting clone ai790297 with xhoi , blunting ends , then digesting with ecori . this fragment was then cloned into pcdna3-mtj1 , which was digested with bamhi , blunt - ended , and digested with ecori . full - length and truncated dmrt2 ( at amino acid 180 ; q9y5r5 ) were amplified from est ai985131 ( incyte ) , and a myc tag was added at the amino terminus . standard deae transfections of plasmids were done in cos-7 cells ( atcc ) and grown in 10% fbs / dmem . cells were fixed 72 h post - transfection in 3% pfa in pbs and myc tags were detected with mouse anti - myc antibodies ( neomarkers , fremont , ca ) and texas - red - x goat anti - mouse antibodies ( molecular probes , eugene , or ) using standard procedures . pfam hidden markov models for 53 dna - binding domains ( see dna - binding domains below ) were used to search proteins in swiss - prot / trembl ( october 2000 release ; 388,909 proteins ) with p - value < 0.0019 ( 0.01/53 ) . swisspfam proteins identified as having any of the 53 domains were also included in our analysis . default parameters were used for hmmtop , phdhtm [ l8 ] , and tmhmm ( version 2 ) . a higher stringency ( -5.0 ) than default was used for psort ii ( alom2 ) . transmembrane segments predicted by individual programs were considered overlapping if ten or more amino acids were shared by each segment . proteins containing transmembrane helices predicted by at least three of the four programs were included in the final set . three predicted tmtfs were discounted as false - positives on the basis of partial or complete overlap of transmembrane helices with other pfam domains ( o01612 , o23045 and q13771 ) . the following pfam models for dna - binding domains were used ( abbreviated as in pfam ) : 7 kda dna - binding ; ap2-domain ; arid ; asnc trans reg ; at hook ; arg represser ; b3 ; bah ; bro ; bac dna - binding ; basic ; bzip ; cbfb nfya ; csd ; cut ; copper - fist ; dm - domain ; e2f tdp ; fork head ; gata ; halz ; hlh ; homeobox ; hsf dna - binding ; hth 3 ; hth 4 ; hth 5 ; irf ; lexa dna - binding ; mbd ; metj ; myb dna - binding ; muts n ; myc - lz ; phd ; rfx dna - binding ; rhd ; runt ; sap ; sigma70 ; srf - tf ; stat ; sigma54 factors ; sigma70 ecf ; t - box ; tbp ; yeast dna - binding ; trans reg c ; zf ( zinc finger)-c2h2 ; zf - c2hc ; zf - c4 ; zf - nf - x1 ; zn - clus . full - length dmrt2 and mtj1 were generated by pcr using pfu polymerase ( stratagene ) and cloned directionally into bamhi / xbai sites of pcdna3 ( invitrogen ) . truncated mtj1 , in which an atg ( methionine ) was added immediately before amino acid 171 ( q61712 ) , was amplified from expressed sequence tag ( est ) ai790297 ( incyte genomics ) and a myc tag was added at the carboxyl terminus . full - length mtj1 was prepared by digesting clone ai790297 with xhoi , blunting ends , then digesting with ecori . this fragment was then cloned into pcdna3-mtj1 , which was digested with bamhi , blunt - ended , and digested with ecori . full - length and truncated dmrt2 ( at amino acid 180 ; q9y5r5 ) were amplified from est ai985131 ( incyte ) , and a myc tag was added at the amino terminus . standard deae transfections of plasmids were done in cos-7 cells ( atcc ) and grown in 10% fbs / dmem . cells were fixed 72 h post - transfection in 3% pfa in pbs and myc tags were detected with mouse anti - myc antibodies ( neomarkers , fremont , ca ) and texas - red - x goat anti - mouse antibodies ( molecular probes , eugene , or ) using standard procedures . we thank j. rine and o. kelly for critical comments on the manuscript , and d. he for assembling overlapping domains . pfam - predicted dna - binding domains , transmembrane segments and bipartite nuclear localization signals are shown for linear protein models and identified by swiss - prot / trembl accession number . colored icons represent various dna - binding domains . predicted transmembrane segments for each program are represented by a filled box . arrows in mtj1 and dmrt2 indicate sites for truncated protein localization experiments shown in figure 2 . orthologs of predicted tmtfs not shown are : luman ( q9ue77 homo sapiens ) , srebp-1 ( q60416 cricetulus griseus , q9wtn3 mus musculus , p56720 rattus norvegicus , q9xx00 caenorhabditis elegans ) , srebp-2 ( q9uh04 h. sapiens , q60429 c. griseus ) , and aflr reg ( p43651 aspergillus parasiticus ) . open reading frames ( orfs ) for o65420 , o43989 , q17928 were extended using additional nucleotide sequence available in the ncbi database ( indicated by stippled rectangles ) . subcellular localizations of predicted and truncated tmtfs in cos-7 cells were detected using anti - myc antibodies . full - length proteins are localized to intracellular membrane compartments , but truncated forms ( ) lacking predicted transmembrane segments accumulate in the nucleus . | backgroundsequestration of transcription factors in the membrane is emerging as an important mechanism for the regulation of gene expression .
a handful of membrane - spanning transcription factors has been previously identified whose access to the nucleus is regulated by proteolytic cleavage from the membrane . to investigate the existence of other transmembrane transcription factors
, we analyzed computationally all proteins in swiss - prot / trembl for the combined presence of a dna - binding domain and a transmembrane segment.resultsusing pfam hidden markov models and four transmembrane - prediction programs , we identified with high confidence 76 membrane - spanning transcription factors in swiss - prot / trembl .
analysis of the distribution of two proteins predicted by our method , mtj1 and dmrt2 , confirmed their localization to intracellular membrane compartments .
furthermore , elimination of the predicted transmembrane segment led to nuclear localization for each of these proteins.conclusionsour analysis uncovered a wealth of predicted membrane - spanning transcription factors that are structurally and taxonomically diverse , 56 of which lack experimental annotation .
seventy - five of the proteins are modular in structure , suggesting that a single proteolysis may be sufficient to liberate a dna - binding domain from the membrane .
this study provides grounds for investigations into the stimuli and mechanisms that release this intriguing class of transcription factors from membranes . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
surgery for pancreatic cancer is probably the most demanding and risky operative procedure in abdominal surgery . remarkable progress has been achieved during the last three decades and pancreatic surgery can nowadays be performed safely with low morbidity and mortality in various specialized hospitals around the world . this is in part a result of regionalization of pancreatic surgery into high volume centers resulting in an increased resection rate in the presence of pancreatic carcinoma which is nowadays approximately 60% and to an extension of indications for resection . consequently , vascular resection , considered a contraindication for pancreatic resection a couple of years ago , is now implemented in the armamentarium of pancreatic surgeons to realize r0-resections when clearance of the portal or superior mesenteric vein can not be realized without vascular resection . the results following synchronous resection of liver metastasis together with resection of periampullary or pancreatic adenocarcinoma or pancreatic resection for m1 carcinoma are not equivocal . although these procedures can be performed safely , the median survival time ranges between 6 and 13 months [ 3 , 4 ] . therefore , synchronous metastasectomy of periampullary cancer is rarely performed when extended disease has already been found preoperatively . however , within daily clinical practice different scenarios occur which may require a decision for resection in an m1 situation . in a young patient with advanced disease , resection may provide a small but important increase of survival . in addition , metastatic disease may become overt when the point of no return has already been passed as it may be the case in the presence of positive interaortocaval lymphnodes , or metastatic disease will be detected during operation despite negative imaging results preoperatively . finally , a strong patient desire may force the surgeon to go further than consented guidelines may summarize existing evidence . since curative r0 resection is the most important prognostic factor for long - term survival even a small potential to achieve r0 resection may provide the legitimisation for extended resection despite the fact that overt systemic disease is already present . so far , there is little data available on the value of synchronous metastasectomy together with pancreatic resection in patients with pancreatic carcinoma . patients who undergo resection of metastases together with pancreatic resection seem to have a higher complication rate than patients with multivisceral liver resections . an extended lymphadenectomy is associated with an increased postoperative complication rate , but has no impact on long - term survival . the fact that despite r0 resection long - term survival does not exceed 25% even in the most experienced pancreatic centers may prove that carcinoma of the pancreas is a systemic disease . further improvement of survival can only be achieved by adjuvant treatment ( picozzi et al . , 2003 ) . it seems logical , that in the presence of metastatic disease a low tumor load may increase the success of adjuvant chemotherapy although the absolute increase in survival time may be very low . we therefore hypothesized that radical resection of m1-pancreatic carcinoma may be justified in well - selected patients as long as these operations can be performed with a low morbidity and mortality . thus , we aimed at reviewing our experiences with pancreatic resections in patients with m1 periampullary cancer of the pancreas after having launched a new pancreatic program in our hospital . there were 280 patients who underwent either pancreatoduodenectomy or left - sided pancreatic resection for histologically proven pancreatic adenocarcinoma or periampullary cancer . among this patient population we identified 20 ( 7,1% ) patients ( 9 men , 11 women ; mean age 60 years ) in our prospective database who underwent either a pancreaticoduodenectomy ( pd ) , or a left - sided distal pancreatectomy together with resection of metastasis for pancreatic adenocarcinoma . resection of metastatic disease was defined as the resection of any tumor deposit outside the regional lymph nodes or resection of infiltrated surrounding organs in order to achieve a r0 resection . magnetic resonance imaging , magnetic resonance cholangiography , or endoscopic retrograde cholangiopancreatography were performed when indicated . in patients who were found intraoperatively to harbour metastatic disease the decision for resection was based on the impression to reach a r0 situation with synchronous resection of metastasis and a good clinical performance status of asa iii or better . patients with suspected metastatic disease preoperatively were operated with potentially curative intention or with palliative intention . reasons for resection were good performance status and patient 's will to receive maximal treatment . all pathologic specimen were reviewed by a single pathologist ( at ) to confirm the diagnosis of pancreatic adenocarcinoma or cancer of the papilla vateri . a pancreatic fistula was defined as a prolonged drainage with amylase activity of more than 10.000 wound infection was defined as any wound requiring reopening for the drainage of pus together with a positive wound culture . mortality was defined as any death during postoperative hospitalization or within 30 days of surgery . follow - up information was obtained through direct contact with the patient and review of hospital charts and operative notes . the results of the study group were compared with a matched - pair control group of 20 patients with pancreatic adenocarcinoma , who were matched according to age and tumor location and who did undergo only pancreaticoduodenectomy or distal pancreatic resection for stadium iib or iii pancreatic adenocarcinoma . all statistical analyses were performed using spss - software ( version 15 ) . the distribution of age at operation , blood loss , and postoperative hospital stay were presented as median with range . comparisons between subgroups were performed with fisher 's exact test and differences between means were tested by the t - test . cases were matched with control group ( resection without multivisceral resection ) in a 1 : 1 fashion . specifically , cases and controls were matched on primary tumor characteristics and age of the patient . morbidity , mortality , and overall survival were compared between cases and control group . the nonparametric product limit method ( kaplan - meier estimations ) two - sided p values were always computed and an effect was considered statistically significant at p .05 . the primary tumor was located in the pancreatic head in 9 ( 45% ) patients , in the pancreatic tail in 9 ( 45% ) , and at the papilla vateri in 2 ( 10% ) . synchronous liver metastasis was present in 14 ( 70% ) patients , 4 ( 20% ) patients had peritoneal metastases , one patient had a metastasis in the transverse mesocolon , 2 ( 10% ) patients had a metastasis in the greater omentum , and 3 ( 15% ) patients had macroscopically lymph node metastases . six patients ( 30% ) had metastatic disease at more than one location . in 7 ( 35% ) metastatic disease was known before operation based on preoperative imaging in 5 of 20 patients ( 25% ) . of the 20 patients , 8 ( 40% ) underwent a pylorus - preserving duodenopancreatectomy and 2 ( 10% ) a classic whipple - procedure . in 8 ( 40% ) patients a distal pancreatic resection was performed and 2 ( 10% ) patients underwent a total pancreatectomy . details of the localisation of the tumor and the metastases , tnm - staging , r - status postoperatively , and type of surgery performed are listed in table 2 . an r0/r1-resection could be achieved in 11 patients ( 55% ) , whereas in the remaining patients only an r2 resection was performed due to remaining metastatic disease in liver . there was no perioperative mortality , and complications occurred in 9 ( 45% ) patients . the median intraoperative blood loss was 1000 ml ( range 3002500 ml ) and the median postoperative length of hospital stay was 20.7 d ( 1171 d ) . this was statistically not different to a matched - pair group of patients with ductal adenocarcinoma of the pancreas without metastatic disease ( tables 3 and 4 ) . all patients received postoperative adjuvant or palliative chemotherapy , two patients underwent neoadjuvant radiochemotherapy prior to surgery . the median postoperative survival was 10.7 months ( range 2.637.8 mo ) which was not significantly different to the control group who had a median survival time of 15.6 months ( p = .11 ; figure 1 ) . all deaths were caused by recurrent cancer.there was no difference in the median survival between patients with liver metastases ( median survival time 11 months ) compared to patients who had metastases at other locations ( median survival 14.1 months ; p = .62 ) . pancreatic cancer still carries a dismal prognosis and the potential to cure a patient can only be achieved when the primary tumor can be completely resected . however , this scenario can only be accomplished in about 5%25% of patients presenting with locally resectable cancer . patients with metastatic pancreatic cancer who receive palliative chemotherapy with gemcitabine have a median survival time of 5.6 months . various protocols with gemcitabine - based regimen or multidrug regimen tested in prospectively randomised phase iii clinical trials have not altered this situation substantially making metastatic pancreatic cancer one of the most frustrating malignancies to investigate and treat . therefore , when metastatic disease is recognized preoperatively , an operative procedure is usually avoided except for surgical palliation . in addition , according to the s-3 guidelines of the german cancer society pancreatic resections should be avoided in the presence of intraoperative metastatic disease . however , locally advanced disease may be resected as long as an r0 resection can be achieved . both statements have a grade of evidence of 3 which means that only systematic reviews and individual case control studies are available to scientifically support this statement . it seems reasonable , that postoperative chemotherapy may be more effective , when no gross tumor is left in situ . in addition , the high recurrence rate even after r0 resection is an indirect prove for early systemic spread which is not treatable by surgery but by chemotherapy . our study gives support to the hypothesis despite the relatively small number of patients that in the presence of metastasis of a pancreatic adenocarcinoma radical resection is possible and safe and that overall survival is comparable to patients who do not have metastatic disease . this relatively good result is in part consequence of restriction of the procedure to well suited patients who were expected to tolerate even a significantly expanded procedure . the mean age of our patients was 58 years , and all patients were either asa 1 or 2 ( data not given ) . therefore , it was expected that these patients would tolerate pancreatic resection with synchronous metastasectomy without significant increase in morbidity or mortality . nevertheless , the median survival of 10.6 months is low , but we believe that we could offer these patients additional life time , although quality of life was not evaluated . however , these procedures should be restricted to well - suited patients and only be performed at centers with significant expertise in pancreatic surgery . a shortcoming of the study may be the fact that patients had various presentation of the m1 category . most of our patients had liver metastasis , but there were also patients with peritoneal or lymph nodes metastases who were classified as m1 or the tumor involved surrounding organs . since there is no significant difference in survival according to the location of the metastasis of pancreatic carcinoma , we believe that an analysis of this group was appropriate . according to the results of at least 3 randomized trials extended lymphadenectomy has not been effective in improving survival of patients with pancreatic adenocarcinoma with 1 and 3 years survival between 51% and 77% and between 16 and 41% , respectively . therefore , in an nonmetastasized pancreatic adenocarcinoma a standard lymphadenectomy is adequate . when locally advanced cancer with suspected venous infiltration is present extensive surgery can be performed safely with morbidity and mortality rates comparable to conventional resections . in a recent analysis of 136 patients with locally advanced pancreatic cancer yekebas et al . could demonstrate that patients with concomitant vascular resections had the same median ( 15 versus 16 mo ; p = .86 ) and two year survival ( 34% versus 36% : p = .9 ) compared to patients who did not undergo vascular resection . even in multivariate analysis vascular infiltration was not considered a negative prognostic factor . even arterial encasement of the celiac trunk in the presence of a pancreatic corpus carcinoma is not considered a contraindication for surgery . recently published a series of 23 patients with pancreatic body cancer with infiltration of the celiac trunk . negative surgical margins were obtained in 91% of patients , postoperative mortality was 0% , and the median survival was 21 months . however , early hepatic recurrence in 6 patients was a hint that this procedure may be indicated for the treatment of less advanced disease . in a recently published study shrikhande et al . reported a series of 29 patients with m1 ductal adenocarcinoma of the pancreas who underwent pancreatic resection with synchronous resection of metastases . of these 11 patients had metastatic disease to the liver and 9 to the peritoneum . the authors concluded , that concomitant resection of primary pancreatic tumor and metastases can be performed safely . however , a risk benefit ratio should be carefully assessed since the overall increase in survival is moderate . in our study the survival time of patients with liver metastasis was 11 months compared to 14.1 months when metastases were located at other regions ( p = .62 ) . in a study of 22 patients who underwent pancreatic resection and synchronous liver resection for metastasised pancreatic adenocarcinoma , gleisner et al . found that the median survival was only 5.9 months , which was not statistically different from patients who underwent palliative bypass alone ( 5.6 months ; p = .46 ) . they concluded from the results of their study that simultaneous resection for patients who present with synchronous liver metastasis from pancreatic adenocarcinoma is not justified . in our study we found a median survival of 10.6 months for the complete group , while the 14 patients with liver metastases had a median survival of 11 months . despite this small increase we believe that in highly selected patients after careful counselling this aggressive therapy is still an option that should be offered to the patient . the fact that there was no significant difference in survival between the study and the matched - pair control group despite an objective difference in the median survival of 5 months is probably due to the limited number of patients . with an increased number of patients we would have expect this difference to reach statistical significance . compared to published results of patients receiving palliative chemotherapy who have a median survival time of approximately 5.48.4 months [ 13 , 14 ] , the increase in survival in patients with m1 ductal adenocarcinoma of the pancreas is about 2 - 5 months . we believe that patients who may benefit must be carefully selected and therapeutic options and expected survival should be discussed with the patients and their families . in the presence of pancreatic neuroendocrine carcinomas extended resections or even debulking operations seem to be associated with increased survival . reported on a series of 23 patients with pancreatic islet - cell carcinomas with synchronous hepatic metastases who underwent synchronous hepatic resections without perioperative mortality . this demonstrates , that extended resections in this subset of metastasized patients with primary pancreatic tumors are justified . the preoperative or intraoperative differentiation between a pancreatic adenocarcinoma and a neuroendocrine carcinoma may be difficult when typical carcinoma associated syndromes are missing preoperatively . however , intraoperatively the differentiation may be easier from the macroscopic point of view , and when histology has not been determined preoperatively , intraoperative frozen section may identify the tumor so operative strategy can be planned appropriately . currently , a monocentric prospective randomized trial on the value of pancreatic resection in the presence of liver metastasis is under way in germany ( nct00855634 , pamevitum ) . the results of this study may give further evidence whether pancreatic resection in this scenario is justified . the results of our study demonstrate , that resection of pancreatic adenocarcinoma with synchronous metastases can be performed safely . justification for combined resection should be made on an individual basis for each patient only . there is probably a small increase in survival compared to patients who undergo palliative chemotherapy and there was no statistically significant difference in survival compared to patients who underwent resection for stadium iib or iii tumors . the decision to resect a patient in an m1 situation should be carefully assessed and may be an option in highly selected cases . | background . for m1 pancreatic adenocarcinomas pancreatic resection is usually not indicated . however , in highly selected patients synchronous metastasectomy may be appropriate together with pancreatic resection when operative morbidity is low .
materials and methods . from january 1 , 2004 to december , 2007 a total of 20 patients with pancreatic malignancies were retrospectively evaluated who underwent pancreatic surgery with synchronous resection of hepatic , adjacent organ , or peritoneal metastases for proven uicc stage iv periampullary cancer of the pancreas .
perioperative as well as clinicopathological parameters were evaluated .
results .
there were 20 patients ( 9 men , 11 women ; mean age 58 years ) identified .
the primary tumor was located in the pancreatic head ( n = 9 , 45% ) , in pancreatic tail ( n = 9 , 45% ) , and in the papilla vateri ( n = 2 , 10% ) .
metastases were located in the liver ( n = 14 , 70% ) , peritoneum ( n = 5 , 25% ) , and omentum majus ( n = 2 , 10% ) .
lymphnode metastases were present in 16 patients ( 80% ) .
all patients received resection of their tumors together with metastasectomy .
pylorus preserving duodenopancreatectomy was performed in 8 patients , distal pancreatectomy in 8 , duodenopancreatectomy in 2 , and total pancreatectomy in 2 .
morbidity was 45% and there was no perioperative mortality .
median postoperative survival was 10.7 months ( 2.637.7 months ) which was not significantly different from a matched - pair group of patients who underwent pancreatic resection for uicc adenocarcinoma of the pancreas ( median survival 15.6 months ; p = .1 ) . conclusion .
pancreatic resection for m1 periampullary cancer of the pancreas can be performed safely in well - selected patients . however , indication for surgery has to be made on an individual basis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
occupational accident is an unexpected and unplanned one , and causes injuries in one or more workers . angle grinders are one of the most dangerous tools used around the world to cut stone , metal and concrete . penetrating head injury from angle grinders is increasingly recognized due to their frequent use in the work place and at home . a series of two penetrating head injuries associated with angle grinder use are presented , as these are rarely reported in indian literature . two construction workers were brought to the emergency room in an altered sensorium after an accident at their construction site . the history revealed that the first patient was cutting stone with an angle grinder without protective guard . two busted disc embedded in the ceiling , the third hit on his fore head and the fourth hit in the occipital region of his co - worker , who was working at five meters away . following the blow on his head from behind , both the patients were immediately shifted to a nearby healthcare center , where the impacted broken disc was removed and the wound was packed . as there was an increased amount of bleeding from the wound site and declining level of consciousness , both the patients were referred for further management . a 35-year - old right - handed healthy male , without any significant past medical history , was brought to the emergency room following the above said mishap . on examination he was also drowsy and responding to deep painful stimuli with a glasgow coma scale score of 10 . there was a lacerated wound of 11 x 4 cms over the forehead with tissue loss [ figure 1 ] . the wound was grossly contaminated with sand , mud and burnt cloth pieces with tobacco which was used for haemostatic purpose in folk medicine . clinical photograph showing lacerated wound in the fore head the trauma series x - rays were normal and focused abdominal sonogram for trauma ( fast ) was negative . a non enhanced computerized tomography ( ct ) serial axial section of 5 mm thickness of the skull revealed bilateral frontal and ethmoid comminuted depressed fracture . the subarachnoid pneumocephalus and hemosinus of frontal and ethmoid were also noted [ figures 2 and 3 ] . the ventricular system and the posterior fossa were normal with no midline shift . ncct of the head showing bilateral frontal and ethmoid comminuted depressed fracture computerized tomography of the head demonstrated bilateral cerebral multiple hemorrhagic contusion , acute sub arachnoid hemorrhage and pneumocephalus the patient was subjected to surgery to elevate the depressed segment , debridement and for dural repair . dural tear was present in right frontal region and brain matter was herniating through it . the site of injury was irrigated and cleaned with limited local debridement and the dura was then closed in a watertight fashion . depressed fontal bone fragments were elevated , the wound closed in layers and a compression dressing was applied . he was given broad spectrum antibiotics , analgesics and tetanus vaccination along with fosphenytoin as antiseizure prophylaxis . the postoperative course was uneventful and the patient was discharged on day seven without any neurological deficit and wound infection . a 55-year - old right - handed euglycemic and normotensive male was brought to the emergency room following the above said accident . on examination he was unconscious and responding to deep painful stimuli with a glasgow coma scale ( gcs ) score of 7 . both pupils were equally reacting to light . in view of poor gcs and to protect airway , he was intubated and mechanically ventilated . his hemodynamics was stabilized and adequate saturation was maintained . during the secondary survey , the patient was found to have fracture neck of humerus and rib fracture involving three to six ribs on right side which were radiologically confirmed . there was a lacerated wound of 12 x 3 cms over the right occipital region with particulate matter . the ct of the head demonstrated bilateral cerebral multiple hemorrhagic contusion , acute sub arachnoid hemorrhage and pneumocephalus . his wounds were debrided and closed in layers . his humerus fracture was fixed and splinted . he was started on mannitol , antiepileptics , broad spectrum antibiotics , analgesics and tetanus vaccination . a 35-year - old right - handed healthy male , without any significant past medical history , was brought to the emergency room following the above said mishap . on examination he was also drowsy and responding to deep painful stimuli with a glasgow coma scale score of 10 . there was a lacerated wound of 11 x 4 cms over the forehead with tissue loss [ figure 1 ] . the wound was grossly contaminated with sand , mud and burnt cloth pieces with tobacco which was used for haemostatic purpose in folk medicine . clinical photograph showing lacerated wound in the fore head the trauma series x - rays were normal and focused abdominal sonogram for trauma ( fast ) was negative . a non enhanced computerized tomography ( ct ) serial axial section of 5 mm thickness of the skull revealed bilateral frontal and ethmoid comminuted depressed fracture . the subarachnoid pneumocephalus and hemosinus of frontal and ethmoid were also noted [ figures 2 and 3 ] . the ventricular system and the posterior fossa were normal with no midline shift . ncct of the head showing bilateral frontal and ethmoid comminuted depressed fracture computerized tomography of the head demonstrated bilateral cerebral multiple hemorrhagic contusion , acute sub arachnoid hemorrhage and pneumocephalus the patient was subjected to surgery to elevate the depressed segment , debridement and for dural repair . dural tear was present in right frontal region and brain matter was herniating through it . the site of injury was irrigated and cleaned with limited local debridement and the dura was then closed in a watertight fashion . depressed fontal bone fragments were elevated , the wound closed in layers and a compression dressing was applied . he was given broad spectrum antibiotics , analgesics and tetanus vaccination along with fosphenytoin as antiseizure prophylaxis . the postoperative course was uneventful and the patient was discharged on day seven without any neurological deficit and wound infection . a 55-year - old right - handed euglycemic and normotensive male was brought to the emergency room following the above said accident . on examination he was unconscious and responding to deep painful stimuli with a glasgow coma scale ( gcs ) score of 7 . both pupils were equally reacting to light . in view of poor gcs and to protect airway , he was intubated and mechanically ventilated . the patient was found to have fracture neck of humerus and rib fracture involving three to six ribs on right side which were radiologically confirmed . there was a lacerated wound of 12 x 3 cms over the right occipital region with particulate matter . the ct of the head demonstrated bilateral cerebral multiple hemorrhagic contusion , acute sub arachnoid hemorrhage and pneumocephalus . his wounds were debrided and closed in layers . his humerus fracture was fixed and splinted . he was started on mannitol , antiepileptics , broad spectrum antibiotics , analgesics and tetanus vaccination . traumatic brain injury ( tbi ) occurring at the workplaces , comprises about seven per cent of all tbi population , and carries major economic upshot in terms of loss of wages and medical expenses . penetrating head injuries constitute only a small part of the total number of traumatic head injuries seen in an emergency room . non missile high - velocity penetrating brain injuries are unusual among civilian population which results in high mortality and morbidity . in civilian setup , the most common non missile penetrating cranial wounds are due to low velocity type caused by knives , nails , scissors , iron rod , fishing harpoon , electrical plug , fan blade , pencil and ceramic stone.[46 ] similar to this case series , abdoli and amirjamshidi reported the penetrating head wounds caused by industrial grinder tool among the construction workers . when the disc rotating at a rate of 10,000 to 15 , 000 revolutions per minute is shattered , the pieces gain sufficient momentum to travel far as a missile and cause havoc . the pathophysiological consequence of penetrating head injuries depends on the kinetic energy and the trajectory of the object into the brain . in the injuries with high impact velocity , the underlying traumatic damage may be severe than presumed as observed in the first case of this report . at times the local variations in thickness and strength of the skull and the angle of the impact determine the severity of the fracture and injury to the brain . impacts striking the skull at nearly perpendicular angles may cause bone fragments to travel along the same trajectory as the penetrating object , to shatter the skull in an irregular pattern , or to produce linear fractures that radiate away from the entry defect . the most appropriate management in the field is to leave the transcranial object in situ and transport the patient to the trauma center carefully . tan and choudhari suggested that in the presence of an obvious penetrating injury with an imbedded foreign body , no attempt should be made to remove the object until the patient has had a thorough clinical and radiological evaluation . thurner and pollak demonstrated the morphology of the wounds sustained using angle grinders that tend to follow the shape of the cutting disc ; most often curvilinear but may vary slightly depending on the angle of skin entry . the volume of tissue loss is directly dependent on the size of the disc used . finding fragments of disc and the material being cut in the wound is pathognomic of angle grinder injuries . following the primary injury or impact , secondary injuries may occur similar to the second case . a thorough secondary survey should be performed following the initial resuscitation as per advanced trauma life support ( atls ) guidelines in order to avoid missed injuries and medico legal consequences . expeditious removal of the penetrating foreign body and focal debridement of the scalp , skull , dura , and involved parenchyma are the goals of surgical treatment . watertight dural closure has long been a mainstay of the surgical management of penetrating brain injuries and serves to prevent cerebral fungus , reduce the instance of csf leak , and provide another important barrier to infection . if seizures are not evident in the acute phase , anticonvulsants may be discontinued at the end of seven days . the duration of use of antiepileptics remains somewhat controversial , but long - term use does not seem to be beneficial . despite the effectiveness of hyperventilation in rapidly reducing intracranial pressure ( icp ) in some patients , its use is not recommended in view of significant reduction in cerebral blood flow which may worsen long - term neurological outcome . the neurological sequelae following the non missile penetrating head injuries are determined by the severity and location of the initial injury as well as the rapidity of the exploration and fastidious debridement . injuries occur for a number of reasons , firstly the disc itself may kick back from the surface it is cutting . this will send the rotating disc toward the operator , parallel to the axis at which it is being used . face is more often at risk of a penetrating wound as happened in the first case since the guard has been removed while the machine was on . our closed survey revealed that the workers were not informed well on the selection and correct fitting of the disc ; and the importance of protective guard for the machine and personal protective equipments for individual operating these machines . the shop owners surveyed said that the wrong type of disc was frequently used , increasing the likelihood of the disc shattering . finally , shattering of the disc can occur when the disc has been incorrectly fitted . the discs rotate between 10,000 15,000 rev / min , giving sufficient momentum to travel far and penetrate deeply as occurred in these cases . as a preventive measure the users have to be taught and trained to use the correct disc size and type , wear appropriate personal protective equipments , use the angle grinder with protective guard and maintain the safety by standing perpendicular to the plane of the cutting wheel , and thereby can greatly reduce the occurrence of such injuries . in addition the healthcare workers have to be oriented well to handle such cases carefully . | penetrating cranial injury is a potentially life - threatening condition .
injuries resulting from the use of angle grinders are numerous and cause high - velocity penetrating cranial injuries .
we present a series of two penetrating head injuries associated with improper use of angle grinder , which resulted in shattering of disc into high velocity missiles with reference to management and prevention .
one of those hit on the forehead of the operator and the other on the occipital region of the co - worker at a distance of five meters .
the pathophysiological consequence of penetrating head injuries depends on the kinetic energy and trajectory of the object . in the nearby healthcare center
the impacted broken disc was removed without realising the consequences and the wound was packed . as the conscious level declined in both , they were referred .
ct brain revealed fracture in skull and changes in the brain in both .
expeditious removal of the penetrating foreign body and focal debridement of the scalp , skull , dura , and involved parenchyma and watertight dural closure were carried out .
the most important thing is not to remove the impacted foreign body at the site of accident .
craniectomy around the foreign body , debridement and removal of foreign body without zigzag motion are needed .
removal should be done following original direction of projectile injury .
the neurological sequelae following the non missile penetrating head injuries are determined by the severity and location of initial injury as well as the rapidity of the exploration and fastidious debridement . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
our experimental plan is schematically diagrammed in figure 1 . the enstem - a cell line ( emd millipore ) was used in this study . nscs are routinely validated by the manufacturer for high levels of expression of the multipotent markers nestin and sox2 , and low - level expression of the pluripotent marker oct-4 , along with the ability to differentiate into multiple neuronal phenotypes and to maintain a normal karyotype after multiple passages . under our growth conditions , these nscs displayed abundant expression of sox2 and nestin and retained their ability to differentiate , as described previously . nscs were grown on poly - l - ornithine ( 20 g / ml ) and laminin ( 5 g / ml ) coated tissue culture treated flasks . cells were grown in neural expansion medium ( millipore ) supplemented with l - glutamine ( 2 mm ) and basic fibroblast growth factor ( bfgf ; 20 ng / ml ) . adherent monolayers of nscs were passaged every other day ( 1:2 ) with accutase as the dissociation agent . for transplantation studies , nscs were labeled with 5-bromo-2'-deoxyuridine ( brdu ) by supplementing the growth media with 4 m brdu for three days prior to transplantation . to verify the brdu labeling index ( i.e. percentage of brdu positive cells ) , cells were plated on chamber slides and processed for brdu detection using standard immunocytochemical approaches . alternatively , transplanted human stem cells were detected using the human specific nuclear antigen marker ( hunu ) . on the day of transplantation , accutase and neural expansion media were pre - warmed in a 37c water bath . cells were placed in incubation medium ( neural expansion media containing 10 m of y-27632 ) for one hour . y-27632 ( rock inhibitor , emd - calbiochem ) was used to improve the survival of nscs post - transplantation . after one hour , incubation media was removed and cells were treated with accutase for 5 minutes . after this brief treatment add an equal volume of neural expansion media to neutralize the accutase and strain the cells through a 70 m cell strainer . count the cells with a haemocytometer and prepare 1.0 x 10 live nscs per microliter in injection medium ( neural expansion media containing 10 m of y-27632 , 40 ng / ml bfgf , 20 ng / ml bdnf ) . cells were stored in neural expansion media ( as described in 1.5 ) and kept on ice until the time of transplantation , and should be used within 6h to minimize cell death by prolonged cold - storage . sedated athymic nude rat(s ) were placed in a laboratory mri scanner in the prone position with the skull at the superior end ( head first ) . it can provide exquisite soft - tissue contrast with high spatial resolution cross - sectional ( axial ) images . the scan focused on the skull region and a set of 22 t2 e60 weighted images with 0.8 mm image thickness were generated . these images provide necessary information to identify the left and right hippocampi within the rat 's brain . following the mri scan ( 24h ) , animals were sedated [ anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) ] and placed in a radiation oncology ct scanner where another cross - sectional ( axial ) image volume was generated . the rat was placed in the same or close to the same position as used for the mri scan and the ct unit was set to scan the skull region . a study of 106 ct images with 0.8 mm image thickness was generated which became the ct treatment planning data . treatment planning the mri and ct image data were transferred to the eclipse ( varian medical systems , palo alto , ca ) treatment planning software via dicom ( digital - imaging and communications in medicine ) . eclipse is a sophisticated and commercially available software used in radiation oncology to model and design the best radiation treatment plan for a patient . it incorporates organ density information that is extracted from the ct image data and used to calculate the radiation dose distribution within the body.cover the animal using a small surgical drape with only the head exposed . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp.center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero.the next step in the process is to determine the irradiation technique to be used . intensity - modulated radiation therapy ( imrt ) and volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc are 2 high - precision irradiation techniques that use a 6 mv photon beam to deliver a radiation dose . the difference between these techniques is that imrt delivers a dose using several static trajectories each corresponding to a different direction but converging at the target volume more suitable for extremely small target volumes . rapidarc on the other hand , delivers a dose dynamically through one or more arcs focused on the target region ( fig . 4).regardless of delivery technique , information on target dose , target conformity and dose constraints to critical organs have to be entered in the dose optimization window in eclipse . this information along with organ volumes is used to tailor the dose distribution , which is achieved by continually varying the beam attenuation during irradiation.once a plan was generated , eclipse provides the calculated dose superimposed to the axial , coronal and sagittal images ( fig . 5 ) as well as in dose - volume histogram ( dvh ) format ( fig . 6 ) . it is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved . the mri and ct image data were transferred to the eclipse ( varian medical systems , palo alto , ca ) treatment planning software via dicom ( digital - imaging and communications in medicine ) . eclipse is a sophisticated and commercially available software used in radiation oncology to model and design the best radiation treatment plan for a patient . it incorporates organ density information that is extracted from the ct image data and used to calculate the radiation dose distribution within the body . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp . center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero . the next step in the process is to determine the irradiation technique to be used . intensity - modulated radiation therapy ( imrt ) and volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc are 2 high - precision irradiation techniques that use a 6 mv photon beam to deliver a radiation dose . the difference between these techniques is that imrt delivers a dose using several static trajectories each corresponding to a different direction but converging at the target volume more suitable for extremely small target volumes . rapidarc on the other hand , delivers a dose dynamically through one or more arcs focused on the target region ( fig . information on target dose , target conformity and dose constraints to critical organs have to be entered in the dose optimization window in eclipse . this information along with organ volumes is used to tailor the dose distribution , which is achieved by continually varying the beam attenuation during irradiation . once a plan was generated , eclipse provides the calculated dose superimposed to the axial , coronal and sagittal images ( fig . 5 ) as well as in dose - volume histogram ( dvh ) format ( fig . 6 ) . it is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved . irradiation process once a plan was approved for delivery , digitally - reconstructed radiograph ( drr ) images were generated from the ct treatment planning data in eclipse . these are orthogonal images weighted on bone density to highlight the skull and other bony landmarks . thereafter , treatment plan and drrs are sent to the treatment delivery computer system via dicom.the delivery system controls a varian trilogy linear accelerator normally used for radiotherapy of humans . the accelerator is equipped with 120 computer controlled thin multileaf collimators ( mlc ) and a diagnostic quality x - ray imaging system included within the on - board imaging ( obi ) system . this delivery system uploads the eclipse - specific information and reconfigures the collimator parameters in the accelerator for delivering the planned dose.at this point the rat was prepared for irradiation , sedated and covered with a paper blanket to keep it warm . after a few minutes , the rat , with its blanket but with the skull exposed , was placed on the treatment table in the same position used to generate the ct scan . this is a critical step because position accuracy is directly correlated to accuracy in dose delivery.once the rat was positioned properly on the treatment table , a set of orthogonal x - ray images is taken using the trilogy 's obi system . these are high resolution images that were subsequently fused to the eclipse generated drrs using the obi image fusion software.the x - ray images and drrs were digitally matched using software that provide information on table position shifts that must be made to achieve co - registration of the images ( fig . after reviewing this information and verifying that the resultant shifts do not inadvertently lead into any collisions between the accelerator and the treatment table , the computer applies the shifts and the treatment table moves automatically to the desired position.at this point , irradiation commences . to deliver a 10 gy dose with a 6-field imrt plan , the beam - on time is approximately 10 minutes while a two - arc rapidarc takes ~3 minutes . after treatment delivery is complete , the rat was removed from the treatment room and allowed to recover in a holding cage kept on a heating pad . once a plan was approved for delivery , digitally - reconstructed radiograph ( drr ) images were generated from the ct treatment planning data in eclipse . these are orthogonal images weighted on bone density to highlight the skull and other bony landmarks . thereafter , treatment plan and drrs are sent to the treatment delivery computer system via dicom . the delivery system controls a varian trilogy linear accelerator normally used for radiotherapy of humans . the accelerator is equipped with 120 computer controlled thin multileaf collimators ( mlc ) and a diagnostic quality x - ray imaging system included within the on - board imaging ( obi ) system . this delivery system uploads the eclipse - specific information and reconfigures the collimator parameters in the accelerator for delivering the planned dose . at this point the rat was prepared for irradiation , sedated and covered with a paper blanket to keep it warm . after a few minutes , the rat , with its blanket but with the skull exposed , was placed on the treatment table in the same position used to generate the ct scan . this is a critical step because position accuracy is directly correlated to accuracy in dose delivery . once the rat was positioned properly on the treatment table , a set of orthogonal x - ray images is taken using the trilogy 's obi system . these are high resolution images that were subsequently fused to the eclipse generated drrs using the obi image fusion software . the x - ray images and drrs were digitally matched using software that provide information on table position shifts that must be made to achieve co - registration of the images ( fig . after reviewing this information and verifying that the resultant shifts do not inadvertently lead into any collisions between the accelerator and the treatment table , the computer applies the shifts and the treatment table moves automatically to the desired position . at this point , irradiation commences . to deliver a 10 gy dose with a 6-field imrt plan , the beam - on time is approximately 10 minutes while a two - arc rapidarc takes ~3 minutes . after treatment delivery is complete , the rat was removed from the treatment room and allowed to recover in a holding cage kept on a heating pad . animals husbandry and surgical preparation for this study , we employed two month old atn rats acquired from national cancer institute ( strain 0n01 cr : nih - rnu ) . animals were kept in sterile cages and maintained in a temperature- and light controlled barrier - a environment with 12-h/12-h light / dark cycle . rats were provided autoclaved food and water ad libitum and care was taken to prevent eye infection by cleaning eyes every week with vetropolycin eye ointment ( western medical supply , arcadia , ca).for stereotaxic surgery , rats were anesthetized with an i.p . injection of anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) . complete anesthesia was induced after 10 - 15 minutes and sedation was monitored using palpable reflex ( toe pinch ) . if required during surgery , 15 - 20% of the original dose may be given to maintain sufficient anesthesia.remove fur from head using electric clippers . clean shaved part using providone / iodine ( 3 times ) followed by 70% alcohol ( 3 times ) before incision .
for this study , we employed two month old atn rats acquired from national cancer institute ( strain 0n01 cr : nih - rnu ) . animals were kept in sterile cages and maintained in a temperature- and light controlled barrier - a environment with 12-h/12-h light / dark cycle . rats were provided autoclaved food and water ad libitum and care was taken to prevent eye infection by cleaning eyes every week with vetropolycin eye ointment ( western medical supply , arcadia , ca ) . for stereotaxic surgery , rats were anesthetized with an i.p . injection of anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) . complete anesthesia was induced after 10 - 15 minutes and sedation was monitored using palpable reflex ( toe pinch ) . if required during surgery , 15 - 20% of the original dose may be given to maintain sufficient anesthesia . clean shaved part using providone / iodine ( 3 times ) followed by 70% alcohol ( 3 times ) before incision . stereotaxic surgery stereotaxic instruments ( digital monitor and frame ) , micromotor drill , dry bed sterilizer and controllers should be kept in a laminar flow hood to prevent possible infection during surgery procedure in atn rats . a stereotaxic stage with an embedded heating pad can be used to keep the animals warm during surgery . we used ' kimberly - clark safeskin purple nitrile sterile exam gloves ' thoughout the surgery . in addition , surgeon used 70% alcohol spray to sterilize hands during surgery.cover the animal using a small surgical drape with only the head exposed . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp.center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero.following these positioning steps , apply lubricating eye ointment to prevent drying and protect them from possible iodine or alcohol spills . use this ointment at least 2 times during surgery.make midline skin incision ( 2 cm ) along scalp using sterile scalpel , and clean using sterile cotton tipped applicator . avoid damage in caudal and neck muscles areas.by using dissecting retractor , hold the periosteum open and clear soft tissue using cotton tipped applicator dipped in 1% hydrogen peroxide ( made in pbs ) . to stop bleeding , maintain firm pressure for at least 1 min using cotton tipped applicator or gauze . apply firm scraping motion to clean skull and dabbing motion for skin until skull sutures , bregma and lambda were visible.precise stereotaxic coordinates , referenced to the bregma , were determined using the rat brain atlas . stem cell transplantation was done at four distinct sites for each hemisphere using the following stereotaxic coordinates : anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain.ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm
once the bregma has been identified , all three coordinates ( ap , ml and dv ) should be zeroed on the digital display / controller ( on the digital stereotaxic frame ) . then proceed to mark the precise transplantation sites ( using the foregoing coordinates ) with a fine point marker pen attached to a small probe holder on the frame.drill a 0.35 mm hole ( no . 1/4 dental burr , carbide vanadium drill bits ) through the skull using the foot pedal controlled micromotor drill ( leica - myneurolab ) . take care to prevent damage to the dura membrane . if bleeding occurs during drilling , . do not apply alcohol or iodine at the drill site , as this will cause irritation to the animal.rats received bilateral intra - hippocampal injections of a suspension of nscs injected in a maximum volume of 1 l using a 5 l hamilton microsyringe ( 30 gauge ) . the precise number of cells within this volume will vary according to experimental specifics . to accomplish this , carefully insert the tip of the needle in the skull until reaching the surface of the brain ( i.e. meninges ) . at this point , the dv coordinate should be zeroed on the digital display / controller , then gently insert the needle to the desired depth ( dv , 3.2 mm ) . wait 1 min before initiating any injection of cells.inject 0.25 l volume / min ( slow release ) using a timer . once a total of 1 l is injected , wait 8 min before retracting needle from transplantation site . after this time , slowly retract the needle from the transplantation site ( 0.5 mm / min ) to prevent capillary reflux of the injection contents back through the needle track.follow steps 3.2(j - k ) for the remaining transplantation sites ( 4 per hemisphere).remove the skin retractor from the skull and use blunt tweezers to gently pull back retracted skin and apply 4 - 5 sterile stainless clips using autoclips surgical suture applier ( leica - myneurolab).after removing the animal from the frame , inject with analgesic ( buprenorphin , 0.1 mg / kg , s.c . , every 12h ) and lactated ringer 's solution ( 5 ml/250 g adult rat , s.c.).place the animal back in its cage that should be kept on a heating pad . place some moistened pellets ( animal chow ) and transgel in a separate petri dish in each animal cage . alternatively , dietgel recovery ( clearh2o , portland , me ) can be used as a source of water and nutrients.monitor animals during their recovery time and apply analgesia ( buprenorphine 0.02 mg / kg ( every 12h for 2 days ) . check for signs of pain , distress , reddening or infection of surgery site . apply providone / iodine or neosporin ointment once daily ( till 2 - 3 days ) to prevent possible infection . if any signs of infection , pain or distress persist following analgesic or antibiotic treatment within 12 hours of surgery , consult veterinarian or animal care staff for further assistance . stereotaxic instruments ( digital monitor and frame ) , micromotor drill , dry bed sterilizer and controllers should be kept in a laminar flow hood to prevent possible infection during surgery procedure in atn rats . a stereotaxic stage with an embedded heating pad can be used to keep the animals warm during surgery . we used ' kimberly - clark safeskin purple nitrile sterile exam gloves ' thoughout the surgery . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp . center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero . following these positioning steps , apply lubricating eye ointment to prevent drying and protect them from possible iodine or alcohol spills . make midline skin incision ( 2 cm ) along scalp using sterile scalpel , and clean using sterile cotton tipped applicator . hold the periosteum open and clear soft tissue using cotton tipped applicator dipped in 1% hydrogen peroxide ( made in pbs ) . to stop bleeding , maintain firm pressure for at least 1 min using cotton tipped applicator or gauze . apply firm scraping motion to clean skull and dabbing motion for skin until skull sutures , bregma and lambda were visible . precise stereotaxic coordinates , referenced to the bregma , were determined using the rat brain atlas . stem cell transplantation was done at four distinct sites for each hemisphere using the following stereotaxic coordinates : anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain.ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm
anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain . ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mm ap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm ap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm once the bregma has been identified , all three coordinates ( ap , ml and dv ) should be zeroed on the digital display / controller ( on the digital stereotaxic frame ) . then proceed to mark the precise transplantation sites ( using the foregoing coordinates ) with a fine point marker pen attached to a small probe holder on the frame . 1/4 dental burr , carbide vanadium drill bits ) through the skull using the foot pedal controlled micromotor drill ( leica - myneurolab ) . if bleeding occurs during drilling , apply firm pressure using cotton tipped applicator . do not apply alcohol or iodine at the drill site , as this will cause irritation to the animal . rats received bilateral intra - hippocampal injections of a suspension of nscs injected in a maximum volume of 1 l using a 5 l hamilton microsyringe ( 30 gauge ) . the precise number of cells within this volume will vary according to experimental specifics . to accomplish this , carefully insert the tip of the needle in the skull until reaching the surface of the brain ( i.e. meninges ) . at this point , the dv coordinate should be zeroed on the digital display / controller , then gently insert the needle to the desired depth ( dv , 3.2 mm ) . once a total of 1 l is injected , wait 8 min before retracting needle from transplantation site . after this time , slowly retract the needle from the transplantation site ( 0.5 mm / min ) to prevent capillary reflux of the injection contents back through the needle track . follow steps 3.2(j - k ) for the remaining transplantation sites ( 4 per hemisphere ) . remove the skin retractor from the skull and use blunt tweezers to gently pull back retracted skin and apply 4 - 5 sterile stainless clips using autoclips surgical suture applier ( leica - myneurolab ) . after removing the animal from the frame , , every 12h ) and lactated ringer 's solution ( 5 ml/250 g adult rat , s.c . ) . place the animal back in its cage that should be kept on a heating pad . place some moistened pellets ( animal chow ) and transgel in a separate petri dish in each animal cage . alternatively , dietgel recovery ( clearh2o , portland , me ) can be used as a source of water and nutrients . monitor animals during their recovery time and apply analgesia ( buprenorphine 0.02 mg / kg ( every 12h for 2 days ) . apply providone / iodine or neosporin ointment once daily ( till 2 - 3 days ) to prevent possible infection . if any signs of infection , pain or distress persist following analgesic or antibiotic treatment within 12 hours of surgery , consult veterinarian or animal care staff for further assistance . fusion of ct ( bony anatomy ) and mri ( soft - tissue ) images within the eclipse software . matching of axial , coronal , and sagittal sections allow for the co - registry of the critical anatomical features of the rat brain derived from each imaging modality . subsequent to image fusion , hippocampi and brain excluding hippocampi are identified and contoured axially defining specific volumetric regions for these organs . these regions provide anatomical and volume information necessary for adjusting the dose distribution to the desired target(s ) . high precision irradiation options using intensity - modulated radiation therapy ( imrt ) or volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc . these techniques deliver 6 mv photon beams either as multiple static trajectories that can converge on very small target volumes ( imrt ) , or dynamically as one or more arcs focused on the target region ( rapidarc ) . data contrasts the percentage of the irradiated versus the non - irradiated hippocampal volume under the single hippocampus treatment plan shown in fig . orthogonal digitally - reconstructed radiograph ( drr ) images generated from the ct treatment planning data in eclipse are fused to orthogonal x - ray images of the rat on the treatment table taken with the trilogy 's on - board imaging ( obi ) system . the drrs are weighted on bone density that highlights the skull and other bony landmarks which facilitate co - registration with the obi images . matching of these sets provide treatment table position shifts that must be made to achieve the co - registration of the ct and x - ray images . , animals were perfused , brains were sectioned and stained with brdu ( to detect transplanted nscs ) and counter stained with hematoxylin . the needle track ( nt , red line ) , indicates the injection trajectory that deposited nscs at the transplant - release site ( tr ) , just below the corpus callosum ( cc ) and above the ca1 . transplanted nscs showed extensive migration from tr throughout the host hippocampus ( dentate gyrus , dg ; dentate hilus , dh ; ca1 and ca3 subfields ; magnification x4 ) . the enstem - a cell line ( emd millipore ) was used in this study . nscs are routinely validated by the manufacturer for high levels of expression of the multipotent markers nestin and sox2 , and low - level expression of the pluripotent marker oct-4 , along with the ability to differentiate into multiple neuronal phenotypes and to maintain a normal karyotype after multiple passages . under our growth conditions , these nscs displayed abundant expression of sox2 and nestin and retained their ability to differentiate , as described previously . nscs were grown on poly - l - ornithine ( 20 g / ml ) and laminin ( 5 g / ml ) coated tissue culture treated flasks . cells were grown in neural expansion medium ( millipore ) supplemented with l - glutamine ( 2 mm ) and basic fibroblast growth factor ( bfgf ; 20 ng / ml ) . adherent monolayers of nscs were passaged every other day ( 1:2 ) with accutase as the dissociation agent . for transplantation studies , nscs were labeled with 5-bromo-2'-deoxyuridine ( brdu ) by supplementing the growth media with 4 m brdu for three days prior to transplantation . to verify the brdu labeling index ( i.e. percentage of brdu positive cells ) , cells were plated on chamber slides and processed for brdu detection using standard immunocytochemical approaches . alternatively , transplanted human stem cells were detected using the human specific nuclear antigen marker ( hunu ) . on the day of transplantation , accutase and neural expansion media were pre - warmed in a 37c water bath . cells were placed in incubation medium ( neural expansion media containing 10 m of y-27632 ) for one hour . y-27632 ( rock inhibitor , emd - calbiochem ) was used to improve the survival of nscs post - transplantation . after one hour , incubation media was removed and cells were treated with accutase for 5 minutes . after this brief treatment add an equal volume of neural expansion media to neutralize the accutase and strain the cells through a 70 m cell strainer . count the cells with a haemocytometer and prepare 1.0 x 10 live nscs per microliter in injection medium ( neural expansion media containing 10 m of y-27632 , 40 ng / ml bfgf , 20 ng / ml bdnf ) . cells were stored in neural expansion media ( as described in 1.5 ) and kept on ice until the time of transplantation , and should be used within 6h to minimize cell death by prolonged cold - storage . sedated athymic nude rat(s ) were placed in a laboratory mri scanner in the prone position with the skull at the superior end ( head first ) . . it can provide exquisite soft - tissue contrast with high spatial resolution cross - sectional ( axial ) images . the scan focused on the skull region and a set of 22 t2 e60 weighted images with 0.8 mm image thickness were generated . these images provide necessary information to identify the left and right hippocampi within the rat 's brain . following the mri scan ( 24h ) , animals were sedated [ anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) ] and placed in a radiation oncology ct scanner where another cross - sectional ( axial ) image volume was generated . the rat was placed in the same or close to the same position as used for the mri scan and the ct unit was set to scan the skull region . a study of 106 ct images with 0.8 mm image thickness was generated which became the ct treatment planning data . treatment planning the mri and ct image data were transferred to the eclipse ( varian medical systems , palo alto , ca ) treatment planning software via dicom ( digital - imaging and communications in medicine ) . eclipse is a sophisticated and commercially available software used in radiation oncology to model and design the best radiation treatment plan for a patient . it incorporates organ density information that is extracted from the ct image data and used to calculate the radiation dose distribution within the body.cover the animal using a small surgical drape with only the head exposed . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp.center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero.the next step in the process is to determine the irradiation technique to be used . intensity - modulated radiation therapy ( imrt ) and volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc are 2 high - precision irradiation techniques that use a 6 mv photon beam to deliver a radiation dose . the difference between these techniques is that imrt delivers a dose using several static trajectories each corresponding to a different direction but converging at the target volume more suitable for extremely small target volumes . rapidarc on the other hand , delivers a dose dynamically through one or more arcs focused on the target region ( fig . 4).regardless of delivery technique , information on target dose , target conformity and dose constraints to critical organs have to be entered in the dose optimization window in eclipse . this information along with organ volumes is used to tailor the dose distribution , which is achieved by continually varying the beam attenuation during irradiation.once a plan was generated , eclipse provides the calculated dose superimposed to the axial , coronal and sagittal images ( fig . 5 ) as well as in dose - volume histogram ( dvh ) format ( fig . 6 ) . it is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved . the mri and ct image data were transferred to the eclipse ( varian medical systems , palo alto , ca ) treatment planning software via dicom ( digital - imaging and communications in medicine ) . eclipse is a sophisticated and commercially available software used in radiation oncology to model and design the best radiation treatment plan for a patient . it incorporates organ density information that is extracted from the ct image data and used to calculate the radiation dose distribution within the body . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp . center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero . the next step in the process is to determine the irradiation technique to be used . intensity - modulated radiation therapy ( imrt ) and volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc are 2 high - precision irradiation techniques that use a 6 mv photon beam to deliver a radiation dose . the difference between these techniques is that imrt delivers a dose using several static trajectories each corresponding to a different direction but converging at the target volume more suitable for extremely small target volumes . rapidarc on the other hand , delivers a dose dynamically through one or more arcs focused on the target region ( fig . information on target dose , target conformity and dose constraints to critical organs have to be entered in the dose optimization window in eclipse . this information along with organ volumes is used to tailor the dose distribution , which is achieved by continually varying the beam attenuation during irradiation . once a plan was generated , eclipse provides the calculated dose superimposed to the axial , coronal and sagittal images ( fig . 5 ) as well as in dose - volume histogram ( dvh ) format ( fig . 6 ) . it is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved . irradiation process once a plan was approved for delivery , digitally - reconstructed radiograph ( drr ) images were generated from the ct treatment planning data in eclipse . these are orthogonal images weighted on bone density to highlight the skull and other bony landmarks . thereafter , treatment plan and drrs are sent to the treatment delivery computer system via dicom.the delivery system controls a varian trilogy linear accelerator normally used for radiotherapy of humans . the accelerator is equipped with 120 computer controlled thin multileaf collimators ( mlc ) and a diagnostic quality x - ray imaging system included within the on - board imaging ( obi ) system . this delivery system uploads the eclipse - specific information and reconfigures the collimator parameters in the accelerator for delivering the planned dose.at this point the rat was prepared for irradiation , sedated and covered with a paper blanket to keep it warm . after a few minutes , the rat , with its blanket but with the skull exposed , was placed on the treatment table in the same position used to generate the ct scan . this is a critical step because position accuracy is directly correlated to accuracy in dose delivery.once the rat was positioned properly on the treatment table , a set of orthogonal x - ray images is taken using the trilogy 's obi system . these are high resolution images that were subsequently fused to the eclipse generated drrs using the obi image fusion software.the x - ray images and drrs were digitally matched using software that provide information on table position shifts that must be made to achieve co - registration of the images ( fig . 7 ) . after reviewing this information and verifying that the resultant shifts do not inadvertently lead into any collisions between the accelerator and the treatment table , the computer applies the shifts and the treatment table moves automatically to the desired position.at this point , irradiation commences . to deliver a 10 gy dose with a 6-field imrt plan , the beam - on time is approximately 10 minutes while a two - arc rapidarc takes ~3 minutes . after treatment delivery is complete , the rat was removed from the treatment room and allowed to recover in a holding cage kept on a heating pad . once a plan was approved for delivery , digitally - reconstructed radiograph ( drr ) images were generated from the ct treatment planning data in eclipse . these are orthogonal images weighted on bone density to highlight the skull and other bony landmarks . thereafter , treatment plan and drrs are sent to the treatment delivery computer system via dicom . the delivery system controls a varian trilogy linear accelerator normally used for radiotherapy of humans . the accelerator is equipped with 120 computer controlled thin multileaf collimators ( mlc ) and a diagnostic quality x - ray imaging system included within the on - board imaging ( obi ) system . this delivery system uploads the eclipse - specific information and reconfigures the collimator parameters in the accelerator for delivering the planned dose . at this point the rat was prepared for irradiation , sedated and covered with a paper blanket to keep it warm . after a few minutes , the rat , with its blanket but with the skull exposed , was placed on the treatment table in the same position used to generate the ct scan . this is a critical step because position accuracy is directly correlated to accuracy in dose delivery . once the rat was positioned properly on the treatment table , a set of orthogonal x - ray images is taken using the trilogy 's obi system . these are high resolution images that were subsequently fused to the eclipse generated drrs using the obi image fusion software . the x - ray images and drrs were digitally matched using software that provide information on table position shifts that must be made to achieve co - registration of the images ( fig . 7 ) . after reviewing this information and verifying that the resultant shifts do not inadvertently lead into any collisions between the accelerator and the treatment table , the computer applies the shifts and the treatment table moves automatically to the desired position . at this point , the beam - on time is approximately 10 minutes while a two - arc rapidarc takes ~3 minutes . after treatment delivery is complete , the rat was removed from the treatment room and allowed to recover in a holding cage kept on a heating pad . animals husbandry and surgical preparation for this study , we employed two month old atn rats acquired from national cancer institute ( strain 0n01 cr : nih - rnu ) . animals were kept in sterile cages and maintained in a temperature- and light controlled barrier - a environment with 12-h/12-h light / dark cycle . rats were provided autoclaved food and water ad libitum and care was taken to prevent eye infection by cleaning eyes every week with vetropolycin eye ointment ( western medical supply , arcadia , ca).for stereotaxic surgery , rats were anesthetized with an i.p . injection of anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) . complete anesthesia was induced after 10 - 15 minutes and sedation was monitored using palpable reflex ( toe pinch ) . if required during surgery , 15 - 20% of the original dose may be given to maintain sufficient anesthesia.remove fur from head using electric clippers . clean shaved part using providone / iodine ( 3 times ) followed by 70% alcohol ( 3 times ) before incision .
for this study , we employed two month old atn rats acquired from national cancer institute ( strain 0n01 cr : nih - rnu ) . animals were kept in sterile cages and maintained in a temperature- and light controlled barrier - a environment with 12-h/12-h light / dark cycle . rats were provided autoclaved food and water ad libitum and care was taken to prevent eye infection by cleaning eyes every week with vetropolycin eye ointment ( western medical supply , arcadia , ca ) . for stereotaxic surgery injection of anesthesia cocktail ( ketamine , 30 mg / kg , xylazine , 2.5 mg / kg and acepromazine , 1 mg / kg ) . complete anesthesia was induced after 10 - 15 minutes and sedation was monitored using palpable reflex ( toe pinch ) . if required during surgery , 15 - 20% of the original dose may be given to maintain sufficient anesthesia . clean shaved part using providone / iodine ( 3 times ) followed by 70% alcohol ( 3 times ) before incision . stereotaxic surgery stereotaxic instruments ( digital monitor and frame ) , micromotor drill , dry bed sterilizer and controllers should be kept in a laminar flow hood to prevent possible infection during surgery procedure in atn rats . a stereotaxic stage with an embedded heating pad can be used to keep the animals warm during surgery . we used ' kimberly - clark safeskin purple nitrile sterile exam gloves ' thoughout the surgery . in addition , surgeon used 70% alcohol spray to sterilize hands during surgery.cover the animal using a small surgical drape with only the head exposed . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp.center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero.following these positioning steps , apply lubricating eye ointment to prevent drying and protect them from possible iodine or alcohol spills . use this ointment at least 2 times during surgery.make midline skin incision ( 2 cm ) along scalp using sterile scalpel , and clean using sterile cotton tipped applicator . avoid damage in caudal and neck muscles areas.by using dissecting retractor , hold the periosteum open and clear soft tissue using cotton tipped applicator dipped in 1% hydrogen peroxide ( made in pbs ) . to stop bleeding , maintain firm pressure for at least 1 min using cotton tipped applicator or gauze . apply firm scraping motion to clean skull and dabbing motion for skin until skull sutures , bregma and lambda were visible.precise stereotaxic coordinates , referenced to the bregma , were determined using the rat brain atlas . stem cell transplantation was done at four distinct sites for each hemisphere using the following stereotaxic coordinates : anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain.ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm
once the bregma has been identified , all three coordinates ( ap , ml and dv ) should be zeroed on the digital display / controller ( on the digital stereotaxic frame ) . then proceed to mark the precise transplantation sites ( using the foregoing coordinates ) with a fine point marker pen attached to a small probe holder on the frame.drill a 0.35 mm hole ( no . 1/4 dental burr , carbide vanadium drill bits ) through the skull using the foot pedal controlled micromotor drill ( leica - myneurolab ) . take care to prevent damage to the dura membrane . if bleeding occurs during drilling , . do not apply alcohol or iodine at the drill site , as this will cause irritation to the animal.rats received bilateral intra - hippocampal injections of a suspension of nscs injected in a maximum volume of 1 l using a 5 l hamilton microsyringe ( 30 gauge ) . the precise number of cells within this volume will vary according to experimental specifics . to accomplish this carefully insert the tip of the needle in the skull until reaching the surface of the brain ( i.e. meninges ) . at this point , the dv coordinate should be zeroed on the digital display / controller , then gently insert the needle to the desired depth ( dv , 3.2 mm ) . wait 1 min before initiating any injection of cells.inject 0.25 l volume / min ( slow release ) using a timer . once a total of 1 l is injected , wait 8 min before retracting needle from transplantation site . after this time , slowly retract the needle from the transplantation site ( 0.5 mm / min ) to prevent capillary reflux of the injection contents back through the needle track.follow steps 3.2(j - k ) for the remaining transplantation sites ( 4 per hemisphere).remove the skin retractor from the skull and use blunt tweezers to gently pull back retracted skin and apply 4 - 5 sterile stainless clips using autoclips surgical suture applier ( leica - myneurolab).after removing the animal from the frame , inject with analgesic ( buprenorphin , 0.1 mg / kg , s.c . , every 12h ) and lactated ringer 's solution ( 5 ml/250 g adult rat , s.c.).place the animal back in its cage that should be kept on a heating pad . place some moistened pellets ( animal chow ) and transgel in a separate petri dish in each animal cage . alternatively , dietgel recovery ( clearh2o , portland , me ) can be used as a source of water and nutrients.monitor animals during their recovery time and apply analgesia ( buprenorphine 0.02 mg / kg ( every 12h for 2 days ) . check for signs of pain , distress , reddening or infection of surgery site . apply providone / iodine or neosporin ointment once daily ( till 2 - 3 days ) to prevent possible infection . if any signs of infection , pain or distress persist following analgesic or antibiotic treatment within 12 hours of surgery , consult veterinarian or animal care staff for further assistance . stereotaxic instruments ( digital monitor and frame ) , micromotor drill , dry bed sterilizer and controllers should be kept in a laminar flow hood to prevent possible infection during surgery procedure in atn rats . a stereotaxic stage with an embedded heating pad can be used to keep the animals warm during surgery . we used ' kimberly - clark safeskin purple nitrile sterile exam gloves ' thoughout the surgery . position the animal head firmly within the stereotaxic frame ( benchmark digital , leica - myneurolab ) by inserting ear bars into the external auditory meatus . take extreme care when sliding the tip of the ear bar into the ear canal . place the incisor bar by hooking the rat 's upper incisors and adjusting the bar height to the standard reference point , then tighten nose clamp . center the position of the head between the ear bars with minimal lateral movement ( 4 mm ) to achieve stereotaxic zero . following these positioning steps , apply lubricating eye ointment to prevent drying and protect them from possible iodine or alcohol spills . make midline skin incision ( 2 cm ) along scalp using sterile scalpel , and clean using sterile cotton tipped applicator . hold the periosteum open and clear soft tissue using cotton tipped applicator dipped in 1% hydrogen peroxide ( made in pbs ) . to stop bleeding , maintain firm pressure for at least 1 min using cotton tipped applicator or gauze . apply firm scraping motion to clean skull and dabbing motion for skin until skull sutures , bregma and lambda were visible . precise stereotaxic coordinates , referenced to the bregma , were determined using the rat brain atlas . stem cell transplantation was done at four distinct sites for each hemisphere using the following stereotaxic coordinates : anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain.ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2mmap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm
anterio - posterior ( ap ) 3.0 mm from bregma , medio - lateral ( ml ) 1.8 mm from midline , and dorso - ventral ( dv ) 3.2 mm from the surface of the brain . ap , 3.6 mm ; ml , 2.5 mm ; dv , 3.2 mm ap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm ap , 4.2 mm ; ml , 3.2 mm ; dv , 3.2 mm once the bregma has been identified , all three coordinates ( ap , ml and dv ) should be zeroed on the digital display / controller ( on the digital stereotaxic frame ) . then proceed to mark the precise transplantation sites ( using the foregoing coordinates ) with a fine point marker pen attached to a small probe holder on the frame . 1/4 dental burr , carbide vanadium drill bits ) through the skull using the foot pedal controlled micromotor drill ( leica - myneurolab ) . . do not apply alcohol or iodine at the drill site , as this will cause irritation to the animal . rats received bilateral intra - hippocampal injections of a suspension of nscs injected in a maximum volume of 1 l using a 5 l hamilton microsyringe ( 30 gauge ) . the precise number of cells within this volume will vary according to experimental specifics . to accomplish this , carefully insert the tip of the needle in the skull until reaching the surface of the brain ( i.e. meninges ) . at this point , the dv coordinate should be zeroed on the digital display / controller , then gently insert the needle to the desired depth ( dv , 3.2 mm ) . once a total of 1 l is injected , wait 8 min before retracting needle from transplantation site . after this time , slowly retract the needle from the transplantation site ( 0.5 mm / min ) to prevent capillary reflux of the injection contents back through the needle track . follow steps 3.2(j - k ) for the remaining transplantation sites ( 4 per hemisphere ) . remove the skin retractor from the skull and use blunt tweezers to gently pull back retracted skin and apply 4 - 5 sterile stainless clips using autoclips surgical suture applier ( leica - myneurolab ) . after removing the animal from the frame , , every 12h ) and lactated ringer 's solution ( 5 ml/250 g adult rat , s.c . ) . place the animal back in its cage that should be kept on a heating pad . place some moistened pellets ( animal chow ) and transgel in a separate petri dish in each animal cage . alternatively , dietgel recovery ( clearh2o , portland , me ) can be used as a source of water and nutrients . monitor animals during their recovery time and apply analgesia ( buprenorphine 0.02 mg / kg ( every 12h for 2 days ) . check for signs of pain , distress , reddening or infection of surgery site . apply providone / iodine or neosporin ointment once daily ( till 2 - 3 days ) to prevent possible infection . if any signs of infection , pain or distress persist following analgesic or antibiotic treatment within 12 hours of surgery , consult veterinarian or animal care staff for further assistance . . fusion of ct ( bony anatomy ) and mri ( soft - tissue ) images within the eclipse software . matching of axial , coronal , and sagittal sections allow for the co - registry of the critical anatomical features of the rat brain derived from each imaging modality . subsequent to image fusion , hippocampi and brain excluding hippocampi are identified and contoured axially defining specific volumetric regions for these organs . these regions provide anatomical and volume information necessary for adjusting the dose distribution to the desired target(s ) . high precision irradiation options using intensity - modulated radiation therapy ( imrt ) or volumetrically - modulated arc therapy ( vmat ) in the form of rapidarc . these techniques deliver 6 mv photon beams either as multiple static trajectories that can converge on very small target volumes ( imrt ) , or dynamically as one or more arcs focused on the target region ( rapidarc ) . data contrasts the percentage of the irradiated versus the non - irradiated hippocampal volume under the single hippocampus treatment plan shown in fig . orthogonal digitally - reconstructed radiograph ( drr ) images generated from the ct treatment planning data in eclipse are fused to orthogonal x - ray images of the rat on the treatment table taken with the trilogy 's on - board imaging ( obi ) system . the drrs are weighted on bone density that highlights the skull and other bony landmarks which facilitate co - registration with the obi images . matching of these sets provide treatment table position shifts that must be made to achieve the co - registration of the ct and x - ray images . animals were perfused , brains were sectioned and stained with brdu ( to detect transplanted nscs ) and counter stained with hematoxylin . the needle track ( nt , red line ) , indicates the injection trajectory that deposited nscs at the transplant - release site ( tr ) , just below the corpus callosum ( cc ) and above the ca1 . transplanted nscs showed extensive migration from tr throughout the host hippocampus ( dentate gyrus , dg ; dentate hilus , dh ; ca1 and ca3 subfields ; magnification x4 ) . considerable research is underway exploring the myriad of ways that stem cells can be used clinically to restore normal functions to damaged , aged and diseased tissues . the eventual realization of these efforts will require a detailed understanding of the behavior of engrafted cells within unique microenvironments that are distinct from undamaged normal tissue . our work has demonstrated that within the irradiated tissue bed , cranially engrafted nscs can functionally restore cognition , where they survive , migrate and differentiate along neural and glial lineages . precisely how these cells mediate recovery of cognition is uncertain at present , but does depend on conducting a series of carefully controlled experimental procedures in a reproducible manner . we have detailed these critical procedures here in efforts to expedite the translational potential of stem cell therapies for ameliorating adverse cognitive effects associated with the clinical management of brain and other forms of cancer . additional considerations that are likely to have a significant impact of the quality of data are highlighted below . transplantation based therapies depend on stem cells as the critical reagent , and accordingly , care must be exercised to properly characterize cultures , maintain sterility and use matched passage numbers for reliability of results . transplanted human stem cells were labeled with brdu prior to surgery , to provide one means for tracking them in vivo . under our experimental conditions , transplanted nscs did not undergo extensive proliferation , so that dilution of the brdu label was not problematic . alternatively , transplanted human stem cells can be distinguished from host cells by immunostaining for human specific markers such as nuclear matrix protein ( h - nuc or hnuma ) or human - specific nuclear antigen ( hunu ) . human stem cells could also be labeled with a variety of fluorescent markers to facilitate their identification within the host brain . attention to irradiation parameters define precise dose delivery techniques , and those described here model current clinical practices in radiation oncology . reproducible transplantation of stem cells in the brain is also critical , and is accomplished using a digital stereotaxic instrument . the capability to precisely micromanipulate a micro - syringe for stem cell implantation in small brain structures like the hippocampus greatly reduces human error . with this apparatus , nscs were transplanted in four distinct sites spanning the anterior to posterior regions of the rat hippocampus . dorso - ventral ( dv ) coordinates were determined based on experience with athymic nude ( atn ) rats such that surgical procedures would not cause damage to the hippocampal formation . a coronal section through the rat brain reveals key structures of the hippocampal formation including the dentate gyrus ( dg ) , the dentate hilus ( dh ) and ca1 and ca3 subfields ( fig . 8) . transplanted nscs , introduced dorsally from the visible needle tract ( nt , redline ) , are visualized as darkly staining ( brown ) cells deposited at the transplant - release site ( tr ) just below the corpus callosum ( cc ) , that subsequently migrate throughout the septo - temporal axis of the hippocampus . | radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors . while beneficial
, cranial irradiation can induce a progressive and debilitating decline in cognition that may , in part , be caused by the depletion of neural stem cells . given
the increased survival of patients diagnosed with brain cancer , quality of life in terms of cognitive health has become an increasing concern , especially in the absence of any satisfactory long - term treatments.to address this serious health concern we have used stem cell replacement as a strategy to combat radiation - induced cognitive decline .
our model utilizes athymic nude rats subjected to cranial irradiation .
the ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator ( linac ) based stereotaxic radiosurgery .
two days following irradiation , human neural stem cells ( hnscs ) were stereotaxically transplanted into the hippocampus .
rats were then assessed for changes in cognition , grafted cell survival and for the expression of differentiation - specific markers 1 and 4-months after irradiation .
our cognitive testing paradigms have demonstrated that animals engrafted with hnscs exhibit significant improvements in cognitive function .
unbiased stereology reveals significant survival ( 10 - 40% ) of the engrafted cells at 1 and 4-months after transplantation , dependent on the amount and type of cells grafted .
engrafted cells migrate extensively , differentiate along glial and neuronal lineages , and express a range of immature and mature phenotypic markers.our data demonstrate direct cognitive benefits derived from engrafted human stem cells , suggesting that this procedure may one day afford a promising strategy for the long - term functional restoration of cognition in individuals subjected to cranial radiotherapy . to promote the dissemination of the critical procedures necessary to replicate and extend our studies ,
we have provided written and visual documentation of several key steps in our experimental plan , with an emphasis on stereotaxic radiosurgey and transplantation . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
plasma membrane transporters are the entry gate for most of the nutrients used by cells to generate energy and building blocks for cellular maintenance , growth and development . in this sense , they may exert important control over all cellular activities , either as simple providers of the nutrient molecules into the cellular interior or because they may signal the presence of the extracellular nutrients to the cellular machinery . a striking example of this sensing role of transporters has been discovered in yeast where multiple nutrient transporters appear to control activation of the protein kinase a pathway at the onset of fermentable growth induction . when yeast cells grow on a fermentable sugar , like glucose , they display high activity of the protein kinase a ( pka ) pathway , causing low levels of storage carbohydrates , low stress tolerance , and high growth and fermentation rates . when such fermenting cells are starved for a single essential nutrient , like nitrogen , phosphate or sulfate , they will arrest growth , reduce their fermentation rate , accumulate high levels of storage carbohydrates and develop high stress tolerance , indicating that the activity of the pka pathway is being downregulated ( thevelein and de winde 1999 ) . re - addition of the lacking nutrient triggers rapid reversal to a high - pka phenotype and one of the earliest read - outs is activation of the pka phosphorylation target trehalase ( hirimburegama et al . transporters have been shown to play an essential role as receptors in this activation process : gap1 for activation with amino acids ( donaton et al . 2006 ) , pho84 for phosphate ( giots et al . 2003 ; popova et al . 2010 ) and sul1,2 for sulfate ( kankipati et al . , in preparation ) . because of their double function as transporter and receptor , we have called such proteins transceptors ( holsbeeks et al . transporters are well known to function either as passive transport systems , carrying molecules down their concentration gradient , or as active transport systems , using energy to carry molecules uphill against their concentration gradient . active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an atp molecule or by carriers which make use of a pre - established electrochemical ion gradient to drive co - transport of the nutrient molecule and a co - transported ion . the latter category comprises symporters and antiporters , which carry the ion in the same or opposite direction , respectively , as the transported substrate . in recent years , much insight has been gained in the structure and functioning of transporters , due to the successes in crystallization of substrate transporter complexes and determination of their 3d - structures , for instance lacy , glpt , fucp and pipt . in spite of this one of these is the identity of the amino acid residues that are responsible for binding the co - transported ion during its passage through the transporter . site - directed mutagenesis of candidate residues , that can bind a co - transported ion and are located in or close to a transmembrane domain , would normally be the method of choice . however , replacement of such residues can abolish ion coupling in transport but can also affect the whole functionality of the transporter , because the residue is in some way important for maintenance of the proper structure , for insertion in the membrane or for another feature of the transporter that is essential for transport . this makes it difficult to draw a definite conclusion about the precise role of the residue . the discovery of the yeast transceptors , in which the signaling function was found to be independent from the transport function , provides a novel approach to assess the general functionality of the transporter and may therefore help in providing evidence that a certain amino acid residue is truly involved in binding the co - transported ion . substrate - induced endocytic internalization and sorting to the vacuole is a well - known mechanism by which cells regulate the level of transporter in the plasma membrane as a function of external substrate availability . the prevailing idea in the literature is that transport of the substrate through the transporter generates in some way a signal and/or makes the transporter susceptible for ubiquitination ( cain and kaiser 2011 ; seron et al . the latter is then thought to serve as the signal for endocytosis , which is followed by sorting and breakdown of the transporter in the vacuole ( dupre et al . 2004 ; gitan and eide 2000 ; liu et al . 2007 ; hicke and dunn 2003 ; nikko et al . 2008 ; eguez et al . 2004 ; lauwers et al . 2010 ; horak 2003 ; shih et al . the compounds that we have developed to study the relationship between transport and signaling in the gap1 transceptor have now been used as novel tools to study the connection between the different events happening in substrate - induced internalization of the transceptors . this has led to the unexpected finding that these events can be uncoupled in several previously unanticipated ways . identification of proton - binding residues in a few model transporters has been inferred mainly from site - directed mutagenesis , crystal structure determinations and the suggested modus operandi of the transporter . the modus operandi of individual protons in a proton - driven uptake system can be dissected in three related aspects : ( 1 ) coupling between substrate binding / dissociation and conformational changes in the transporter ; ( 2 ) coupling between the substrate binding / dissociation and protonation / deprotonation of residues ; and ( 3 ) coupling between protonation / deprotonation and conformational changes in the transporter . most frequently these are glu / asp / his residues , and to a lesser extent lys / arg / tyr . the lactose permease of escherichiacoli , lacy , serves as a paradigm when assessing proton - driven transport ( abramson et al . 2004 ) . a tightly interconnected hydrogen bond and salt bridge cluster composed of glu325 , lys319 and his322 ( tm10 ) , arg302 ( tm9 ) , glu269 ( tm8 ) , and tyr236 and asp240 ( tm7 ) , can be found in the crystal structure . glu325 , his322 and arg302 are thought to be directly involved in proton translocation ( kaback et al . 2001 ) . due to the lack of a potential hydrogen bond donor in the immediate vicinity , glu325 is believed to be protonated , and thus prevents h escape from the cluster , maintaining coupling with the sugar - binding site ( smirnova et al . the involvement and importance of protons can be seen in a brief description of what is believed to be the main transport mechanics : ( 1 ) the co - apo conformation is immediately protonated . the h is shared by glu269 and his322 ; ( 2 ) the substrate is initially recognized by trp151 , arg144 and glu126 . this will lead to a disruption of the salt bridge between arg144 and glu126 , bringing his322 in contact with glu325 . this may induce proton transfer from his322 to glu325 , leading to a rapid conformational change and to the cytoplasm facing conformation , ci ; ( 3 ) the substrate is released into the cytoplasm ; ( 4 ) the salt bridge between arg144 and glu126 is re - established . it has been suggested that arg302 could interact with glu325 to drive proton release from glu325 because mutants in either residue exhibit the same specific defect in proton - coupled lactose translocation reactions , with no effect on sugar binding , exchange or counterflow ( sahin - toth and kaback 2001 ) . a similar protonation / deprotonation mechanism has been proposed to play an important role in proton - dependent oligopeptide transporter ( pot ) family members . the crystal structures of oligopeptide transporters from the bacteria shewanella oneidensis ( peptso ) ( newstead et al . 2011 ) , streptococcus thermophilus ( peptst ) ( solcan et al . 2012 ) and geobacillus kaustophilus ( gkpot ) ( doki et al . they have suggested mechanisms involved in proton - coupled peptide transport . in case of the gkpot , the positive charge of arg43 may facilitate the deprotonation of glu310 during the transition to the occluded , apo state , which allows the formation of a salt bridge between arg43 and glu310 . mutagenesis of glu310 to gln resulted in an inactive transporter , suggesting that glu310 is involved in h binding / translocation , by blocking the transition of the transporter between the inward- and outward - open states ( doki et al . furthermore , glu32 has been proposed to be of importance ( based on molecular dynamics simulations ) in the transition mechanism between outward - facing and occluded states . it has been proposed that glu32 is another protonation site , and the h translocation occurs between glu32 and glu310 ( similar to the h translocation from his322-glu269 to glu325 in lacy ) . for the human pot members pept1 and pept2 , his57 and his87 , respectively , have been suggested as primary protonation sites based on results of site - directed mutagenesis ( fei et al . 2003 ) , whereas in case of the peptso his61 was suggested to be the primary protonation site based on the crystal structure determination ( newstead et al . the proposed mechanism for fucose : h transport is also believed to involve residues that undergo a protonation / deprotonation cycle . along the transport pathway residues asp46 and glu135 ( 3rd helical turn of tm1 and tm4 , respectively ) are thought to be involved in this protonation / deprotonation cycle . transport studies , involving site - directed mutagenesis , have confirmed that these residues play an important role . asp46 is believed to be essential for proton - dependent active transport , whereas glu135 might be involved in substrate recognition ( dang et al . 2010 ) . moreover , the proposed mechanism and involvement of these two residues is as follows : ( 1 ) in the co - apo conformation , l - fucose can only bind following protonation of asp46 . this protonation step will neutralize the negative charge and thus lower the energy barrier for the l - fucose entry / transport ; ( 2 ) proton translocation from asp46 to glu135 will result in abolishment of the hydrogen bond between glu135 and tyr365 enabling binding of the substrate with the protonated glu135 ; ( 3 ) the protonation and binding of the substrate to glu135 will trigger rigid body rotation of the n- and c - domains , resulting in the ci conformation of the transporter . the transport cycle is completed with the deprotonation of glu135 and the release of the substrate into the cytoplasm . in the case of the p. indica phosphate transporter , ( pipt ) , , asp324 is proposed to be protonated , which gives preference to phosphate binding . in case of the saccharomyces cerevisiae high - affinity inorganic phosphate transporter , pho84 , site - directed mutagenesis studies have shown that asp358 may play a role in proton - coupled phosphate transport activity ( samyn et al . pho84 has been shown to be a transceptor for activation of the pka pathway and previous work has shown that transport of substrate is not required for the induction of signaling by the receptor function of pho84 ( popova et al . interestingly , mutagenesis of asp358 in pho84 abolished transport but left signaling largely unaffected ( samyn et al . 2012 ) . this shows that mutagenesis of asp358 does not significantly compromise proper membrane insertion and general functionality of pho84 , reinforcing the suggestion from the site - directed mutagenesis that asp358 is specifically required for proton binding . in addition , it confirmed that substrate transport is not required for signaling by the pho84 transceptor . a similar result has been obtained , recently , for the sul1 and sul2 sulfate transporters . site - directed mutagenesis of a putative proton - binding residue abolished transport without affecting signaling . this supports a role for this residue in proton binding , shows that also in this case substrate transport is not required for signaling and provides a strong argument that sul1,2 function as sulfate transceptors ( kankipati et al . mutagenesis of asp178 in pho84 also allowed partial uncoupling of transport and signaling ( samyn et al . this has also been achieved for the gap1 transceptor by deletion of nhx1 or pmp3 , two gap1-interacting proteins . deletion of nhx1 or pmp3 strongly reduced amino acid uptake by gap1 , but did not affect signaling at all ( van zeebroeck et al . all these data support that the transport and signaling functions of the transceptors are not dependent on each other . hence , site - directed mutagenesis of putative residues involved in binding the co - transported ion can be a very efficient approach to separate the signaling from the transport function and in this way establish the capacity of a transporter to function as a transceptor.fig . 1model of the pho84 phosphate transceptor with two putative proton - binding residues allowing uncoupling of transport and signaling . the protein has 12 predicted transmembrane domains and functions as a phosphate / proton symporter . d178 and d358 are two conserved putative proton - binding residues , predicted to be located adjacent to the phosphate translocation pathway . mutagenesis of d178 to e or n reduces v
max with about 50 % , but does not lower signaling as measured by phosphate - induced activation of the pka target trehalase . mutagenesis of d358 to n strongly reduces transport to 20 % but does not affect signaling . mutagenesis of d358 to e completely abolishes transport but only causes a 50 % drop in signaling model of the pho84 phosphate transceptor with two putative proton - binding residues allowing uncoupling of transport and signaling . the protein has 12 predicted transmembrane domains and functions as a phosphate / proton symporter . d178 and d358 are two conserved putative proton - binding residues , predicted to be located adjacent to the phosphate translocation pathway . mutagenesis of d178 to e or n reduces v
max with about 50 % , but does not lower signaling as measured by phosphate - induced activation of the pka target trehalase . mutagenesis of d358 to n strongly reduces transport to 20 % but does not affect signaling . mutagenesis of d358 to e completely abolishes transport but only causes a 50 % drop in signaling amino acid residues with a negatively charged side chain also play a role in the transport mechanism of yeast mitochondrial carriers ( kunji and robinson 2010 ) . again , the most common residues found in the substrate - binding site are e , and also d. these residues are d130 in the yeast phosphate carrier ( phelps and wohlrab 1991 ) , e600 in the yeast aspartate / glutamate carrier ( cavero et al . 2000 ) and e24 in the yeast gtp / gdp carrier ( vozza et al . 2004 ) . despite belonging to the class of sodium - dependent secondary transport proteins , the plasma membrane glutamate transporters are somewhat odd class members . these transporters utilize the downhill movement of na and k ions to transport glutamate ( kanner and bendahan 1982 ) , but in addition , it has been suggested that protons are co - transported together with glutamate ( zerangue and kavanaugh 1996 ) . in case of the excitatory amino acid carrier ( eaac1 ) , glutamate can only bind to the protonated form of the transporter , and upon translocation the released glutamate and h will cause relocation of its binding sites to the basic , unprotonated state ( watzke et al . 2000 ) . site - directed mutagenesis and kinetic measurements support the conclusion that e373 serves as a proton acceptor ( grewer et al . furthermore , initially h295 was thought to be involved also in proton co - transport . this notion has been refuted since the replacement of h295 with glutamine , an amino acid residue that can not be protonated , generates a fully functional transporter with transport kinetics that are close to those of the wild - type eaac1 . h295 is now deemed to play a rather secondary role as protonation - dependent modulator , where the protonated h295 dramatically decreases the affinity of the transporter for glutamate ( tao and grewer 2005 ) . the melibiose permease of e. coli accomplishes uphill transport by utilizing na , li or h as cosolute ( tsuchiya et al . several mutagenesis studies have shown that residues d19 , d55 , d59 and d124 are important for na - dependent affinity and transport of melibiose ( pourcher et al . 1991 , 1993 ; zani et al . more recently , substrate - induced irdiff spectroscopy was applied to reevaluate the importance of these four residues ( granell et al . the results suggested that only d55 and d59 are essential ligands for na , since only the d55c and d59c mutants do not exhibit any structural variation upon incubation with na . several transporters , which were originally not assigned to sequence - related families , have in common the leut - like fold . to date , from the eight transporters having the leut - like fold ( perez and ziegler 2013 ) , a detailed description of the alternating - access mechanism and the na - binding sites for the leut from the neurotransmitter : sodium symporter ( nss ) family ( weyand et al . 2008 ) , mhp1 from the nucleobase : cation symporter-1 ( ncs1 ) family ( krishnamurthy and gouaux 2012 ; krishnamurthy et al . 2009 ) and betp from the betaine / carnitine / choline transporter ( bcct ) family ( perez et al . based on the crystal structure and molecular dynamics analysis , the leut from aquifex aeolicus reveals two sodium - binding sites , namely na1 and na2 . the na in na1 is coordinated by five residues , namely a22 ( tm1 ) , t254 ( tm6 ) , n27 ( tm1 ) , n286 ( tm7 ) and t254 ( tm6 ) . the coordination of the na in na2 is executed by g20 ( tm1 ) , v23 ( tm1 ) , a351 ( tm8 ) , t354 ( tm8 ) and s355 ( tm8 ) . in conclusion , putative amino acid residues responsible for the binding of co - transported ions have generally been suggested on the basis of site - directed mutagenesis studies of candidate residues located in or close to transmembrane domains . however , this is not sufficient to make a definite conclusion since replacement of an amino acid residue can affect transport activity also because it disturbs the general structure or functioning of the transporter . advanced biophysical techniques are generally required to gain additional evidence for a role of a specific residue in binding the co - transported ion . identification of proton - binding residues in a few model transporters has been inferred mainly from site - directed mutagenesis , crystal structure determinations and the suggested modus operandi of the transporter . the modus operandi of individual protons in a proton - driven uptake system can be dissected in three related aspects : ( 1 ) coupling between substrate binding / dissociation and conformational changes in the transporter ; ( 2 ) coupling between the substrate binding / dissociation and protonation / deprotonation of residues ; and ( 3 ) coupling between protonation / deprotonation and conformational changes in the transporter . most frequently these are glu / asp / his residues , and to a lesser extent lys / arg / tyr . the lactose permease of escherichiacoli , lacy , serves as a paradigm when assessing proton - driven transport ( abramson et al . 2004 ) . a tightly interconnected hydrogen bond and salt bridge cluster composed of glu325 , lys319 and his322 ( tm10 ) , arg302 ( tm9 ) , glu269 ( tm8 ) , and tyr236 and asp240 ( tm7 ) , can be found in the crystal structure . glu325 , his322 and arg302 are thought to be directly involved in proton translocation ( kaback et al . 2001 ) . due to the lack of a potential hydrogen bond donor in the immediate vicinity , glu325 is believed to be protonated , and thus prevents h escape from the cluster , maintaining coupling with the sugar - binding site ( smirnova et al . the involvement and importance of protons can be seen in a brief description of what is believed to be the main transport mechanics : ( 1 ) the co - apo conformation is immediately protonated . the h is shared by glu269 and his322 ; ( 2 ) the substrate is initially recognized by trp151 , arg144 and glu126 . this will lead to a disruption of the salt bridge between arg144 and glu126 , bringing his322 in contact with glu325 . this may induce proton transfer from his322 to glu325 , leading to a rapid conformational change and to the cytoplasm facing conformation , ci ; ( 3 ) the substrate is released into the cytoplasm ; ( 4 ) the salt bridge between arg144 and glu126 is re - established . it has been suggested that arg302 could interact with glu325 to drive proton release from glu325 because mutants in either residue exhibit the same specific defect in proton - coupled lactose translocation reactions , with no effect on sugar binding , exchange or counterflow ( sahin - toth and kaback 2001 ) . a similar protonation / deprotonation mechanism has been proposed to play an important role in proton - dependent oligopeptide transporter ( pot ) family members . the crystal structures of oligopeptide transporters from the bacteria shewanella oneidensis ( peptso ) ( newstead et al . 2011 ) , streptococcus thermophilus ( peptst ) ( solcan et al . 2012 ) and geobacillus kaustophilus ( gkpot ) ( doki et al . they have suggested mechanisms involved in proton - coupled peptide transport . in case of the gkpot , the positive charge of arg43 may facilitate the deprotonation of glu310 during the transition to the occluded , apo state , which allows the formation of a salt bridge between arg43 and glu310 . mutagenesis of glu310 to gln resulted in an inactive transporter , suggesting that glu310 is involved in h binding / translocation , by blocking the transition of the transporter between the inward- and outward - open states ( doki et al . furthermore , glu32 has been proposed to be of importance ( based on molecular dynamics simulations ) in the transition mechanism between outward - facing and occluded states . it has been proposed that glu32 is another protonation site , and the h translocation occurs between glu32 and glu310 ( similar to the h translocation from his322-glu269 to glu325 in lacy ) . for the human pot members pept1 and pept2 , his57 and his87 , respectively , have been suggested as primary protonation sites based on results of site - directed mutagenesis ( fei et al . 2003 ) , whereas in case of the peptso his61 was suggested to be the primary protonation site based on the crystal structure determination ( newstead et al . the proposed mechanism for fucose : h transport is also believed to involve residues that undergo a protonation / deprotonation cycle . along the transport pathway residues asp46 and glu135 ( 3rd helical turn of tm1 and tm4 , respectively ) are thought to be involved in this protonation / deprotonation cycle . transport studies , involving site - directed mutagenesis , have confirmed that these residues play an important role . asp46 is believed to be essential for proton - dependent active transport , whereas glu135 might be involved in substrate recognition ( dang et al . 2010 ) . moreover , the proposed mechanism and involvement of these two residues is as follows : ( 1 ) in the co - apo conformation , l - fucose can only bind following protonation of asp46 . this protonation step will neutralize the negative charge and thus lower the energy barrier for the l - fucose entry / transport ; ( 2 ) proton translocation from asp46 to glu135 will result in abolishment of the hydrogen bond between glu135 and tyr365 enabling binding of the substrate with the protonated glu135 ; ( 3 ) the protonation and binding of the substrate to glu135 will trigger rigid body rotation of the n- and c - domains , resulting in the ci conformation of the transporter . the transport cycle is completed with the deprotonation of glu135 and the release of the substrate into the cytoplasm . in the case of the p. indica phosphate transporter , ( pipt ) , , asp324 is proposed to be protonated , which gives preference to phosphate binding . in case of the saccharomyces cerevisiae high - affinity inorganic phosphate transporter , pho84 , site - directed mutagenesis studies have shown that asp358 may play a role in proton - coupled phosphate transport activity ( samyn et al . pho84 has been shown to be a transceptor for activation of the pka pathway and previous work has shown that transport of substrate is not required for the induction of signaling by the receptor function of pho84 ( popova et al . interestingly , mutagenesis of asp358 in pho84 abolished transport but left signaling largely unaffected ( samyn et al . 2012 ) . this shows that mutagenesis of asp358 does not significantly compromise proper membrane insertion and general functionality of pho84 , reinforcing the suggestion from the site - directed mutagenesis that asp358 is specifically required for proton binding . in addition , it confirmed that substrate transport is not required for signaling by the pho84 transceptor . a similar result has been obtained , recently , for the sul1 and sul2 sulfate transporters . site - directed mutagenesis of a putative proton - binding residue abolished transport without affecting signaling . this supports a role for this residue in proton binding , shows that also in this case substrate transport is not required for signaling and provides a strong argument that sul1,2 function as sulfate transceptors ( kankipati et al . mutagenesis of asp178 in pho84 also allowed partial uncoupling of transport and signaling ( samyn et al . this has also been achieved for the gap1 transceptor by deletion of nhx1 or pmp3 , two gap1-interacting proteins . deletion of nhx1 or pmp3 strongly reduced amino acid uptake by gap1 , but did not affect signaling at all ( van zeebroeck et al . all these data support that the transport and signaling functions of the transceptors are not dependent on each other . hence , site - directed mutagenesis of putative residues involved in binding the co - transported ion can be a very efficient approach to separate the signaling from the transport function and in this way establish the capacity of a transporter to function as a transceptor.fig . 1model of the pho84 phosphate transceptor with two putative proton - binding residues allowing uncoupling of transport and signaling . the protein has 12 predicted transmembrane domains and functions as a phosphate / proton symporter . d178 and d358 are two conserved putative proton - binding residues , predicted to be located adjacent to the phosphate translocation pathway . mutagenesis of d178 to e or n reduces v
max with about 50 % , but does not lower signaling as measured by phosphate - induced activation of the pka target trehalase . mutagenesis of d358 to n strongly reduces transport to 20 % but does not affect signaling . mutagenesis of d358 to e completely abolishes transport but only causes a 50 % drop in signaling model of the pho84 phosphate transceptor with two putative proton - binding residues allowing uncoupling of transport and signaling . the protein has 12 predicted transmembrane domains and functions as a phosphate / proton symporter . d178 and d358 are two conserved putative proton - binding residues , predicted to be located adjacent to the phosphate translocation pathway . mutagenesis of d178 to e or n reduces v
max with about 50 % , but does not lower signaling as measured by phosphate - induced activation of the pka target trehalase . mutagenesis of d358 to n strongly reduces transport to 20 % but does not affect signaling . mutagenesis of d358 to e completely abolishes transport but only causes a 50 % drop in signaling amino acid residues with a negatively charged side chain also play a role in the transport mechanism of yeast mitochondrial carriers ( kunji and robinson 2010 ) . again , the most common residues found in the substrate - binding site are e , and also d. these residues are d130 in the yeast phosphate carrier ( phelps and wohlrab 1991 ) , e600 in the yeast aspartate / glutamate carrier ( cavero et al . 2000 ) and e24 in the yeast gtp / gdp carrier ( vozza et al . 2004 ) . despite belonging to the class of sodium - dependent secondary transport proteins , the plasma membrane glutamate transporters are somewhat odd class members . these transporters utilize the downhill movement of na and k ions to transport glutamate ( kanner and bendahan 1982 ) , but in addition , it has been suggested that protons are co - transported together with glutamate ( zerangue and kavanaugh 1996 ) . in case of the excitatory amino acid carrier ( eaac1 ) , glutamate can only bind to the protonated form of the transporter , and upon translocation the released glutamate and h will cause relocation of its binding sites to the basic , unprotonated state ( watzke et al . 2000 ) . site - directed mutagenesis and kinetic measurements support the conclusion that e373 serves as a proton acceptor ( grewer et al . furthermore , initially h295 was thought to be involved also in proton co - transport . this notion has been refuted since the replacement of h295 with glutamine , an amino acid residue that can not be protonated , generates a fully functional transporter with transport kinetics that are close to those of the wild - type eaac1 . h295 is now deemed to play a rather secondary role as protonation - dependent modulator , where the protonated h295 dramatically decreases the affinity of the transporter for glutamate ( tao and grewer 2005 ) . the melibiose permease of e. coli accomplishes uphill transport by utilizing na , li or h as cosolute ( tsuchiya et al . several mutagenesis studies have shown that residues d19 , d55 , d59 and d124 are important for na - dependent affinity and transport of melibiose ( pourcher et al . 1991 , 1993 ; zani et al . more recently , substrate - induced irdiff spectroscopy was applied to reevaluate the importance of these four residues ( granell et al . the results suggested that only d55 and d59 are essential ligands for na , since only the d55c and d59c mutants do not exhibit any structural variation upon incubation with na . several transporters , which were originally not assigned to sequence - related families , have in common the leut - like fold . to date , from the eight transporters having the leut - like fold ( perez and ziegler 2013 ) , a detailed description of the alternating - access mechanism and the na - binding sites for the leut from the neurotransmitter : sodium symporter ( nss ) family ( weyand et al . 2008 ) , mhp1 from the nucleobase : cation symporter-1 ( ncs1 ) family ( krishnamurthy and gouaux 2012 ; krishnamurthy et al . 2009 ) and betp from the betaine / carnitine / choline transporter ( bcct ) family ( perez et al . based on the crystal structure and molecular dynamics analysis , the leut from aquifex aeolicus reveals two sodium - binding sites , namely na1 and na2 . the na in na1 is coordinated by five residues , namely a22 ( tm1 ) , t254 ( tm6 ) , n27 ( tm1 ) , n286 ( tm7 ) and t254 ( tm6 ) . the coordination of the na in na2 is executed by g20 ( tm1 ) , v23 ( tm1 ) , a351 ( tm8 ) , t354 ( tm8 ) and s355 ( tm8 ) . in conclusion , putative amino acid residues responsible for the binding of co - transported ions have generally been suggested on the basis of site - directed mutagenesis studies of candidate residues located in or close to transmembrane domains . however , this is not sufficient to make a definite conclusion since replacement of an amino acid residue can affect transport activity also because it disturbs the general structure or functioning of the transporter . advanced biophysical techniques are generally required to gain additional evidence for a role of a specific residue in binding the co - transported ion . a major breakthrough in transceptor analysis was the identification of nontransported substrate analogues that were able to trigger the signaling function of the transceptor : l - leu - gly for gap1 ( van zeebroeck et al . 2009 ) , glycerol-3-phosphate and other organic phosphate esters for pho84 ( popova et al . 2010 ) and d - glucosamine 2-sulfate for sul1,2 ( kankipati et al . these molecules provided first of all a major new argument for the receptor function of the transporters . with these in addition , the compounds turned out to be interesting new tools for studying the signaling function of the transceptors independently of their transport function , but have also turned out to be powerful tools to investigate other outstanding questions with respect to transporter functionality and regulation . the molecular mechanisms underlying substrate - induced endocytosis of nutrient transporters have been studied in great detail in yeast , with gap1 serving as the main model system ( jauniaux and grenson 1990 ; lauwers et al . the first well - established change in the gap1 permease following addition of amino acid is its ubiquitination , which is followed by sorting to the multivesicular body ( mvb ) and degradation in the vacuole / lysosome . prevention of ubiquitination by mutagenesis of the n - terminal lys 9 and lys 16 residues , abolishes endocytosis , which has been taken as evidence that ubiquitination serves as a signal for endocytosis . this reasoning has been extended to many other transporters and ubiquitination is generally conceived as the main signal triggering endocytosis ( barberon et al . 2008 ; hicke and dunn 2003 ; liu et al . 2007 ; gitan and eide 2000 ; dupre et al . 2004 ) . the initial trigger for recruitment of the rsp5 ubiquitin ligase is not well understood , but is generally thought to be caused in some way by the transport of the substrate through the carrier . this has been concluded from the behavior of mutant forms of the transporters that displayed strongly reduced uptake capacity and were no longer endocytosed . such results have been obtained for the smf1 metal transporter ( liu and culotta 1999 ; jensen et al . 2009 ) , the fur4 uracil permease ( seron et al . 1999 ) , the pho84 phosphate transporter ( petersson et al . 1999 ; lundh et al . 2009 ) , the ftr1 iron transporter ( felice et al . 2005 ) , gap1 ( cain and kaiser 2011 ) and the sulfate transporter sul2 ( jennings and cui 2012 ) in s. cerevisiae and for the uric acid / xanthine transporter , anuapa , in aspergillus nidulans ( gournas et al . 2010 ) . for the latter transporter , 3-methylxanthine , was identified as a competitive inhibitor of transport , unable to induce endocytosis . this was taken as evidence that interaction of the substrate with the anuapa transporter was not enough to trigger endocytosis and that transport was required . the possibility that a substrate could be transported through a nutrient permease without triggering endocytosis has apparently never been considered . the discovery of the nontransported signaling agonist l - leu - gly indicated that this compound physically interacted with gap1 , which raised the question whether it would also be able to trigger endocytosis . interestingly , l - leu - gly triggered ubiquitination and endocytosis in a similar way as regular amino acids ( van zeebroeck et al . this indicates for the first time that a full transport cycle is not required to trigger endocytosis . classical receptors are well known to undergo ligand - induced endocytosis ( sorkin and von zastrow 2002 ) and transceptors thus behave for substrate - induced endocytosis in a similar way as classical receptors , as was also observed for signaling . this emphasizes again the apparent similarity between the behavior of transceptors and classical receptors ( kriel et al . also in mammalian cells , the surprising similarity between the mechanisms involved in substrate - induced transporter endocytosis and ligand - induced receptor endocytosis has been pointed out ( miranda and sorkin 2007 ) . the same is true for l - asp--phe , but this dipeptide is unable to trigger signaling . it was also unable to trigger endocytosis , but unexpectedly induced oligo - ubiquitination ( van zeebroeck et al . this provides the first indication that oligo - ubiquitination of a transporter may not be enough to trigger its endocytosis and that therefore an additional event is required . an unexpected discovery was also that some amino acids , l - lysine , l - histidine and l - tryptophan , are very well transported by gap1 but they are not able to trigger signaling ( van zeebroeck et al . examination of ubiquitination and endocytosis with these amino acids unexpectedly revealed that l - lysine transport leads to ubiquitination but not endocytosis ( fig . l - lysine even counteracts endocytosis triggered by a regular amino acid , like l - citrulline . l - histidine , on the other hand , triggered efficient ubiquitination and endocytosis ( van zeebroeck et al . these results demonstrated that signaling , ubiquitination and endocytosis can be uncoupled in different ways . there is no evidence that endocytosis can happen without ubiquitination , since gap1 mutated in the n - terminal lysines 9 and 16 , that function as ubiquitin attachment sites , is completely deficient in endocytosis ( soetens et al . 2specific compounds allow uncoupling of transport , ubiquitination , endocytosis and signaling in the gap1 transceptor . a the uptake of an amino acid triggers a ( series of ) conformational changes , of which one allows the ubiquitination of gap1 . b the dipeptide l - leu - gly is not transported but is able to trigger all subsequent events like a regular amino acid . c the dipeptide l - asp--phe is also not transported but is only able to trigger the conformation that allows ubiquitination . d
l - lysine is transported but is not able to trigger signaling nor endocytosis e
l - histidine can not trigger signaling but can induce the whole endocytic internalization process just like a regular amino acid specific compounds allow uncoupling of transport , ubiquitination , endocytosis and signaling in the gap1 transceptor . a the uptake of an amino acid triggers a ( series of ) conformational changes , of which one allows the ubiquitination of gap1 . b the dipeptide l - leu - gly is not transported but is able to trigger all subsequent events like a regular amino acid . c the dipeptide l - asp--phe is also not transported but is only able to trigger the conformation that allows ubiquitination . d
l - lysine is transported but is not able to trigger signaling nor endocytosis . however , it can also induce the conformation that elicits ubiquitination . e
l - histidine can not trigger signaling but can induce the whole endocytic internalization process just like a regular amino acid these new results on substrate - induced signaling , ubiquitination and endocytosis of transceptors may also indicate that different substrates follow a somewhat different passageway when carried through the transporter , and/or that they trigger different conformational changes during their passage . alternatively , the different substrates may trigger the same series of conformational changes but with different kinetics . to trigger a downstream event , a specific conformation would have to persist long enough to allow proper interaction with a signal transmission protein or a protein that in some way supports initiation of ubiquitination or endocytosis . several examples have been reported where transporters appeared to carry out an additional regulatory function triggered by sensing of the nutrient ( gojon et al . 2011 ; hundal and taylor 2009 ; rogato et al . however , as opposed to the situation with the yeast transceptors controlling activation of the pka pathway , no common principles , either for mechanisms involved or targets affected , have been identified . in spite of this , the use of substrate analogues may turn out to be a very useful approach to gain first of all strong evidence for the presence of an additional receptor function in the transporter and second to learn about the mechanisms involved in the signaling function . substrate - induced internalization of nutrient transporters has also been documented in other organisms . as mentioned previously , detailed studies have been made on the uric acid / xanthine transporter , anuapa , in a. nidulans ( gournas et al . also in this case , substrate analogues have been used to gain insight in the underlying mechanisms . in mammalian cells , evidence has also been obtained for ubiquitination as a signal for the initiation of substrate - induced endocytosis in several types of nutrient transporters ( melikian 2004 ; zahniser and sorkin 2009 ; vina - vilaseca et al . the discovery of a nutrient receptor function in a set of yeast plasma membrane transporters that are induced by starvation for their substrate is providing unexpected insight and new approaches and tools to study the mechanisms involved in transport and regulation of the transporter protein level in the membrane . it can be expected that further elucidation of the mechanisms involved in signaling by these transceptors and their connection with transceptor downregulation will provide further unanticipated findings that would have been overlooked if the proteins would just have been studied as transporters . | in the yeast saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor .
these transporters are induced when yeast cells are starved for their substrate , which triggers entry into stationary phase and acquirement of a low protein kinase a ( pka ) phenotype .
re - addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the pka pathway , the latter being mediated by the nutrient transceptors . at the same time , the transceptors are ubiquitinated , endocytosed and sorted to the vacuole for breakdown .
investigation of the signaling function of the transceptors has provided a new read - out and new tools for gaining insight into the functionality of transporters .
identification of amino acid residues that bind co - transported ions in symporters has been challenging because the inactivation of transport by site - directed mutagenesis is not conclusive with respect to the cause of the inactivation .
the discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling .
inactivation of transport with maintenance of signaling in transceptors supports that a true proton - binding residue was mutagenised .
determining the relationship between transport and induction of endocytosis has also been challenging , since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate .
the use of analogues with different combinations of transport and signaling capacities has revealed that transport , ubiquitination and endocytosis can be uncoupled in several unexpected ways .
the results obtained are consistent with transporters undergoing multiple substrate - induced conformational changes , which allow interaction with different accessory proteins to trigger specific downstream events . |
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mirnas are endogenous , small ( 1825 nt ) , non - protein - coding rna molecules . originally discovered in c. elegans mirnas bind to transcripts of target protein - coding genes in a sequence - specific manner , functioning primarily to decrease the transcript and/or protein levels of their targets . however , mirna - target interactions resulting in increased protein levels have also been noted , . in recent decades , mirnas have been increasingly recognized as important genetic regulators in the mammalian system . moreover , numerous mirnas have been reported to function as both oncogenes and tumor suppressors , regulating tumor initiation and progression at all levels . the biogenesis of mirnas is a multistep process that is tightly regulated within the cell . figure 1 depicts the steps in canonical mirna processing from transcription in the nucleus to the interaction with mrnas in the cytoplasm . genes that encode mirnas can be located in intergenic regions or within the exons or introns of other genes . transcription of these mir - genes is performed predominantly by rna polymerase ii ( pol ii ) , though rna pol iii performs transcription in some cases , . a variety of pol ii associated transcription factors direct mirna transcription , thereby regulating mirna expression at the level of transcription . the rna product that is transcribed is called a primary mirna ( pri - mirna ) . these pri - mirnas vary in length from hundreds to thousands of base pairs and exist in diverse stem - loop structures . following transcription , the pri - mirna is cleaved within the nucleus by the type iii rnase drosha , in association with digeorge syndrome critical region gene 8 ( dgcr8 ) , at the stem of the loop structure to release a 70 nt precursor mirna ( pre - mirna ) . this pre - mirna is then exported out of the nucleus by the dsrna - specific transporting complex composed of exportin-5 and gtp - bound ras - related nuclear protein ( ran - gtp ) . mirnas are processed through a complex series of highly regulated steps in the nucleus and the cytoplasm , from transcription through to their functional roles as transcript and protein level regulators . abbreviations : rna pol ii , rna polymerase type ii ; pri - mirna , primary microrna ; pre - mirna , precursor microrna ; dgcr8 , drosha - digeorge syndrome critical region gene 8 ; xpo5 , nuclear export factor exportin 5 ; ran , ras - related nuclear protein ; gtp , guanosine tri - phosphate ; ago , argonaute ; trbp , tar ( hiv-1 ) rna - binding protein 2 ; pact , protein kinase , interferon - inducible double stranded rna ; dicer , dicer 1 ribonuclease type iii ; mir * , passenger strand from mature mirna duplex ; mirisc , microrna rna - induced silencing complex ; p - body , processing body . in the cytoplasm , the pre - mirna is further processed by dicer and its cofactor trans - activation - responsive rna - binding protein ( trbp ) to release the 22 nt , mature mirna duplex . one strand of this duplex , termed the guide strand , is then preferentially incorporated into the mirna - inducible silencing complex ( mi - risc ) . although this process is not completely understood , the guide strand is almost always the strand with the 5 terminus that is least thermodynamically stable . the key functional element of mirisc is the argonaute protein ( in mammals , one of ago1 - 4 ) . in addition to argonaute , several other proteins may be involved in mirisc , including the p - body marker fragile x mental retardation 1 protein ( fmrp ) and the de - capping activator rck / p54 , . once a mature mirna is incorporated , mirisc can then bind to a target mrna at a sequence - specific binding site . a particular mirna may bind to hundreds of different genes , sometimes even onto multiple sites for a given target mrna . binding occurs with either perfect complementarity or , more often , imperfect complementarity . in these instances of imperfect complementarity , there is often a short , 68 nt seed region , located near the 5 end of the mirna , which appears to be of paramount importance in terms of dictating binding to specific target mrnas . these seed regions are often conserved among species and form the basis for many of the current in silico target prediction algorithms,. mirnas can regulate expression of their targets through either mrna degradation or translational inhibition . in instances of perfect or near - perfect mirna - mrna complementarity , degradation of target mrnas can be mediated by ago2 , the only argonaute protein with slicer activity . in cases of imperfect complementarity , all 4 argonaute proteins are capable of inhibiting protein translation as part of mirisc . in addition , mirna - target relationships with imperfect binding can also result in mrna degradation through a non - sequence - specific mechanism within cytoplasmic processing bodies ( p - bodies ) . in most cases , the net effect of mirisc binding to a target mrna is a decrease in its protein levels . however , recent reports have demonstrated a few instances wherein protein levels is actually increased . for example , mir-10a can bind the 5 untranslated region ( utr ) of the mrna transcript for several ribosomal genes , increasing the expression of these genes . as the list of mirnas has grown , so has our knowledge regarding their biological functions . indeed , mirnas have been found to play a role in most , if not all , cellular processes , including many pathways related to cancer development and progression . mirnas function as tumor suppressor genes or oncogenes , with some mirnas mediating contradictory roles in different diseases . aberrant mirna expression and function have been described in a wide variety of human malignancies , with chromosomal amplifications / deletions , point mutations , or epigenetic alterations as potential causes . comprehensive mirna expression profiling has been performed in a variety of human cancers , yielding a number of interesting observations . these include expression signatures that are capable of distinguishing cancer cells from normal cells or one cancer from another ; predicting response to a particular drug ; or predicting patient outcome , . indeed , mirna signatures capable of predicting patient outcome have been developed for a number of human cancers including lung cancer , , breast cancer , brain caner , and chronic lymphocytic leukemia . prognostic , predictive , and biological roles have also been described for mirnas in npc , as discussed below . the first study on the global profiling of mirnas in npc was published in 2008 by paul ahlquist 's group at the national cancer institute ( nci ) . using a microarray - based approach to profile 31 laser - capture microdissected npc samples and 10 normal nasopharyngeal epithelial samples in particular , mir-29c was significantly down - regulated in npc , and several mir-29c targets involved in extracellular matrix synthesis and function were identified and validated . subsequently , other groups have identified a number of dysregulated mirnas in both nasopharyngeal tumor and blood samples from patients with npc ( table 1 ) . in addition to alterations in mirna expression in npc , we and others have demonstrated phenotypic roles for mirnas in nasopharyngeal tumorigenesis using in vitro and in vivo models . the first mirna characterized in the context of npc was mir-29c , which was the main focus of the first mirna profiling reported by sengupta et al .. in this initial study , the authors demonstrated that mir-29c plays a potential tumor suppressive role , targeting mrnas that encode extracellular matrix proteins ( collagens 3a1 , 4a1 , and 15a1 , and laminin 1 ) . they postulated that suppression of mir-29c will subsequently increase the migration and invasion of npc cells through up - regulation of these components of the extracellular matrix . however , they stopped short of testing this hypothesis in the initial study , and further reports supporting these claims have not been published to date . subsequent studies by two other groups corroborated the role of mir-29c as a tumor suppressor in npc , but these reports differed in their causal mechanisms and putative targets . while liu et al . demonstrated that down - regulation of mir-29c resulted in the promotion of npc cell migration and invasion through increased expression of t - cell lymphoma invasion and metastasis 1 ( tmp1 ) , zhang et al . showed that mir-29c knock - down resulted in increased resistance to radiotherapy and cisplatin through up - regulation of the anti - apoptotic regulators mcl-1 and bcl-2 . thus , much like other mirnas , mir-29c can act through a number of pathways to suppress the proliferation , survival , and motility of npc cells . in 2011 , we reported that mir-375 is a potential tumor suppressor in head and neck cancers , including npc . we discovered that metadherin ( mtdh ) , a newly emerging oncogene , was an important target for down - regulation by mir-375 and that mtdh overexpression was particularly detrimental to npc patients , resulting in an increased risk of distant relapse . not only has mir-9 been identified as a potential circulating biomarker of advanced npc , several potential targets and functions of mir-9 have also been reported . recently , two studies describing putative tumor suppressive mechanisms for mir-9 in npc have been published , . reported that hypermethylation and subsequent underexpression of mir-9 led to up - regulation of its putative target c - x - c chemokine receptor type 4 ( cxcr4 ) , resulting in increased cell growth , migration , and invasion through activation of the mitogen - activated protein kinase ( mapk ) pathway . in contrast , gao et al . postulated a role for mir-9 in modulating the immune response to npc by targeting several interferon ( ifn)-induced genes , multiple members of the major histocompatibility complex ( mhc ) class i molecule , and a number of interleukins and related genes . other potential , functionally active mirnas in npc include mir-26a , , mir-98 , mir-155 , , mir-200a / b , mir-205 , , and mir-216b . proposed targets of these mirnas represent regulators of important processes such as the epithelial - to - mesenchymal transition ( emt ) , as well as signaling pathways including notch , phosphatidylinositol-4,5-bisphosphate 3-kinase ( pi3k)-akt , and mapk . intriguingly , the function , pathways , and targets affected by many of the mirnas involved in nasopharyngeal tumorigenesis overlap . two such mirnas are mir-26a and mir-218 , which both target enhancer of zeste homolog 2 ( ezh2 ) , resulting in decreased oncogenic properties of migration , invasion , and cell survival , . this overlapping functional impact of multiple mirnas adds an increased layer of complexity when attempting to elucidate their biological role ; however , this property could also increase our confidence in the biological importance of affected genes and pathways , given that there are multiple mechanisms that malignant cells exploit to activate or inhibit the same gene or pathway . the mirnas included in this table satisfy each of the following criteria : ( 1 ) a statistically significant alteration in their expression was identified in specimens from patients with npc ; and ( 2 ) some degree of validation ( either in additional samples , using an alternate analytical method , or functional validation ) was reported . patients with npc vs. healthy controls : for these mirnas , levels were significantly increased ( up - regulated ) or decreased ( down - regulated ) in tumors and plasma from patients with npc compared with nasopharyngeal tissues and plasma from healthy controls . prognostic association : expression higher ( positive ) or lower ( negative ) in tumor and plasma samples from patients with poor prognosis . epstein - barr virus ( ebv ) , which is present in the vast majority of npc , also encodes a number of its own mirnas . twenty - five ebv - encoded pre - mirnas , which are processed into 44 mature mirna sequences , have been verified to date . these mirna emanate from two major regions of the ebv genome : ( 1 ) the bamhi - a rightward transcripts ( bart ) and ( 2 ) the open reading frame of the bhrf1 gene . originally cloned from ebv - infected burkitt 's lymphoma cells , bhrf1-encoded mirnas do not appear to be expressed in ebv - positive npc primary tissues , . indeed , in the first report of comprehensive ebv mirna profiling in primary npc , the majority of samples showed substantial expression of all 35 interrogated bart - encoded mirnas but no expression of the 4 interrogated bhrf1-encoded mirnas . in contrast , npc - derived cell lines c666 - 1 and hone - akata expressed all 4 bhrf1-derived mirnas in addition to the 35 interrogated bart - encoded mirnas . viral targets include latent membrane protein 1 ( lmp1 ) , putatively targeted by several mirnas from the bart region ( ebv - mir - bart1 , 9 , 16 , 17 ) , , , and lmp2a , a target of ebv - mir - bart22 . subsequent functional experiments suggest that modulation of these viral proteins by bart - encoded mirna can influence multiple cellular properties including cell proliferation , survival , and evasion of host immune response. host targets of bart - encoded mirnas include the pro - apoptotic effectors p53 up - regulated modulator of apoptosis ( puma ) , bcl-2 interacting mediator of cell death ( bim ) , and translocase of outer mitochondrial membrane 22 homolog ( tomm22 ) , as well as several genes thought to influence host immune response , including mhc class i - related chain b ( micb ) , importin 7 ( ipo7 ) , , and dicer . overall , ebv - encoded mirnas play a complementary role to the viral proteins expressed in npc , contributing to evasion of the host immune response and promoting the survival and proliferation of npc cells . the first study on the global profiling of mirnas in npc was published in 2008 by paul ahlquist 's group at the national cancer institute ( nci ) . using a microarray - based approach to profile 31 laser - capture microdissected npc samples and 10 normal nasopharyngeal epithelial samples in particular , mir-29c was significantly down - regulated in npc , and several mir-29c targets involved in extracellular matrix synthesis and function were identified and validated . subsequently , other groups have identified a number of dysregulated mirnas in both nasopharyngeal tumor and blood samples from patients with npc ( table 1 ) . in addition to alterations in mirna expression in npc , we and others have demonstrated phenotypic roles for mirnas in nasopharyngeal tumorigenesis using in vitro and in vivo models . the first mirna characterized in the context of npc was mir-29c , which was the main focus of the first mirna profiling reported by sengupta et al .. in this initial study , the authors demonstrated that mir-29c plays a potential tumor suppressive role , targeting mrnas that encode extracellular matrix proteins ( collagens 3a1 , 4a1 , and 15a1 , and laminin 1 ) . they postulated that suppression of mir-29c will subsequently increase the migration and invasion of npc cells through up - regulation of these components of the extracellular matrix . however , they stopped short of testing this hypothesis in the initial study , and further reports supporting these claims have not been published to date . subsequent studies by two other groups corroborated the role of mir-29c as a tumor suppressor in npc , but these reports differed in their causal mechanisms and putative targets . while liu et al . demonstrated that down - regulation of mir-29c resulted in the promotion of npc cell migration and invasion through increased expression of t - cell lymphoma invasion and metastasis 1 ( tmp1 ) , zhang et al . showed that mir-29c knock - down resulted in increased resistance to radiotherapy and cisplatin through up - regulation of the anti - apoptotic regulators mcl-1 and bcl-2 . thus , much like other mirnas , mir-29c can act through a number of pathways to suppress the proliferation , survival , and motility of npc cells . in 2011 , we reported that mir-375 is a potential tumor suppressor in head and neck cancers , including npc . we discovered that metadherin ( mtdh ) , a newly emerging oncogene , was an important target for down - regulation by mir-375 and that mtdh overexpression was particularly detrimental to npc patients , resulting in an increased risk of distant relapse . not only has mir-9 been identified as a potential circulating biomarker of advanced npc , several potential targets and functions of mir-9 have also been reported . recently , two studies describing putative tumor suppressive mechanisms for mir-9 in npc have been published , . reported that hypermethylation and subsequent underexpression of mir-9 led to up - regulation of its putative target c - x - c chemokine receptor type 4 ( cxcr4 ) , resulting in increased cell growth , migration , and invasion through activation of the mitogen - activated protein kinase ( mapk ) pathway . in contrast , gao et al . postulated a role for mir-9 in modulating the immune response to npc by targeting several interferon ( ifn)-induced genes , multiple members of the major histocompatibility complex ( mhc ) class i molecule , and a number of interleukins and related genes . other potential , functionally active mirnas in npc include mir-26a , , mir-98 , mir-155 , , mir-200a / b , mir-205 , , and mir-216b . proposed targets of these mirnas represent regulators of important processes such as the epithelial - to - mesenchymal transition ( emt ) , as well as signaling pathways including notch , phosphatidylinositol-4,5-bisphosphate 3-kinase ( pi3k)-akt , and mapk . intriguingly , the function , pathways , and targets affected by many of the mirnas involved in nasopharyngeal tumorigenesis overlap . two such mirnas are mir-26a and mir-218 , which both target enhancer of zeste homolog 2 ( ezh2 ) , resulting in decreased oncogenic properties of migration , invasion , and cell survival , . this overlapping functional impact of multiple mirnas adds an increased layer of complexity when attempting to elucidate their biological role ; however , this property could also increase our confidence in the biological importance of affected genes and pathways , given that there are multiple mechanisms that malignant cells exploit to activate or inhibit the same gene or pathway . the mirnas included in this table satisfy each of the following criteria : ( 1 ) a statistically significant alteration in their expression was identified in specimens from patients with npc ; and ( 2 ) some degree of validation ( either in additional samples , using an alternate analytical method , or functional validation ) was reported . patients with npc vs. healthy controls : for these mirnas , levels were significantly increased ( up - regulated ) or decreased ( down - regulated ) in tumors and plasma from patients with npc compared with nasopharyngeal tissues and plasma from healthy controls . prognostic association : expression higher ( positive ) or lower ( negative ) in tumor and plasma samples from patients with poor prognosis . epstein - barr virus ( ebv ) , which is present in the vast majority of npc , also encodes a number of its own mirnas . twenty - five ebv - encoded pre - mirnas , which are processed into 44 mature mirna sequences , have been verified to date . these mirna emanate from two major regions of the ebv genome : ( 1 ) the bamhi - a rightward transcripts ( bart ) and ( 2 ) the open reading frame of the bhrf1 gene . originally cloned from ebv - infected burkitt 's lymphoma cells , bhrf1-encoded mirnas do not appear to be expressed in ebv - positive npc primary tissues , . indeed , in the first report of comprehensive ebv mirna profiling in primary npc , the majority of samples showed substantial expression of all 35 interrogated bart - encoded mirnas but no expression of the 4 interrogated bhrf1-encoded mirnas . in contrast , npc - derived cell lines c666 - 1 and hone - akata expressed all 4 bhrf1-derived mirnas in addition to the 35 interrogated bart - encoded mirnas . viral targets include latent membrane protein 1 ( lmp1 ) , putatively targeted by several mirnas from the bart region ( ebv - mir - bart1 , 9 , 16 , 17 ) , , , and lmp2a , a target of ebv - mir - bart22 . subsequent functional experiments suggest that modulation of these viral proteins by bart - encoded mirna can influence multiple cellular properties including cell proliferation , survival , and evasion of host immune response. host targets of bart - encoded mirnas include the pro - apoptotic effectors p53 up - regulated modulator of apoptosis ( puma ) , bcl-2 interacting mediator of cell death ( bim ) , and translocase of outer mitochondrial membrane 22 homolog ( tomm22 ) , as well as several genes thought to influence host immune response , including mhc class i - related chain b ( micb ) , importin 7 ( ipo7 ) , , and dicer . overall , ebv - encoded mirnas play a complementary role to the viral proteins expressed in npc , contributing to evasion of the host immune response and promoting the survival and proliferation of npc cells . in summary , the interrogation of mirnas in the context of npc has provided both clinical and biological insight into the behavior of this disease . further development of strategies to measure and manipulate mirnas and their targets in a clinical setting would be required before such findings can be translated into improvements in the management of this disease . | micrornas ( mirnas ) provide insight into both the biology and clinical behavior of many human cancers , including nasopharyngeal carcinoma ( npc ) .
the dysregulation of mirnas in npc results in a variety of tumor - promoting effects .
furthermore , several mirnas are prognostic markers for npc .
in addition to cellular mirnas , npc samples also often contain mirnas encoded by epstein - barr virus , and these mirnas may impact npc biology by targeting both cellular and viral genes .
given their numerous putative roles in npc development and progression , a thorough understanding of the impact of mirna dysregulation in npc is expected to shed light on useful biomarkers and therapeutic targets for the clinical management of this disease . in this review , we describe the efforts to date to identify and characterize such mirnas in the context of npc . |
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despite ever - increasing literature on hip fracture , there are no authoritative and evidence - based guidelines for the management of displaced intracapsular femoral neck fractures ( fnf).1 a general lack of consensus exists among orthopedic trauma surgeons in the management of these fractures.2 the decision making in hip fracture treatment depends on age , patient 's co - morbidities , pre - fracture mobility status , associated injuries , bone quality , fracture configuration and pre - existing degenerative status of the joint.36 the present consensus is that fnf in patients below 60 years of age should be treated with internal fixation , and arthroplasty should be reserved for elderly patients above 80 years , 60 to 80 years old patients with displaced fnf still remain a grey zone.7 the surgical duration , bleeding , need of blood transfusion , infection and immediate postoperative mortality is considered less with internal fixation compared to arthroplasty.8 however , fixation failure and reoperation rates are higher with internal fixation ; thus , majority of orthopedic surgeons prefer prosthetic replacement.911 present guidelines favors total hip arthroplasty ( tha ) in active , independent patients aged > 60 years and primary hemiarthroplasty ( ha ) for elderly , moribund patients with dependent living status.12 the functional results of arthroplasty however best done can not equal that of a united fnf . many of the daily functional activities of asian patients require squatting and sitting cross - legged which can not be performed with arthroplasty , and thus , every efforts of joint conservation should be aimed for . the controversial factors in cancellous screw fixation are : method of reduction - open or closed , number of screws used two , three or four , the configuration of screws parallel or nonparallel , vertical or triangular fashion , the positioning of screws in the head center of head or in the periphery and the addition of bone grafting several biomechanical analytical studies have assessed the stability after simulated fracture fixation on cadaveric femora with variable results.1319 the conventional ao fixation using three screws in apex proximal triangular configuration is generally practiced by several surgeons for fixing fnf.2021 in a radiographic review study of patients who underwent cannulated cancellous screw fixation for fnf , six different types of screw configurations were found : triangular configurations , consisting of two parallel screws with a third screw placed either superiorly , inferiorly , anteriorly or posteriorly ; and linear configurations with two or three screws in a vertical line.22 in a multinational survey of 298 orthopedic surgeons , it was found that 73% agreed on the use of three cannulated screws and more than half used the triangle with base inferior construct.2 patwa et al . in a biogeometric study of indian femurs found that inferior half of femoral neck is narrower than superior half and recommended apex distal configuration for screw fixation.20 both in experimental and clinical studies , controversies exist regarding the ideal screw fixation method that can provide good stability and good clinical results respectively . the area covered by the fixation device is important in any fracture fixation ; more the area on either side of fracture , better is the stability . we felt that , by adding the fourth screw and keeping the screws in peripheral portion of the head and neck , the area of fixation can be easily increased in fnf fixation . simple geometric projections and calculations revealed a dramatic increase in the volume occupied by the four peripheral screws compared to three screws [ table 1 ] . with the aim of providing higher success rate in a large group of patients using a single uniform procedure , we started four screw fixation . we report the outcome of four quadrant parallel peripheral screw fixation technique in active , independent patients aged 50 years with displaced fnf . the three or four screw configurations , corresponding projected geometric designs and the area covered . the converging patterns have lower volume than parallel patterns with four quadrant parallel peripheral pattern having the maximum area covered with the screws one hundred and eighty patients with displaced fracture neck femur aged 50 years or more who were operated for displaced femoral neck fracture in our center from january 2005 to december 2008 were included in the study . the patient selection criteria for four cannulated screw fixation were : age 50 years or more , displaced fracture neck of femur ( garden grade iii and iv ) , presence of primary compressile trabeculae in hip anteroposterior ( ap ) radiograph , no radiographic evidence of pathological fracture , and independently mobile patient without neuromuscular disorder or cognitive impairment and without any metabolic bone disease . sixty four patients who completed a minimum of two year followup were included in the study . the mean age of patients was 60 years range ( range 50 - 73 years ) . the mean time interval between injury and surgery was 48 hours ( range eight hours - seven days ) . fnf reduction was attempted by gentle traction with hip in neutral extension , neutral or 10 to 15 of abduction , neutral or 10 of internal rotation with patient on fracture table [ figure 1 ] . a cross sectional contact area of more than 75% without varus deformity was considered as satisfactory reduction . ( a ) x - ray right hip joint showing garden iv femoral neck fracture , ( b and c ) anteroposterior and lateral fluoroscopic views showing closed reduction the inferior screws were placed first . the starting point for the inferior screws was usually at the level of lesser trochanter . since the lateral surface of femur is convex , the starting point often slips anterior or posterior . to avoid this , a pilot hole was made with 3 mm k wire perpendicular to shaft surface in the exact needy location i.e. , posterior to mid - lateral cortex at the level of lesser trochanter for inferior posterior ( ip ) screw . the pilot hole was enlarged with 3.5- or 4-mm drill bit , which was directed in oblique direction as required to make an oval hole in the cortex . a nonthreaded 2-mm guide wire was taken in the hand and pushed into the desired screw trajectory : parallel and just superior to the inferior border of the neck in the anteroposterior view , parallel and just anterior to the posterior border of the neck in the lateral view [ figure 2a ] . with bigger pilot hole , the guide wire could be easily manipulated in both the planes in the required direction . after positioning the guide wire in the desired path , the same was drilled into the head 2 mm short of the articular margin . no drilling or tapping was done in the head or neck region . a 7-mm diameter , 16-mm partially threaded cannulated cancellous screw of adequate length was passed over the guide wire and tightened noting the compression across fracture site [ figure 2b ] . if the neck diameter was smaller , a 6-mm diameter screw was used . for inferior anterior ( ia ) screw , pilot drill hole was made exactly at the same level of ip screw , anterior to the mid lateral cortex of femur . the guide wire was passed free hand , parallel to the ip screw in the ap view and parallel and just posterior to the anterior cortex of the neck in the lateral view [ figure 3 ] . the superior posterior ( sp ) screw was placed just below and parallel to the superior border of the neck in the ap view and parallel to the ip screw in the lateral view [ figure 4 ] . the superior anterior ( sa ) screw was placed parallel to the sp screw in the ap plane and parallel to the ia screw in the lateral view [ figure 5 ] . fluoroscopic views ( a and b ) showing inferior posterior quadrant guide wire and screw fluoroscopic views ( a - c ) showing inferior anterior quadrant guide wire and screw fluoroscopic views ( a and b ) showing superior posterior quadrant guide wire and screw fluoroscopic views ( a - c ) showing superior anterior quadrant guide wire and screw in this fashion , all the four quadrants i.e. , inferior posterior , inferior anterior , superior posterior and superior anterior quadrants of the femoral heads were stabilized with the neck / trochanter fragment . the screws were placed as much parallel as possible and as peripheral in the neck as possible . we term this fixation as four quadrant parallel peripheral ( fqpp ) fixation [ figure 6 ] . the screws could diverge from the neck into the head but convergence of the screws towards head center was not accepted . the mean duration of surgery was 42 minutes ( range 30 - 55 minutes ) . peroperative photograpyh showing screws head configuration at entry point in four quadrant fixation postoperatively , the limb was kept in 10 - 15 of abduction with knee flexed at 10 - 20. postoperative radiographs were obtained on the first postoperative day . the patient was encouraged to do static quadriceps , terminal knee extension and active assisted / active straight leg raising ( slr ) exercises once anesthesia weans off . patient was advised for opposite side turns in the bed in the immediate postop period . twenty four hours after surgery , patient was made to sit up on the bed side and nonweight bearing ( nwb ) walking was started . on 2 postop day , transfer to high seat , mobile western commode was allowed . the patients were followedup clinically and radiologically after three weeks , six weeks , three months , six months , twelve months and after two years . partial weight bearing ( pwb ) was allowed after three weeks and full weight bearing ( fwb ) was allowed after radiographic union was evident . radiologically fracture union was defined as continuity of at least three cortices in ap and lateral views without any fracture gap . clinically fracture was considered as healed when there was no local tenderness and patient could do full weight bearing without any support . once the fracture was healed , patients were encouraged to sit on the floor cross legged and to do squatting . the functional outcome was evaluated with a new six - point score [ table 2 ] . the one year score was taken as final functional outcome . a six - point functional outcome scoring system for asians after hip surgery fnf reduction was attempted by gentle traction with hip in neutral extension , neutral or 10 to 15 of abduction , neutral or 10 of internal rotation with patient on fracture table [ figure 1 ] . a cross sectional contact area of more than 75% without varus deformity was considered as satisfactory reduction . ( a ) x - ray right hip joint showing garden iv femoral neck fracture , ( b and c ) anteroposterior and lateral fluoroscopic views showing closed reduction the inferior screws were placed first . the starting point for the inferior screws was usually at the level of lesser trochanter . since the lateral surface of femur is convex , the starting point often slips anterior or posterior . to avoid this , a pilot hole was made with 3 mm k wire perpendicular to shaft surface in the exact needy location i.e. , posterior to mid - lateral cortex at the level of lesser trochanter for inferior posterior ( ip ) screw . the pilot hole was enlarged with 3.5- or 4-mm drill bit , which was directed in oblique direction as required to make an oval hole in the cortex . a nonthreaded 2-mm guide wire was taken in the hand and pushed into the desired screw trajectory : parallel and just superior to the inferior border of the neck in the anteroposterior view , parallel and just anterior to the posterior border of the neck in the lateral view [ figure 2a ] . with bigger pilot hole , the guide wire could be easily manipulated in both the planes in the required direction . after positioning the guide wire in the desired path , the same was drilled into the head 2 mm short of the articular margin . no drilling or tapping was done in the head or neck region . a 7-mm diameter , 16-mm partially threaded cannulated cancellous screw of adequate length was passed over the guide wire and tightened noting the compression across fracture site [ figure 2b ] . if the neck diameter was smaller , a 6-mm diameter screw was used . for inferior anterior ( ia ) screw , pilot drill hole was made exactly at the same level of ip screw , anterior to the mid lateral cortex of femur . the guide wire was passed free hand , parallel to the ip screw in the ap view and parallel and just posterior to the anterior cortex of the neck in the lateral view [ figure 3 ] . the superior posterior ( sp ) screw was placed just below and parallel to the superior border of the neck in the ap view and parallel to the ip screw in the lateral view [ figure 4 ] . the superior anterior ( sa ) screw was placed parallel to the sp screw in the ap plane and parallel to the ia screw in the lateral view [ figure 5 ] . fluoroscopic views ( a and b ) showing inferior posterior quadrant guide wire and screw fluoroscopic views ( a - c ) showing inferior anterior quadrant guide wire and screw fluoroscopic views ( a and b ) showing superior posterior quadrant guide wire and screw fluoroscopic views ( a - c ) showing superior anterior quadrant guide wire and screw in this fashion , all the four quadrants i.e. , inferior posterior , inferior anterior , superior posterior and superior anterior quadrants of the femoral heads were stabilized with the neck / trochanter fragment . the screws were placed as much parallel as possible and as peripheral in the neck as possible . we term this fixation as four quadrant parallel peripheral ( fqpp ) fixation [ figure 6 ] . the screws could diverge from the neck into the head but convergence of the screws towards head center was not accepted . the mean duration of surgery was 42 minutes ( range 30 - 55 minutes ) . peroperative photograpyh showing screws head configuration at entry point in four quadrant fixation postoperatively , the limb was kept in 10 - 15 of abduction with knee flexed at 10 - 20. postoperative radiographs were obtained on the first postoperative day . the patient was encouraged to do static quadriceps , terminal knee extension and active assisted / active straight leg raising ( slr ) exercises once anesthesia weans off . patient was advised for opposite side turns in the bed in the immediate postop period . twenty four hours after surgery , patient was made to sit up on the bed side and nonweight bearing ( nwb ) walking was started . on 2 postop day , transfer to high seat , mobile western commode was allowed . the patients were followedup clinically and radiologically after three weeks , six weeks , three months , six months , twelve months and after two years . partial weight bearing ( pwb ) was allowed after three weeks and full weight bearing ( fwb ) was allowed after radiographic union was evident . radiologically fracture union was defined as continuity of at least three cortices in ap and lateral views without any fracture gap . clinically fracture was considered as healed when there was no local tenderness and patient could do full weight bearing without any support . once the fracture was healed , patients were encouraged to sit on the floor cross legged and to do squatting . the functional outcome was evaluated with a new six - point score [ table 2 ] . the one year score was taken as final functional outcome . a six - point functional outcome scoring system for asians after hip surgery screw back out indicating fracture collapse and coxa breva was seen as early in the immediate postoperative x - ray . the average distance of screw back out was 6 mm ( 2 - 12 mm ) . x - ray left hip joint anteroposterior view ( a ) showing femoral neck fracture , ( b , c ) anteroposterior and lateral views showing union after four quadrant parallel peripheral screw fixation nonanatomical healing or malunion was observed in 45 cases ( 70% ) . various components of malunion observed were coxa breva ( n = 35 ) , coxa valga ( n = 22 ) , rotational ( n = 18 ) , translational ( n = 12 ) and coxa vara ( n = 1 ) [ figure 8 ] . if there was cortical reduction at one border of the neck with step in another border , it was considered as rotational malunion . if there was a cortical step at both superior and inferior borders of neck , it was considered as translational malunion . x - ray hip joint anteroposterior view showing healed femoral neck fracture ( a ) coxa breva ( b ) coxa valga ( c ) rotational malunion ( d ) translational malunion ( e ) coxa vara the functional score was excellent in 50 hips ( 78.2% ) , good in 13 hips ( 20.3% ) and poor in one hip ( 1.5% ) . screw removal was done in the second patient resulting in complete pain relief . except for these two patients , all patients , except for the tha patient , could do cross legged sitting in the floor and squatting . cross legged sitting was complete in 48 hips ( flexed knee close to the floor ) and partial in the rest . squatting was complete in 54 hips ( thigh close to the abdomen ) and partial in the rest . fifty two patients achieved maximum functional score at the end of 6 months which was maintained in the subsequent followup . eleven patients continued to show improvement in sitting cross legged and squatting after 6 months that improved their final score at 1 year . x - ray hip joint anteroposterior view ( a and b ) showing head resorption and total hip arthroplasty since intracapsular fracture neck of femur heals by primary healing , along with stability in coronal and sagittal planes , absolute rotational stability is necessary across the fracture site.2327 the proximity of polyaxial hip joint and the location of fracture at junction of lower limb and trunk results in variety of forces acting across the fracture , even in a bed bound patient . the commonly used screw fixation construct in fnf may be imperfect with one or more of technical flaws : unacceptable reduction ( less contact area ) , lack of parallelism , convergence towards head center , crowding of screws in small area , inadequate screw length , repeated drilling into the head weakening screw purchase and leaving fracture gap . loading on imperfect mechanical construct can result in uncontrolled collapse , tilting of head into varus , loss of contact , nonunion and screw penetration into the joint . the key therefore is to provide good stable construct that can withstand the routine strains , still maintain contact between the fracture ends , provide stability and allow healing.28 the fqpp construct gives better initial stability , allows better controlled collapse , improves stability with passing time because of increased contact and promotes union . the four screws which are placed in the periphery of the neck act as four struts or pillars and provide excellent stability . even if the screw purchase is suboptimal in osteoporotic bones , fqpp screws act as internal splints . the good peripheral or circumferential fixation does not allow the head to settle in varus or any angulations and eliminates the detrimental rotational strain . since the screws are parallel , they permit controlled collapse maintaining the contact at any point of time . the problems of three screw fixation can be of twofold : the adequacy of fixation especially in older population and ability for controlled collapse . this is especially common when there is lack of parallelism between the three screws and the apex screw is not in the center of two basal screws.29 during daily activities , the loads on femoral head alternate anteriorly and posteriorly . if there is less support on the anterior quadrants , loading can result in head tilting into varus anteriorly with screw penetration in the posterior quadrant of the head . if there is less support on the posterior quadrants , loading can result in head tilting into varus posteriorly with screw penetration in the anterior quadrant of the head . in fqpp this could be seen as early as 24 hours after surgery , because patients were allowed to do bed side activities and early slr exercises . this controlled collapse or fracture settling is similar to that of distal radius fracture or intertrochanteric fracture and can be considered as a good sign provided the reduction and screws orientation are maintained . in the 70 year old patient who developed femoral head fragmentation and resorption , histo - pathological study of the retrieved loose bony fragments did not show any pathology . we have evaluated the clinical outcome with a new six point hip functional score [ table 2 ] . the existing scoring systems either do not consider sitting cross legged and squatting or cumbersome for quick clinical application . assessment at any point of time in the postoperative period should convey the functional status since the results could not be expressed as an improvement over the preoperative score , which is not possible in fracture cases . this new hip clinical score is based on the routine clinical observations or questions that are done with patient during their clinical visit . pain , range of motion in the form of sitting cross legged and squatting , functional activity in the form of walking , stair climbing and individual mobility outside the house were assessed . in elderly patients , basic functional assessment may be sufficient rather than advanced functional assessment , as the individual functional capacity is highly variable depending on underlying general fitness . the assessment with the new score is fast and can be done by surgeon or any trained health professional . being a single digit numeric score , we feel it easily reflects the postsurgical functional results . the results of our study are relevant in general and in particular to indian population . many indian patients come from rural background , where they do physical work even in advanced age . with healed fnf , unsupervised physical work , sitting in floor , squatting and regular life style can be allowed . unlike replacement arthroplasty , prolonged medical supervision or followup is not necessary . most of the patients do not have insurance cover , the cost of fnf fixation surgery is less than that of ha or tha . while factors like high cost , lack of surgical expertise , lack of operation theatre facilities and fear for dislocation may force surgeon against tha option , fear for fixation failure and nonunion may force surgeons against fixation option , leaving the lone ha option which is not suitable for all age groups . with high success rate , fqpp can be picked as first choice over tha or ha , especially in the age group between 50 to 70 years . while revising a failed closed screw fixation surgery into tha is almost like a virgin hip surgery , converting painful or failed hemiarthroplasty into tha is difficult and akin to revision tha on several occasions with suboptimal results . the advantages of fqpp technique are many : good clinical outcome , a modification of existing technique which surgeons are familiar , small learning curve , simple routine theatre setup , short surgical time and low expenditure . the technique may appear demanding , but with adherence to key surgical steps , it can be mastered easily . the strengths of our study are that it is a prospective study , has relatively large sample population and has adequate followup . the first and foremost myth is that an accurate anatomical reduction is essential for fracture union and fnf can not malunite . as shown from this study , anatomical reduction in fnf the changes in anatomy are increased neck shaft angle , few millimeters of displacement or collapse and few degrees of rotation . in fnf , this nonanatomical healing can hamper some function like full cross legged sitting in the floor and full squatting , but patients can walk comfortably without pain , limp and walking aid . the tips in getting acceptable reduction in fnf are minimal traction , neutral or mild internal rotation , neutral or mild abduction and understanding the three dimensional anatomy before manipulation . heavy traction , vigorous manipulation and excessive internal rotation are common pitfalls that prevent reduction . except for varus orientation , malreductions like valgus orientation repeated manipulations should not be done to reduce the opening of anterior or posterior cortex , which could be easily achieved with screw compression . a cross sectional fracture contact area of 75% or more without coxa vara should be the aim of reduction . the second myth is about the synovial fluid bathing the fracture surfaces resulting in fnf nonunion . amongst synovial joints all intraarticular fractures involving femoral condyle and tibial condyle can heal with simple splinting . while these intraarticular fractures can heal by secondary healing without any surgical intervention the third myth is that most if not all fnf results in avn femoral head . vigorous traction and manipulations , multiple un - physiological manipulations and open reduction may result in avn . the fracture per se does not result in avn at least in older population , where the injury is of low velocity nature and the capsule is intact . in india , no patient developed avn after fixation in our study . in conclusion , four quadrant parallel peripheral screw fixation technique gives good clinical results in displaced femoral neck fractures in large group of patients aged 50 years or above . this technique , a slight variation of the regular three screw fixation construct , provides excellent stability necessary for primary or endosteal healing of these fractures . with high healing rates , minimal complications and the availability of rescue surgery in the form of prosthetic replacement , this fixation should be attempted in all possible cases of femoral neck fractures in older population . | background : the treatment options for displaced femoral neck fracture in elderly are screw fixation , hemiarthroplasty and total hip arthroplasty based primarily on age of the patient .
the issues in screw fixation are ideal patient selection , optimal number of screws , optimal screw configuration and positioning inside the head and neck of femur .
the problems of screw fixation may be loss of fixation , joint penetration , avascular necrosis of femoral head , nonunion , prolonged rehabilitation period and the need for second surgery in failed cases .
we hereby present results of a modified screw fixation technique in femoral neck fractures in patients 50 years of age.materials and methods : patients 50 years of age ( range 50 - 73 years ) who sustained displaced femoral neck fracture and fulfilled the inclusion criteria were enrolled in this prospective study .
they were treated with closed reduction under image intensifier control and cannulated cancellous screw fixation .
accurate anatomical reduction was not aimed and a cross sectional contact area of > 75% without varus was accepted as good reduction .
four screws were positioned in four quadrants of femoral head and neck , as parallel and as peripheral as possible .
radiological and functional results were evaluated periodically .
sixty four patients who could complete a minimum followup of two years were analyzed.results:radiologically , all fractures healed after mean duration of 10 weeks ( range 8 - 12 weeks ) .
there was no avascular necrosis .
nonanatomical healing was observed in 45 cases ( 70% ) .
all patients except one had excellent functional outcome and could do cross - legged sitting and squatting .
chondrolysis with progressive head resorption was seen in one case , which was converted to total hip arthroplasty.conclusion:closed reduction and cannulated cancellous screw fixation gives satisfactory functional results in large group of elderly patients .
the four quadrant parallel peripheral ( fqpp ) screw fixation technique gives good stability , allows controlled collapse , avoids fixation failure and achieves predictable bone healing in displaced femoral neck fracture in patients 50 years of age . |
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injection of immunoglobulins in its initial form was developed during the 1940s for use in the war effort and was first used for primary immunodeficiency ( pid ) as a subcutaneous ( sc ) injection in 1952 by dr . since then , neither an optimal dose nor serum trough level has been established . from the 1950s through the 1970s , janeway and others administered igg by intramuscular ( i m ) injections with doses of 25 mg / kg / week or 100 mg / kg / month , resulting in serum levels of 110 to 260 mg / dl , even though the landmark british medical research council ( mrc ) study found that higher doses resulted in better outcomes . in the early 1980s , introduction of igg preparations that could be safely given intravenously ( intravenous igg , ivig ) made it possible to administer higher doses without severe reactions . as ivig and newer sc preparations of igg ( scig ) were being adopted , the consensus regarding the optimal dose gradually grew to 400800 mg / kg / month to achieve serum levels of 500 mg / dl or greater . one potential reason for the continued evolution of the optimal igg dose may be that the objectives of treatment have been changing through the decades , as studies showed that higher iv doses which yielded higher serum trough levels were more effective in reducing the incidence of infection . the world health organization ( who ) defines health to be a state of complete physical , mental and social well being and not merely the absence of disease or infirmity . given that serious infections are efficiently prevented by igg replacement , other outcomes that measure productivity , well being , and health utilization in addition , immunologists often raise the dose to try to prevent progression of chronic lung disease and other end - organ complications of pid . the pid committee of the american academy of allergy , asthma , and immunology ( aaaai ) concluded in a 2006 report that the dose of igg should be titrated to achieve a trough level greater than 500 mg / dl in agammaglobulinemic patients and that trough levels greater than 800 mg / dl have the potential to improve pulmonary outcomes . there have been few dose - ranging studies with igg . in most recent licensing trials , the dose of igg is based on the patients ' previous treatment regimens without specifying how those doses were chosen by the patients ' individual physicians . a survey of immunologists conducted recently by the aaaai found that those immunologists who devote less than 10% of their practice to pid usually target a serum level of 500 to 750 mg / dl , whereas more experienced immunologists whose pid patient population exceeds 10% of their practice were significantly more likely to target serum levels above 750 mg / dl ( p = 0.02 ) . in 2010 , the canadian blood services published their review of dose - ranging studies and agreed that seven of the eight articles they reviewed concluded that outcomes were improved with higher doses of ivig . nevertheless , the authors concluded that although higher doses and serum trough levels appear to be associated with improved clinical response , clinical outcomes such as frequency of infections and days missed from school and work should be used to monitor the effectiveness of igg therapy . recently , lucas et al . showed , in a cohort of 90 patients mostly with common variable immunodeficiency ( cvid ) , that the ranges of both igg dose and igg trough level were very large and that the goal of replacement therapy should be to improve clinical outcome and not to reach a particular igg trough level . recent pooled analyses of multiple studies showed continuous relationships between igg levels and incidence of pneumonia or other infections , with no sharp break points or plateaus [ 11 , 12 ] . no guidelines have identified a dose or serum level of igg at which no additional clinical benefit is achieved , yet several payors have developed their own guidelines to limit reimbursement for igg to only the dose needed to achieve a given serum igg level regardless of the patient 's clinical response . other factors , such as periodic short supplies of igg , have also limited the prescribing of higher doses of igg and raising of igg trough levels in pid patients to achieve optimal clinical responses . the purpose of this analysis was to determine if there is a relationship between the use of a higher sc dose of igg and improvements in patient outcomes such as missed work / school days , hospital days , days of antibiotic use , and infection rate . european and us health authorities required two different dosing levels for hizentra in their registration trials . this presented an opportunity to compare the outcomes from these independent cohorts of patients treated with different doses . this is the first analysis comparing patient outcomes achieved with two different sc dosing levels of hizentra , a recently approved 20% igg preparation for sc use.table icomparison of the hizentra us and eu clinical registration trialshizentra trial enrolling patients primarily from the euhizentra trial enrolling patients from the usapre - trial requirements 6 months of stable outcomes 6 months of stable outcomes stable iv or sc igg dosing stable iv igg dosing 3 trough levels 500 mg / dl 3 trough levels 500 mg / dlinclusion / exclusion criteriasame same wash - in / wash - out period12 weeks12 weekshizentra dose during wash - in / wash - out periodequivalent to pre - trial igg dose1.3 times the pre - trial igg doseefficacy period28 weeks52 weekshizentra dose during efficacy periodequivalent to pre - trial igg dose ( 1:1 dose)1.5 times the pre - trial igg dose(1.5:1 dose)outcomes measuredsame ( igg trough levels , rate of infections , hospitalization days , days of antibiotic use , and missed work / school days)same ( igg trough levels , rate of infections , hospitalization days , days of antibiotic use , and missed work / school days)based on a us fda - mandated dose adjustment coefficient determined in pharmacokinetic sub - study to maintain area under the concentration - time curve following switch from ivigeu european union , iv intravenous , sc subcutaneous , us united states comparison of the hizentra us and eu clinical registration trials based on a us fda - mandated dose adjustment coefficient determined in pharmacokinetic sub - study to maintain area under the concentration - time curve following switch from ivig eu european union , iv intravenous , sc subcutaneous , us united states the two independent patient cohorts were from two clinical trials conducted roughly in parallel , one in the usa and the other primarily in the european union ( eu ) , both of which shared many features of patient selection and study design as part of a single clinical development program ( table i ) . both studies used an open - label , multi - center , single - arm design . patients qualified for the study if they were male or female , aged from 2 to 65 years , and diagnosed with primary humoral deficiency as defined by the pan - american group for immunodeficiency ( pagid ) and the european society for immunodeficiencies ( esid ) . prior to being enrolled in the european study , patients had to be successfully maintained on a stable ( variation in dose not to exceed 10% ) iv or sc dose of igg of 200800 mg / kg / month , to have at least three documented pre - study serum igg trough levels of 500 mg / dl , and to have experienced an acceptable clinical response as judged by their physician . study patients were converted to an equivalent dose of hizentra administered weekly by the sc route . this patient population is referred to as the 1:1 dose cohort . after a 12-week wash - in / wash - out phase , patients entered the 28-week efficacy period where the dose was held constant ( 1:1 dose ) and outcomes were monitored . the second cohort of patients came from a study that was conducted in the usa from november 2006 through october 2008 . the study had similar pre - enrollment requirements for a stable pre - study dose with an acceptable clinical outcome . as requested by the us food and drug administration ( fda ) , the dose for the 52-week efficacy phase was individually adjusted to give values for area under the curve of serum igg vs. time ( auc ) during scig therapy , which were equal to those recorded while the patients were still on the iv regimens prescribed by their physicians before enrollment . this resulted in a mean 1.5-fold increase in the scig dose compared to the previous ivig dose . additional details on the study designs are presented in the individual publications fully describing each study 's effect on traditional endpoints such as infection rates and safety [ 14 , 16 ] . the following secondary efficacy outcomes were assessed in both trials : missed work / school days due to infection , hospitalization days due to infection , and days of antibiotic therapy . these outcomes were reported as rate ( days / patient / year ) for the wash - in / wash - out and efficacy periods and served as the basis for this report . local tolerability was assessed differently in the two studies . in the eu study , only patient 's perception 2472 h after the administration was recorded , while in the us study , the investigator assessed local tolerability within 1545 min of the end of the infusion in addition to the patient 's perception . differences in the distribution of categorical variables between the two cohorts were compared using chi - square test . mean age , weight , igg dose , and serum igg trough levels were compared using student 's t - test . the rates of infection , hospitalization , antibiotic use , and missed work / school days were compared between cohorts using poisson regression , with and without controlling for key demographic differences between the cohorts , i.e. , sex , age group ( 216/17 years ) , and pid type ( xla / cvid and other ) using the days within the study as offset . the factor pid type was dropped from the multi - factorial analysis for hospitalization due to convergence problems . all analyses were performed using sas version 9.2 of the sas system , sas institute inc . , the characteristics of the two patient cohorts are summarized in table ii . because similar study protocols were used to recruit patients from different continents , the two patient populations were generally similar . however , the eu population included more male , pediatric , and x - linked agammaglobulinemia ( xla ) patients.table iisummary of baseline and dosing characteristics of the patients in each trialparameter1:1 dose cohort ( eu ) ( n = 46)1.5:1 dose cohort ( us ) ( n = 38)p - value of differences between cohortsgender ( % ) female33550.0368 male6745age ( years ) mean ( sd)21.5 ( 15.6)36.3 ( 19.5)0.0003age group ( % ) 211 years3780.0134 216 years5024 > 16 years5076weight ( kg ) mean ( sd)52.1 ( 24.7)70.0 ( 21.3)0.0006pid diagnosis ( % ) cvid61950.0014 xla375 arag20mean weekly doses before study start ( mg / kg)122144n / amedian igg levels before study start ( mg / dl)n / a mean ( sd)749 ( 157)1,010 ( 257)weekly dose of hizentra during the efficacy period ( mg / kg ) mean ( range ) [ sd]120 ( 59243 ) 213 ( 72379 ) < 0.0001mean trough level during the efficacy period ( mg / dl ) mean ( sd)810 ( 144)1,254 ( 322)<0.0001arag autosomal recessive agammaglobulinemia , cvid common variable immunodeficiency , eu european union , n / a not applicable , sd standard deviation , us united states , xla x - linked agammaglobulinemiachi - square teststudent 's t - testmean of individual patient median values summary of baseline and dosing characteristics of the patients in each trial arag autosomal recessive agammaglobulinemia , cvid common variable immunodeficiency , eu european union , n / a not applicable , sd standard deviation , us united states , xla x - linked agammaglobulinemia mean of individual patient median values in the eu study , the mean weekly igg dose administered during the efficacy period was 120 mg / kg ( range 59243 mg / kg ) , resulting in a mean trough level of 810 mg / dl ( standard deviation [ sd ] 144 mg / dl ) . in the us study , the mean weekly igg dose administered during the efficacy period was 213 mg / kg ( range 72379 mg / kg ) , resulting in a mean trough level of 1,253 mg / dl ( sd 321 mg / dl ) . of note , patients in the eu cohort had received lower mean igg doses during their previous therapy ( mean sd 107 29 mg / kg / week for ivig treatment and 132 50 mg / kg / week for scig ) compared with patients in the us cohort ( 145 48 mg / kg / week ) . no serious bacterial infections were reported in either study ( table iii ) . however , the rate of other infections was 2.76 events / patient / year in the 1.5:1 dose cohort vs. 5.18 events / patient / year in the 1:1 dose cohort ( p < 0.0001 ) . the rate of days hospitalized in the 1.5:1 dose group was 0.2 days / patient / year vs. 3.48 days / patient / year in the 1:1 dose group ( p < 0.0001 ) . the proportion of patients who became hospitalized was lower in the 1.5:1 dose group ( 2.6 compared to 8.7 for the 1:1 cohort ) ; however , the difference observed was not statistically significant ( p = 0.2423 ) . the rate of days of antibiotic use was 48.50 days / patient / year in the 1.5:1 dose group vs. 72.75 days / patient / year in the 1:1 dose group ( p < 0.001 ) . work / school days were missed at a rate of 2.06 days / patient / year in the 1.5:1 dose group vs. 8.00 in the 1:1 dose group ( p < 0.001 ) . the proportion of patients missing one or more work / school days per year was lower in the 1.5:1 dose cohort , but the difference observed did not reach statistical significance ( p = 0.2637 ) . the results of the eu study were substantially affected by the data of a child with recurrent pneumonia who missed 71 work / school days and spent 63 days in hospital [ 14 , 17 ] . the results of this analysis were similar to those for the entire populations ( table iii).table iiicomparison of health outcomes in the two cohorts of pid patients treated with different doses of subcutaneous hizentra1:1 dose cohort ( eu ) ( n = 46)1.5:1 dose cohort ( us ) ( n = 38)analysis of differences between cohortsunivariate comparisonsmulti - factorial comparisons adjusting for covariablesrate of serious infections ( events / patient / year)00rate of infections ( events / patient / year)5.182.76<0.0001<0.0001sensitivity analysis5.16<0.0001<0.0001rate of days of hospitalization due to infection ( days / patient / year)3.480.20<0.0001<0.0001sensitivity analysis0.950.0003n / apercent of patients hospitalized due to infection8.72.60.2423n / asensitivity analysis6.670.3925rate of days with antibiotics for treatment or prophylaxis of infection ( days / patient / year)72.7548.5<0.0001<0.0001sensitivity analysis 66.62<0.0001<0.0001rate of days missed off work / school due to infection ( days / patient / year)8.02.06<0.0001<0.0001sensitivity analysis 5.25<0.0001<0.0001percent of patients missing 1 day off work / school per year due to infection43.531.60.2637n / asensitivity analysis 42.20.3179eu european union , n / a not applicable , us united statessensitivity analysis of health outcomes after exclusion of one patient from the 1:1 cohort ( eu ) who suffered from recurrent pneumoniap - value from poisson regressionp value from chi - square testthe factor pid type was dropped from the multi - factorial analysis for hospitalization due to convergence problemsanalysis not performed due to non - convergence of the model comparison of health outcomes in the two cohorts of pid patients treated with different doses of subcutaneous hizentra eu european union , n / a not applicable , us united states sensitivity analysis of health outcomes after exclusion of one patient from the 1:1 cohort ( eu ) who suffered from recurrent pneumonia p - value from poisson regression p value from chi - square test the factor pid type was dropped from the multi - factorial analysis for hospitalization due to convergence problems analysis not performed due to non - convergence of the model an analysis of rate of infection by month of the year was performed to account for the different duration of the two studies . the results showed an even distribution of onset of infections over the year in the us study ( infection rate range , 1.143.82 infections / patient / year ) , while a seasonal variability was noted in the eu study . in the eu study , the rate of infection by month of onset ranged between 2.27 and 11.21 infections / patient / year , with the lowest rates of infection observed in june ( 2.27 infections / subject / year ) and july ( 2.71 infections / subject / year ) and the highest rates in january ( 11.21 infections / subject / year ) and december ( 9.34 infections / subject / year ) . however , the overall rates of infection did not show any particular trend ; thus , the rates for september , october , and november were similar to those for february , april , and august ( 5.176.65 ) . the rate of non - serious infections was significantly different between the two cohorts . to analyze further the reasons for this difference , an analysis of infection rates in patients experiencing more than four infections during the efficacy periods of the two studies was performed ( table iv ) . a substantial contribution to the annualized rate of infections in the eu study was due to 12 patients who experienced a total of 79 infection episodes in the efficacy period , resulting in a statistically significant difference between the two cohorts ( 12.05 in the 1:1 cohort and 8.38 in the 1.5:1 cohort ; p = 0.0430 ) . in the 1.5:1 cohort ( usa ) , seven patients experienced a total of 51 infection episodes ( table iv).table ivanalysis of infection rates in the two cohorts of pid patients1:1 dose cohort ( eu ) ( n = 46)1.5:1 dose cohort ( us ) ( n = 38)analysis of differences between cohortsnumber ( % ) of patients without infections in efficacy period10 ( 21.7)7 ( 18.4)0.7064number ( % ) of patients with 14 infection episodes in efficacy period24 ( 52.2)24 ( 63.2)0.3113number ( % ) of patients with > 4 infection episodes in efficacy period12 ( 26.1)7 ( 18.4)0.4032annualized rate of infection in patients with > 4 infection episodes in efficacy period12.058.380.0430eu european union , n / a not applicable , us united statesp value from chi - square testp value from poisson regression analysis of infection rates in the two cohorts of pid patients eu european union , n / a not applicable , us united states p value from chi - square test p value from poisson regression because the two cohorts differed in gender , age , and pid type , a poisson regression was conducted by controlling these differences as covariates in the analysis across the efficacy period of each study . the results of this analysis confirmed the significant differences in all four study outcomes ( p < 0.0001 , table iii ) . in addition , an analysis controlling for the length of the observation period was performed to compare the 1.5:1 and 1:1 cohorts up to the 28-week time point . during the first 28 weeks of the efficacy period in the 1.5:1 cohort , the rates of infection ( 2.7 events / patient / year ) , hospitalization ( 0 days / patient / year ) , antibiotic use ( 49.5 days / patient / year ) , and missed work / school days ( 1.61 days / patient / year ) were similar to those observed during the entire 54-week efficacy period . consequently , all efficacy outcomes observed in the 1.5:1 dose group were improved compared to those in the 1:1 dose group , regardless of the length of the observation period . the most common adverse event reported in each study was local reactions at the injection site . the rate of local reactions was 0.59 events / infusion in the 1.5:1 cohort and 0.06 events / infusion in the 1:1 cohort . most of the local reactions were of mild or moderate intensity ( 99.7% in the 1.5:1 cohort and 99.1% in the 1:1 cohort ) . the difference in the rate of local reactions between the studies was likely due to the different assessment of local tolerability . two independent trials of hizentra were conducted in the usa and the eu with similar designs except that regulatory authorities in the usa required that patients be converted to a sc dose that was 1.5 times higher than the iv dose they were previously using . the us fda requested that scig dose be adjusted to achieve an equivalent auc of igg serum levels when patients were converted from monthly iv dosing to weekly sc dosing . calculations based on igg serum levels obtained from patients under ivig indicated that it would require a sc dose that was 1.5 times the iv dose to achieve an equivalent auc . on the other hand , the european medicines agency ( ema ) accepted the direct conversion of the monthly iv dose to a weekly sc dose by simple division of the monthly dose into weekly increments , as long as this resulted in non - inferior igg levels compared to the trough on previous therapy . indeed , experts disagreed when asked if igg efficacy is driven by peak , trough , or auc serum levels . as a result , two parallel registration trials with similar designs were initiated with different scig dose levels , creating a unique opportunity to explore the effect of igg dose on treatment outcomes in stable well - managed pid patients . by comparing the two cohorts , we found that the 1:1 and 1.5:1 doses of scig were equally effective in achieving freedom from serious life - threatening bacterial infections . however , we showed that in the 1.5:1 dose ( usa ) cohort , there were significantly better function ( fewer work / school days missed ) , significantly less health care utilization ( fewer hospitalization days and less antibiotic use ) , and a significantly lower rate of non - serious infections . this two - cohort approach offers some valuable insights and strongly suggests a positive effect of 1.5:1 igg dose on outcomes . these findings should be interpreted with caution as the comparison of results from two independent studies differs from that of results collected in a single randomized trial . factors not controlled statistically or by randomization may be contributing to the differences observed ( baseline pid severity , practice differences between the eu and the usa , etc . ) . as the differences in behaviors ( work / school attendance , antibiotic use ) correlated with differences in infection rate , it seems unlikely that the improvement in clinical outcomes in the us study was purely a function of the differences in patient behavior between the usa and the eu . also , there were several differences between the two studies that might limit a direct side - by - side comparison . first , the us study was longer than the eu study ( 54-week vs. 28-week efficacy period , respectively ) . second , the pre - specified primary endpoints were different . in the us study , the primary endpoint was the annual rate of serious bacterial infections , while in the eu study , the primary endpoint was the total serum igg trough level . third , the eu study had more pediatric and xla patients , while the us study had a larger sample size and a higher dropout rate ( 43% vs. 16% for the eu study [ 14 , 16 ] ) . nevertheless , by controlling the gender , age , and pid type as covariates , the significant differences in all four study outcomes were confirmed , thereby strongly suggesting that the differences in study design and population were very unlikely responsible for the better outcomes observed in the us cohort . fourth , us patients were treated with higher doses for a longer period of time as they had received higher pre - study igg doses ( 144 vs. 122 mg / kg in the eu study ) . however , this difference does not change the overall result that the more igg patients received , the better indices of health they had . an alternate explanation for these findings could be that the pid patients enrolled in the usa had milder disease or had better outcomes in their pre - study period compared to the eu cohort . unfortunately , no efficacy assessment was done prior to study initiation to determine if the two groups were similar at baseline . in spite of these research design limitations , it is interesting to note that these findings do not contradict previous dose - ranging studies . indeed , of the ten previously published igg dose response studies [ 3 , 1826 ] summarized in table v , only the first two studies published in 1984 did not show a dose - response relationship . response function at the top of the dose range above which no further outcome improvement was achieved . , in a meta - analysis that included 17 studies with 676 patients treated with ivig and 2,127 patient - years of follow - up , that pneumonia risk was directly related to trough level of igg and that this risk could be progressively reduced by higher trough igg levels of up to at least 1,000 mg / dl . similarly , berger showed that the incidence of non - serious infections was inversely proportional to the steady state serum igg levels achieved in different scig studies .table va summary of the dose comparison studies of igg in pid published to datepublicationrouteigg preparationnumber of patientsstudy designoutcomes monitoredresultsdose response relationship foundidentified upper dose with no incremental benefithill , 1971 not availablenot available176open - label0.05 g / kg was superior to 0.025 g / kg ( reduced frequency of febrile episodes , otitis media , and pneumonia)nonomontanaro , 1984 ivgamimune 10%11randomizedno patient outcomes , time from 500 mg / kg dose to serum level under 400 mg / dl150 and 500 mg / kg were not differentiatednon / aochs , 1984 ivgamimune 10%35two - arm randomized , 12 monthnumber of infections , antibiotic use , days missed100 and 400 mg / kg are equally effectivenon / abernatowska , 1987 ivendoglobulin12two - arm randomized cross - over with retrospective vhistory , 12 monthfever , infections , antibiotic use , lung function500 mg / kg is superior to 150 mg / kg , especially in children with more symptomsyesnoroifman , 1987 ivsandoglobulin12two - arm randomized cross - overinfections , lung function600 mg / kg is superior to 200 mg / kg ; > 500 mg / dl is superior to < 500 mg / dlyesnoliese , 1992 iv , imvarious29retrospective chart reviewpneumonia , days hospitalized400 mg / kg q3 weeks iv is superior to 200 mg / kg q3 weeks iv or 100 mg / kg q3 weeks imyesnopruzanski , 1996 iviveegam21three - arm randomizedinfections600 or 400 mg / kg is superior to 200 mg / kg;yesnoquartier , 1999 ivvarious31retrospective chart reviewhospitalization for infection>800 mg / dl is superior to 500800 mg / dl or < 500 mg / dlyesnoeijkhout , 2001 ivimmunoglobulin iv43two - arm randomized cross - over , 9 monthinfection rate and duration600/800 mg / kg is superior to 300/400 mg / kg ; 940 mg / dl is superior to 630 mg / dlyesnoroifman , 2003 ivgamunex , 10%73findings based on post - hoc analysis of datainfection rate>900 mg / dl is superior to > 700900 mg / dl ; > 700900 mg / dl is superior to < 700 mg / dlyesnogamimune - n 10%73favre , 2005 ivsandoglobulin7observational , 116 patient - yearsinfections400 mg / kg q3 weeks is superior to 200 mg / kg q3 weeksyesnothis studyschizentra84open - label , multi - center , single - arminfections , missed work / school days due to infection , hospitalization days due to infection , and days of antibiotic therapymean weekly igg dose of 120 mg / kg improved secondary efficacy outcomes compared to 213 mg / kgyes , for some outcomesnoiv intravenous , i m intramuscular , n / a not applicablethis pertains to the flat portion of the dose response curve where an increase in the dose is not associated with any additional clinical responseno response or outcomes were monitored , only effect of dose on serum levelsthe dose response relationship , in that the highest dose had inferior outcomes to the lowest dose a summary of the dose comparison studies of igg in pid published to date iv intravenous , i m intramuscular , n / a not applicable this pertains to the flat portion of the dose response curve where an increase in the dose is not associated with any additional clinical response no response or outcomes were monitored , only effect of dose on serum levels the dose response relationship , in that the highest dose had inferior outcomes to the lowest dose none of these studies assessed if the outcomes achieved by patients from higher - dose groups were comparable to healthy ( non - pid ) individuals with a normal rate of infection or comparable to individuals with a state of complete physical , mental and social well being . in our study , the pid patients in the 1.5:1 dose group have missed 2.06 work / school days / patient / year due to infection alone . the us bureau of labor statistics reports that , on average , in 2010 , us workers missed 2.3 days / year due to any infections , other illnesses , or injuries this would indicate that pid patients treated with the 1.5:1 dose may still have sub - optimal experience of normal activity if other reasons for missed activity are considered . while all pid studies focus on the lack of disease , specifically a reduction in the rate of infections , not all include measures assessing the extent to which a patient achieves the who definition of health or some other measure of well being or function . efficacy measures such as antibiotic use , hospitalization rate , and missed work / school days serve as clinical and economic indicators in clinical trials ; however , they do not represent the total spectrum of the patient benefit which can be achieved through optimal igg dose . other associated patient benefit effects may be occurring with 1.5:1 igg doses , even if they were not measured . for example , a child may not miss school with low - grade infections but can not partake in the more physical aspects of classroom and playground life . an adult may not be so sick as to warrant time off work , but persistently lacks the energy for a normal social life . the 1.5:1 dose is therefore expected to be associated with a greater normalcy of life experiences , moving patients closer to a life without breakthrough infections or chronic lack of well being . moreover , the long - term sequelae of frequent infections accumulate over time , leading to structural changes in lung or sinus tissue . preventing such sequelae may lead to further improvements in long - term patient outcomes and potential reductions in long - term health care costs . higher igg doses may also help reduce the frequency of chronic infections . living a life with fewer infections may offer opportunities for pid patients which may otherwise not be afforded . successful treatment can no longer be measured solely by the degree to which serious bacterial infections are prevented . the findings that we report here suggest that increasing the doses of igg might produce improvements in such outcomes , at least in some patients with frequent infections . as the agenda of treatment of pid patients move forward beyond life or death interventions , the goals of igg therapy may move toward optimization of health state outcomes and the full achievement of health and a normal life . the higher - dose cohort in this comparison recorded improved indices of well being compared to those treated with traditional doses of igg . this study , despite its limitations , suggests that increasing the scig doses is associated with significantly fewer infections , days of hospitalization , days of antibiotic use , and days of missed activities , resulting in reduced resource utilization . | the recommended dose of igg in primary immunodeficiency ( pid ) has been increasing since its first use . this study aimed to determine if higher subcutaneous igg doses resulted in improved patient outcomes by comparing results from two parallel clinical studies with similar design .
one patient cohort received subcutaneous igg doses that were 1.5 times higher than their previous intravenous doses ( mean 213 mg / kg / week ) , whereas the other cohort received doses identical to previous subcutaneous or intravenous doses ( mean 120 mg / kg / week ) .
while neither cohort had any serious infections , the cohort maintained on higher mean igg dose had significantly lower rates of non - serious infections ( 2.76 vs. 5.18 episodes / year , p < 0.0001 ) , hospitalization ( 0.20 vs. 3.48 days / year , p < 0.0001 ) , antibiotic use ( 48.50 vs. 72.75 days / year , p < 0.001 ) , and missed work / school activity ( 2.10 vs. 8.00 days / year , p < 0.001 ) .
the higher - dose cohort had lower health care utilization and improved indices of well being compared to the cohort treated with traditional igg doses . |
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technological advancement has brought various forms of information and communication technology ( ict ) within the reach of children . in the united states , over 80% of adolescents own at least one form of new media technology ( e.g. , cell phone , personal data assistant , and computer for internet access ) . data from india regarding the use of ict among children and adolescents are limited . the use and access to ict is not uniform and there is a vast difference among various sociodemographic groups , known as digital divide . digital divide , or the digital split , is a social issue referring to the differing amount of information between those who have access to the internet ( especially broadband access ) and those who do not have access . the organization for economic cooperation and development defines digital divide as the gap between individuals , households , businesses , and geographic areas at different socioeconomic levels with regard to both their opportunities to access icts and to their use of the internet for a wide variety of activities . the potential of using internet as a health promotion tool can not be ignored . unlike the paper - based health promotion materials of the past , the internet has triggered a growth in provision of more interactive health promotion messages , which are likely to enhance the potential for behavior change . various channels of communication are used to promote health among children and ict can serve as an innovative way of delivering the desired health education to bring about behavioral change . information regarding the use of internet is essential to explore the feasibility of use of this mode in information dissemination . puducherry is one the better performing union territories in india with a robust public health system , presence of motivated human resources for health , and a high literacy rate provides opportunity to test innovations in health . hence , an exploratory study was conducted to find out the determinants and prevalence of use of internet among school students of puducherry . an exploratory study was carried out in three schools from urban areas and two schools from rural areas of puducherry between september 2011 and november 2011 . these schools were selected purposively , as they cater to the children from the service areas of the urban and rural health centers ( jiuhc and jirhc ) of jawaharlal institute of post graduate medical education and research , puducherry , which provides health services in these areas . prior permission and informed consent was taken from the principals of the respective schools before starting the study . being an exploratory study , all the students belonging to the standards vi to ix ( n = 421 ) were included in the study . the students of standards x to xii were not included as the school principals refused to give consent , because of the tight teaching schedule that was in practice for them . the students from younger standards were excluded , because the tool to be used was a self - administered one and the teachers expressed doubts regarding the ability of students to answer it . a structured self - administered questionnaire was developed and pretested due to unavailability of a standard instrument . this sought information on sociodemographic details ( age , gender , standard of education , occupation of father , and type of school ) , use of internet , and place of accessing internet , hours of use , and reasons for use . after consulting with the school authorities , the students were made to assemble in a class room and were briefed about the questionnaire . the name of the student or the school was not included in the questionnaire for the same purpose . univariate analysis for determining the association between use of internet and various factors like age categories , gender , area of residence , type of school attended , and occupation of the father was done using chi - square test . logistic regression analysis was done to estimate the odds ratio and 95% confidence intervals ( cis ) for identifying independent determinants of internet use . all tests were two - tailed and statistical significance was defined as a value of p < 0.05 . an exploratory study was carried out in three schools from urban areas and two schools from rural areas of puducherry between september 2011 and november 2011 . these schools were selected purposively , as they cater to the children from the service areas of the urban and rural health centers ( jiuhc and jirhc ) of jawaharlal institute of post graduate medical education and research , puducherry , which provides health services in these areas . prior permission and informed consent was taken from the principals of the respective schools before starting the study . being an exploratory study , all the students belonging to the standards vi to ix ( n = 421 ) were included in the study . the students of standards x to xii were not included as the school principals refused to give consent , because of the tight teaching schedule that was in practice for them . the students from younger standards were excluded , because the tool to be used was a self - administered one and the teachers expressed doubts regarding the ability of students to answer it . a structured self - administered questionnaire was developed and pretested due to unavailability of a standard instrument . this sought information on sociodemographic details ( age , gender , standard of education , occupation of father , and type of school ) , use of internet , and place of accessing internet , hours of use , and reasons for use . after consulting with the school authorities , a suitable date was fixed for data collection . the students were made to assemble in a class room and were briefed about the questionnaire . the name of the student or the school was not included in the questionnaire for the same purpose . univariate analysis for determining the association between use of internet and various factors like age categories , gender , area of residence , type of school attended , and occupation of the father was done using chi - square test . logistic regression analysis was done to estimate the odds ratio and 95% confidence intervals ( cis ) for identifying independent determinants of internet use . all tests were two - tailed and statistical significance was defined as a value of p < 0.05 . among the 421 students included in the study , nearly three - fourth ( 72.2% ) were boys and almost half ( 52.2% ) were from rural area . most ( 57.5% ) of the fathers of these children were employed as unskilled laborers . internet was being used by 100 ( 23.8% ) students and most ( 63% ) of them had accessed it from internet cafes . more than half of them ( 62% ) had used the internet for playing games and other forms of entertainment . the mean ( standard deviation ) hours of internet use per day was 0.9 ( 0.7 ) hours . the use of internet was found to be significantly higher ( p < 0.05 ) among boys ( 30.9% ) , students from urban areas ( 39% ) and from government schools ( 26.3% ) . the proportion of early adolescents using internet was more compared with late adolescents ; however , this was not statistically significant ( p = 0.059 ) [ table 1 ] . determinants of internet use among school children after logistic regression analysis , the use of internet was found to be significantly higher in mid- adolescents ( aor : 2.205 ; 95% ci [ 1.284 - 3.785 ] ) , among boys ( aor : 3.835 ; 95% ci [ 1.533 - 9.594 ] ) and students from urban areas ( aor : 4.694 ; 95% ci [ 2.612 - 8.436 ] [ table 2 ] . internet penetration is found in nearly 24% of the students in the study population ; however , there is a wide disparity in internet use in terms of age group , gender , and geographic location in our study population . after adjusting for other factors using logistic regression , it is found that if a student is living in an urban area , he / she is 4 and 1/2 times more likely to use internet as compared with a rural counterpart . sumanjeet singh reports that the internet access in urban households of india is 10 times that of rural households in 2008 . aspects like lack of connectivity , cost of owning a computer are some of the factors which augment the divide . in contrast in china , 69% of the rural students use internet against 88% of urban students . the internet use among boys is nearly 4 times ( aor : 3.8 ) more than girls . this gender divide in internet use is seen even in developed regions like the united states up to the late nineties ; however , the gap was bridged in 2003 . gender divide in internet use in india among school children is not well - documented . the older children ( mid - adolescents ) are 2 times more likely to use internet when compared with the younger ones ( early adolescents ) . this could be because , the younger students are not yet taught computers in their syllabus and they are not allowed in internet cafes . health behaviors account for an estimated 60% of the risk associated with chronic illnesses such as diabetes , cardiovascular diseases , and some cancers . computer - tailored interventions have become an increasingly common strategy for altering health risk behaviors such as tobacco use , poor diet , and lack of exercise that are linked to chronic disease worldwide . adolescence is the stage in life where habits are formed and this group needs to be targeted as they can not only alter their own behavior but also channel the health promotion message to their respective families and communities . the internet use among the school children in both urban and rural areas is low . if at all we would like to use ict for health promotion , then it would be more appropriate for urban children . | background : the advent of internet has triggered a growth in provision of more interactive health promotion messages , which are likely to enhance the potential for behavior change especially among children .
we intended to find out the determinants and prevalence of use of internet among school students of puducherry.materials and methods : an exploratory study was carried out among all the students belonging to the standards vi to ix ( n = 421 ) in three schools from urban areas and two schools from rural areas of puducherry .
a pretested , self - administered questionnaire which sought information on sociodemographic details ( age , gender , standard of education , occupation of father , and type of school ) , use of internet , and place of accessing internet , hours of use , and reasons for use was utilized .
the statistical significance between the categorical variables was done using chi - square test .
all significance tests were two - tailed and statistical significance was defined as a value of p < 0.05.results:majority ( 72.2% ) were boys .
less than one - third ( 23.8% ) of the students used internet .
the use of internet was found to be significantly higher in mid - adolescents ( adjusted or : 2.205 ) ; among boys ( adjusted or : 3.835 ) and students from urban areas ( adjusted or : 4.694).conclusion : there is a geographical and gender divide observed among the school children in the use of internet . taking the steps to bridging the digital divide will help utilize internet as a media for health promotion . |
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breast cancer is the leading cause of cancer mortality in women worldwide and is one of the major contributors to the global health burden ( 13 ) . mounting clinical data suggest that the development of metastatic spread of the disease is responsible for at least 90% of the cancer - associated mortality , and the survival rate falls from 90% for localized breast cancer to 20% for metastatic breast cancer ( 4 ) . lymphatic metastasis is one of the most important pathways of breast cancer systemic metastasis , and is closely related to prognosis and therapy plans for breast cancer patients . frequently , the initial sites of metastasis are the regional lymph nodes ( 5 , 6 ) , and migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis , a process that generates new lymphatic vessels from pre - existing lymphatics or lymphatic endothelial progenitors ( 79 ) . some researchers suggest that the vascular endothelial growth factor - c ( vegf - c)/vascular endothelial growth factor receptor 3 ( vegfr3 ) signaling system is the most efficient pathway in regulating lymphangiogenesis . vegf - c , also called lymphatic vessel growth factor , belongs to the vegf family , and involves in tumor lymph - angiogenesis by inducing lymphatic endothelial proliferation and vessel enlargement to facilitate the shedding of tumor cells into the surrounding lymphatic vessels ( 10 , 11 ) . vegf - c expression has recently been reported to be correlated with lymph node metastasis in breast ( 12 , 13 ) , gastric ( 14 ) , colorectal ( 15 ) , lung ( 16 ) , prostate ( 17 ) , head and neck ( 18 ) , and gallbladder cancer ( 19 ) . however , in breast cancer , the definite role of vegf - c has not yet been elucidated . some studies reported that vegf - c expression correlated with lymph node metastasis and patients poor survival ( 13 , 20 ) , while some others not ( 21 , 22 ) . to date , insufficient samples and some other factors have resulted in controversial results of different clinical studies . to derive a more precise estimation of the relationship between vegf - c expression and clinical outcomes in patients with breast cancer , we performed a meta - analysis of 14 prospective or retrospective cohort studies with a total of 1,573 breast cancer patients . a systematic literature search of medline ( 1960 through february , 2013 ) , embase ( 1988 through february , 2013 ) , web of science ( 1960 through february , 2013 ) databases , and two chinese databases ( wanfang and chinese national knowledge infrastructure databases , 1960 through february , 2013 ) was conducted by two study investigators ( d.c . and b.l . ) independently for all relevant articles about the prognostic value of vegf - c expression in breast cancer patients . immunohistochemistry , breast neoplasma(s ) , breast carcinoma , metastasis , and prognosis . studies eligible for inclusion in this meta - analysis should meet the following criteria : 1 ) measures vegf - c expression in the cancer tissue with immunohis - tochemistry ( ihc ) , 2 ) patients have pathologically confirmed breast cancer , 3 ) patients provides information on survival time according to vegf - c expression ; 4 ) patients has a follow up time exceeding 5 years . when an individual author published several articles obtained from the same patient population , only the newest or most complete article was included in the analysis . the exclusion criteria of the meta - analysis were : ( a ) animal studies ; ( b ) meta - analyses , letters , reviews , meeting abstracts , or editorial comments ; ( c ) studies with duplicate data or incomplete data . two reviewers independently extracted the following data from each study : first author s name , year of publication , type of cohort study , country of origin , total number of cases ( n ) , follow - up time , and numbers of the patients with positive and negative expression of vegf - c , etc . review manager 5.0 program ( version 5.1.0 ; the cochrane collaboration , oxford , england ) and stata ( version12.0 , stata corporation , tx , usa ) were used to perform all the statistical analysis . two models of pooling data for dichotomous outcomes were conducted : the random - effects model and the fixed - effects model . the pooled statistical analysis was calculated using the fixed effects model , but a random - effect model was performed when the p value of heterogeneity test was < 0.1 . the odds ratio and 95% ci were calculated for each study , and the combined or and 95% ci were calculated for the studies . or was the proportion of the exposed population in whom disease has developed over the proportion of the un - exposed population in whom disease has developed in a case - control study . by convention , or > 1 implies a worse prognosis in vegf - c positive group . the significance of the pooled or was determined by the z test and a p value of less than 0.05 was considered significant . in order to assess the between - study heterogeneity , the i statistic to quantify the proportion of the total variation due to heterogeneity the potential publication bias was assessed by begg s funnel plot and egger s test ( 23 , 24 ) . a systematic literature search of medline ( 1960 through february , 2013 ) , embase ( 1988 through february , 2013 ) , web of science ( 1960 through february , 2013 ) databases , and two chinese databases ( wanfang and chinese national knowledge infrastructure databases , 1960 through february , 2013 ) was conducted by two study investigators ( d.c . and b.l . ) independently for all relevant articles about the prognostic value of vegf - c expression in breast cancer patients . immunohistochemistry , breast neoplasma(s ) , breast carcinoma , metastasis , and prognosis . studies eligible for inclusion in this meta - analysis should meet the following criteria : 1 ) measures vegf - c expression in the cancer tissue with immunohis - tochemistry ( ihc ) , 2 ) patients have pathologically confirmed breast cancer , 3 ) patients provides information on survival time according to vegf - c expression ; 4 ) patients has a follow up time exceeding 5 years . when an individual author published several articles obtained from the same patient population , only the newest or most complete article was included in the analysis . the exclusion criteria of the meta - analysis were : ( a ) animal studies ; ( b ) meta - analyses , letters , reviews , meeting abstracts , or editorial comments ; ( c ) studies with duplicate data or incomplete data . two reviewers independently extracted the following data from each study : first author s name , year of publication , type of cohort study , country of origin , total number of cases ( n ) , follow - up time , and numbers of the patients with positive and negative expression of vegf - c , etc . review manager 5.0 program ( version 5.1.0 ; the cochrane collaboration , oxford , england ) and stata ( version12.0 , stata corporation , tx , usa ) were used to perform all the statistical analysis . two models of pooling data for dichotomous outcomes were conducted : the random - effects model and the fixed - effects model . the pooled statistical analysis was calculated using the fixed effects model , but a random - effect model was performed when the p value of heterogeneity test was < 0.1 . the odds ratio and 95% ci were calculated for each study , and the combined or and 95% ci were calculated for the studies . or was the proportion of the exposed population in whom disease has developed over the proportion of the un - exposed population in whom disease has developed in a case - control study . by convention , or > 1 implies a worse prognosis in vegf - c positive group . the significance of the pooled or was determined by the z test and a p value of less than 0.05 was considered significant . in order to assess the between - study heterogeneity , the i statistic to quantify the proportion of the total variation due to heterogeneity the potential publication bias was assessed by begg s funnel plot and egger s test ( 23 , 24 ) . 1 . a total of 152 potentially relevant articles were reviewed , and 138 articles were excluded due to their irrelevance to the current analysis and insufficient data . therefore , the final meta - analysis was performed on the basis of the remaining 14 studies that met the criteria set forth in the search strategy and study selection . the main features of eligible studies are summarized in table 1 . among those 14 studies , 12 studies reported data on the lymph node metastasis , and 5 studies reported data on the vegf - c expression on overall survival and disease - free survival . there were 1,240 cases providing the data on lymph node metastasis , among which 629 cases had metastasis and 611 cases were without metastasis , or 772 cases were with positive vegf - c expression and 518 cases were negative vegf - c expression . flow diagram of the literature search and trial selection process the cumulative metastasis rate and vegf - c expression rate of breast cancer were 50.73% ( 629/1240 ) and 62.26% ( 772/1240 ) , respectively . the vegf - c positive cases had a cumulative metastasis rate of 56.87% ( 439/772 ) that was higher than 36.68% ( 190/518 ) in vegf - c - negative cases . of those 12 studies on lymph node metastasis , there was high between - study heterogeneity ( i=75 % ) . meta - analysis of random - effects model was adopted , and showed vegf - c expression was associated with lymph node metastasis in patients with breast cancer ( random - effects , or=2.14 ; 95 % ci : 1.213.77 , p<0.009 ) ( fig . there were 5 studies with a total of 414 breast cancer cases relating the overall survival ( os ) and disease - free survival ( dfs ) . of those 5 studies on os and dfs , there were low between - study heterogeneities ( i=10 % for os ; i=0 % for dfs ) . meta - analysis of fixed - effects model showed vegf - c expression was associated with poorer os in patients with breast cancer ( fixed - effects , or = 2.46 , 95% ci : 1.464.14 , p < 0.001 ) ( fig . in addition , meta - analysis of fixed - effects model also showed vegf - c expression was associated with poorer dfs in patients with breast cancer ( fixed - effects , or = 2.10 , 95% ci : 1.32 3.35 , p = 0.002 ) ( fig . we performed the funnel plots and egger s test to assess the publication bias on studies related to lymph node metastasis . funnel plot did not reflect obvious asymmetry in this meta - analysis ( figure 3 ) . also , no indication of publication bias was found ( begg s p value = 0.373 and egger s p value = 0.192 ) . since there are not more than five prognostic studies about os and dfs , publication bias of the included studies was not performed . baseline characteristics of the 14 eligible studies in the meta - analysis forrest plot ( random - effects model ) of odds ratios ( ors ) for the association of vegf - c expression with lymph node metastasis in patients with breast cancer forest plot ( fixed - effects model ) of odds ratios ( ors ) for 5 contributing studies assessing the effect of vegf - c expression on survival in patients with breast cancer . ( a ) overall survival ; ( b ) disease - free survival begg s funnel plot for publication bias test with pseudo 95% confidence limits for the studies related to lymph node metastasis there are no doubts that the members of vegf family are important cancer players , thus the field of vegf research is very promising in the area of cancer research . vegf - c has been reported to be a lymphatic - specific growth factor , which is the first ligand to be identified for vegfr-3 ( 25 ) . since the expression of vegfr-3 the major function of vegf - c appears to be the regulation of lymphatic vessel growth ( 26 ) . to date , many studies have reported that the overexpression of vegf - c significantly correlated with lymph node metastasis and lymphangio - genesis in primary tumours , such as thyroid ( 27 ) , prostate ( 28 ) , gastric ( 29 ) , colorectal ( 30 ) , ovarian ( 31 ) , and breast cancers ( 32 ) . however , other reports could not confirm such correlations , or opposite relationships were found ( 33 ) . at present , there is no consensus on the association between increased vegf - c expression detected by ihc and poor survival in patients with breast cancer . more accurate evaluation of the impact of vegf - c expression on patient survival is needed . the findings from our study suggest that vegf - c expression is associated with the prognosis of patients with breast cancer , and patients with higher vegf - c expression have poorer survival . in breast cancer , metastasis occurs primarily through the lymphatic system , and the extent of lymph node involvement is a key prognostic factor for the disease ( 7 , 34 ) . there has been accumulating evidence showing that vegf - c is the central regulator of lymphangiogenesis ( 35 ) . as a result of the sample size limitation for the individual studies , broad agreement on the association between vegf - c expression and lymph node metastasis in breast cancer has not yet been reached . in the present study , our analyses , combining 12 independent studies that included 1,240 cases with breast cancer , revealed that vegf - c expression is positively associated with lymph node metastasis . these findings might be important for prognosis and treatment of breast cancer , in addition to improve the understanding of biology . on the other hand , there is significant effect of vegf - c expression on os and dfs in the complete patient sample set , including 5 independent studies . our study suggested that vegf - c expression was associated with poorer dfs in patients with breast cancer . in addition , vegf - c expression was also associated with poorer os in patients with breast cancer . the findings from the present study suggest that vegf - c expression is associated with the prognosis of breast cancer patients , and patients with higher vegf - c expression have poorer survival . our results should be interpreted cautiously since some limitations exist in this present meta - analysis . therefore , the publication bias may have occurred , even though the use of a statistical test did not show it . second , the number of included studies was relatively small with only about 1,240 cases . moreover , other clinical factors such as age and different chemotherapies in each study might lead to bias . determining whether or not these factors influence the results of this meta - analysis would need further investigation . third , the effect from our meta - analysis could be overestimated because there are two retrospective cohort studies which had high risk of reporting biases . therefore , adequately prospective studies with large sample size are required to further assess the precise prognostic effect of vegf - c expression in breast cancer . finally , the studies included in this meta - analysis were from different populations , it is possible that demographic factors can confound our results . despite the limitations listed above , our study shows a significant correlation between vegf - c expression and lymph node metastasis in breast cancer patients . moreover , vegf - c expression might be a potential prognostic factor for survival in patients with breast cancer , if detected by immunochemistry . in order to become a useful prognostic factor at the level of individual patient and in the context of targeted therapy , these results need to be confirmed by an adequately designed prospective study , and larger clinical trials with widely accepted assessment methods are necessary to define the precise prognostic significance for vegf - c in breast cancer patients . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors . | abstractbackgroundvascular endothelial growth factor ( vegf)-c , as a lymphangiogenic factor , plays important roles in the progression of several malignancies .
however , its clinical prognostic value in breast cancer still remains controversial .
we performed a meta - analysis of available studies to assess the association between vegf - c expression and the ou - tcomes of breast cancer patientsmethodswe searched eligible studies in three english databases ( medline , embase , and web of science ) and two chinese databases ( wanfang and chinese national knowledge infrastructure databases ) .
key words used in the research included
vegf - c , breast cancer ,
immunohistochemistry , breast neoplasma(s ) , breast carcinoma , metastasis , and prognosis .
fourteen studies with a total of 1 , 573 breast cancer cases were finally included into the meta - analysis . the pooled odds ratios ( ors ) with the corresponding 95% confidence interval ( 95% cis ) for lymph node metastasis , overall survival , and disease - free survival
were calculated by using fixed - effects or random - effects models .
heterogeneity and publication bias were also assessed.resultsmeta-analysis of random - effects model showed vegf - c expression was associated with lymph node metastasis in patients with breast cancer ( random - effects , or = 2.14 ; 95 % ci 1.213.77 , p = 0.009 ) .
vegf - c expression was associated with poorer overall survival ( fixed - effects , or = 2.46 , 95% ci : 1.464.14 , p < 0.001 ) and disease - free survival ( fixed - effects , or = 2.10 , 95% ci : 1.323.35 , p = 0.002 ) in patients with breast cancer.conclusionvegf-c expression is positively associated with lymph node metastasis in breast cancer , and vegf - c detection in breast cancer might be an effective and feasible means to predict outcome . |
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high blood pressure can induce coronary artery disease , congestive heart failure , stroke , impaired vision , and kidney disease ; untreated hypertension affects all organ systems and can shorten people 's life expectancy . common clinical strategies to achieve a lowering of blood pressure include the use of beta adrenergic antagonists , calcium channel blockers , diuretics , angiotensin ii ( ang ii ) receptor antagonists and ang converting enzyme ( ace ) inhibitors . activation of the renin - ang system ( ras ) both systemically and locally seems to be of importance for cardiovascular function . the octapeptide ang ii definitively plays a central role . in a reversal , for example , of left ventricular hypertrophy , so far the most important independent risk factor for an adverse outcome , blocking of the ras with ace inhibition has been shown to be particularly effective . a more complete blockade of the ang ii type 1 receptor would offer more effective attenuation of the unfavorable effect of ang ii . in recent years , there has been a growing interest and demand in using medicinal plants for treating and preventing various diseases including cardiovascular diseases . traditional medicines of plants origin have received much attention due to several factors such as easy availability , affordable cost , safety , and efficacy as well as cultural acceptability . teucrium polium l. ( tp ) is a flowering plant belonging to the family labiate and is found abundantly in south western asia , europe , and north africa . teucrium species have been used as medicinal herbs for over 2000 years as diuretic , diaphoretic , tonic , antipyretic , antispasmodic , antidiabetic , and many of them are used in folk medicine . there is increasing evidence of cardiovascular effects of tp such as positive inotropic and chronotropic , decreasing of blood pressure , and lowering blood lipid . nevertheless , the exact effect of tp extract on the vascular system has not been clarified . therefore , in the present study , we aimed to investigate the effects of the hydroalcholic extract of tp on ang ii - induced hypertension and related heart rate ( hr ) variability . stems and leaves of tp were collected in october 2012 from khorasan province , ferdows , iran , and identified by ferdowsi university herbarium ( voucher no . 152 - 2016 - 4 ) and then dried at room temperature . aerial parts ( 300 g ) of the plant were soaked in ethanol ( 50% ) for 48 h and paper filter was used to filter the solute after mixing . the solution was then dried using a 40c oven for 72 h. the dried extract was dissolved in the distilled water to make desired doses . male wistar rats , 200 - 250 g were housed in colony rooms with 12/12 h light / dark cycle at 21c 2c and had free access to food and water . the experimental protocol was approved by ethical committee at mashhad university of medical sciences ( process number 900559 ) . the following reagents were used : ang ii and urethane ( sigma , usa ) , losartan ( los ) ( a gift from daru pakhsh , i.r . forty - eight male wistar rats were divided into six groups as following order ( n = 8 in each group )
control group ; received saline ( intravenous [ i.v])ang group ; received ang ii ( 300 ng , i.v)los group ; received los ( 10 mg / kg , i.v 0.5 ml ) 30 min before injection of ang iitp 100 group ; received 100 mg / kg of tp extract ( i.p . ) 30 min before injection of ang iitp 200 group ; received 200 mg / kg of tp extract ( i.p . ) 30 min before injection of ang iitp 400 group ; received 400 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii . control group ; received saline ( intravenous [ i.v ] ) ang group ; received ang ii ( 300 ng , i.v ) los group ; received los ( 10 mg / kg , i.v 0.5 ml ) 30 min before injection of ang ii tp 100 group ; received 100 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii tp 200 group ; received 200 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii tp 400 group ; received 400 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii . rats were anesthetized with urethane ( 1.4 g / kg , i.p . with 0.7 g / kg as a supplementary dose ) . temperature was kept at 37.5c with a heating lamp . a polyethylene catheter-50 filled by heparinized saline the catheter connected to a pressure transducer then mean arterial pressure ( map ) , and hr , were continuously recorded by a power lab system ( i d instrument , australia ) . methods was 0.5 ml . the data of the blood pressure and hr were expressed as mean standard error of the mean time course alterations of hr and arterial pressure was plotted . the maximum change was compared with the control or ang group ( independent t - test ) values . stems and leaves of tp were collected in october 2012 from khorasan province , ferdows , iran , and identified by ferdowsi university herbarium ( voucher no . 152 - 2016 - 4 ) and then dried at room temperature . aerial parts ( 300 g ) of the plant were soaked in ethanol ( 50% ) for 48 h and paper filter was used to filter the solute after mixing . the solution was then dried using a 40c oven for 72 h. the dried extract was dissolved in the distilled water to make desired doses . male wistar rats , 200 - 250 g were housed in colony rooms with 12/12 h light / dark cycle at 21c 2c and had free access to food and water . the experimental protocol was approved by ethical committee at mashhad university of medical sciences ( process number 900559 ) . the following reagents were used : ang ii and urethane ( sigma , usa ) , losartan ( los ) ( a gift from daru pakhsh , i.r . forty - eight male wistar rats were divided into six groups as following order ( n = 8 in each group )
control group ; received saline ( intravenous [ i.v])ang group ; received ang ii ( 300 ng , i.v)los group ; received los ( 10 mg / kg , i.v 0.5 ml ) 30 min before injection of ang iitp 100 group ; received 100 mg / kg of tp extract ( i.p . ) 30 min before injection of ang iitp 200 group ; received 200 mg / kg of tp extract ( i.p . ) 30 min before injection of ang iitp 400 group ; received 400 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii . control group ; received saline ( intravenous [ i.v ] ) ang group ; received ang ii ( 300 ng , i.v ) los group ; received los ( 10 mg / kg , i.v 0.5 ml ) 30 min before injection of ang ii tp 100 group ; received 100 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii tp 200 group ; received 200 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii tp 400 group ; received 400 mg / kg of tp extract ( i.p . ) 30 min before injection of ang ii . rats were anesthetized with urethane ( 1.4 g / kg , i.p . with 0.7 g / kg as a supplementary dose ) . temperature was kept at 37.5c with a heating lamp . a polyethylene catheter-50 filled by heparinized saline then mean arterial pressure ( map ) , and hr , were continuously recorded by a power lab system ( i d instrument , australia ) . the data of the blood pressure and hr were expressed as mean standard error of the mean time course alterations of hr and arterial pressure was plotted . the maximum change was compared with the control or ang group ( independent t - test ) values . baseline map and hr before injection of saline were recorded then saline injected into the jugular vein . injection of saline had no significant effects on map ( before : 95 1.6 mmhg and after : 99 2.17 mmhg ) or hr ( before : 264 7.32 beats / min and after : 262 7.1 beats / min ) . the time course changes of map and hr after injection of ang are shown in figure 1 . maximal change in map was statistically significant compared with the control group ( : 32.37 3.37 , p < 0.001 ) . moreover , significant hr changes were shown after injection of ang ( : 72.79 5.1 , p < 0.001 ) [ figure 2 ] . time courses of changes in mean arterial pressure ( map ) and heart rate ( hr ) in response to injection of angiotensin ii ( ang ii ) ( 300 ng ) in groups of control , ang ii and losartan . for each group , n = 8 . ( a ) map , * * * p < 0.001 compared to control and + + + p < 0.001 compared to ang group . ( b ) hr , * * * p < 0.001 compared to control and + + + p < 0.001 compared to ang group typical recording of blood pressure and heart rate in response to angiotensin ii injection . the vertical lines indicate the injection time losartan pretreatment ( 10 mg / kg , i.v ) inhibited cardiovascular effects induced by ang ii so the cardiovascular responses to ang ii after pretreatment with los did not differ significantly with those observed before injection of ang ii ( map : 9.05 5.63 mmhg and hr : 15.76 5.7 beats / min ) . in 4 min after injection of ang ii , maximum significant changes of map and hr were observed in los group compared with simultaneous changes in other two groups , independent t - test , p < 0.001 . meanwhile , map changes in los group were significantly lower than those of control and ang groups ( repeated measures anova , p < 0.001 for both ) [ figure 1a ] . as well as hr changes in los group were lower compared to ang group ( repeated measures anova , p < 0.001 ) [ figure 1b ] . pretreatment of normotensive rats with different doses of tp ( 100 , 200 , and 400 mg / kg , i.p . ) reduced significantly map compared to baseline ( paired t - test , p < 0.001 for all three doses , data not shown ) but had no effect on hr . the cardiovascular responses to ang ii injection antagonized by tp so that in these groups the maximum changes of map were significantly lower than those of induced in ang group ( map : 14.73 3.94 , 13.44 4.93 , 14.19 1.9 compared to 32.37 3.37 mmhg , respectively , t - test , p < 0.001 for all three groups ) . moreover , map changes in all three groups of tp extract were significantly lower than ang group ( repeated measures anova , p < 0.001 ) [ figure 3a ] and hr changes in two groups of tp 100 and tp 200 showed a significant reduction compared to ang group ( repeated measures anova , p < 0.001 and p < 0.01 , respectively ) [ figure 3b ] . time courses of changes in mean arterial pressure ( map ) and heart rate ( hr ) in response to injection of angiotensin ii ( ang ii ) ( 300 ng ) in groups of ang ii and three doses of teucrium polium l. ( tp ) extract ( tp 100 , tp 200 and tp 400 ) . for each group , n = 8 . ( a ) map , * * * p < 0.001 compared to ang group . ( b ) hr however , no significant hr changes were observed between these pretreated groups and ang group in the maximum level of effect [ hr : 55.6 3.8 , 53.62 5.25 , 60.12 8.87 compared to 72.79 5.10 beats / min , respectively ; figure 3 ] . baseline map and hr before injection of saline were recorded then saline injected into the jugular vein . injection of saline had no significant effects on map ( before : 95 1.6 mmhg and after : 99 2.17 mmhg ) or hr ( before : 264 7.32 beats / min and after : 262 7.1 beats / min ) . the time course changes of map and hr after injection of ang are shown in figure 1 . maximal change in map was statistically significant compared with the control group ( : 32.37 3.37 , p < 0.001 ) . moreover , significant hr changes were shown after injection of ang ( : 72.79 5.1 , p < 0.001 ) [ figure 2 ] . time courses of changes in mean arterial pressure ( map ) and heart rate ( hr ) in response to injection of angiotensin ii ( ang ii ) ( 300 ng ) in groups of control , ang ii and losartan . for each group , n = 8 . ( a ) map , * * * p < 0.001 compared to control and + + + p < 0.001 compared to ang group . ( b ) hr , * * * p < 0.001 compared to control and + + + p < 0.001 compared to ang group typical recording of blood pressure and heart rate in response to angiotensin ii injection . losartan pretreatment ( 10 mg / kg , i.v ) inhibited cardiovascular effects induced by ang ii so the cardiovascular responses to ang ii after pretreatment with los did not differ significantly with those observed before injection of ang ii ( map : 9.05 5.63 mmhg and hr : 15.76 5.7 beats / min ) . in 4 min after injection of ang ii , maximum significant changes of map and hr were observed in los group compared with simultaneous changes in other two groups , independent t - test , p < 0.001 . meanwhile , map changes in los group were significantly lower than those of control and ang groups ( repeated measures anova , p < 0.001 for both ) [ figure 1a ] . as well as hr changes in los group were lower compared to ang group ( repeated measures anova , p < 0.001 ) [ figure 1b ] . pretreatment of normotensive rats with different doses of tp ( 100 , 200 , and 400 mg / kg , i.p . ) reduced significantly map compared to baseline ( paired t - test , p < 0.001 for all three doses , data not shown ) but had no effect on hr . the cardiovascular responses to ang ii injection antagonized by tp so that in these groups the maximum changes of map were significantly lower than those of induced in ang group ( map : 14.73 3.94 , 13.44 4.93 , 14.19 1.9 compared to 32.37 3.37 mmhg , respectively , t - test , p < 0.001 for all three groups ) . moreover , map changes in all three groups of tp extract were significantly lower than ang group ( repeated measures anova , p < 0.001 ) [ figure 3a ] and hr changes in two groups of tp 100 and tp 200 showed a significant reduction compared to ang group ( repeated measures anova , p < 0.001 and p < 0.01 , respectively ) [ figure 3b ] . time courses of changes in mean arterial pressure ( map ) and heart rate ( hr ) in response to injection of angiotensin ii ( ang ii ) ( 300 ng ) in groups of ang ii and three doses of teucrium polium l. ( tp ) extract ( tp 100 , tp 200 and tp 400 ) . for each group , n = 8 . ( a ) map , * * * p < 0.001 compared to ang group . ( b ) hr however , no significant hr changes were observed between these pretreated groups and ang group in the maximum level of effect [ hr : 55.6 3.8 , 53.62 5.25 , 60.12 8.87 compared to 72.79 5.10 beats / min , respectively ; figure 3 ] . in the present study , the injection of normal saline with the same volume of tp extract had not significant effects on blood pressure and hr of the control group . all three doses of tp extract ( 100 , 200 , and 400 mg / kg ) significantly reduced map in normotensive condition as well as inhibited of increase in blood pressure after injection of ang ii which indicated a relaxation effect on vascular smooth muscles . angiotensin ii regulates blood pressure and plasma volume via aldosterone - regulated sodium excretion , sympathetic nervous activity , and thirst responses . as previous studies have been shown , ang ii - mediated hypertension could be induced through different signaling events ; stimulation of phospholipase c and phosphatidylinositol hydrolysis , increased intracellular free calcium concentration , activation of protein kinase c , increased intracellular free concentrations of na , and decreased intracellular free concentrations of mg , activation of tyrosine kinases , implication of mitogen - activated protein kinase pathways or phospholipase d activation , so in our study the hypotensive effect of tp extract could be attributed to affect on each of these pathways . besides , former reports have also demonstrated that tp extract had relaxation effects on ileum and vascular smooth muscles . moreover , tp contains important ingredients such as salvigenin , cirsiliol , pinen- and , sabinene , myrcene , germacrene d , limonene , -caryophyllene , spathulenol which can influence on vascular smooth muscle tone . for instance , in many studies , the antispasmodic effect of some components such as - and -pinen , cirsiliol , spathulenol , limonene , and salvigenin from other members of lamiacee family have been demonstrated . altogether , it seems that the tp ingredients can induce the hypotensive effects in rats . on the other hand , abundant evidence now suggests that a key mechanism by which ang ii influences blood pressure is via its ability to produce reactive oxygen species ( ros ) . most investigations of ang ii hypertension and oxidant stress have focused on the vasculature as a key player , and in particular the notion that increased levels of o2 lead to diminished bioactivity of nitric oxide and thus vasoconstriction . an important role for ros - mediated vascular smooth muscle hypertrophy and remodeling in ang ii - dependent hypertension has also received considerable attention so considering to known antioxidant properties of tp , it can impress on hypertension through this mechanism . our data showed that in spite of the effect of tp on lowering of blood pressure it could not improve tachycardia induced by ang , but even if tp could reduce hr , it seems activation of baroreflex , which may be had a role in blood pressure reduction conceal impact of tp on hr , in addition , since the baroreflex response is impaired in anesthesia therefore , no changes of hr may at least in part , be due to the blunted of baroreflex activity . taking together upon to our findings tp can prevent hypertension induced by activation of ras through antagonizing the effects of ang ii and according to its various ingredients as well as different implicated pathways involve in this type of hypertension , the mechanisms of tp action are different . | background : antispasmodic and vasorelaxant effects of teucrium polium l. ( tp ) were mentioned in former studies , so we attempted to evaluate the eventual preventive effect of tp in an acute experimental model of hypertension induced by angiotensin ii ( ang ii).methods : forty - eight male wistar rats were divided randomly into six groups ( n = 8) ; control group ( c ) , which received only saline , group ang ii ; which received ang ii ( 300 ng / min , iv ) , group losartan ( los ) ; which received los ( 10 mg / kg , iv ) before ang ii injection , three groups of tp 100 , tp 200 , and tp 400 ; which received different doses of tp extract ( 100 , 200 and 400 mg / kg , ip , respectively ) before ang ii application .
after cannulation of the femoral artery , mean arterial blood pressure ( map ) and heart rate ( hr ) was continuously measured and recorded during the experiments .
comparisons were performed using t - test with spss software , version 16 ( spss , chicago , il).results : map and hr in ang group were significantly higher than the control group ( p < 0.001 ) , map in group los significantly was lower than ang group ( p < 0.001 ) and pretreatment with three doses of tp extract also inhibited increasing of map after ang ii injection ( p < 0.001 ) .
los also inhibited the increase of hr due to ang ii ( p < 0.001 ) , but none of three doses of tp extract had a protective effect on tachycardia induced by ang ii.conclusions:it seems tp extract could be effective in preventing of high blood pressure induced by ang ii pathway activation but could not have remarkable efficacy for improving the created tachycardia . |
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cannabinoid cb1 receptors and relevant endocannabinoids are widely distributed throughout the brain in mediating affective , cognitive , and motor behaviors [ 1 , 2 ] . much of the current neurochemical evidence suggests that their functional roles are mainly to modulate presynaptic neurotransmission impinging on postsynaptic neurons [ 36 ] . midbrain raphe serotonergic neurons are known as an important substrate of cannabinoids , which have been demonstrated operating in a plethora of neurological responses [ 69 ] . thus , the explicit understanding of cannabinoid modulations on midbrain raphe serotonergic neuronal activity has been for many years one of the central issues in the cannabinoid research . the dorsal ( drn ) raphe nuclei ( mrn ) are two principal sources of serotonergic projections to the forebrain . the drn serotonergic projections are likely associated with cognitive and affective activity [ 10 , 11 ] while mrn serotonergic neurons are more clinging to motor behavior [ 12 , 13 ] . some of the forebrain neurons , particularly gabaergic and glutamatergic afferents , in turn innervate raphe nuclei forming feedback circuits that control extreme fluctuations of 5-ht availability . the convergence of evidence suggests that gabaergic and glutamatergic afferents put different weights on controlling serotonergic activity in two raphe nuclei . gabaergic afferents have a strong and tonic inhibitory influence on serotonergic neurons in the drn but little to the mrn [ 1517 ] . however , less is known about the fundamental mechanism underlying neural circuit used by cannabinoids for 5-ht release in the forebrain . the primary goal of the present study was to determine how 5-ht efflux in somatodendritic and axon terminal regions is altered differently in response to administrations of cannabinoids . this study was carried out in the drn and one of serotonergic ascending projection sites , namely , nucleus accumbens ( nacc ) . the second goal was to test the hypothesis that there are direct and indirect effects of cannabinoids on 5-ht efflux in the nacc . specifically , the direct effect is simply to inhibit axonal terminal activity via the activation of cb1 receptors , resulting in reduction in 5-ht efflux . in contrast , the indirect effect is to disinhibit postsynaptic 5-ht neurons at the drn involving in gabaergic neurotransmission , resulting in an increase in 5-ht efflux . our results clearly support a conclusion that when cannabinoids are administered systemically in freely behaving animals , the actual 5-ht efflux is determined by the net effect of two opposite actions , resulting in only a small increase in 5-ht efflux in the nacc . adult male sprague - dawley rats purchased from charles river laboratories ( raleigh , nc , usa ) were pair housed with food and water available ad libitum in a temperature- and humidity - controlled facility and were maintained on a 12 h light / dark cycle ( lights on at 08:00 am ) . all animal use procedures were in strict accordance with the national institutes of health guide for the care and use of laboratory animals , and the study was specifically approved by the institutional animal care & use committee at florida atlantic university ( permit number / id : a10 - 05 ) . rats weighing from 300 to 350 g were anesthetized with a combination of xylazine ( 4 mg / kg i.p . ) and ketamine ( 80 mg / kg i.p . ) , and then guide cannulae ( 21-gauge stainless steel tubing ) were preimplanted as described previously in detail using standard techniques for stereotaxic surgery . the coordinates for guide cannulae in the drn were ap 1.2 relative to interaural zero , ml 4.0 , and dv 1.0 below the skull surface at a 32 angle lateral to midline ; and in the nacc , ap 10.7 , ml 1.4 , and dv 1.0 below the skull surface . the evening before the experiments , rats were briefly anesthetized with isoflurane , and aseptic dialysis probes were inserted through the guide cannulae . the target coordinates for the tip of the probe were as follows : in the drn , ap 1.2 mm relative to interaural zero , ml 0.6 mm relative to midline , and dv 5.56.4 mm below the skull surface ; in the nacc , ap 10.7 mm , ml 1.4 mm , and dv 6.08.5 mm . rats were then placed in a test chamber and attached to a fluid swivel that allowed animals to move freely . dialysis probes were perfused overnight with artificial cerebrospinal fluid ( acsf ) containing 140 mm nacl , 3.0 mm kcl , 1.5 mm cacl2 , 1.0 mm mgcl2 , 0.25 mm nah2po4 , and 1.0 mm na2hpo4 . samples were collected at 30-min intervals and analyzed by high - performance liquid chromatography with electrochemical detection ( hplc - ec ; eicom htec-500 ) in conjugation with an autoinjector ( cma 200 ) . in the 5-ht assay , mobile phase ( 0.1 m phosphate buffer at ph 6.0 , 500 mg / l 1-decanesulfonic acid , 50 mg / l edta , and 1.0% methanol ) was pumped at a rate of 0.50 ml / min . in the gaba assay , samples were prederivatized with o - phthaldialdehyde and 2-methy-2-propanethiol for 10 min at room temperature and then analyzed on a c18 column ( 100 3.2 mm , basi , usa ) . the mobile phase consisted of 40% acetonitrile , 0.1 m sodium acetate buffer , and 50 mg the flow rate of gaba separation was set at 0.7 ml / min . to investigate the neural circuit involving in cb1 receptors , drugs were examined by administering to rats through two distinct routes : systemic administration and local infusion . for the systemic administration , win 55,212 - 2 , cp 55940 , am251 , and sr 147778 being dissolved in saline containing 10% tween-80 and 20% dimethyl sulfoxide ( dmso ) were injected intraperitoneally at a volume of 1 ml / kg in body weight . win 55,212 - 2 mesylate and am251 were purchased from tocris cookson ( ellisville , mo , usa ) . control animals were administered with the vehicle that was used to dissolve the drugs . for the local infusion , drugs dissolved in the infusion medium were delivered directly into the drn or nacc . except for experiments in figure 3 , win 55,212 - 2 was usually dissolved in the artificial cerebrospinal fluid ( acsf ) containing 1.5% ethanol and 1.5% cremophor . cremophor and ethanol were purchased from sigma . in figure 3 , win 55,212 - 2 was dissolved in the acsf containing 1 m citalopram in addition to 1.5% ethanol and 1.5% cremophor . note that the purpose of this set of experiments was to test whether cannabinoids could directly inhibit 5-ht efflux released from dendrites in the drn or from axon terminals in the nacc . however , 5-ht basal release was already low in the regular microdialysis samples at which an inhibitory response to drugs is not easily detected by measuring reduction in 5-ht efflux . thus , citalopram , a selective serotonin reuptake inhibitor ( ssri ) , was employed to elevate the baseline so that reduction in 5-ht efflux , if any , could be reliably measured with the microdialysis approach . previous studies have demonstrated that citalopram added into the infusion medium at the concentration of 1 m would not alter the quality of the drug response . lastly , bicuculline at the concentration of 100 m was dissolved in the acsf before infusing into the drn . bicuculline was purchased as the methobromide - h2o salt from tocris cookson ( ellisville , mo , usa ) . in the control animals , the drn or nacc was locally infused with the vehicle ( isotonic ) that was used to dissolve the drugs . to reduce intersubject variability in group analysis , the data presented in figures are expressed as mean ( s.e.m . ) percentage of changes from averaged baseline measurements . mean baseline 5-ht levels were calculated as the average of four successive samples before drug administration . statistical analysis was performed by the two - way ( drug treatment time ) factorial anova . if significant interactions of drug treatment time course were found , further statistical analysis was carried out using the one - way anova followed by post hoc scheffe test in determining the significance of the respective time points . the first set of experiments was to determine whether systemic injection of win 55,212 - 2 ( win55 hereafter ) had an effect on 5-ht efflux in the brain . the doses used in this study were based on behavioral investigations at which the drug was effective to produce conditioning place preference [ 22 , 23 ] . as shown in figure 1(a ) , win55 at 2.5 and 5 mg / kg ( i.p . ) failed to elicit a significant effect on extracellular 5-ht in the drn . the conclusion was supported by the two - way anova test revealing that there was no significant effect of treatment doses ( fdose ( 2,21 ) = 1.598 , p = 0.2258 ) , time ( ftime ( 5,105 ) = 1.324 , p = 0.2599 ) , or interaction of time doses ( fint ( 10,105 ) = 0.54 , p = 0.8583 ) . in contrast , the same injection produced a dose- and time - dependent increase in the nacc ( figure 1(b ) ; fdose ( 2,26 ) = 11.854 , p = 0.0002 ; ftime ( 5,130 ) = 5.766 , p = 0.0002 ) . the post hoc scheffe test showed that the significant effect was apparent 30 min after injection . cp55940 ( cp55 hereafter ) , which is known for its high affinity to bind cannabinoid receptors , was used to reexamine the nacc response to cannabinoid injection . the dose used in this study was 0.25 mg / kg and 0.5 mg / kg . cp55 at 0.5 mg / kg produced over 50% increases in the nacc ( figure 1(c ) ) but not in the drn ( data not shown ) . the anova test revealed a significant effect of drug doses ( fdose ( 2,24 ) = 7.185 , p = 0.0036 ) and time ( ftime ( 5,120 ) = 6.766 , p = 0.0001 ) . the post hoc scheffe test showed that the significant increase occurred between 0.5 and 1.5 h after the injection . next , we tested whether the win55-induced increases in 5-ht efflux in the nacc could be attributable to the activation of cb1 receptors . sr 147778 is a newly synthesized chemical that has a highly potent antagonistic activity at cb1 receptors [ 25 , 26 ] . as shown in figure 2(a ) , sr 147778 ( 5 mg / kg , i.p . ) alone had no effect on 5-ht in the nacc . injection of sr 147778 30 min before win55 ( 5 mg / kg i.p . ) blocked the win55-evoked increases in 5-ht efflux in the nacc ( ftreatment ( 1,9 ) = 9.457 , p = 0.0132 ) . am251 pretreatment 30 min before win55 significantly blocked the win55-elicited increases in 5-ht in the nacc ( figure 2(b ) ; ftreatment ( 1,11 ) = 31.804 , p = 0.0002 ) . it has been suggested that endogenous opioidergic system may be one of the substrates responsible for the effect of cb1 receptor agonists in the cns [ 2729 ] . since opioids also cause regional selective effects on 5-ht efflux , we tested whether endogenous opioids are involved in the win55-induced increases in 5-ht in the nacc . if this was the case , the opioid receptor antagonist naloxone should be able to antagonize the effect . as shown in figure 2(c ) , naloxone alone ( 10 mg / kg , s.c . ) had no effect on 5-ht efflux . naloxone pretreatment failed to block the win55-induced increases in 5-ht efflux ( ftreatment ( 1,14 ) = 1.579 , p = 0.2295 ) , suggesting that endogenous opioids did not mediate the effect of cannabinoids on 5-ht efflux . we suggest that the existence of a neural circuit is responsible for regional selective effects of cannabinoids on 5-ht efflux in the cns . to test this hypothesis , cannabinoids were directly applied to the drn or nacc through microdialysis probes while 5-ht efflux was determined using the same probes . in this set of experiment , 1 m citalopram was included into the infusion medium to elevate the basal levels of 5-ht efflux . note that citalopram at such a low concentration would not alter the response of serotonergic neurons to drug administration . as shown in figure 3(a ) , win55 at 100 , 300 , and 1000 m infusing into the drn produced a robust increase in 5-ht efflux in a concentration - dependent manner ( fdose ( 3,19 ) = 5.273 , p = 0.0081 ) . in contrast , win55 infusion directly into the nacc caused a concentration - dependent reduction in 5-ht efflux in the nacc ( figure 3(b ) ; fdose ( 3,20 ) = 5.219 , p = 0.008 ) . thus , win55 produced two opposite effects on 5-ht efflux in the nacc , depending on the route of drug administrations . we suggest that the systemic win55-induced increases may be due to disinhibition of serotonergic neurons in the drn . if this was the case , we expected that local infusion of win55 to the drn would also elicit an increase in 5-ht in the nacc , similar to the systemic injection . to test this , we employed a dual - probe microdialysis technique . as shown in figure 4(a ) , win55 infusion into the drn caused a concentration - dependent increase in the nacc ( fdose ( 2,18 ) = 4.256 , p = 0.0307 ) . based on evidence that the direct effect of cannabinoids is inhibitory [ 31 , 32 ] , we further suggest that the win55-induced increases may be due to inhibition of gabaergic neurotransmission on the serotonergic neurons in the drn . if this was the case , bicuculline , a gabaa receptor antagonist , locally infusing into the drn would block the inhibitory impact of gabaergic neurotransmission and thereby disrupt the effect of win55 on the increased 5-ht efflux . as shown in figure 4(b ) , bicuculline pretreatment in the drn abolished the systemic win55 ( 5 mg / kg , i.p)-elicited increases in 5-ht efflux in the nacc ( ftreatment ( 1,14 ) = 3.153 , p = 0.0975 ) . lastly , to confirm the relationship between gabaergic presynaptic responses and cannabinoid administration , we measured changes in gaba efflux in the drn . as shown in figure 5 , there was a significant reduction in gaba efflux in the drn following injection of win55 and cp55 ( ftreatment ( 2,13 ) = 43.843 , p < 0.0001 ) . by measuring changes in 5-ht and gaba efflux in the drn and/or nacc , this present study demonstrated that there exists a neural circuit in the drn through which cannabinoids regulate serotonergic neuronal activity in the nacc . our data provide evidence demonstrating that cannabinoids can exert both indirect and direct effects on serotonergic neurons ; the direct effect of cannabinoids is inhibitory while the indirect effect via presynaptic circuit is excitatory . despite having opposite effects , we showed that systemic administration of exogenous cannabinoids such as win55 and cp55 has a modest excitatory net effect on 5-ht efflux in the nacc of drug - nave animals . this is consistent with the reports that the neurophysiologic function of cannabinoids is considered to be a neuromodulator that usually exerts their effect on both local principal neurons and all types of afferents [ 6 , 32 , 33 ] , resulting in a site - specific response . in the drn , gabaergic and glutamatergic components are two major afferents that synapse onto the serotonergic neurons , controlling 5-ht availability for the forebrain [ 14 , 35 ] . cb1 receptors are found to be widely expressed in the raphe afferents in addition to serotonergic cells . thus , cannabinoids can exert their effects on all regulatory afferents and principal neurons that form a neural circuit including gabaergic , glutamatergic , and serotonergic components . as such , the change in 5-ht efflux in response to systemic cannabinoids can not be exclusively a result of the direct action on serotonergic neurons but a complicated integration of inhibitory modulation of both inhibitory and excitatory neurotransmissions impinging on postsynaptic serotonergic neurons . thus , the effect of cannabinoids on 5-ht release would be a compromised result of excitatory and inhibitory influences , which may explain the findings that there were only modest changes in 5-ht efflux observed in the present study . the present findings that cannabinoids exert both direct and indirect effects on serotonergic neurons are well consistent with the previous reports that cannabinoids can either reduce [ 36 , 37 ] , elevate [ 38 , 39 ] , or bidirectionally alter serotonergic activity . theoretically , the inhibition of glutamatergic afferents would cause a reduction in 5-ht efflux while the inhibition of gabaergic afferents would result in an increase in 5-ht efflux . therefore , their net effects on serotonergic neurons are likely determined by their relative potency of tonic activity . physiologically , gabaergic afferents exert spontaneous inhibitory tone on serotonergic neurons while glutamatergic afferents almost have no measurable role in terms of spontaneous action . note that spontaneous action of glutamatergic afferents is apparent only at a specific behavioral state such as responding to auditory stimulation . therefore , it is conceivable that modulation of gabaergic afferents would be more prominent on 5-ht efflux than the glutamatergic influence . in general , presynaptic modulation of gabaergic inputs has been suggested to be the key mechanism responsible for several illicit drugs such as opioids , alcohol , and gamma hydroxybutyrate [ 18 , 41 , 42 ] . in the present study , we suggest that cannabinoids may use the same neural mechanism for their central action , which inhibits gabaergic neurotransmissions to the drn , thus resulting in disinhibition of serotonergic neurons ( disinhibition theory ) . this may explain our neurochemical data that 5-ht efflux is elevated in the drn - nacc pathway following the systemic administration of exogenous cannabinoids . we demonstrate that changes in 5-ht efflux are dependent not only on doses of cannabinoid injection but also on brain areas examined . specifically , systemic injection produced approximately a 50100% increase from baseline 5-ht in the nacc . in contrast , there was an insignificant effect in the drn following the same injection . the mechanism for the area - dependent efflux is not clearly understood but likely ascribed to regional circuits in the controlling of postsynaptic 5-ht efflux ( see discussion further below ) and also due to possible difference in the release of 5-ht in the two regions ( see a review by ) . the release of 5-ht from the serotonergic neurons is believed to take place at axon terminals where 5-ht - containing vesicles can be found . in the drn , there are numerous recurrent axon collaterals projected from other raphe nuclei , by which serotonergic neurons can be negatively regulated . recent development of in vivo neurochemistry has suggested that dendrites are also equipped with 5-ht - containing vesicles , and many neuroactive chemicals can influence a dendritic release [ 45 , 46 ] . this suggests that 5-ht efflux measured by microdialysis in the drn is derived from many sources involved in distinct mechanisms including dendritic release , recurrent axon collateral release , and axon terminal release . although it is difficult to determine the amounts of each release contributing to the 5-ht efflux in the drn , it is conceivable that dendritic release may be an important component in the extracellular measurement . much of available evidence suggests that the properties of dendritic release may be different from that at axons in the forebrain . for instance , the spontaneous 5-ht release is relatively higher in the drn compared with that from axon terminals [ 17 , 47 ] . it is known that the receptor subtype expression is different between the somatodendritic sites and axon terminals of serotonergic neurons [ 15 , 48 ] . altogether , drugs that cause changes in 5-ht efflux at somatodendritic sites of the drn would not necessarily produce the same response in forebrain areas that contain exclusively the axon terminals . we found that the neurochemical response to cannabinoids is depending on the route of drug administration . unlike systemic injection , local application of cannabinoids to the nacc failed to produce the increase in 5-ht . instead it should be kept in mind that citalopram at the concentration of 1 m was added in the infusion medium into the nacc . after the addition of citalopram , the basal level of 5-ht efflux before drug administration was elevated to ~4 pg / sample , almost 10 times greater than the physiological level . since citalopram effectively blocks reuptake of extracellular 5-ht released from local circuitry , a small reduction in 5-ht efflux can be , therefore , sensitively determined . this pharmacological approach has been widely employed in the in vivo 5-ht research using microdialysis techniques . furthermore , it has been demonstrated that a local application of citalopram at such a low concentration did not alter responsivity of serotonergic neurons to drug administration when the data were expressed as percentage changes relative to baseline . for comparison , citalopram was also infused into the drn for investigating the effects of cannabinoids on 5-ht efflux in the drn . as a result , win55 caused a concentration - dependent increase in the drn but a decrease in the nacc . this further supports that neural circuits that control 5-ht release in response to cannabinoids are different in two regions , regardless the addition of citalopram in the infusion medium . previous in vitro studies demonstrated that cannabinoids decrease 5-ht release from axon terminals [ 3 , 37 ] . this effect can be ascribed to a direct inhibitory activation of cb1 receptors located on the axon terminals . alternatively , excitatory presynaptic influence from glutamatergic afferents may be inhibited by cannabinoid application , as suggested in one study . however , there is no evidence implicating that 5-ht axon terminal in this region is tonically controlled by glutamatergic afferents . thus , reduction in 5-ht efflux can be considered to be a direct inhibitory effect of cannabinoids on serotonergic axon terminals as suggested in the previous studies [ 23 , 37 ] . this is consistent with the observation that cb1 receptors are expressed in the axon terminals that might directly respond to cannabinoids . in the present study , we found that the local infusion of win55 into the drn produced an increase in 5-ht in both the drn and nacc . these observations are in agreement with findings that excitatory stimulation of the drn serotonergic cells results in an increase in extracellular 5-ht in the nacc [ 49 , 50 ] . our data suggest that a cb1 receptor - mediated neural circuit that directly controls 5-ht release and transmission through the drn - nacc pathway would be activated by cannabinoids in the drn . the effect was apparent in response to 300 m and 1000 m , but not 100 m win55 . in this regard , relatively high concentrations of win55 were used during the local infusion for two reasons . first , the microdialysis membrane served as a barrier to the region of the brain . it has been demonstrated with a 1-mm microdialysis probe that only ~7% of infused substances can cross the barrier as examined in an in vitro study . second , the viscosity of the organic solvent in which win55 was dissolved was relatively high , which might have dampened the diffusion of the drug across the probe membrane . thus , it was necessary to utilize high concentrations in the infusion medium that drugs could effectively follow concentration gradients diffusing into local brain tissue . finally , we presented evidence that gabaergic neurotransmission in the drn is the major neural target for cannabinoid regulation , which is responsible for changes in 5-ht efflux of the drn - nacc pathway . this view is strengthened by the observation that gabaergic neurons have a strong tonic inhibition on serotonergic neurons in the drn [ 17 , 49 ] . thus , although the action of cannabinoids in the circuit appears to have multiple sites including gabaergic and glutamatergic and serotonergic neurons [ 6 , 39 , 51 ] , the net consequence would be that serotonergic neurons are disinhibited resulting in an increase in 5-ht efflux in the regions receiving drn projections . this hypothesis was experimentally approved in this study by testing with bicuculline , a gabaa receptor antagonist , in which blocking gaba activation in the drn abolished the increase in 5-ht in the nacc ( figure 4 ) . previous studies have shown that cannabinoids could reduce gaba efflux or gaba - mediated spontaneously inhibitory postsynaptic currents in many brain regions including but not limited to frontal cortex , pallidus , substantia nigra , and hippocampus [ 5255 ] . in the present study , we measured changes in gaba efflux in the drn , demonstrating that the reduction in gaba efflux was in correlation with the increase in 5-ht efflux , further supporting the role of gabaergic neurotransmission in the drn - nacc serotonergic pathway . collectively , systemic administration of cannabinoids may exert two opposite effects on serotonergic neurotransmission , resulting in a small increase in 5-ht efflux in the nacc . | in vivo microdialysis was used in this study to reveal the role of cannabinoids in regulating serotonin ( 5-ht ) efflux in the nucleus accumbens ( nacc ) and dorsal raphe nucleus ( drn ) .
the cannabinoid cb1 receptor agonists win55212 - 2 and cp55940 systematically administered to rats caused significant increases in 5-ht efflux in the nacc but failed to have an effect in the drn . to reveal mechanisms underlying regionally selective responses , we tested the hypothesis that cannabinoids have both direct and indirect effects on 5-ht efflux , depending on the location of cb1 receptors in the neural circuit between drn and nacc .
we showed that the direct effect of cannabinoids caused a reduction in 5-ht efflux whereas the indirect effect resulted in an increase .
furthermore , the indirect effect was blocked by the gabaa receptor antagonist bicuculline in the drn , suggesting that the action is likely due to a presynaptic inhibition on gabaergic activity that exerts a tonic influence on neuronal circuits regulating 5-ht efflux .
involvement of gabaergic neurons was confirmed by measuring changes in gaba efflux . taken together , our study suggests that cannabinoids may have direct and indirect effects on the 5-ht regulatory circuits , resulting in regionally selective changes of 5-ht efflux in the brain . |
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because of the large diameter of the cervical cord in relative to the spinal canal and the significant range of motion , even a small epidural abscess can cause devastating results1 ) . published a thorough meta - analysis of cervical osteomyelitis cases , conducting a most comprehensive literature - based review of the etiology , diagnosis , and treatment of cervical vertebral osteomyelitis ( cvo)2 ) . the authors listed alcoholism , dental work , liver disease , diabetes , previous history of surgery , drug abuse , and tuberculosis as a medical comorbidities or serious medical illness predisposing cvo and reported that number of patients requiring surgical intervention ranged from 18 to 43%(without distinction of cervical or non - cervical ) , and among surgically treated patients , most preferred approach was direct anterior debridement . after debridement , no further treatment or anterior fusion , anterior and posterior fusion were considered as a further treatment options . however , although standard management for cervical vertebral osteomyelitis is medical treatment using proper antibiotics , this article did not proposed practical guidelines depend on each patients with cervical osteomyelitis which should be required surgical treatment , in that there are various surgical strategy and options to choose especially in cervical osteomyelitis because of several factors such as degree of the infectious progress , site of spinal cord compression , and expected kyphotic deformity due to bony destruction or disc space involvement . thus we analyzed clinical cases of cervical osteomyelitis treated in the single institute , which were caused by various causative factors , and studied the strategic factors that should be considered . also , we studied treatment results of antibiotics after operation for cervical osteomyelitis . we retrospectively reviewed 14 patients who had been treated surgically for cervical osteomyelitis from may 2000 to july 2008 ( table 1 ) . we investigated their clinical course , comorbid medical diseases , antibiotic regimen , surgical methods , and their laboratory and radiologic findings including plain x - ray , computed tomography ( ct ) , magnetic resonance imaging ( mri ) and clinical outcomes ( preoperative and post - operative visual analogue scale ( vas ) score , american spinal injury association ( asia ) impairment scale ) . in most cases , the diagnosis of cervical osteomyelitis can be accomplished by using plain x - ray films and computerized tomography scans . nevertheless , preferential use of t1 gadolinium enhanced magnetic resonance imaging is helpful in identifying epidural compressive processes in patients suffered from neurological deficit . in our study , all patients were evaluated with mri to confirm diagnosis , additionally using ct images for estimating bony destruction and instability . also , laboratory results such as wbc with differential counting , esr ( erythrocyte sedimentation rate ) , crp ( c - reactive protein ) was performed for confirmation of diagnosis , severity of disease , clinical response to antibiotics , and used as guideline for decision to discontinue antibiotics11 ) . indications for surgical treatment included neurological deficit due to spinal cord compression , progressive deformity and instability of the cervical spine , intractable pain despite of conservative treatment , poorly controlled infectious condition with antibiotics administration . this study included ten men and four women , whose mean age was 48.8 ( range , 21 - 69 ) years old . 5 patients had primary spontaneous spondylodiscitis , 5 patients had post operative spondylodiscitis and 4 patients had tuberculosis in cervical spine . the average symptom durations were 9 days for primary pyogenic spondylodiscitis and 21.5 days for tuberculosis in the cervical spine . in post the causative microorganisms in pyogenic infection were methicillin resistant s. aureus ( 5 ) , p. aeruginosa ( 1 ) , methicillin resistant coagulase negative streptococcus ( 1 ) , p. aeruginosa changed to mrsa(1 ) and 2 patients showed no growth on culture studies . the patients enrolled in this study had no risk factors such as immunocompromised status , intravenous drug user , or have undergone an oropharyngeal procedure except for 3 patients with diabetes mellitus and 2 patients who suffered esophageal injury during cervical anterior interbody fusion surgery . 7 ( 70% ) patients among them improved post - operatively ( table 1 ) . the antibiotics were administered a mean of 38.6 ( range , 23 - 75 ) days with culture sensitive intravenous ( iv ) antibiotics and then were treated a mean of 58.2 ( range , 13 - 106 ) days with oral antibiotics for primary pyogenic spondylodiscitis . however , patients with spondylodiscitis were treated longer , as follows : mean 21 ( 6.6 - 48.6 ) weeks with culture sensitive iv antibiotics and then 43.3 ( 9 - 70 ) days with oral antibiotics . generally , iv antibiotics was replaced with oral antibiotics , which was mostly 2 generation cephalosporin , if serum crp levels were maintained below 5mg / l for 4 weeks and the esr and white blood cell ( wbc ) count were within normal limits . iv antibiotics covering anaerobes were also administered to post - operative infected patients , especially in case of esophageal perforation . in the 4 cases of post - tuberculosis infections , we used an antituberculotic regimen ( isoniazid ; inh(h ) , ethambutol ; emb ( e ) , rifampicin ; rmp ( r ) , pyrazinamide ; pza(z ) , herz ) for a mean 383.8 days . we classified spinal lesion types as follows : cord compression without deformity ( 2 ) , kyphotic deformity without cord compression ( 3 ) , both spinal cord compression and kyphotic deformity ( 6 ) , and neither cord compression or kyphotic deformity ( 3 ) . eleven patients underwent single stage surgical treatment including cervical anterior interbody fusion with aibg ( 5 ) , infection site debridement with or without irrigation catheter insertion ( 5 ) , and simultaneous performed cervical anterior interbody fusion at the c5 - 6 - 7-t1 levels and transthoracic thoracic interbody fusion at the t5 - 6 - 7 levels with aibg and plate reinforcement for cervicothoracic tuberculosis ( 1 ) . the 2 stage operation included 1 case of anterior debridement and 2 cases of anterior debridement with aif at the time of first operation and posterior fixation with instruments was performed during the second operation ( table 2 ) . in one patient with post - operative infection after cervical aif c4 - 5 - 6 - 7 due to esophageal rupture ( case 11 ) , follow up x - ray showed fracture of the previously inserted anterior cervical plate after anterior debridement for infection control . therefore , we removed the fractured anterior cervical plate because solid fusion was done already . in the other patient ( case 10 ) who had an intraoperative esophageal rupture and extensive mediastinitis , we could not perform the 2 operation for stabilization due to continuous pus discharge and uncontrollable infection after 1st operation for esophageal repair and debridement . thus , kyphotic deformity with cord compression developed 5 months after the 1 operation , but the infection was finally cured with antibiotics . 10 ( 71.4% ) patients had neurologic deficits at the time of diagnosis and 7 patients ( 70% ) among them improved postoperatively . this study included ten men and four women , whose mean age was 48.8 ( range , 21 - 69 ) years old . 5 patients had primary spontaneous spondylodiscitis , 5 patients had post operative spondylodiscitis and 4 patients had tuberculosis in cervical spine . the average symptom durations were 9 days for primary pyogenic spondylodiscitis and 21.5 days for tuberculosis in the cervical spine . in post the causative microorganisms in pyogenic infection were methicillin resistant s. aureus ( 5 ) , p. aeruginosa ( 1 ) , methicillin resistant coagulase negative streptococcus ( 1 ) , p. aeruginosa changed to mrsa(1 ) and 2 patients showed no growth on culture studies . the patients enrolled in this study had no risk factors such as immunocompromised status , intravenous drug user , or have undergone an oropharyngeal procedure except for 3 patients with diabetes mellitus and 2 patients who suffered esophageal injury during cervical anterior interbody fusion surgery . 7 ( 70% ) patients among them improved post - operatively ( table 1 ) . the antibiotics were administered a mean of 38.6 ( range , 23 - 75 ) days with culture sensitive intravenous ( iv ) antibiotics and then were treated a mean of 58.2 ( range , 13 - 106 ) days with oral antibiotics for primary pyogenic spondylodiscitis . however , patients with spondylodiscitis were treated longer , as follows : mean 21 ( 6.6 - 48.6 ) weeks with culture sensitive iv antibiotics and then 43.3 ( 9 - 70 ) days with oral antibiotics . generally , iv antibiotics was replaced with oral antibiotics , which was mostly 2 generation cephalosporin , if serum crp levels were maintained below 5mg / l for 4 weeks and the esr and white blood cell ( wbc ) count were within normal limits . iv antibiotics covering anaerobes were also administered to post - operative infected patients , especially in case of esophageal perforation . in the 4 cases of post - tuberculosis infections , we used an antituberculotic regimen ( isoniazid ; inh(h ) , ethambutol ; emb ( e ) , rifampicin ; rmp ( r ) , pyrazinamide ; pza(z ) , herz ) for a mean 383.8 days . we classified spinal lesion types as follows : cord compression without deformity ( 2 ) , kyphotic deformity without cord compression ( 3 ) , both spinal cord compression and kyphotic deformity ( 6 ) , and neither cord compression or kyphotic deformity ( 3 ) . eleven patients underwent single stage surgical treatment including cervical anterior interbody fusion with aibg ( 5 ) , infection site debridement with or without irrigation catheter insertion ( 5 ) , and simultaneous performed cervical anterior interbody fusion at the c5 - 6 - 7-t1 levels and transthoracic thoracic interbody fusion at the t5 - 6 - 7 levels with aibg and plate reinforcement for cervicothoracic tuberculosis ( 1 ) . the 2 stage operation included 1 case of anterior debridement and 2 cases of anterior debridement with aif at the time of first operation and posterior fixation with instruments was performed during the second operation ( table 2 ) . in one patient with post - operative infection after cervical aif c4 - 5 - 6 - 7 due to esophageal rupture ( case 11 ) , follow up x - ray showed fracture of the previously inserted anterior cervical plate after anterior debridement for infection control . therefore , we removed the fractured anterior cervical plate because solid fusion was done already . in the other patient ( case 10 ) who had an intraoperative esophageal rupture and extensive mediastinitis , we could not perform the 2 operation for stabilization due to continuous pus discharge and uncontrollable infection after 1st operation for esophageal repair and debridement . thus , kyphotic deformity with cord compression developed 5 months after the 1 operation , but the infection was finally cured with antibiotics . 10 ( 71.4% ) patients had neurologic deficits at the time of diagnosis and 7 patients ( 70% ) among them improved postoperatively . cervical spine spondylodiscitis is a rare , but serious manifestation of spinal infection9 ) . in cervical vertebral osteomyelitis and abscess thus , the surgical strategy should focus on spinal biomechanical stabilization , neural decompression , and infectious tissue removal14,19 ) . there are various predisposing risk factors for spondylodiscitis which include diabetes mellitus , rheumatic diseases , immunosuppressive diseases , previous invasive treatment modalities ( pharyngeal surgery , tonsillectomy etc ) , septic condition ( pneumonia , urinary tract infection , etc ) , and intravenous drug abuse3,4,5,6,15,20,22 ) . in our series , there were 3 patients with diabetes mellitus and 2 patients with esophageal injuries during cervical anterior surgery . there are no solid guidelines for antibiotics for cervical osteomyelitis , but generally a minimum of 6 to 8 weeks is sufficient for pyogenic infections17 ) . the average duration of iv antibiotic treatment for post - operative infections was 21 ( range , 6.6 - 48.6 ) weeks . it was definitely a longer duration than previous reports because of the small sample size , which included some chronic uncontrolled infections . a standard antibiotic treatment regimen can not be derived from published results and the choice of regimen usually depends on clinician 's preferences or policy13,16 ) . since we inference that a patient had spondylitis through his or her clinical symptoms , imaging studies , and laboratory results , we usually began broad spectrum iv antibiotics such as a combination of third generation cephalosporin , penicillin with -lactamase inhibitor , and aminoglycoside before the culture sensitivity was reported . we switched from iv antibiotics to oral antibiotics if the serum crp level was maintained below 5mg / l for 4 weeks . and we used an oral antibiotic for 1 to 2 months for complete infection control . in cases of elderly patients with poor general conditions or rapid neurological deterioration or a chronic uncontrolled infectious process first an operation was performed for neural decompression or early identification of the causative organism and infection control . then , a second operation for stabilization or preventing an expected kyphotic deformity was performed as soon as possible . in elderly patients , reduced operative times and intraoperative blood loss during each operation can diminish the perioperative complication10 ) . because unnecessary excessive corpectomies could cause iatrogenic instability and result in unneeded secondary operations for stabilization or higher complication rate in multi - segmental fusions8,18 ) , meticulous preoperative interpretation of both t1 gadolinium enhanced mri and sagittal reconstruction ct scans should be necessary to determine the exact extent of infection involvement . it is important to distinguish infection involvement from simple bone swelling in order to decide the corpectomy level . in case 1 ( fig . 1 ) , t1 gadolinium enhanced mr images revealed whole vertebral enhancement but sagittal reconstruction ct scan showed that bony destruction was limited to the lower part of the c6 vertebrae and the upper part of the c7 vertebrae . therefore , we decided to perform segmental cervical anterior debridement and fusion with an autologous bone graft at the c6/7 level . in contrast , both t1 gadolinium mri and sagittal reconstruction ct scan exhibited extensive bony destruction , especially middle column of the c5 and c6 in case 7 ( fig . 2 ) . therefore , we should perform corpectomies both at the c5 and c6 vertebrae . in post - operative infection cases , it is important to check the esophageal injury . it can be detected by esophagography and neck or chest ct with contrast media23 ) . if there is an esophageal injury , we chose primary esophageal repair , irrigation , debridement , and silastic or catheter drainage for 1 operation and used broad spectrum antibiotics with the addition of anaerobes - covering antibiotics such as clindamycin or metronidazole simultaneously . the esophagography should be followed - up regularly until it is confirmed that there is no leakage from the repair site . then , a secondary operation for stabilization should be planned as soon as possible because an extensive infectious process can cause devastating results such as progressive kyphotic deformity with cord compression ( case 10 , fig . 3 ) . anterior debridement and fusion has advantages for relieving neurological deterioration and ceasing the infectious process in the early stage . in patients with epidural or paravertebral abscesses compressing the cord but no preexisting instability , we prefer to choose the anterior approach without posterior stabilization for debridement and fusion if indicated however , if there is extensive bony destruction and disc space invasion causing instability , we believe that three column stabilization should be performed by combined anterior fusion and posterior fixation7 ) . the combined approach also minimizes pseudoarthrosis , graft dislodgement , deformity progression , and anterior plate failure which contributes to worse prognosis in complicated pathological disorders like multicolumn involvement or extensive multilevel invasion with infection12 ) . first of all , the number of patient who was enrolled in this study was too small and heterogeneously mixed with pathogen to draw conclusion . surgical discipline for tuberculosis and bacterial disease should be differentiated because bony destruction process by tuberculosis could be different from bacterial origin . however as mentioned earlier in this literature , cervical osteomyelitis is a rare disease and also the portion of patients demanding surgical treatment is small . this study consisted of primary and secondary ( postoperative ) infections , also bacterial and non - bacterial ( tuberculosis ) infections , lacking consistency . on the other hand , heterogeneity of subjects made in possible to analysis variable aspect of cervical osteomyelitis and their surgical management to each situation . we , in this article , want to discuss or investigate the detailed surgical techniques including approach , level of corpectomy , and special consideration in overall . for example , distinct characteristics of tuberculous osteomyelitis are that more preserved bony quality , relative disc preservation , and more prominent extra - vertebral abscess , which needs distinguished strategies in surgical decision making . there are no solid guidelines for antibiotics for cervical osteomyelitis , but generally a minimum of 6 to 8 weeks is sufficient for pyogenic infections17 ) . the average duration of iv antibiotic treatment for post - operative infections was 21 ( range , 6.6 - 48.6 ) weeks . it was definitely a longer duration than previous reports because of the small sample size , which included some chronic uncontrolled infections . a standard antibiotic treatment regimen can not be derived from published results and the choice of regimen usually depends on clinician 's preferences or policy13,16 ) . since we inference that a patient had spondylitis through his or her clinical symptoms , imaging studies , and laboratory results , we usually began broad spectrum iv antibiotics such as a combination of third generation cephalosporin , penicillin with -lactamase inhibitor , and aminoglycoside before the culture sensitivity was reported . we switched from iv antibiotics to oral antibiotics if the serum crp level was maintained below 5mg / l for 4 weeks . and we used an oral antibiotic for 1 to 2 months for complete infection control . in cases of elderly patients with poor general conditions or rapid neurological deterioration or a chronic uncontrolled infectious process , we preferred to plan a staged operation to reduce perioperative morbidities . first an operation was performed for neural decompression or early identification of the causative organism and infection control . then , a second operation for stabilization or preventing an expected kyphotic deformity was performed as soon as possible . in elderly patients , reduced operative times and intraoperative blood loss during each operation can diminish the perioperative complication10 ) . because unnecessary excessive corpectomies could cause iatrogenic instability and result in unneeded secondary operations for stabilization or higher complication rate in multi - segmental fusions8,18 ) , meticulous preoperative interpretation of both t1 gadolinium enhanced mri and sagittal reconstruction ct scans should be necessary to determine the exact extent of infection involvement . it is important to distinguish infection involvement from simple bone swelling in order to decide the corpectomy level . in case 1 ( fig . 1 ) , t1 gadolinium enhanced mr images revealed whole vertebral enhancement but sagittal reconstruction ct scan showed that bony destruction was limited to the lower part of the c6 vertebrae and the upper part of the c7 vertebrae . therefore , we decided to perform segmental cervical anterior debridement and fusion with an autologous bone graft at the c6/7 level . in contrast , both t1 gadolinium mri and sagittal reconstruction ct scan exhibited extensive bony destruction , especially middle column of the c5 and c6 in case 7 ( fig . 2 ) . therefore , we should perform corpectomies both at the c5 and c6 vertebrae . in post - operative infection cases , it is important to check the esophageal injury . it can be detected by esophagography and neck or chest ct with contrast media23 ) . if there is an esophageal injury , we chose primary esophageal repair , irrigation , debridement , and silastic or catheter drainage for 1 operation and used broad spectrum antibiotics with the addition of anaerobes - covering antibiotics such as clindamycin or metronidazole simultaneously . the esophagography should be followed - up regularly until it is confirmed that there is no leakage from the repair site . then , a secondary operation for stabilization should be planned as soon as possible because an extensive infectious process can cause devastating results such as progressive kyphotic deformity with cord compression ( case 10 , fig . 3 ) . anterior debridement and fusion has advantages for relieving neurological deterioration and ceasing the infectious process in the early stage . in patients with epidural or paravertebral abscesses compressing the cord but no preexisting instability , we prefer to choose the anterior approach without posterior stabilization for debridement and fusion if indicated however , if there is extensive bony destruction and disc space invasion causing instability , we believe that three column stabilization should be performed by combined anterior fusion and posterior fixation7 ) . the combined approach also minimizes pseudoarthrosis , graft dislodgement , deformity progression , and anterior plate failure which contributes to worse prognosis in complicated pathological disorders like multicolumn involvement or extensive multilevel invasion with infection12 ) . first of all , the number of patient who was enrolled in this study was too small and heterogeneously mixed with pathogen to draw conclusion . surgical discipline for tuberculosis and bacterial disease should be differentiated because bony destruction process by tuberculosis could be different from bacterial origin . however as mentioned earlier in this literature , cervical osteomyelitis is a rare disease and also the portion of patients demanding surgical treatment is small . further study including larger group of patients would be necessary for more reliable conclusion . this study consisted of primary and secondary ( postoperative ) infections , also bacterial and non - bacterial ( tuberculosis ) infections , lacking consistency . on the other hand , heterogeneity of subjects made in possible to analysis variable aspect of cervical osteomyelitis and their surgical management to each situation . we , in this article , want to discuss or investigate the detailed surgical techniques including approach , level of corpectomy , and special consideration in overall . for example , distinct characteristics of tuberculous osteomyelitis are that more preserved bony quality , relative disc preservation , and more prominent extra - vertebral abscess , which needs distinguished strategies in surgical decision making . in cases of elderly patients with poor general conditions or rapid neurological deterioration or a chronic uncontrolled infectious process , we preferred to plan a staged operation to reduce perioperative morbidities . first an operation was performed for neural decompression or early identification of the causative organism and infection control . then , a second operation for stabilization or preventing an expected kyphotic deformity was performed as soon as possible . in elderly patients , reduced operative times and intraoperative blood loss during each operation can diminish the perioperative complication10 ) . because unnecessary excessive corpectomies could cause iatrogenic instability and result in unneeded secondary operations for stabilization or higher complication rate in multi - segmental fusions8,18 ) , meticulous preoperative interpretation of both t1 gadolinium enhanced mri and sagittal reconstruction ct scans should be necessary to determine the exact extent of infection involvement . it is important to distinguish infection involvement from simple bone swelling in order to decide the corpectomy level . in case 1 ( fig . 1 ) , t1 gadolinium enhanced mr images revealed whole vertebral enhancement but sagittal reconstruction ct scan showed that bony destruction was limited to the lower part of the c6 vertebrae and the upper part of the c7 vertebrae . therefore , we decided to perform segmental cervical anterior debridement and fusion with an autologous bone graft at the c6/7 level . in contrast , both t1 gadolinium mri and sagittal reconstruction ct scan exhibited extensive bony destruction , especially middle column of the c5 and c6 in case 7 ( fig . 2 ) . therefore , we should perform corpectomies both at the c5 and c6 vertebrae . in post - operative infection cases , it is important to check the esophageal injury . it can be detected by esophagography and neck or chest ct with contrast media23 ) . if there is an esophageal injury , we chose primary esophageal repair , irrigation , debridement , and silastic or catheter drainage for 1 operation and used broad spectrum antibiotics with the addition of anaerobes - covering antibiotics such as clindamycin or metronidazole simultaneously . the esophagography should be followed - up regularly until it is confirmed that there is no leakage from the repair site . then , a secondary operation for stabilization should be planned as soon as possible because an extensive infectious process can cause devastating results such as progressive kyphotic deformity with cord compression ( case 10 , fig . anterior debridement and fusion has advantages for relieving neurological deterioration and ceasing the infectious process in the early stage . in patients with epidural or paravertebral abscesses compressing the cord but no preexisting instability , we prefer to choose the anterior approach without posterior stabilization for debridement and fusion if indicated however , if there is extensive bony destruction and disc space invasion causing instability , we believe that three column stabilization should be performed by combined anterior fusion and posterior fixation7 ) . the combined approach also minimizes pseudoarthrosis , graft dislodgement , deformity progression , and anterior plate failure which contributes to worse prognosis in complicated pathological disorders like multicolumn involvement or extensive multilevel invasion with infection12 ) . first of all , the number of patient who was enrolled in this study was too small and heterogeneously mixed with pathogen to draw conclusion . surgical discipline for tuberculosis and bacterial disease should be differentiated because bony destruction process by tuberculosis could be different from bacterial origin . however as mentioned earlier in this literature , cervical osteomyelitis is a rare disease and also the portion of patients demanding surgical treatment is small . this study consisted of primary and secondary ( postoperative ) infections , also bacterial and non - bacterial ( tuberculosis ) infections , lacking consistency . on the other hand , heterogeneity of subjects made in possible to analysis variable aspect of cervical osteomyelitis and their surgical management to each situation . we , in this article , want to discuss or investigate the detailed surgical techniques including approach , level of corpectomy , and special consideration in overall . for example , distinct characteristics of tuberculous osteomyelitis are that more preserved bony quality , relative disc preservation , and more prominent extra - vertebral abscess , which needs distinguished strategies in surgical decision making . anterior cervical spine surgery is the preferable treatment option in patients with neurological deterioration , extensive bony destruction with expected kyphotic deformity , and uncontrolled infection being managed only with antibiotics . the anterior - posterior combined approach , which combines the anterior approach for rapid neural decompression and identification of causative microorganism and the posterior approach for stabilization of the preexisting instability or expected kyphotic deformity , could be a preferable choice for multilevel diseases . also , sufficient and proper uses of antibiotics with follow - up of laboratory results are important for thorough treatment of cervical osteomyelitis . | objectiveto study practical guidelines and strategies in the treatment of cervical osteomyelitis.methodswe retrospectively reviewed 14 patients who underwent surgical treatment for cervical osteomyelitis from may 2000 to july 2008 .
we investigated their clinical course , antibiotic regimen , surgical methods , and laboratory and radiologic findings including x - ray , ct and mri.results5 patients had primary spondylodiscitis , 5 patients had post operative spondylodiscitis and 4 patients had tuberculosis in cervical spine .
the causative microorganisms were mrsa ( 5 ) , p. aeruginosa ( 1 ) , methicillin resistant coagulase negative streptococcus ( 1 ) , p. aeruginosa changed to mrsa ( 1 ) , and 2 patients showed no growth on culture studies .
patients were treated 13.8 weeks ( range , 5.4 - 25.8 ) with iv antibiotics and then treated for 58.2 days ( range , 13 - 106 ) with oral antibiotics .
antituberculotic medications were used for a mean of 383.8 days .
patients were treated with anterior debridement and fusion ( 5 ) , irrigation and debridement ( 5 ) , simultaneous cervical anterior interbody and transthoracic thoracic interbody fusion ( 1 ) .
3 patients underwent the planned 2-staged operation , which included an anterior debridement with or without fusion for the 1st operation and posterior instrumentation for 2nd operation .
10 patients ( 71.4% ) had neurologic deficits at the time of diagnosis and 7 patients ( 70% ) among them improved post-operatively.conclusionanterior cervical spine surgery is the preferable treatment option in patients with neurological deterioration , extensive bony destruction with expected kyphotic deformity , and uncontrolled infection being managed only with antibiotics .
antibiotics are also important for thorough treatment . |
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perivascular epithelioid cell tumors ( pecomas ) are rare mesenchymal tumors comprised of immunohistochemically and histologically distinctive perivascular epithelioid cells arranged radially around a vascular lumen and are characterized by abundant cytoplasmic periodic acid - schiff - positive glycogen ( 1 , 2 ) . these cells express melanocytic ( melan - a and microphthalmia transcription factor ) and smooth muscle ( hmb-45 and actin ) markers . pecomas include various histologic subtypes of angiomyolipomas , lymphangioleiomyomatosis , clear cell ( sugar ) and myomelanocytic tumors in addition to sarcoma of the perivascular cells and pigmented melanotic tumors ( 1 ) . they have been observed in various organs but are most frequently seen in the kidney , uterus and retroperitoneum and less frequently in the gastrointestinal tract or other organs ( 3 , 4 ) . to the best of our knowledge , ovarian pecoma with pulmonary metastasis has not been described previously in radiologic or pathologic terms . we herein report , for the first time , the imaging and histopathologic features of pulmonary metastatic pecoma arising from the ovaries . a 48-year - old female presented with mild abdominal discomfort for several months and a history of total hysterectomy for leiomyoma ( 4 years previously ) . physical examination and laboratory findings were unremarkable . contrast - enhanced abdominal computed tomography ( ct ) scan was acquired using a 640 slice scanner ( aquilion one , toshiba ) by injecting 120 ml of intravenous contrast iohexol ( omnipaque , general electric healthcare ) . ct scans revealed two large , well - defined , multiloculated , solid and cystic masses in the pelvic cavity ( figure 1a ) . normal ovaries were not detected , and the small bowel loops and distal colon were displaced by this mass . both masses exhibited a thick wall or capsule , multiple internal septations and highly enhancing solid nodules . the highly enhancing solid portions of the pelvic masses encased the bilateral ovarian veins and exhibited a twisted appearance mimicking prominent vascular structures ( figure 1b ) . the septations and solid portions were highly enhanced ( approximately 150 hu on post - contrast images ) and exhibited mild washout on the delayed ct scan , on which they were measured approximately 110 hu ( figure 1c ) . on non - contrast ct scans , the cystic portions of the masses were homogeneously low - density and measured approximately 35 - 40 hu , suggesting that internal hemorrhage had occurred or that high - density fluid had filled the mass . there was no evidence of internal calcification or a fat component . pelvic magnetic resonance imaging ( mri ) was also performed with a 1.5 tesla machine ( signa hdxt , ge healthcare , milwaukee , wi , usa ) , using a body phased - array coil . bilateral ovarian masses presented as cystic and solid masses . on t1-weighted images , they were heterogeneously hypointense and hyperintense , but the hyperintense portion did not exhibit signal drop on fat - suppressed t1-weighted images , and also showed typical fluid - fluid level with signal decay on t2-weighted axial images , thus indicating internal hemorrhage ( figure 1d ) . on contrast enhanced t1-weighted images , the solid portions of the masses were highly enhanced . as shown on ct scans , bilateral ovarian vessels were encased by an upwardly extended mass ( figure 1d and 1e ) just like twisted bread stick appearance ( figure 2 ) . however , the signal void was well preserved in these involved vessels , so neither vascular flow impairment nor obstruction was evident . chest contrast - enhanced ct scan revealed multiple , variable - sized , well - defined nodules diffusely distributed throughout both lungs ( figure 1f ) . these nodules were highly enhanced , measuring approximately 140 hu on post - contrast scans . 18f - fluorodeoxyglucose positron emission tomography / computed tomography ( discovery ste , ge medical system , milwaukee , wi , usa ) demonstrated mild hypermetabolism within the pelvic and lung masses ( maximum standardized uptake value = 2.2 and 2.7 , respectively ) ( figure 1 g ) . based on the above imaging findings , rare ovarian neoplasm with perivascular extension along both gonadal vessels was suggested as a differential diagnosis . we included epithelial origin malignancies such as clear cell carcinoma and non - epithelial origin sex cord tumors such as ovarian sclerosing stromal tumor when considering hypervascular solid and cystic adnexal masses . even though vascular tumors are rare in the female genital tract , particularly in the ovary , vascular tumor such as hemangioma with extravascular growth pattern directly involving the bilateral ovaries could be possible . microscopic examination revealed that the tumor was composed of nests of epithelioid cells with cleared - out cytoplasm . immunohistochemical staining revealed that the tumor cells were diffusely positive for smooth muscle actin and strongly multifocally positive for human melanin black-45 ( hmb-45 ) ( figure 1i ) . results from histologic and immunohistochemical analyses of tissues obtained from the lung were identical to those of the pelvic masses . the term perivascular epithelioid cell ( pec ) was first proposed by bonetti et al . ( 5 ) in 1992 , and the term pecoma was first introduced by zamboni et al . this type of tumor consists of three components : thick - walled often hyalinized blood vessels , smooth muscle cells , and adipose tissue . angiomyolipoma of the kidney and liver , clear cell sugar tumor of the lung , and lymphangiomyomatosis are all pecomas ( 7 ) . there have been approximately 100 cases of pecomas previously reported in the literature , either as case reports or small sample studies . due to its rarity and the low number of case reports , the most common site for pecoma has not clearly been determined . however , according to a recent study ( 4 ) , the kidney and uterus are the most prevalent organs . to the best of our knowledge , the present case is the first occurrence of malignant ovarian pecoma with pulmonary metastasis . because of the rarity of pecomas , imaging findings of these tumors in various organs are scarce . however , a few recent studies have reported the following common imaging features of pecomas : well - defined border , regular shape , large size without a lobular appearance , complete capsule with a well - defined margin , and frequent mildly necrotic areas ( 3 , 4 , 8) . the enhancement pattern is typically inhomogeneous , probably due to the heterogeneous composition of the tumor ( 9 ) . ( 10 ) reported a rapid wash - in and slow wash - out pattern of enhancement in renal pecomas . other reports suggested that arteriovenous hypervascularity in contrast - enhanced ct and mr images is a feature of pecomas ( 9 , 11 ) . ( 2 ) recently reported imaging findings for a pulmonary malignant pecoma , which were characterized by well - defined , multiple round nodules exhibiting heterogeneous and intense enhancement ( the solid portion was associated with strong enhancement , i.e. , 115 hu on post - contrast images ) . two large , well - defined , multi - loculated ovarian masses were detected , accompanying multiple septal and peripheral enhancing solid portions that typically showed intense enhancement in the portal phase and slight delayed washout . although the majority of previously reported pecomas appear to behave in a benign fashion , a malignant course with local recurrence and distant metastasis has also been reported ( 2 , 4 , 12 ) . malignant pecomas are associated with two or more of the following worrisome features : high mitotic rate ( > 1/50 high - power fields ) , atypical mitotic figures , marked pleomorphism and nuclear atypia , hypercellularity , infiltrative growth pattern , large tumor size ( > 5 cm ) , and the presence of necrosis or vascular invasion ( 8) . the most notable aspects of our case were that both ovarian vessels were encased or contiguous with the masses . the bilateral pelvic masses contiguously extended along both ovarian vessels and exhibited twisted bread stick like appearance due to their wrapping around these vascular structures . however , vascular obstruction or flow impairment was not evident even though completely surrounded by masses . despite the large sizes of the bilateral ovarian masses , neither clearly discernible lymphadenopathy nor ascites solid metastases to the pulmonary parenchyma alone as the sole form of distant metastatic spread is very unusual without malignant pleural nodules or effusions . the majority of malignant pecomas exhibit no invasion or infiltration of the surrounding structures although contiguous venous invasion has been reported ( 4 ) . in conclusion , due to its extreme rarity , an accurate pre - operative diagnosis of ovarian pecoma is thought to be impossible . little is known about the ct and mri characteristics of these tumors , which contributes to the difficulty of establishing a diagnosis . however , in cases of ovarian hypervascular masses that encase ovarian vessels , pecoma should be considered as a differential diagnosis . | perivascular epithelioid cell tumor ( pecoma ) is a rare mesenchymal tumor composed of immunohistochemically and histologically distinctive perivascular epithelioid cells . here
, we report on the computed tomography ( ct ) and magnetic resonance imaging ( mri ) findings of ovarian pecoma with pulmonary metastasis .
the tumor was visible as a multilocular hemorrhagic mass that encased the ovarian vessels .
these findings were different to those of other common ovarian tumors . |
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research on cardiopulmonary resuscitation ( cpr ) has been intensively carried out over recent years and led to improved rates of successful resuscitation . however , the topic of interest is not only the ultimate outcome of whether patients are discharged from the hospital , but also the neurological outcome . in this context a processed eeg analysis computing the bispectral index ( bis ) is one method for monitoring the level of consciousness during anaesthesia . the bis value is a single numeric value on a scale from 0 to 100 . while values of 85100 indicate consciousness , sedation occurs at levels ranging from 65 to 85 and general anaesthesia is titrated to values between 40 and 60 . the spontaneous eeg will be inhibited once cerebral perfusion pressure is reduced below a critical value as a result of the protective mechanism that reduces the metabolic demands of neuronal ischaemia . a review of the literature revealed only eight case reports of using bis monitoring during cpr published in the last 6 years , but none described bis monitoring in only one patient undergoing double cardiac resuscitation within a few days . we report the case of a 69-year - old man suffering twice from cardiac arrest within 8 days , both monitored with bis , which revealed different results with different outcomes . the first cardiac arrest occurred on the haematological ward . on arrival of the cpr team , the patient was gasping for air , had enlarged pupils , and primary pulseless electrical activity was the first heart rhythm identified on electrocardiography . after immediate intubation and ventilation , the patient received a total of 4 mg of epinephrine during cpr . spontaneous circulation returned after 13 min and the patient was transferred to the intensive care unit ( icu ) , where he needed extensive catecholamine treatment for the next 6 h. bis monitoring was started 11 min after the cardiac arrest and showed an initial bis value of 87 and an average value of 78 5 , a suppression ratio of 0 and a sufficient sqi ( signal quality index , derived from impedance measurement of electrodes ) , which is considered to be above 50 ( fig . 1 ) . the sedation regimen consisted of 20 mg etomidate in the first 15 min of cpr ; additionally propofol ( between 80 and 160 mg / h ) and fentanyl ( 157 g / h ) were administered after return of spontaneous circulation . in the following 2 h the patient suffered several times from brief cardiac arrest , which required short periods of cpr and 200 meq of sodium bicarbonate to stabilize metabolism . eight hours after the first cpr the catecholamine dosage could be stopped , and the patient emerged from anaesthesia without showing neurological deficiencies . a mini - mental state examination showed 27 of 30 points , and the cerebral performance category score was 2 . to detect the cause of the cardiac arrest , computed tomography ( ct ) angiography scan of the body was performed and showed incomplete , bilateral pulmonary embolism of single segmental arteries as the probable cause of the cardiac arrest , while radiological examination disclosed no pathological findings in the brain . the patient was discharged from the icu on day 4 after admission under stable cardiopulmonary conditions , but suffering from acute renal failure . 1bis monitoring during the first cardiac arrest , when the patient survived without any neurological deficiency . note that the value of the suppression ratio was 0 during the entire external cardiac compression bis monitoring during the first cardiac arrest , when the patient survived without any neurological deficiency . note that the value of the suppression ratio was 0 during the entire external cardiac compression eight days after discharge , the patient collapsed again and suffered from respiratory insufficiency with dyspnoea , cyanosis and an oxygen saturation of 70% . it was possible to stabilize the patient under biphasic positive airway pressure ( bipap ) ventilation . no ct scan for the diagnosis of pulmonary embolism followed due to the lack of therapeutic consequences . after the first stabilization with non - invasive artificial ventilation the patient s respiration and circulation became increasingly insufficient , requiring intubation and catecholamine treatment . sedatives and analgesics consisted of midazolam ( 10 mg / h ) and fentanyl ( 157 g / h ) . later on , the patient again needed cpr after diagnosis of pulseless electrical activity on the ecg , probably caused by a second thromboembolic event . the initial bis value was 72 and decreased to 13 during cpr with an average value of 46 22 ( fig . 2 ) . the suppression ratio increased from 11 to 68 and emg decreased from 39 to 28 . despite intensive treatment with high catecholamine doses , it was not possible to combat the haemodynamic insufficiency and thus intensive care physicians decided to stop cpr . bis values initially reached high levels and decreased after 15 min of cardiac decompression . the burst suppression pattern occurred immediately at the beginning of resuscitation and suppression continuously rose until the end of cardiac compression . the last increase of bis possibly represents improved cerebral activity following a rise of mean arterial pressure caused by a final bolus of 5 mg epinephrine prior to stopping cpr the second cardiac arrest was followed by an unsuccessful cardiac resuscitation . bis values initially reached high levels and decreased after 15 min of cardiac decompression . the burst suppression pattern occurred immediately at the beginning of resuscitation and suppression continuously rose until the end of cardiac compression . the last increase of bis possibly represents improved cerebral activity following a rise of mean arterial pressure caused by a final bolus of 5 mg epinephrine prior to stopping cpr bis monitoring has shown its value in operating rooms around the world in preventing awareness during surgery and in the control of anaesthesia [ 1 , 2 ] . there are a few reports of using bis as a monitor in cardiac arrest and there is considerable interest in whether it could be useful to predict the outcome of cpr . we report a case of a 69-year - old patient with the unique situation of having had two cardiac arrests within 8 days , receiving bis monitoring twice during the cpr and showing two different courses of bis values , resulting in two completely different outcomes . during the first event the patient had immediately high bis values under cpr and a good neurological outcome . the cpr team considered stopping resuscitation due to the length of the resuscitation and the patient s palliative status , but the patient showed return of spontaneous circulation . assuming that high bis values result from good cerebral perfusion , the conclusion would be that high bis values might be the result of a sufficient mechanical resuscitation . additionally , bis monitoring of the suppression ratio can also detect suppression patterns indicating cerebral ischaemia . during the first course of cpr the electrical activity of the patient s brain did not show any suppression ratio , when bis monitoring started . this fact highlights the existing activity of basic and functional metabolism of the brain . in the second course of cpr 8 days later , the patient had low bis values under cpr , which further decreased . the most interesting point seems to be that bis monitoring may be a good parameter of sufficient cardiac output under cardiac massage in cases of pulmonary thromboembolism . during the first event fragmentation of the pulmonary thrombus apparently occurred resulting in good cerebral perfusion and bis values . at the second event cardiac massage failed to result in sufficient cerebral perfusion . previous reports have suggested bis values above 40 as favourable for the patient s outcome . additionally , increasing bis values in the early stage of both during and after resuscitation are associated with good survival and sufficient cerebral performance [ 5 , 6 ] . unlike former case reports , the bispectral index , suppression ratio and emg values are presented for discussion in the context of a double cpr event in the same patient . there are several concerns in the overestimation of pure bis values , which could limit the prognostic value of bis monitoring . various manipulations to the head and body during cpr can lead to high emg values , which can cause an artificial increase of the bis and may diminish the assessment of bis values . emg levels between 40 and 60 may influence the bis values and cause false high bis values . a low signal quality and narcotics can also influence the bis value , especially propofol , anaesthesia gas and other anaesthetics [ 810 ] . the bis values of our patient increased despite sedation with etomidate and fentanyl during the first cpr and decreased during the second cpr under an even lower amount of sedatives . additionally , etomidate in particular is known to cause muscle fasciculations , which can produce an emg artifact . however , the administered drugs did not have a strong influence on the bis and emg values in the dose in which they were applied in both cprs , and the emg remained at the same level despite the application of a small dose of etomidate . one limitation of interpreting bis values may be a low eeg quality , but the signal quality was sufficient and high bis values resulted in survival and a good cerebral outcome after the first cpr . initially lower and further decreasing bis values were associated with the death of the patient at the second cpr ( fig . 2 ) . the patient showed bis values around 77 , which resulted in a good outcome ( fig . 1 ) , while values between 55 and 10 during cpr resulted in an unfavourable outcome ( fig . 2 ) . other authors described bis values above 40 as acceptable during cpr [ 3 , 12 ] . interestingly , the pattern of the suppression ratio showed a relatively high value at the beginning of monitoring , which continuously increased until the end of resuscitation during the second cardiac arrest . there may be a possible reciprocal correlation between the high suppression ratio values and the success of cpr . presumably , the suppression ratio may give more information in combination with the bis value than the latter value alone . since the suppression ratio value can not be influenced by the emg , it may be a more reliable parameter than the bis value , which is calculated from many subparameters including the emg . however , increasing bis values after stabilization of the circulation correlated with favourable outcome in this particular case , while decreasing values ended with death . besides cerebral monitoring during anaesthesia bis may monitor the success and quality of external cardiac compression especially in patients suffering from thromboembolism in the pulmonary circulation . | research on cardiac resuscitation has led to various changes in the techniques and drug administration involved in modern advanced life support . besides improving primary cardiac survival ,
interest is increasingly focused on a favourable neurological outcome .
however , until now there has been no on - site equipment to support the clinical observations of the cardiopulmonary resuscitation ( cpr ) team .
bispectral index ( bis ) monitoring has been used for avoiding awareness during anaesthesia for many years .
we report a case of a 68-year - old patient suffering twice from cardiac arrest due to thromboembolism within a few days .
while the first cardiac resuscitation was survived without neurological consequences , the patient died after the second event .
both resuscitation events were monitored using the bis .
we discuss the course of bis values and their possible contribution to the prediction of outcome . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
coeliac disease ( cd ) is a common autoimmune condition affecting the small bowel ; it is a gluten - sensitive enteropathy in genetically susceptible individuals . gluten is a constituent of wheat and gluten - like molecules are present in related foodstuffs including barley , rye and oats . advances in clinical awareness and serological testing have influenced its prevalence over the last decade [ 1 , 3 ] . it is reported 1.5 times more frequently in women compared with men and is also associated with a range of other autoimmune conditions [ 1 , 3 ] . from a genetic perspective there is a strong association with the hla - dq2 and the hla - dq8 haplotype , which are expressed in over 90% of patients . the pathogenesis of cd is thought to be related to an increase in intestinal permeability [ 4 , 5 ] and the accumulation of gliadin in the lamina propria , which stimulates both the adaptive and innate immune responses . this manifests clinically in constitutional and malabsorptive symptoms , including diarrhoea , bloating and weight loss . the diagnosis of cd relies on clinical presentation , detection of serum iga autoantibodies and the demonstration of villous atrophy on duodenal biopsy ( in accordance with marsh 's histological criteria ) . most patients have a rapid recovery to normal health following a gluten - free diet ( gfd ) , with improvement of symptoms seen within weeks to month . in the majority the complications of this disease are related to malnutrition and an association with enterocyte - associated t cell lymphoma ( eatl ) . we present the clinical course of diagnosis and management in a case of refractory coeliac disease ( rcd ) . this report highlights the particular therapeutic challenges faced by this phenomenon and the potential utility for parenteral nutrition in the management of such cases . we present the case of a 68-year - old lady who developed persistent non - bloody diarrhoea and vomiting whilst travelling in india . she had a 10-day stay with her family , spending time in both urban and rural settings and drinking only bottled water and unpasteurised buffalo milk . on return to the uk , her symptoms did not resolve and she was referred to the gastroenterology team at ealing hospital . her initial clinical investigations revealed a positive tissue transglutaminase assay ( 234 u / ml , normal range 010 u / ml ) and normocytic anaemia ( haemoglobin 10.9 g / l , mean corpuscular volume 87.1 fl ) . endoscopic biopsies from the second part of the duodenum showed shortened broad - based villi with an increased number of intraepithelial lymphocytes , consistent with a diagnosis of cd . she commenced a strict gfd , with a later addition of steroid therapy ( budesonide ) . this did not control her symptoms , and when seen at follow - up ( 6 months after her initial presentation ) , she complained of worsening diarrhoea and steatorrhoea , with bowels opening up to 16 times a day . she also complained of colicky abdominal pain , significant weight loss ( from 62 to 49 kg ) and was noted to be peripherally oedematous up to her knees . given the alarming rate of progression of her symptoms , she was admitted from the clinic to hospital . clinically , we questioned whether this was progression of her cd or the presentation of a second pathology . her admission investigations were consistent with severe malnutrition and pancreatic exocrine insufficiency , with an albumin of 12 g / l and reduced faecal elastase of 86 g / g . she was initially treated with a strict gfd , high - energy high - protein diet , fresubin energy drinks and creon supplementation , with no significant improvement . while she was an inpatient , we proceeded to exclude other causes of malnutrition . indeed , stool cultures were negative for bacteria , ova and cysts ; tuberculosis was excluded with a negative quantiferon test ; she was negative for hiv and was empirically treated for small bowel bacterial overgrowth with cyclical ciprofloxacin and metronidazole , with no effect . her imaging included a normal computed tomography of the chest , abdomen and pelvis and a magnetic resonance imaging of the small bowel showing only small bowel diverticula . upper gastrointestinal endoscopy showed chronic gastritis ( negative for helicobacter pylori ) and colonoscopy was unremarkable , with no evidence of neoplasia in her biopsies . gene array studies of the biopsies obtained from d2 confirmed polyclonal t cell receptor arrangement , further reducing the likelihood of lymphoma . thus overall , we were unable to find a second pathology explaining her intestinal failure , leaving rcd as her diagnosis of exclusion . she was then given a short trial ( 5 days ) of total parenteral nutrition ( tpn ) via a hickman line . the tpn regimen was kabiven 14 ( day 1 : 768 ml at 32 ml / h , day 2 : 1,560 ml at 65 ml / h , day 3 : 1,920 ml at 80 ml / h , day 4 : 1,920 ml at 80 ml / h , day 5 : 1,920 ml at 80 ml / h ) . table 1 details the constituents of each bag of kabiven 14 . while on tpn , her fluid balance , weight and blood sugar levels were closely monitored . in addition , she had daily monitoring of her haemoglobin , sodium , potassium , calcium , phosphate , magnesium , liver function tests , urea and creatinine . following this period of tpn , her diarrhoea and steatorrhoea settled , her peripheral oedema improved and her albumin climbed to 34 she was transferred to a specialist intestinal failure unit , where she received just 2 further days of tpn before being safely discharged back to the community . a short period of tpn thus induced remission , characterised by a complete resolution of symptoms and biochemical improvement , which were sustained at follow - up . rcd is characterised by recurrent or persistent symptoms and villous atrophy despite strict adherence to a gfd for at least 6 months when other causes on non - responsive cd and malignancy have been excluded [ 3 , 8 , 9 ] . this is essentially a diagnosis of exclusion , made only after eliminating other causes of malabsorption , gluten contamination in diet and malignancy . rcd can be categorised into primary ( persistent symptoms without initial response to diet ) and secondary ( relapse following initial response to diet ) . type 1 rcd displays a predominantly normal intraepithelial lymphocyte population and type 2 rcd shows an aberrant population , whereby the intraepithelial lymphocytes demonstrate a loss of surface t cell receptor , cd3 or cd8 expression , and have a greater potential to progress and evolve into an aggressive eatl . this explains the poor prognosis associated with type 2 ( 5-year survival between 44 and 58% ) , in contrast to type 1 , which generally runs a benign course [ 12 , 13 ] . immunohistochemistry for our patient showed the presence of cd3 and cd8 , consistent with increased phenotypically normal intraepithelial lymphocytes in type 1 rcd ( fig . 2 ) . following the exclusion of other causes of malabsorption such as giardiasis , topical sprue , hiv and whipple 's disease currently there are no standardised treatment regimens for rcd due to the rarity of the condition . clinicians are usually guided by the limited number of cases reported in the literature which have successfully responded to different managements . there are a number of potential therapies already described in the literature , including the use of steroids and steroid - sparing agents such as azathioprine . in the majority of rcd type 1 cases , steroid use is effective in inducing clinical remission and mucosal recovery ; however , use in rcd type 2 shows clinical response in 75% , but does not induce mucosal recovery or prevent the progression to eatl . although majority of the literature focuses on immunosuppressive therapies for rcd treatment , nutritional support is an essential part of management which should not be overlooked . elemental and liquid diets can be used to ensure total gluten withdrawal ; however , malabsorption and protein - losing enteropathy can be so severe in some cases that patients may require intravenous nutritional support . studies have shown that up to 60% of patients who were hospitalised for rcd required tpn due to severe weight loss , malnutrition , severe electrolyte abnormalities , complex nutritional deficiencies and/or steatorrhoea [ 12 , 13 , 15 ] . tpn has the ability to provide adequate nutrition and replacement of the necessary proteins whilst allowing time for intestinal mucosal recovery . all rcd patients would benefit from the involvement of an experienced dietician who can provide tpn regimes tailored to each patient 's requirement . in our case , g / l and her haemoglobin improved from 6.7 to 12 g / dl at follow - up 6 months later . clinically , her diarrhoea slowly subsided within 1 week of starting tpn , and her weight increased from 39 kg on admission to 43 kg at follow - up . we have described a case of severe rcd where symptoms persisted despite a strict gfd , treatment with steroids , antibiotics for small bowel bacterial overgrowth and pancreatic supplementation for pancreatic insufficiency . there is limited evidence and guidance on the most effective treatment options for rcd patients . however , our case illustrates the importance of the consideration of tpn in patients presenting with rcd ; a short period of tpn in our patient induced remission of cd , enabling successful recovery , weight gain and total control of symptoms . we declare no support from or financial relationships with any organizations that might have an interest in the submitted work . | refractory coeliac disease ( rcd ) is characterised by recurrent or persistent malabsorptive symptoms and villous atrophy , despite strict adherence to a gluten - free diet for at least 6 months and where other causes of malabsorption including malignancy have been excluded .
there is limited evidence and guidance on the effective management of these patients .
we describe a case of severe rcd in our hospital , with symptoms controlled effectively only by total parenteral nutrition ( tpn ) .
this 68-year - old woman initially presented to the clinic with persistent non - bloody diarrhoea and vomiting .
a diagnosis of coeliac disease was confirmed with a positive tissue transglutaminase assay and histology .
a strict gluten - free diet was ineffective and she represented 6 months later with 13 kg weight loss ( 16.7% ) , ongoing abdominal pain and diarrhoea , with bowels opening 16 times a day .
she was oedematous , had an albumin of 12
g / l and required hospital admission .
she was treated for pancreatic insufficiency and presumptively for small bowel bacterial overgrowth with no resolution of symptoms .
we ruled out infectious causes and investigated for small bowel malignancy ; all results were negative .
small bowel enteroscopy showed ulcerative jejunitis .
she was given 5 days of tpn , following which her symptoms improved and albumin normalised . this was sustained with symptom resolution and weight gain seen at follow - up .
tpn successfully and rapidly induced remission in this case .
thus , a short period of tpn should be considered as a potential component of management in patients with severe rcd . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
igg4-related disease is a unique systemic inflammatory condition characterized by tumorous swelling of affected organs and high - serum igg4 concentrations [ 13 ] . further studies have confirmed that igg4-related disease can involve a variety of organs including the salivary glands ( chronic sclerosing sialadenitis ) [ 5 , 6 ] , lacrimal glands ( mikulicz 's disease ) [ 7 , 8 ] , and retroperitoneum ( retroperitoneal fibrosis ) [ 9 , 10 ] . the bile duct lesion is called igg4-related sclerosing cholangitis ( igg4-sc ) or igg4-associated cholangitis [ 12 , 13 ] ( the former is used hereafter ) . since we reported that igg4-sc is a distinct entity which should be differentiated from primary sclerosing cholangitis ( psc ) , clinical and pathological features have been clarified [ 12 , 13 ] . in this paper , we describe the concept , pathology , differential diagnosis , and pathogenesis of igg4-sc . the former should be differentiated from psc , whereas the latter radiologically resembles hilar cholangiocarcinoma . of note a study by the mayo clinic found that only 4 of 53 patients ( 7.5% ) with igg4 cholangiopathy had cholangitis without autoimmune pancreatitis . whether or not igg4-sc can involve only peripheral small bile ducts like small - duct psc is an interesting issue . given that a recent study revealed that peripheral igg4 cholangiopathy was always associated with large duct lesions , it seems safe to assume that igg4-sc predominantly affects large bile ducts , which are detectable by cholangiographic or radiological examinations . recent papers have introduced igg4-related autoimmune hepatitis , which accounts for 3% of cases of type 1 autoimmune hepatitis in the japanese population . the term igg4 hepatitis should be only used for patients who do not have radiological biliary abnormalities and are found to have chronic hepatitis based on liver biopsies . given that one cases of igg4 hepatitis was complicated later by sclerosing cholangitis during followup , igg4 hepatitis might also belong to a spectrum of igg4 cholangiohepatitis . in our experience , patients with igg4-sc usually present with obstructive jaundice due to a pancreatic head mass ( autoimmune pancreatitis ) or severe biliary stricture [ 12 , 13 ] . other patients are sometimes discovered to have igg4-sc during a workup for other igg4-related conditions such as sialadenitis , retroperitoneal fibrosis and kidney lesions . weight loss or new - onset diabetes mellitus due to pancreatitis is another potential symptom . there is no doubt that elevated serum igg4 levels are the most specific indicator . other sensitive but not specific markers include hyper -globulinemia ( observed in 50% of patients ) , hyper igg ( 6070% ) , antinuclear antibodies ( 4050% ) , rheumatoid factor ( 20% ) , and eosinophilia ( 1525% ) [ 18 , 19 ] . autoantibody against ss - a ( ro ) or ss - b ( la ) , antimitochondria antibody , and antineutrophilic cytoplasmic antibody ( anca ) are all exceptional ( < 5% ) [ 18 , 19 ] . studies on autoimmune pancreatitis have provided further data on autoantibodies which might participate in the pathogenesis . antibodies against lactoferrin ( lf ) and carbonic anhydrase ( ca ) ii are frequently detected in cases of autoimmune pancreatitis ( 73% and 54% , resp . ) . interestingly , a strong positive correlation between increases in serum igg4 levels and anti - ca - ii antibody levels has been reported . anti - ca - iv , another autoantibody , was detected in 10 of 29 ( 34% ) patients with autoimmune pancreatitis . given that lf and cas are expressed in some exocrine organs , these autoantibodies may be related to systemic manifestations of igg4-related disease . of note is that autoantibodies of the igg4 subclass have not been detected in patients with igg4-related disease so far . it is not difficult to make a diagnosis of igg4-sc if surgically resected specimens are available . the gold standard for the diagnosis of igg4-sc is histology including characteristic features on h&e and extensive infiltration by igg4 plasma cells on immunostaining . pathological features can be summarized as follows : ( 1 ) diffuse lymphoplasmacytic infiltration , ( 2 ) storiform fibrosis , ( 3 ) obliterative phlebitis , ( 4 ) eosinophilic infiltration , and ( 5 ) numerous igg4 plasma cells [ 11 , 23 ] . features unusual for igg4-sc are neutrophilic infiltration with or without abscesses , xanthogranulomatous change , and mucosal erosive change . we speculate that endothelium may express chemotactic factors , but this has not been examined so far . serology , imaging , other organ involvement , and biopsy need to be considered for the diagnosis of nonsurgical cases . given that most patients ( > 90% ) with igg4-sc have autoimmune pancreatitis , it seems most important to examine changes in the pancreas . autoimmune pancreatitis is radiologically suspected in most cases , and the diagnosis can be confirmed by the serological examination of igg4 . histological detection of igg4 plasma cells is usually not necessary for cases showing typical radiological features ( sausage - like diffuse swelling , peripancreatic capsule - like rim , and irregular narrowing of the pancreatic duct ) and high - serum igg4 levels . but , if there are any unusual features on imaging or igg4 levels are not elevated , biopsies should be considered to detect igg4 plasma cells . most institutions use 135 or 140 mg / dl as a cut - off point for serum igg4 levels , with more than 300 mg / dl being highly specific for igg4-related disease [ 4 , 25 ] . it is challenging to diagnose igg4-sc not associated with pancreatitis . in fact , most patients have been surgically treated for suspected biliary malignancy . in our experience , detecting igg4 plasma cell is recommended even if the patients have high - serum igg4 concentrations . vater 's ampulla biopsy is least invasive and technically easiest , and especially useful for patients discovered endoscopically to have ampullary swelling [ 2629 ] . another potential approach is a liver needle biopsy which can detect igg4 plasma cells infiltrating into peripheral small portal tracts [ 15 , 30 , 31 ] . this is particularly useful for patients with intrahepatic biliary abnormalities on cholangiograms , but not useful for patients with only intrapancreatic bile duct stricture . the last choice is a bile duct biopsy , the usefulness of which might depend on the ability and experience of the endoscopists . the biggest advantage of this method is that not only igg4 plasma cells but also other histological features such as storiform fibrosis and eosinophilic infiltration are detectable . the specificity and sensitivity of the diagnosis by these three biopsies are summarized in table 2 . normally , more than 10 igg4 plasma cells are used as the diagnostic threshold for biopsy samples . some institutions use steroid trials for the diagnosis [ 3234 ] , whereas other groups , especially from japan , are against doing so . given that igg4-sc needs to be differentiated from malignant tumours ( cholangiocarcinoma ) different from other typical autoimmune diseases , much attention should be paid to diagnostic steroid trials . large bile ducts affected by igg4-sc show transmural inflammation with storiform fibrosis and obliterative phlebitis ( figure 1 ) , whereas inflammation is more extensive on the luminal side with erosion or xanthogranulomatous changes in psc . clinically , a young age ( < 40 years ) and history of inflammatory bowel disease are features suggestive of psc , whereas igg4-sc is more likely in patients with other sclerosing lesions including autoimmune pancreatitis and retroperitoneal fibrosis . serologically , igg4 levels are most useful , but it should be noted that 9% of psc patients show elevated igg4 levels . reported that psc and igg4-sc can be differentiated based on a detailed examination of cholangiograms . infiltration of igg4 plasma cells or the presence of periportal fibroinflammatory nodules in needle biopsy samples is suggestive of igg4-sc ( figure 1 ) , whereas ductopenia and periductal concentric fibrosis are more commonly seen in psc [ 15 , 31 ] . it is still unclear whether or not psc can be differentiated from burned - out igg4-sc . zhang et al . examined tissue infiltration of igg4 plasma cells in explanted liver with psc . twenty - three of 98 livers ( 23% ) showed more than 10 igg4 cells per hpf which might be less specific given that most igg4-related lesions show more than 100 igg4 cells / hpf in surgical specimens . in addition , another group revealed that 2 out of 41 ( 5% ) explanted livers with psc showed more than 100 igg4 cells / hpf . these two studies suggested that explanted livers with psc can sometimes show moderate degrees of igg4 cell infiltration ( > 10 cells / hpf ) and rarely exhibit marked infiltration ( > 100 cells / hpf ) around large bile duct lesions . importantly , the other histological features of these cases were not typical of igg4-sc but consistent with psc [ 38 , 39 ] . the histological diagnosis of igg4-sc is not enough just based on the number of igg4 plasma cells . this is a rare disease entity characterized by numerous lymphoid follicles around hilar or perihilar bile ducts [ 40 , 41 ] . follicular cholangitis is different from psc in that the inflammatory cell infiltration is more extensive and biliary epithelial damage is not conspicuous . igg4 plasma cell infiltration or obliterative phlebitis is usually not conspicuous , different from igg4-sc . in our experience , hilar cholangiocarcinoma is the most important and difficult differential diagnosis of igg4-sc in the clinical field , particularly for patients without autoimmune pancreatitis . serum igg4 levels can be mildly elevated in patients with cholangiocarcinoma , but titers of more than 300 mg / dl are highly suggestive of igg4-sc . as described above , histological examination to detect igg4 cell infiltration this is one significant difference from psc [ 12 , 13 ] . at the moment , it is difficult to conclude the recommended dose and duration of steroid therapy for igg4-sc because of a lack of published data . a japanese study on autoimmune pancreatitis recommended an initial dose of 0.6 mg / kg / day , which was then reduced to a maintenance dose over a period of 36 months . in that study , disease relapses appeared to be reduced but not eliminated by maintenance treatment with low - dose steroid . recent papers have provided data suggesting that the th2-type immune response is activated in igg4-related disease including igg4-sc [ 4448 ] . quantitative real - time pcr using rna extracted from frozen tissue of affected organs including bile ducts revealed significantly higher ratios of il-4/ifn- , il-5/ifn- , and il-13/ifn- in igg4-related disease tissues than in tissues from patients with classical autoimmune diseases . lymphocytes expressing il-4 were clearly demonstrated by in situ hybridization . recent papers also showed that peripheral blood mononuclear cells collected from patients with igg4-related disease produced predominantly th2-type cytokines such as il-4 , il-5 , il-10 , and il-13 after t - cell stimulation . interestingly , the number of regulatory t - cells ( tregs ) is characteristically increased in both tissue and blood of patients with igg4-related disease . our investigation revealed that the mrna expression of forkhead box p3 ( foxp3 , a tregs - specific transcriptional factor ) was higher in igg4-related disease than in classical autoimmune diseases . two regulatory cytokines , il-10 and transforming growth factor- ( tgf- ) , are significantly overexpressed [ 49 , 50 ] . furthermore , cd4cd25foxp3 tregs could be detected within affected tissues by immunohistochemistry , in numbers significantly higher than in autoimmune and nonautoimmune disease controls . the number of foxp3 cells was significantly correlated with the number of igg4 plasma cells in igg4-related cholangitis . miyoshi et al . examined the number of tregs in the blood and reported that the number of cd4cd25 tregs was significantly higher in patients with aip than in patients with chronic pancreatitis and was correlated with the level of igg4 in serum . they speculated that hyporeaction of nave tregs might be involved in the development of igg4-related disease , whereas hyperreaction of cd4cd25 tregs could reflect igg4-related disease progression . the possible involvement of h pylori in the pathogenesis of aip was reported in 2005 . gastric h. pylori infection triggers aip in genetically predisposed subjects via molecular mimicry between human ca - ii and alpha - carbonic anhydrase of h. pylori . frulloni et al . found that 94% of patients with aip had antibodies against a plasminogen - binding protein of h. pylori . the amino acid sequence of the plasminogen - binding protein exhibited homology with that of the ubiquitin - protein ligase e3 component n - recognin 2 , an enzyme expressed in pancreatic acinar cells . however , the involvement of h. pylori in the pathogenesis of other igg4-related lesions has not been reported so far . igg4-sc is a unique cholangiopathy which should be differentiated from classical psc or biliary malignancy . | igg4 cholangiopathy can involve any level of the biliary tree which exhibits sclerosing cholangitis or pseudotumorous hilar lesions .
most cases are associated with autoimmune pancreatitis , an important diagnostic clue . without autoimmune pancreatitis , however , the diagnosis of igg4-cholangiopathy is challenging .
indeed such cases have been treated surgically .
igg4-cholangiopathy should be diagnosed based on serological examinations including serum igg4 concentrations , radiological features , and histological evidence of igg4 + plasma cell infiltration .
steroid therapy is very effective even at disease relapse . a th2-dominant immune response or the activation of regulatory t cells seems to be involved in the underlying immune reaction .
it is still unknown why igg4 levels are specifically elevated in patients with this disease .
igg4 might be secondarily overexpressed by th2 or regulatory cytokines given the lack of evidence that igg4 is an autoantibody . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the lymphatic system complements functions of the blood vascular system by regulating tissue fluid balance , facilitating interstitial protein transport , and serving immunological functions . fluid and macromolecules that exit blood capillaries are collected from the interstitial space by lymphatic capillaries and returned back to the blood circulation through the network of larger lymphatics . lymphatics are also responsible for absorption of fat from the gut . by directing leukocytes and antigens from tissues to lymph nodes , although the lymphatic and blood vascular systems rely on each other for the maintenance of tissue homeostasis , they are structurally and functionally distinct entities . whereas the main function of large lymphatics is efficient transport of lymph back into the blood circulation , the lymphatic microvasculature is responsible for the uptake of components from the interstitium . given their central role in regulating interstitial fluid pressure and cell trafficking , it is surprising that lymphatic endothelial cells ( lecs ) have until recently been poorly characterized , at least from a molecular point of view . this scenario is changing rapidly following the development of techniques for the isolation of pure lecs and the characterization of their molecular properties . lymphatic capillaries are blind - ending vessels , comprised of a single , nonfenestrated endothelial cell layer , that is optimally adapted for the uptake of fluid , macromolecules , and cells . although lecs have many properties in common with the endothelium of blood vessels , they also have very distinct structural features that have been best characterized at the ultrustructural level . lymphatic capillaries generally possess a more irregular and wider lumen than blood capillaries , and their endothelium is extremely attenuated . in contrast to blood vessels , lymphatic capillaries have an incomplete basement membrane and are not invested by pericytes . they are generally observed in a partially or fully collapsed state ( schmid - schnbein , 1990a ; aukland and reed , 1993 ) . unique to lymphatic capillaries are also overlapping intercellular junctions that are formed by the extensive superimposition of adjacent lecs . an increase in interstitial fluid pressure causes these junctions to open , thereby permitting the easy passage of fluid and particles into the vessel . as fluid enters the lumen , pressure differences across the vessel wall decrease and the junctions begin to close , preventing retrograde flow back into the interstitium ( fig . 1 ) ( schmid - schnbein , 1990b ; ikomi and schmid - schnbein , 1996 ) . lecs are attached to interstitial collagen by anchoring filaments , composed of elastic fibers ( leak and burke , 1966 ; gerli et al . , 1990 ) , which preserve functionality of lymphatics when interstitial pressure rises by preventing vessel collapse . the composition and organization of the ecm are thus also likely to play a critical role in lymphangiogenesis . the lymphatic capillary is uniquely adapted for the uptake of fluid , lipids , macromolecules , and cells from the interstitium . in contrast to the blood capillary , the lymphatic capillary has poorly developed basal lamina ( bm ) and is devoid of pericytes ( p ) . lymphatic endothelium is highly attenuated , and cells are connected directly to the interstitial collagen via anchoring filaments ( af ) . t , t cell ; d , dendritic cell ; apc , antigen presenting cell . during development and wound healing , angiogenesis generally preceeds lymphangiogenesis , implying the existence of distinct yet spatially and temporally coordinated regulatory mechanisms . two members of the vegf family , vegf - c and vegf - d , have been demonstrated to play a critical role in lymphangiogenesis via activation of vegfr-3 , which is expressed mainly by lecs in normal adult tissues ( joukov et al . , 1996 ; lee et al . , 1996 ; vegfr-3 signaling is important for development of the embryonic lymphatic system , lymphatic regeneration in the adult , and tumor lymphangiogenesis ( alitalo and carmeliet , 2002 ) . vegf - c and vegf - d , when fully proteolytically processed , can also activate vegfr-2 ( joukov et al . , 1997 ) , but whether vegfr-2 plays a direct role in lymphangiogenesis is less clear . vegf - c also binds to a nonkinase receptor neuropilin-2 ( nrp2 ) ( karkkainen et al . , 2001 ) , a classic receptor for class iii semaphorins , which regulate chemorepulsive guidance of developing axons . recent studies in nrp2-deficient mice demonstrated impeded development of lymphatic capillaries in most tissues , suggesting a role for nrp2 in lec proliferation and , perhaps , guidance . nrp2 may cooperate with vegfr-3 to mediate vegf - c dependent lymphangiogenesis ( yuan et al . , 2002 ) . finally , ang2 is expressed by lecs ( petrova et al . , 2002 ; podgrabinska et al . , 2002 ) and is required for the proper development of the lymphatic system ( gale et al . , 2002 ) . mice deficient in ang2 displayed disorganization and hypoplasia of lymphatic capillaries , and collecting lymphatic vessels were not properly invested by smooth muscle . as a result , ang2 knockout mice developed severe lymphedema . interestingly , the lymphatic phenotype caused by ang2 deficiency was rescued by ang1 , suggesting redundant roles for these molecules in lymphatic development . the homeobox transcription factor prox-1 appears to be required for the commitment of endothelial cells to the lymphatic differentiation program ( wigle and oliver , 1999 ; wigle et al . , prox-1 expression in embryos localizes to a subpopulation of endothelial cells in embryonic veins , which are commited to the lymphatic pathway . functional inactivation of prox-1 in mice results in the arrest of lymphatic vessel development . in adult tissues , prox-1 is expressed exclusively by lecs , and overexpression of prox-1 in blood endothelial cells ( becs ) down - regulated bec - specific transcripts and up - regulated lec - specific transcripts , thus conferring the lymphatic endothelial phenotype on these cells ( hong et al . most recent evidence suggested that the adaptor protein slp76 and the tyrosine kinase syk , which are expressed primarily in hematopoietic cells , may also contribute to the anatomical separation of the blood and lymphatic vasculature ( abtahian et al . , 2003 ) . many attempts have been made in the past to isolate and culture lecs from a variety of species ( pepper , 2001 ) . all of these studies have described isolation of the cells from large lymphatic vessels and have employed crude mechanical methods of cell separation . since large lymphatics are supplied by a rich network of nutritive blood vessels , the purity of the isolated cell populations has remained in question . furthermore , given the heterogeneity of endothelial cells from different vascular beds , large vessel endothelial cells are likely to be inappropriate for the study of lymphatic capillary structure function relationships . the identification of cell surface markers that reliably distinguish lymphatic endothelium from blood vascular endothelium ( sleeman et al . , 2001 ) has led to the development of superior techniques for the isolation of pure lymphatic and blood vascular endothelial cells . lecs have been isolated by positive selection using antibodies to podoplanin ( kriehuber et al . , 2001 ) , vegfr-3 ( makinen et al . , 2001 ) , or lyve-1 ( podgrabinska et al . , 2002 ) , and by a negative selection with antibodies to cd34 ( hirakawa et al . , 2003 ) lecs were distinguished by their homotypic association , selective responsiveness to vegf - c in terms of growth , survival and morphogenesis , differential ecm requirements , and the distinct gene expression profile . lecs established by the different methods , however , exhibited certain differences in gene expression that may be attributed to the different source of tissues employed , i.e. , adult versus neonatal skin . lecs isolated using vegfr-3 antibodies may be partly contaminated with becs , since vegfr-3 can also be expressed by the blood vascular endothelium ( partanen et al . , 1999 ) . finally , isolated lecs were propagated under different conditions , which may further account for the variations in phenotype . it remains to be determined which purification strategy and culture conditions allow for optimal preservation of the lymphatic endothelial phenotype in vitro . the molecular signature of lecs appears to reflect their unique functional characteristics and provides novel insight into the molecular basis of lymphatic function ( petrova et al . , 2002 ; podgrabinska et al . , 2002 ; hirakawa et al . , 2003 ) . for example , lecs express remarkably high levels of genes implicated in protein metabolism , sorting and trafficking ( podgrabinska et al . , 2002 ) . genes with particularly high representation were those encoding proteins that control specificity of vesicle targeting and fusion , such as members of the snare family , rab gtpases , aaa atpases , and sec - related proteins ( mellman and warren , 2000 ) , indicating the existence of a robust vesicular transport system . the lymphatic endothelium is characterized by an abundance of membrane invaginations and cytoplasmic vesicles ( leak , 1972 , 1976 ) , yet their functional significance has not been established . intercellular clefts are considered to be a major pathway for the movement of fluid and proteins into lymphatics ( schmid - schnbein , 1990b ) . however , some early studies also demonstrated the presence of interstitially injected molecular tracers within intracellular vesicles of lecs ( leak , 1972 , 1976 ) . in agreement with these findings , the results of the gene profiling studies suggest that , in addition to intercellular transport , transendothelial pathways may also be used as a mechanism for entry of molecules into lymphatics ( podgrabinska et al . , 2002 ) . this raises the intriguing possibility that lymphatics may have the capacity to selectively remove molecules from the interstitium and thereby actively control the composition of lymph and interstitial fluid . the importance of the lymphatic system as a pathway for metastasis has been well recognized . metastasis of most cancers occurs initially through the lymphatics and the extent of lymph node involvement is one of the most important prognostic indicators of patient outcome . traditionally , the lymphatic system has not been considered to be actively involved in the process of metastasis . tumor cells are believed to be passively carried into the lymphatic vessels with the interstitial fluid and proteins ( hartveit , 1990 ) , and the prevailing view has been that lymphangiogenesis is not a part of tumorigenesis ( carmeliet and jain , 2000 ; leu et al . , 2000 ; recent studies , however , have demonstrated enlarged lymphatic vessels and lymphangiogenesis in peritumoral areas of several human tumors using lymphatic endothelial markers ( stacker et al . , 2002 ; pepper et al . , 2003 ) . the number of tumor - associated lymphatics has been correlated with lymph node metastases , yet intratumoral lymphatics have so far been observed only in human head and neck cancers and in melanoma . the relative importance of preexisting versus newly - formed lymphatic vessels to lymphogenous metastasis is not understood . although preexisting peritumoral lymphatics are likely to be sufficient for tumor spread , recruitment of lymphatic vessels into the close proximity of a tumor may increase the propensity of tumors to metastasize . increased lymphatic vessel density and/or presence of intratumoral lymphatics should therefore be regarded as an additional pathway rather than a necessity for metastasis . notably , a large number of studies demonstrated a striking correlation between the vegf - c expression in human tumors and lymph node metastases ( stacker et al . , 2002 ; pepper et al . , 2003 ) . moreover , recent experimental studies using vegf - c overexpressing tumor cells have provided direct evidence for the causal role of vegf - c in tumor lymphangiogenesis and lymphogenous metastasis ( mandriota et al . , 2001 ; skobe et al . , 2001 ) . although an increase in lymphatic vessel density may promote tumor spread simply by creating more opportunities for metastatic tumor cells to leave the primary tumor site , lymphatic vessels may also play a more active role in metastasis . for example , soluble factors constitutively expressed by lecs may facilitate tumor cell invasion of lymphatic vessels . activation of lecs by vegf - c or other factors produced by a tumor could promote release of chemokines , which may attract tumor cells into the lymphatics . as the migration of cancer cells to regional lymph nodes resembles physiological migration of leukocytes , it is conceivable that the chemokine - mediated normal mechanisms of lymphocyte homing may also be used for metastatic dissemination . thus far , the importance of two chemokine receptors ( ccrs ) in lymph node metastasis has been established : cxcr4 and ccr7 . cxcr4 was found to be up - regulated in malignant melanoma and in breast cancer , whereas its ligand cxcl12 is highly expressed in lymph nodes and other target organs for breast cancer metastasis . a neutralizing antibody to cxcr4 inhibited metastases to lymph nodes and other organs , demonstrating a critical role for this chemokine / receptor system in mediating tumor cell homing ( muller et al . , 2001 ccr7 and its ligands chemokines ccl19 and ccl21 are of crucial importance for the migration of lymphocytes and dendritic cells to lymph nodes . ccr7 was also found to be highly expressed by human malignant melanoma and breast cancer cells ( muller et al . , 2001 ) , and its expression has been associated with lymph node metastasis in gastric cancer ( mashino et al . , overexpression of ccr7 in a mouse model of melanoma enhanced metastases to lymph nodes , which could be blocked by neutralizing its ligand ccl21 ( wiley et al . , 2001 ) . ccl21 and several other chemokines are constitutively expressed by lecs ( kriehuber et al . , 2001 ; , 2001 ; podgrabinska et al . , 2002 ) , suggesting an active role for lecs in governing cell migration in normal physiology and in cancer . however , a direct role for lymphatic endothelium in the process still remains to be demonstrated . mechanisms mediating tumor cell transmigration across the lymphatic endothelium into this lymphatic vessels also remain obscure . based on the differences in their structure , it has been assumed that the entry of cells into lymphatics is easier than into blood vessels . there is no direct experimental evidence in support of either concept . thus far , very few cell adhesion molecules expressed by lecs have been identified . several genes encoding proteins that constitute adherens junctions , such as desmoplakin , plakoglobin , plakophillin 2 , h - cadherin , and zona occludens 2 , were identified in lecs by gene profiling ( petrova et al . , 2002 ; podgrabinska et al . , 2002 ) . another junctional adhesion molecule belonging to the immunoglobulin superfamily , jam-2 , was found to be expressed in tight junctions of lymphatic vessels and was shown to facilitate lymphocyte transmigration ( aurrand - lions et al . , 2001 ; johnson - leger et al . , 2002 ) . the nature of the lymphatic endothelial junctions may indeed facilitate cell entry and the identification of adhesion molecules typical for lymphatic endothelium may be particularly important for the understanding of leukocyte trafficking and tumor metastasis via lymphatics . in this regard , macrophage mannose receptor ( mr ) i expressed by lecs has been shown to mediate adhesion of lymphocytes to lymphatics in lymph nodes ( irjala et al . , 2001 ) . mr on lecs supports lymphocyte binding to lymphatic vessels in an l - selectin dependent fashion , and this interaction has been suggested to control lymphocyte exit from the lymph nodes . mr was also found to be selectively expressed by cultured lecs ( petrova et al . , 2002 ; podgrabinska et al . , 2002 ) , and its presence on afferent lymphatics suggests its possible involvement also in lymphocyte exit from peripheral tissues . common lymphatic endothelial and vascular endothelial receptor-1 ( clever-1 ) is another recently identified adhesion molecule implicated in binding of lymphocytes to lecs in lymph nodes ( irjala et al . , 2003b ) . because clever-1 is an inducible vascular adhesion molecule , mr and clever-1 expression have also been detected on peri- and intratumoral lymphatic vessels in human head and neck , and breast carcinomas ( irjala et al . , 2003a ) . notably , expression of mr on intratumoral lymphatic vessels was associated with increased lymph node metastases in breast cancer . these pioneering studies aid in shaping the new concept of a more active role of lymphatic vessels in cancer . exquisitely detailed descriptive studies performed almost 100 years ago provided the basis for our understanding of the structure function relationships in the lymphatic system . today , we can truly speak of a renaissance in the field , owing to the identification of lymphatic specific markers and growth factors , as well as the sophistication of the techniques for isolation of pure lecs . the groundwork has thus been laid for study of the molecular mechanisms underlying the characteristic functions of lymphatic vasculature . better understanding of the lymphatic endothelial properties and how they may be altered in inflammation and in cancer may open a new door to therapeutic interventions . | the lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins , and is an important point of entry for leukocytes and tumor cells . the traditional view that lymphatic capillaries are passive participants in these tasks
is currently being challenged .
this overview highlights recent advances in our understanding of the molecular mechanisms underlying the formation and function of lymphatic vessels . |
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common causes include atrial fibrillation , infective endocarditis , anti - phospholipid antibody syndrome and trauma . bilateral renal artery thrombosis due to acute pancreatitis is rare and only one case has been reported so far . a 66-year - old female presented with abdominal pain , nausea , vomiting and fatigue for 5 days . other significant medical history included hypertension for 5 years and use of 8002400 mg ibuprofen for low back pain for the last 3 years . the pertinent findings on physical examination at admission included temperature of 37c , pulse rate of 88 beats / min ( regular ) , blood pressure of 160/90 mmhg , tenderness to palpation in the epigastrium with no rebound , guarding or distention and 2 + pitting edema in both lower extremities . the rest of the laboratory findings are shown in table 1 . a computed tomography ( ct ) scan of abdomen and pelvis without contrast revealed normal pancreas , bilateral renal cortical scarring and mesenteric edema . 10/lesr46 mm / h04 mm / hsodium134 mmol / l136145 mmol / lpotassium5 mmol / l3.55.1 mmol / lbun12.85 mmol / l2.57.1 mmol / lcreatinine362 mol / l5397 mol / lalbumin22 g / l3448 g / lalt50 u / l055 u / last35 u / l034 u / lbilirubin6.84 mol / l3.4220.52 mol / lcalcium2.2 mmol / l2.152.65 mmol / llactate1.6 mmol / l0.52.2 mmol / lldh398 u / l125243 u / lcreatine kinase73 u / l29168 u / lc31.52 g / l0.831.93 g / lc40.34 g / l0.150.57 g / lurine analysisprotein 100 specific gravity 1.017 wbc 75 rbc 6 no rbc / wbc / granular casturine sodium<20 mmol / l serum and urine laboratory values at admission hemodialysis was initiated because of anuria and hyperkalemia on day 4 . percutaneous biopsy of the left kidney was performed the same day ( figure 1 ) . there were four interlobular - sized muscular arteries containing intraluminal thrombi with organization , although no vasculitis was present . based on the biopsy findings of thrombosis without inflammation , renal artery thrombosis was suspected . on day 5 a ct scan of the abdomen / pelvis with contrast was done which showed bowel perforation and bilateral renal artery thrombosis ( figure 2 ) . extensive pancreatic necrosis was noted on laparotomy and therefore she underwent necrosectomy of the pancreas . a tc-99 m mag-3 scan was also done , which confirmed renal ischemia ( figures 3 and 4 ) . doppler study of the lower extremities and neck revealed thrombosis of the bilateral deep veins and internal jugular vein ( attached to dialysis catheter ) , respectively . work up for vasculitis including ana , anti - dsdna , anca , hepatitis panel , antiscl-70 were negative . the etiology of renal artery thrombosis was postulated to be due to the hypercoagulable state induced by acute necrotizing pancreatitis and proximity of the inflamed pancreas to the renal arteries . there are no signs of renal recovery at 6-month follow - up and the patient continues to be on hemodialysis . table 2.work up for hypercoagulabilitylaboratory testvaluereference rangeplasma homocysteine(total)14 mol / l515 mol / l2-glycoprotein 1 ab , igg<4 u / ml<10 u / ml negative2-glycoprotein 1 ab , igm<4 u / ml<10 u / ml negative2-glycoprotein 1 ab , iga<4 u / ml<10 u / ml negativeanti - cardiolipin iga0 apl011 aplanti - cardiolipin igg5 apl014 aplanti - cardiolipin igm4 apl012 apllupus anticoagulant screen44 s2845 sfactor v leiden r506q mutationnegativenegativeprothrombin ( f2)g20210a mutationnegativenegativeanti - thrombin iii activity8685128%anti - thrombin antigen9282136%protein c activity8484171%protein c total antigen7763153%protein s activity5654132%protein s total antigen7163126%
fig . 1.h&e staining showing intraluminal thrombus with organization within inter lobular muscular arteries ( a ) . 3.tc-99m mag-3 scan showing delayed uptake of tracer in both kidneys except upper poles ( a ) . 4.renogram showing delayed perfusion phase and function phase consistent bilateral reno - vascular obstruction and parenchymal damage . ( right kidney = b , left kidney = a. ) work up for hypercoagulability h&e staining showing intraluminal thrombus with organization within inter lobular muscular arteries ( a ) . no inflammation noted in the vessel walls ( b ) . filling defect within right renal artery consistent with thrombosis ( a ) . tc-99 m mag-3 scan showing delayed uptake of tracer in both kidneys except upper poles ( a ) . renogram showing delayed perfusion phase and function phase consistent bilateral reno - vascular obstruction and parenchymal damage . the true incidence of renal artery thrombosis is difficult to estimate because of the vague clinical presentation and rarity of the condition . the common symptoms include diffuse abdominal pain , flank pain , nausea , vomiting and renin - mediated elevation in blood pressure . elevation in creatinine can be a late feature as creatinine levels may not rise until 50% of the kidney function is lost . common laboratory findings include leukocytosis , proteinuria , hematuria , elevated lactate dehydrogenase ( ldh ) with minimal or no rise in serum aminotransferase . the differential diagnosis for this disorder includes causes of acute surgical abdomen and non - surgical causes such as mesenteric ischemia , nephrolithiasis and pyelonephritis . the risks and benefits of the use of intravenous contrast with concomitant renal failure have to be carefully assessed . however , if there is high pre - test probability of renovascular thrombosis , benefits outweigh risks as prompt intervention could be lifesaving and also help recover renal function . a radioisotope scan can show segmental or generalized decrease in renal perfusion and can be used as an additional non - invasive modality to confirm the diagnosis . renal arterial duplex doppler ultrasound study has low sensitivity for the diagnosis of renal arterial thrombosis . the work - up should also include echocardiogram to identify the source of thromboembolism and laboratory testing for hypercoagulability . the etiology of thrombosis is multifactorial in our patient : ( i ) injury to endothelium and increase in the levels of procoagulants like fibrinogen and factor v and viii due to release of proteolytic enzymes , ( ii ) increased thromboxane levels , platelet - endothelium interaction and platelet activation and adhesion and ( iii ) anatomic proximity of renal vessels to the inflamed pancreas . the presence of deep vein thrombosis in the lower extremities and thrombus in the right jugular vein support the hyper coagulable state in our patient . anticoagulation with heparin should be initiated for renal artery thrombosis if revascularization is not an option . the optimal duration of anticoagulation is uncertain ; however , since we postulated that the etiology of our patient 's thrombosis was due to pancreatitis , we decided to treat her for 3 months . the decision to perform endovascular or surgical revascularization will be based on the presence of collateral circulation to the kidneys and time since the event . patients with chronic atherosclerotic disease of the aorta and renal arteries usually have adequate collateral circulation . these patients can have relatively preserved renal function even with complete occlusion of the renal arteries . the optimal period for revascularization of thrombosed renal arteries is yet unclear ; however , revascularization data from other arterial territories would suggest earlier revascularization for optimal results . revascularization therapy can be performed endovascularly or surgically based on the timing of diagnosis and patient comorbidities [ 6 , 13 ] . endovascular revascularization can be performed using intra - arterial streptokinase / urokinase infusion , t - pa power - pulse spray rheolytic thrombectomy and transluminal balloon angioplasty with or without stenting [ 6 , 14 , 15 ] . surgical techniques include aorto - renal bypass graft , in situ repair , arteriotomy with thrombectomy and auto - transplantation of the kidney to the hypogastric vessels . revascularization was not attempted in our patient because of the delay in diagnosis and unstable post - surgical course following the pancreatic necrosectomy . | renal artery thrombosis is a rare , but serious and often under - diagnosed condition .
we report a case of bilateral renal artery thrombosis secondary to acute necrotizing pancreatitis . a 66-year - old female presented with abdominal pain and acute kidney injury ( aki ) .
a renal biopsy showed organized intraluminal thrombi and a computer tomography scan of the abdomen showed bilateral renal artery thrombosis .
emergent laprotomy showed necrosed pancreas .
doppler studies showed deep vein thrombosis of the lower extremities and internal jugular vein thrombosis .
workup for hypercoagulability was unremarkable .
the final diagnosis was aki secondary to bilateral renal artery thrombosis probably due to hypercoagulability of acute necrotizing pancreatitis . |
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a 67-year - old woman presented with blurring of the left eye . the patient presented with best - corrected visual acuity of count fingers at 2 oculus dexter ( od ) and 20/40 oculus sinister ( os ) . a relative afferent pupillary defect was observed in the right eye . there were nuclear sclerosis ( lens opacities classification system ( locs ) iii grading = 1 ) in both eyes . a small optic disc and double ring sign were observed in the right eye and left was normal . in order to evaluate the thickness of each layers of retina 5-line raster scan , which makes five line scans with 6 mm width in horizontal and each separated by 250 m , and optic disc cube scan were generated . we selected the results with minimum signal strength seven and the third row of the five line scans that passed the fovea to compare both eyes . to compare and analyze each layers of the retina , 21-points of each raster scans were selected using the caliper tool in the device . each points were located at 250 ; 500 ; 750 ; 1,000 ; 1,250 ; 1,500 ; 1750 ; 2,000 ; 2,250 ; and 2500 m apart from the fovea to nasal and to temporal side . our oct device did not provide thickness maps of each layers of retina , which was the main concern in this study . therefore , for quantitative analysis of retinal layers , segmentations were done to three parts , which were rnfl , inner retinal layer ( irl ) , and orl ; on the basis of internal limiting membrane , posterior boundary of the rnfl , posterior boundary of the outer plexiform layer , and anterior boundary of the cone outer segment tip line . rnfl thickness ( rnflt ) , irl thickness ( irlt ) , and orl thickness ( orlt ) were measured manually using the caliper tool in the device . the wilcoxon 's signed - rank test and t - test were used for the comparison of rnflt , irlt , and orlt ; between both eyes . the average of rnflt of onh was thinner than the opposite eye [ fig . 1 ] and the thickness analysis of each five line scans were represented by a graph and fundus photograph map , respectively [ fig . 2 ] . rnflt and irlt of onh were significantly thinner than the opposite eye , for all five lines ( p < 0.05 ) . however , orlt did not show significant difference between both eyes for all five lines ( p > 0.05 ) . in addition , to compare the thickness of retinal layers of the nasal and temporal areas from the fovea , each 50 points from the fovea to the nasal and temporal sides were divided into two groups . rnflt and irlt of the right eye were significantly thinner than the left eye on the temporal and nasal side ( p < 0.000 ) , but orlt did not show significant difference between both eyes ( p = 0.879 , 0.164 ) . the differences of rnflt and irlt between both eyes are greater on the nasal side than the temporal side as shown in fig . 2 ( difference of rnflt between both eyes on the temporal and nasal side : 2.04 vs 9.02 m , irlt : 20.86 vs 45.02 m ) . the average thickness of rnfl was thinner in optic nerve hypoplasia ( onh ) ( 45 vs 75 m ) and diffuse thinning of rnfl in onh is apparent by rnfl thickness ( rnflt ) analysis program of cirrus hd - oct comparative fundus photograph maps and graphs of each retinal layers of both eyes . each graphs and fundus photographs with blocks are the comparison of five line scans and point comparison of both eyes , respectively ; comparing ( a ) rnflt , ( b ) irlt , and ( c ) orlt . each points have been represented by the two colors on the thicker side ( blue - right , red - left ) or gray color , if both sides have the same thickness . rnflt and irlt of the left eye is significantly thicker than the right eye , for all five lines ( p < 0.000 ; graph a , b , fundus photograph map a , b ) . however , orlt shows no significant difference between both eyes for all five lines ( p > 0.05 , graph c ) onh results from congenital deficiency of retinal ganglion cells and axons that lead to disorganization of the retinal ganglion cell layer , thinning of rnfl , and a small optic disc . these results can not be exactly explained , but they are thought of as a result of any insult to the visual pathway before the completion of development , especially in the early developmental period . the diagnosis of onh remains primarily on a clinical basis , but it becomes difficult when the degree of hypoplasia is less severe or even segmental in nature , because the thinning of rnfl and disc appearance are variable . therefore , oct , which is an in vivo , noninvasive , valid imaging method that provides accurate , quantitative , and reproducible cross - sectional measurements of the optic nerve and macula , is useful for diagnosing any optic disc and retinal anomalies . nevertheless , there have been few reports on detecting onh using oct worldwide . in our patient , the outcomes obtained through using sd - oct and manual segmentation in our report demonstrated the thinning of rnfl and irl and preserved orl in the patient with congenital , unilateral onh ; which coincided with a conventional histological report . our patient showed that best corrected visual activity ( bcva ) of onh was count fingers at 2. visual potential of onh is related to the degree of papillomacular bundle defect . our report demonstrated uniformly thin rnflt and irlt on both sides of the fovea and showed more significant difference on the nasal side . these results may be related to va of our patient . since it can be difficult to assess the visual potential by the appearance of disc alone , sdoct can be helpful for evaluating the visual capacity of onh . in conclusion , sdoct findings of onh , namely the analysis of the thickness of the retinal layers in our case showed significantly thinner rnflt and irlt than the normal eye , but orlt did not show any difference between onh and the normal . | we investigated a case of unilateral optic nerve hypoplasia using spectral domain optical coherence tomography ( sdoct ) .
optical coherence tomography was done on both eyes using 5-line raster scan for the fovea to analyze the retinal nerve fiber layer thickness , inner retinal layer thickness , outer retinal layer thickness , and optic disc cube scan for the disc .
retinal nerve fiber layer thickness , inner retinal layer thickness , and outer retinal layer thickness were manually measured at 21-points of each five lines , and results were compared between both eyes .
retinal nerve fiber layer thickness and inner retinal layer thickness of optic nerve hypoplasia were significantly thinner than the opposite eye , but there was no significant difference in the thickness of the outer retinal layer between both eyes . |
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injuries to the esophageal wall , such as perforations and anastomotic leaks , are serious complications of surgical and endoscopic interventions and are associated with high rates of morbidity and mortality . mortality occurs in up to 20 % of conservatively managed and 64 % of surgically managed anastomotic leakages in affected patients 1
2 . avoiding the invasiveness of repeated surgery , implantation of self - expanding metal stents ( sems ) or plastic stents is a well - known option for closing defects and is successful in 77 % to 84 % of cases 3 . however , stent migration , prolonged treatment lasting several weeks with impaired food intake , and difficulty in removing the stent are common side effects of this form of treatment 4 . additional techniques for closing esophageal defects include standard clips 5 and clipping devices such as the over - the - scope clip ( otsc ) 6
7 , and there have been a few reported cases describing the use of suturing devices 8 . in patients with mediastinal abscesses , all of the techniques mentioned above should be accompanied by transcutaneous mediastinal drainage , with computed tomography ( ct ) guidance , in order to avoid enlargement of the abscess and sepsis . since 2006 , a new treatment have been available , in the form of endoscopically placed vacuum sponge therapy . the technique was initially used in the lower gastrointestinal tract in patients with pararectal abscesses due to anastomotic insufficiencies after rectal surgery . it involves placing an open - core sponge in the abscess cavity , connected to a drainage tube with a negative - pressure pump 9
10 . in recent years , this approach has also been adapted for use in the upper gastrointestinal tract and it is used as an alternative in the treatment of patients with upper gastrointestinal perforations or leakages . only very few institutions have published reports on their experience with this new technique to date . between april 2012 and october 2014 , 10 patients ( 5 men and 5 women ) aged 57 94 years were treated at our institution using endoscopic vacuum therapy ( evt ) in the upper gastrointestinal tract ( table 1 ) . the leak was located in the esophagus in eight patients and in the very proximal esophagus and piriform recessus in two patients . all of the patients had severe signs of perforation , such as mediastinitis and/or cutaneous emphysema . in six patients , the abscess cavity was large enough for the sponge to be placed into the cavity , while in the other four patients , the sponge was placed intraluminally . in one patient ( no . 10 ) , access to a large mediastinal cavity had to be achieved using balloon dilation of the small opening before sponge placement . the system consists of an overtube , which is placed into the cavity or in the esophageal lumen after intubation with the endoscope . after the endoscope has been removed , the small sponge is positioned in the cavity using a pusher device over the overtube . correct placement is checked endoscopically after removal of the overtube , and suction is applied with a negative - pressure pump ( infov.a . , san antonio , texas , usa ) after the tube has been redirected through the nose . the settings used for the pump were a negative pressure of 100 to 125 mmhg , high intensity , and continuous suction . placement of the sponge and sponge changes were performed under general anesthesia in seven patients due to the proximity of the perforation , with a risk of airway impairment . in two patients with perforations of the piriform recess , continuous sedation was necessary throughout the whole treatment period to prevent pain in the deep hypopharynx and impairment of the airway . enteral nutrition was ensured with a percutaneous endoscopic gastrostomy ( peg ) catheter in three patients and with transnasal enteral feeding tubes in seven patients . all of the conscious patients were able to drink , and patients with extraluminal sponge placement were able to eat soft food . the leak was located in the esophagus in eight patients and in the very proximal esophagus and piriform recessus in two patients . all of the patients had severe signs of perforation , such as mediastinitis and/or cutaneous emphysema . in six patients , the abscess cavity was large enough for the sponge to be placed into the cavity , while in the other four patients , the sponge was placed intraluminally . in one patient ( no . 10 ) , access to a large mediastinal cavity had to be achieved using balloon dilation of the small opening before sponge placement . the system consists of an overtube , which is placed into the cavity or in the esophageal lumen after intubation with the endoscope . after the endoscope has been removed , the small sponge is positioned in the cavity using a pusher device over the overtube . correct placement is checked endoscopically after removal of the overtube , and suction is applied with a negative - pressure pump ( infov.a . , san antonio , texas , usa ) after the tube has been redirected through the nose . the settings used for the pump were a negative pressure of 100 to 125 mmhg , high intensity , and continuous suction . placement of the sponge and sponge changes were performed under general anesthesia in seven patients due to the proximity of the perforation , with a risk of airway impairment . in two patients with perforations of the piriform recess , continuous sedation was necessary throughout the whole treatment period to prevent pain in the deep hypopharynx and impairment of the airway . enteral nutrition was ensured with a percutaneous endoscopic gastrostomy ( peg ) catheter in three patients and with transnasal enteral feeding tubes in seven patients . all of the conscious patients were able to drink , and patients with extraluminal sponge placement were able to eat soft food . the defect in the esophageal wall was successfully closed in seven of the 10 patients ( 70 % ) . the time between perforation or detection of an anastomotic insufficiency and the start of vacuum therapy was less than 24 hours in four patients . the median treatment time was 5 days in patients with endoluminal sponge placement and 14 days in patients with extraluminal sponge placement . two patients died of vascular disease ( one heart attack with cardiogenic shock , one stroke with respiratory insufficiency ) not related to the sponge treatment , 3 and 6 weeks , respectively , after successful closure of the esophageal defects . in the first patient with failed closure of a large anastomotic insufficiency after thoracoabdominal resection of the esophagus , vacuum therapy was started after two failed courses of treatment with fully covered metal stents . the wall of the abscess cavity was therefore consolidated , and it was not possible to stimulate granulation tissue through the sponge treatment . the patient also developed increasing numbers of fistulas between the abscess cavity and the peripheral bronchial airways . after a large number of sponge exchanges ( n = 39 ) , the patient died due to complications after a surgical rescue intervention . the second patient died of fulminant sepsis , which had commenced already before the first sponge placement . the third patient with failed sponge treatment underwent surgery early after the sponge placement , because of the development of a large pleural empyema causing sepsis . all of the interventions were performed without severe side effects besides the three failed treatments mentioned above . the residual parts of the sponges were easily extracted using a rat - tooth forceps , with no damage to the surrounding structures . in one patient , an esophageal stenosis in the area of the prior sponge therapy had to be dilated using bougies . one patient with a very proximal perforation ( no . 4 ) developed respirator - associated pneumonia during the course of treatment , due to artificial ventilation required for 5 days . in the first patient with failed closure of a large anastomotic insufficiency after thoracoabdominal resection of the esophagus , vacuum therapy was started after two failed courses of treatment with fully covered metal stents . the wall of the abscess cavity was therefore consolidated , and it was not possible to stimulate granulation tissue through the sponge treatment . the patient also developed increasing numbers of fistulas between the abscess cavity and the peripheral bronchial airways . after a large number of sponge exchanges ( n = 39 ) , the patient died due to complications after a surgical rescue intervention . the second patient died of fulminant sepsis , which had commenced already before the first sponge placement . the third patient with failed sponge treatment underwent surgery early after the sponge placement , because of the development of a large pleural empyema causing sepsis . all of the interventions were performed without severe side effects besides the three failed treatments mentioned above . the residual parts of the sponges were easily extracted using a rat - tooth forceps , with no damage to the surrounding structures . in one patient , an esophageal stenosis in the area of the prior sponge therapy had to be dilated using bougies . one patient with a very proximal perforation ( no . 4 ) developed respirator - associated pneumonia during the course of treatment , due to artificial ventilation required for 5 days . the main causes of defects developing in the esophageal wall are anastomotic insufficiencies and iatrogenic perforations after endoscopic interventions such as endoscopic submucosal dissection or dilation . other conditions , such as a perforation by a foreign body or spontaneous rupture of the esophagus ( boerhaave syndrome ) , only account for a small number of cases . minimally invasive treatment options include clipping with standard or over - the - scope clips , and placement of metal or plastic stents . however , clips are limited to perforations up to 25 mm long 5 , and stent placement is associated with the risks of stent migration , pain and discomfort , hemorrhage , tissue ingrowth , and food obstruction 11
12 . surgical repair with suturing of defects , with or without interpostion of muscle flaps or esophagectomy , was the treatment of choice for many years in patients with defects of the esophageal wall . it is accompanied by a mortality rate of 10 % to 12 % 13
14 . due to the bacterial contamination of the mediastinum and the development of septic conditions , the mortality rate in patients in whom the start of treatment is delayed by > 24 hours reaches 20 % , in comparison with 7 % in patients in whom treatment starts earlier 14 . in our group of patients we discovered a mortality of 20 % during treatment ( patients no . 1 and 3 ) . in both patients the treatment started after more than 24 hours , in patient no . 1 , after 5 months . cleansing of the contaminated mediastinum is one of the key points in treating esophageal perforations , and mediastinal abscesses and stent therapy should therefore be combined with mediastinal drainage in all cases in which there are detectable mediastinal abscesses . in the present group of patients , the start of therapy was > 24 hours in six patients , and two of the three patients with treatment failure belonged to this group . a major advantage of vacuum therapy is the ability to cleanse the perforation cavity using a minimally invasive approach ; this is required in order to avoid sepsis and death . in the present study , it was possible to cleanse even consolidated wound cavities , with walls covered with fibrinous tissue , to produce fresh granulation tissue during the first 3 to 5 days of vacuum - assisted treatment ( fig . 1 5 ) . this effect has been well known since the first endoscopic lesions were treated with vacuum therapy in the rectum 15 . the effectiveness of sponge cleansing has also led to its use in infected pancreatic pseudocysts in two cases , with good resolution of the cavities 16
17 .
initial endoscopic view into the mediastinum . the entrance to the mediastinal cavity on day 1 .
a small residual recess can be seen in the proximal esophagus on day 29 . a few other types of lesions outside of the esophagus and rectum have also been treated successfully with this method , such as anastomotic insufficiencies in the stomach 18 , after pancreaticoduodenectomy 19 , in patients with boerhaave syndrome 20 , after duodenal perforation 21
and after bariatric surgery with gastric roux - y - bypass
22 ( table 2 ) . a special feature of the present study is that a high proportion of the patients ( 70 % ) were suffering from leakages in the proximal part of the esophagus ; in two patients , the perforation tear had even reached the piriform recess . the complexity of perforations in the proximal esophagus is greater than in the distal esophagus , because surgery is much more invasive and stent placement involves the risk of impairing the airway . in the present group of patients , management of the airway in patients with very proximally located perforations ( patients 4 and 7 , table 1 ) was only achieved using general anesthesia during the whole course of treatment , for 5 days . this strategy is accompanied by a risk of the development of respirator - associated pneumonia , as was observed in patient no . 4 . in other patients , it was possible to carry out sponge changes only under general anesthesia , due to the proximity of the lesions to the airway . this and the fact that placement of the sponge requires a high degree of endoscopic skill indicates that endoscopic vacuum therapy should only be performed in high - volume centers with expertise in interventional endoscopy and intensive - care medicine . treatment decisions should be made by an interdisciplinary team including visceral surgeons , radiologists , and endoscopists in order to discuss the different treatment options . the major disadvantage in evaluating this promising new technique is the absence of comparative studies , due to the complexity of the subject and the large number of different treatment options possible such as various types of surgical interventions and different types of stent and clip devices . therefore it is not possible to recommend this new method as first - line treatment in management of all leaks and perforations of the esophagus . brangewitz et al . reported on a retrospective comparative study including 32 patients who received evt and 39 patients who underwent stent treatment , with mortality rates of 15 % and 25 % , respectively 23 . the other comparative study available ( schniewind et al . ) compared the outcomes of patients with postsurgical esophageal defects and systemic inflammation and reported statistically significant differences in the mortality rates among patients with surgical repair , stent placement , or endoscopic vacuum therapy : 50 % , 83 % , and 12 % , respectively 24 . . limitations of the present study include the small sample size of 10 patients , differences in the underlying causes of the esophageal wall defects , and a lack of comparison with other treatment modalities . a large - scale multicenter study would be mandatory in order to overcome these limitations , because the occurrence of esophageal perforations and leaks is fortunately a rare event . evt for defects in the esophageal wall is a valuable tool in the management of this high - mortality condition and should be taken into consideration by surgeons and gastroenterologists when discussing treatment options in these patients . prospective and comparative studies are required in order to further evaluate the significance of this new minimally invasive approach . | background and study aims : injuries to the esophageal wall , such as perforations and anastomotic leaks , are serious complications of surgical and endoscopic interventions .
since 2006 , a new treatment has been introduced , in the form of endoscopically placed vacuum sponge therapy .
patients and methods : between april 2012 and october 2014 , 10 patients ( 5 men and 5 women ) aged 57 to 94 years were treated at our institution using endoscopic vacuum therapy ( evt ) in the upper gastrointestinal tract .
results : the defect in the esophageal wall was successfully closed in seven of the 10 patients ( 70 % ) .
no severe complications occurred .
conclusions : evt is a valuable tool for management of defects in the esophageal wall and should be considered as a treatment option for patients with this condition . |
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yolk sac tumor ( yst ) is a rare neoplasm , which affects children and adolescents . majority of the ysts occur in the gonads , but about 20% have been found in variety of extragonadal sites such as mediastinum , retroperitoneum , neck and presacral areas . teilum described this as a malignant germ cell neoplasm that showed differentiation towards extra embryonic yolk sac endoderm as an endodermal sinus tumor . the majority of ovarian tumor of this type should have a distinctive pattern with differentiation toward yolk sac structure . present case describes the cytological features of yst occurring in paravertebral region , which to the best of our knowledge has not been reported till now in the literature . a 4-year - old male child admitted with a mass in the left gluteal region of 2 months duration and paravertebral swelling since 15 days . the patient also had a history of urinary retention and bowel incontinence following incision and drainage on left gluteal region mass by a medical practitioner . ultrasonography abdomen showed enlarged liver with multiple well - defined mixed echogenic foci in both lobes of the liver , metastatic deposits , along with a large ill - defined ( 10 cm 5 cm 7 cm ) hypoechoic mass in the deep pelvic region extending posteriorly into gluteal region . serum alpha - fetoprotein level was elevated ( 1,020 iu / ml , normal < 30 iu / ml ) . fine needle aspiration ( fna ) was performed from the gluteal and paravertebral masses using a 22-g needle . air - dried smears were stained with may - grnwald - giemsa and papanicolaou stain . the cells were arranged in cohesive clusters of variable size and shape against a mucoid background . some of the clusters had a distinct papillary configuration [ figure 1 ] . the tumor cells contained ovoid nuclei with irregular nuclear membranes , fine to coarse clumped chromatin and prominent nucleoli [ figure 2 ] . some of the tumor cells revealed intra cytoplasmic inclusions of pink homogeneous material [ figure 3 ] , which were periodic acid schiff ( pas ) positive . based on the cytological findings , a diagnosis of malignant germ cell tumor was made , but schiller - duval bodies were not identified . a wide excision biopsy was performed which showed the features of yst , exhibiting variable histological patterns ( reticular , solid , pseudopapillary and polyvesicular vitelline ) along with schiller - duval bodies . patient was referred to the oncology department where chemotherapeutic agents ( vincristine , bleomycin , and cis - platinum ) were started and he responded well with decrease in size of the sacro - coccygeal mass . tumor cells arranged in papillary configuration ( pap , 200 ) papillary fragments revealing round to oval cells with hyper - chromatic nuclei and prominent nucleoli and abundant vacuolated cytoplasm ( pap , 400 ) clusters and papillary fragments of atypical cells containing abundant cytoplasm . malignant germ cell tumors account for about 3% of pediatric malignancies and among these tumors yst account for most of the cases . the tumor was not a generally recognized entity until the 1960s , when teilum described the tumor in the testes of the young children and in the ovaries , and drew attention to their origin as these tumors are derived from totipotential cells that differentiate primarily into extraembryonic structures that resemble the endodermal sinus of the rat placenta . the armed forces institute of pathology classification considered the lesion to be the infantile variant of embryonal carcinoma , and the lesion was given separate status among germ cell tumors in who classification in 1977 . endodermal sinus tumor was reported to arise primarily from testis and ovaries , but some of the cases may occur in extragonadal sites such as the vulva , vagina , pineal region , broad ligament , prostate , cervix , mediastinum , saccro - coccygeal and retroperitoneum region , but by far the commonest site for extragonadal lesions is the sacro - coccygeal region . the cytological findings of the endodermal sinus tumor have been reported previously revealing clusters of cells with a glandular appearance , large pleomorphic cells , vacuolated cytoplasm and pas positive , diastase resistant hyaline globules . the cytoplasm contained prominent rough endoplasmic reticulum , numerous mitochondria , free ribosomes , glycogen granules , some pinocytic vesicles and dense membrane bound inclusions with the appearance of the lysosomes . the nuclei had an irregular configuration with small clumps of heterochromatin in the periphery along with one or more nucleoli . in our case , a 4-year - old male child presented with endodermal sinus tumor of extragonadal region , predominantly consisting of cohesive clusters of pleomorphic cells , arranged in papillary like configuration containing abundant intracytoplasmic pink vacuoles . endodermal sinus tumor has highly variable histological patterns ( reticular , solid , pseudopapillary and polyvesicular vitelline ) reflecting toward extra - embryonic yolk sac structures which are not appreciated in cytological samples . since the tumor 's cytological spectrum is very broad , difficult differential diagnosis of yolk sac tumor are poorly differentiated adenocarcinoma , dysgerminoma and embryonal carcinoma . the distinction between yst and embryonal carcinoma on the basis of fnac may be difficult because of the epithelial appearance of both the tumors . the tumor cells of embryonal carcinoma show pronounced nuclear pleomorphism and blurred nuclear membranes , cellular cohesion with groupings in ragged - edged clusters , sometimes attached to branching capillaries , is helpful in making the diagnosis whereas dysgerminoma discloses single cells and loose groups which contain cytoplasmic glycogen vacuoles and a large nucleus with presence of lymphocytes , plasma cells , epithelioid cells , furthermore , an amorphous background , called tigroid background . the presence of intracellular and extracellular hyaline globules that are pas stain positive and diastase resistant and correspond to alpha - fetoprotein production . alpha - fetoprotein positivity of the neoplastic cells , showing nuclear pleomorphism and vacuolated bubbly cytoplasm are the most consistent cytologic hallmarks of this neoplasm . yolk sac tumor tends to recur locally and also have a high incidence of metastatic disease at the time of presentation . our patient also showed metastasis in the liver at the time of presentation along with sacro - coccygeal swelling . the outlook is improving with the use of combination chemotherapy , and long - term responses are being noted . adjuvant vincristine , actinomycin - d and cyclophosphamide chemotherapy has been most extensively used in children with endodermal sinus tumors . patients with sacro - coccygeal tumors have a greater probability for survival than those with tumors at other sites . early detection and therapy is important because it is highly aggressive tumor that shows good response to surgery and chemotherapy . yolk sac tumors commonly occur during childhood and adolescence and arise in the sacro - coccygeal region , carry a poor prognosis if adequate chemotherapy is not given . yst can be diagnosed accurately by means of fine needle aspiration biopsy ( fnab ) . a rapid and specific diagnosis obtained by fnab may be extremely beneficial since it may save considerable expenses and spare the patient a host of diagnostic procedures . fnab diagnosis in these cases may also help in planning definitive therapy , which may include chemotherapy and radiotherapy , in addition to surgical resection . | yolk sac tumor ( yst ) is a rare neoplasm that affects children and adolescents .
fine needle aspiration biopsy is an extremely useful procedure for the diagnosis of yst .
main objective is to describe the characteristic cytological features of fine needle aspirates of yst .
we report a case of yst in a 4-year - old male child occurring at paravertebral region showing cytomorphological details . |
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nowadays , endoscopic retrograde cholangiopancreatography ( ercp ) is one of the minimally invasive most frequently performed procedures in the diagnosis and treatment of biliary - pancreatic diseases . if performed by experienced professionals , complications related to this total around 2.5% to 8% , with a mortality rate ranging from 0.5% to 1% . an exceptional ercp complication is represented by subcapsular hepatic hematoma . since 2000 , when subcapsular hepatic hematoma was first described , world literature has not reported more than 20 cases . in this case report , we describe : a further rare case of subcapsular hepatic hematoma manifested only with abdominal pain as first symptom , in accordance with most clinical presentations of similar cases already described ; an analysis of the possible pathophysiological mechanism and a review of present research literature . we introduce the case of a 52-year - old woman with a history of recurring upper abdominal pain ; the last episode required hospitalization after a diagnosis of obstructive jaundice by common bile duct stones . the patient presented jaundice , increase of cholestasis indices , hyperbilirubinemia ( mainly direct ) , ultrasonographic finding of numerous gallstones , and ectasia of the common bile duct to the distal portion , where several gallstones were observed . she had a negative personal history for noteworthy medical and surgical diseases , previous surgery , hemostasis disorders or spontaneous bleeding . her laboratory tests showed white blood cells 4.5 1.000/l ( 410 1.000/l ) , red blood cells 4.03 million/l ( 4.35.5 million/l ) , hemoglobin 12.9 g / dl ( 12.515.5 g / dl ) , platelets 192 1.000/l ( 150450 1.000/l ) , bilirubin 6.9 mg / dl ( 0.31.2 mg / dl ) , aspartate aminotransferase 144 u / l ( 240 u / l ) , alanine aminotransferase 164 u / l ( 449 u / l ) , prothrombin time ratio 1.02 ( 0.81.2 ) , international normalized ratio 1.02 ( 0.81.2 ) , and activated partial thromboplastin time ratio 1.16 ( 0.81.2 ) . as a preliminary procedure to subsequent laparoscopic cholecystectomy , the patient underwent a biliary drainage by endoscopic biliary sphincterotomy performed on a 0.035 inch diameter , 450 cm length straight - tip guidewire ( producer brand ) . this rather complicated procedure led to the extraction of multiple small common bile duct stones , but the clearance could not be completed because of respiratory problems in patient that allowed only the placement of a nasobiliary tube . on the first day , a trans - nasobiliary tube cholangiography was performed ; as a result , in addition to the tube 's endpoint , located in an intrahepatic branch of the right lobe , another 2 massive common bile duct stones were detected . on the second day , a second endoscopic operational procedure was performed and it turned out difficult too ; through this procedure , a complete clearance of common bile duct was achieved . at 24 hours from this procedure , patient began complaining of intense mainly upper abdominal pain , radiating to the right shoulder region , with no peritonism or other signs or symptoms , and pain medication was not working ; laboratory tests revealed 2.1 g / dl bilirubin ( 1.6 direct g / dl ) , aspartate aminotransferase 73 u / l ( 240 u / l ) , alanine aminotransferase 87 u / l ( 449 u / l ) , c - reactive protein 9.15 mg / dl ( 00.5 mg / dl ) , other parameters were within normal limits ( including serum amylase ) . at 36 hours the clinical picture progressed to hemodynamic shock with hypotension , anuria , and hemoglobin levels declining from 11.7 to 8.4 g / dl ( 12.515.5 mg / l ) . she was treated with transfusion of packed red blood cells and plasma , as well as intravenous administration of crystalloids and antibiotics . urgent computed tomography ( ct ) scan demonstrated a 15 cm 11 cm subcapsular hepatic hematoma filled with air and liquid on the surface of the right hepatic lobe ( figures 1 and 2 ) . urgent angiography was performed with evidence of active bleeding starting from a small branch of right hepatic artery , which was selectively embolized . the postembolization course was regular and hemodynamically stable , without fever , without abdominal pain , and the resumption of normal hemoglobin values during the following week . during a follow - up abdominal ct , performed 10 days after embolization , a marked reduction of hematoma was observed ( figure 3 ) . ct scan of upper abdomen showing a subcapsular hepatic hematoma ( hypodense area indicated by the arrows ) in which is present a source of active bleeding ( circle ) . ct scan of middle abdomen showing a subcapsular hepatic hematoma on the surface of the right lobe of liver ( hypodense area indicated by the arrows ) in which is present a source of active bleeding ( circle ) . ct scan of upper abdomen showing a reduction of subcapsular hepatic hematoma ( hypodense area indicated by the arrows ) , mainly on the surface of the right lobe of liver . a clinically significant bleeding complication associated with ercp and sphincterotomy records an approximately 2% rate , according to the different cases , although an endoscopically visible but not clinically detectable bleeding is more commonly detected , ranging from 10% to 30% of all sphincterotomies . by skilled experienced hands , bleeding or hematoma associated with ercp and involving liver , spleen , intestinal wall , or abdominal cavity turn out to be an extremely rare but potentially serious event which requires prompt recognition and treatment . subcapsular hepatic hematoma represents an exceptional event , of which world literature described 21 cases ( table 1 ) . etiology was not completely clarified , although , in accordance with most authors , subcapsular hepatic hematoma would rather seem the result of small - caliber intrahepatic vessels breaking which is determined by guide wire during endoscopy procedure ; hereafter , blood filtration through nip parenchymal liver in centrifugal direction and the presence of a solid capsule would complete pathophysiology . this would justify the presence of air in the hematoma and frequent infections due to the use of a guide wire with a nonsterile technique . after a detailed analysis of worldwide studies and , particularly , in our case , we too came to the conclusion that subcapsular hepatic hematoma stems from accidental puncture by guidewire of intrahepatic biliary tree and resulting breakage of small caliber intrahepatic vessels . patient characteristics from the reports of subcapsular hepatic hematoma post - ercp analyzing data in previous report cases , 86% cases report abdominal pain as first clinical expression , often associated with anemia ( 28.6% ) and fever ( 19% ) . the onset of these only symptoms , in association with immediate or subsequent hypotension , should suggest the presence of subcapsular hepatic hematoma . however , in agreement with most authors , the incidence of this complication might be underestimated , as most patients might show no symptoms and instrumental post - ercp monitoring is uncommon . under no circumstances can laboratory tests be considered major indicators of subcapsular hepatic hematoma , with the exception of a decrease in hematocrit and hemoglobin levels ; on the other hand , instrumental investigations such as ultrasound , ct , or magnetic resonance imaging are to be considered diagnostic methods . most cases are treated conservatively , this represents treatment choice in the case of a patient who is persistently hemodynamically stable . in such cases , we recommend the use of prophylactic antibiotics , given the risk of infected hematoma . surgical treatment should be taken into account in case of deterioration of general clinical conditions , hemodynamic instability , infected hematoma , or high risk of hematoma breakage . this treatment consists in draining hematoma , hemostasis if possible , and follow - up by ct , in order to monitor progress . a viable alternative to surgical treatment is represented by selective or superselective embolization of involved vessels or percutaneous drainage of hematoma . in the cases reported , ( table 1 ) with reference to the needs of different kinds of treatment in some cases , 47.6% patients were treated conservatively , 23.8% patients with percutaneous drainage , 23.8% patients with embolization , and 19% patients required surgery . in our case , hemodynamic stability regained only with infusion therapy and angiographic findings of active bleeding led us to choose treatment by embolization , with resolution of clinical picture . to conclude subcapsular hepatic hematoma after ercp is a rare and alarming complication that must be known and taken into account in the differential diagnosis of symptomatic cases after ercp . its diagnosis is based on clinical data ( abdominal pain , anemia , hypotension , and fever ) , laboratory data ( decrease in hematocrit and hemoglobin levels ) , and especially on imaging ( ultrasound , ct , or magnetic resonance imaging ) . treatment is often conservative but , in some cases , embolization or percutaneous drainage or surgery turn out to be necessary . | abstractendoscopic retrograde cholangiopancreatography ( ercp ) is one of the most frequently performed procedures for the diagnosis and treatment of biliary - pancreatic diseases .
ercp - related complications total around 2.5% to 8% , with a mortality rate ranging from 0.5% to 1% .
an exceptional ercp complication is subcapsular hepatic hematoma , and few cases are reported worldwide.we present the case of a 52-year - old woman with a history of recurring upper abdominal pain and a clinical and ultrasonographic diagnosis of obstructive jaundice due to common bile duct stones .
after 2 difficult endoscopic biliary procedures , common bile duct stones clearance was obtained .
post - ercp course was symptomatic with upper abdominal pain and anemization with hemodynamic instability.ct scan demonstrated a 15 cm 11 cm subcapsular hepatic hematoma filled with air and liquid on the surface of the right hepatic lobe .
the patient was successfully treated with the embolization of a small branch of right hepatic artery angiographically identified as the cause of bleeding.subcapsular hepatic hematoma after ercp is a rare complication that must be taken into account in the differential diagnosis of symptomatic cases after ercp .
its diagnosis is based on clinical and laboratory data and especially on imaging ( ultrasound , ct , or mri ) .
treatment is often conservative but , in some cases , embolization or percutaneous drainage or surgery may be necessary . |
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neuroblastoma ( nb ) is the most common extracranial solid pediatric malignancy representing 810% of all childhood malignancies . nb has been described as an extremely heterogeneous because of its highly variable biologic behavior , ranging from spontaneous regression to a progressive course with fatal outcome . about 90% of patients are diagnosed with neuroblastoma before the age of 5 years ( median age at diagnosis of 18 months ) . nearly 65% of tumours arise in the abdomen and they are usually localized to the medulla of the adrenal gland . most patients are symptoms - free , but some may present with constitutional symptoms ( malaise , fever , and weight loss ) , an enlarging mass , pain , abdominal distention , lymphadenopathy , or respiratory embarrassment secondary to compression or hepatomegaly . biochemical diagnostic findings include increased levels of serum or urine catecholamines or its metabolites and/or nonspecific biomarkers such as lactate dehydrogenas , ferritin , and neuron - specific enolase that may be associated with advanced stage or prolapse . for diagnostic imaging , computed tomography ( ct ) scan and/or magnetic resonance imaging ( mri ) are the gold standard for detection of the tumour site , size and degree of involvement . the standard lines of treatment in management of neuroblastoma include chemotherapy , radiotherapy and/or surgical resection . neuroblastoma arising from suprarenal medulla infiltrating the right liver with radiological finding suggestive for mesenchymal hamartoma is rare . a six - month - old female infant was referred to our center by right liver mass mostly mesenchymal hamartoma on radiological basis for surgical intervention . she was admitted in children hospital for pyrexia of unknown origin with no response to antibiotics and antipyretics . fever was associated with right hypochondrial pain and abdominal enlargement without jaundice for one month . on examination , there was right hypochondrial tenderness and hepatomegaly with liver span equals 8 cm ( normal liver span for this age is 55.5 cm ) . laboratory investigations showed microcytic hypochromic anemia ( haemoglobin = 8.4 gm / dl ) , leukocytosis ( white blood cells count = 13.1 10 ) , thrombocytosis ( platelets = 620 10 ) , slightly elevated alpha - feto protein ( afp ) ( afp = 26.6 ng / ml ) and normal liver function tests ( lfts ) . abdominal ultrasonography ( us ) showed well - defined heterogeneous mass with cystic degeneration and foci of calcification affecting most of the right liver measuring about 7.4 8.4 cm . abdominal computed tomography ( ct ) with contrast showed large well defined , non - enhanced hypodense cystic lesion occupying most of the the right liver ( segment v , vi , vii ) measuring ( 8 8 8.3 cm ) with exophytic component enchroaching upon right suprarenal region and displacing the right kidney inferiorly with no enlarged abdominal lymph node ( ln ) . the mass was highly suggestive for the right liver mesenchymal hamartoma as shown in ( fig . the liver was completely healthy with large mass mainly in segment vi and vii adherent to transverse colon with displacement of the right kidney downward . after surgical assessment of the tumor , right adrenalectomy with en - bloc resection of the adjacent liver segments was performed with safety margin about 1 cm of liver tissue after dissection of the mass from the right renal vessels . grossly the mass was friable measuring about 9 8 8 cm . cut section revealed large areas of hemorrhage and necrosis with infiltration of adjacent liver tissue . microscopically the tumor cells are small with hyperchromatic nuclei and scanty cytoplasm , and are arranged in sheets separated by thin fibro - vascular septa between the nests of tumor cells . postoperative course was uneventful and the patient was discharged after 6 days in good general condition . the patient was referred to pediatric oncology department for receiving adjuvant chemotherapy in the form of cisplatin and adriamycin . neuroblastoma is the most common extracranial solid pediatric malignancy representing 8 - 10% of all childhood malignancies . it accounts for approximately 15% of all cancer - related deaths in the pediatric population . neuroblastoma has been described as an extremely heterogeneous because of its highly variable biologic behavior , ranging from spontaneously regression to a progressive course with ultimate fatal outcome . our case was six - month - old infant at the time of diagnosis which is a common age for diagnosis of neuroblastoma as reported in literature . more than 50% of patients with neuroblastoma are diagnosed as high - risk neuroblastoma with features including large unresectable and widely metastatic tumour resulting in poor prognosis as regard long - term survival . nearly 65% of tumours arise in the abdomen with half of those localized to the medulla of the adrenal gland as reported in our case . in our report , the girl presented by fever of unknown origin for 2 weeks followed by abdominal enlargement and pain as reported in previous studies . biochemical diagnostic tests of neuroblastoma include serum or urinary level of catecholamines or its metabolites ( dopamine , vanillylmandelic acid , and homovanillic acid ) and/or nonspecific biomarkers such as lactate dehydrogenase , ferritin , and neuron - specific enolase . none of these laboratory tests were done preoperatively because the liver mass was not suspicious of neuroblastoma on radiological basis . for diagnostic imaging , abdominal ct scan of the abdomen is the gold standard for detection of the tumour site and the degree of involvement . a magnetic resonance imaging is more sensitive in cases of spinal extention . in our case , the abdominal ct revealed large cystic lesion in the right liver just displacing the right kidney and the right suprarenal gland and it was highly suggestive for mesenchymal hamartoma which is classic for it as reported in the literature , while radiologic findings of neuroblastoma include focal or multifocal solid tumor with calcification in 40%50% of cases , which is not essential for diagnosis . other diagnostic tests include , but not done in our case , iodine-131-meta - iodobenzylguanidine ( mibg ) which is useful in detection of both primary tumour and metastasis but not widely used in clinical practice and flurodeoxyglucose positron emission tomography ( fdg - pet ) which maybe valuable in localizing soft tissue metastasis . confirmation of the diagnosis is confirmed pathologically by tissue biopsy either by complete resection or open biopsy in case of unresectable tumours and/or bm aspiration . no preoperative us or ct guided biopsy from the mass was done to avoid dissemination and seeding of the tumour cells as hepatoblastoma was considered in the differential diagnosis of our case . the standard lines of treatment in management of neuroblastoma include chemotherapy , radiotherapy and/or surgical resection . many new promising therapies were introduced based on molecular mechanisms for differentiation , cell survival and apoptosis . the surgical resection offers definitive therapy with excellent outcome for management of locoregional low - risk neuroblastoma , while high - risk patients are treated with intensive multimodality therapies . in our case , the decision was surgical resection from the start if the tumour was resectable for final pathological diagnosis as the provisional diagnosis was whether hepatoblastoma or mesenchymal hamartoma . a six - month - old female infant was referred to our center by right liver mass mostly mesenchymal hamartoma on radiological basis for surgical intervention . she was admitted in children hospital for pyrexia of unknown origin with no response to antibiotics and antipyretics . fever was associated with right hypochondrial pain and abdominal enlargement without jaundice for one month . , there was right hypochondrial tenderness and hepatomegaly with liver span equals 8 cm ( normal liver span for this age is 55.5 cm ) . laboratory investigations showed microcytic hypochromic anemia ( haemoglobin = 8.4 gm / dl ) , leukocytosis ( white blood cells count = 13.1 10 ) , thrombocytosis ( platelets = 620 10 ) , slightly elevated alpha - feto protein ( afp ) ( afp = 26.6 ng / ml ) and normal liver function tests ( lfts ) . abdominal ultrasonography ( us ) showed well - defined heterogeneous mass with cystic degeneration and foci of calcification affecting most of the right liver measuring about 7.4 8.4 cm . abdominal computed tomography ( ct ) with contrast showed large well defined , non - enhanced hypodense cystic lesion occupying most of the the right liver ( segment v , vi , vii ) measuring ( 8 8 8.3 cm ) with exophytic component enchroaching upon right suprarenal region and displacing the right kidney inferiorly with no enlarged abdominal lymph node ( ln ) . the mass was highly suggestive for the right liver mesenchymal hamartoma as shown in ( fig . the liver was completely healthy with large mass mainly in segment vi and vii adherent to transverse colon with displacement of the right kidney downward . after surgical assessment of the tumor , right adrenalectomy with en - bloc resection of the adjacent liver segments was performed with safety margin about 1 cm of liver tissue after dissection of the mass from the right renal vessels . grossly the mass was friable measuring about 9 8 8 cm . cut section revealed large areas of hemorrhage and necrosis with infiltration of adjacent liver tissue . microscopically the tumor cells are small with hyperchromatic nuclei and scanty cytoplasm , and are arranged in sheets separated by thin fibro - vascular septa between the nests of tumor cells . postoperative course was uneventful and the patient was discharged after 6 days in good general condition . the patient was referred to pediatric oncology department for receiving adjuvant chemotherapy in the form of cisplatin and adriamycin . neuroblastoma is the most common extracranial solid pediatric malignancy representing 8 - 10% of all childhood malignancies . it accounts for approximately 15% of all cancer - related deaths in the pediatric population . neuroblastoma has been described as an extremely heterogeneous because of its highly variable biologic behavior , ranging from spontaneously regression to a progressive course with ultimate fatal outcome . our case was six - month - old infant at the time of diagnosis which is a common age for diagnosis of neuroblastoma as reported in literature . more than 50% of patients with neuroblastoma are diagnosed as high - risk neuroblastoma with features including large unresectable and widely metastatic tumour resulting in poor prognosis as regard long - term survival . nearly 65% of tumours arise in the abdomen with half of those localized to the medulla of the adrenal gland as reported in our case . in our report , the girl presented by fever of unknown origin for 2 weeks followed by abdominal enlargement and pain as reported in previous studies . biochemical diagnostic tests of neuroblastoma include serum or urinary level of catecholamines or its metabolites ( dopamine , vanillylmandelic acid , and homovanillic acid ) and/or nonspecific biomarkers such as lactate dehydrogenase , ferritin , and neuron - specific enolase . none of these laboratory tests were done preoperatively because the liver mass was not suspicious of neuroblastoma on radiological basis . for diagnostic imaging , abdominal ct scan of the abdomen is the gold standard for detection of the tumour site and the degree of involvement . a magnetic resonance imaging is more sensitive in cases of spinal extention . in our case , the abdominal ct revealed large cystic lesion in the right liver just displacing the right kidney and the right suprarenal gland and it was highly suggestive for mesenchymal hamartoma which is classic for it as reported in the literature , while radiologic findings of neuroblastoma include focal or multifocal solid tumor with calcification in 40%50% of cases , which is not essential for diagnosis . other diagnostic tests include , but not done in our case , iodine-131-meta - iodobenzylguanidine ( mibg ) which is useful in detection of both primary tumour and metastasis but not widely used in clinical practice and flurodeoxyglucose positron emission tomography ( fdg - pet ) which maybe valuable in localizing soft tissue metastasis . confirmation of the diagnosis is confirmed pathologically by tissue biopsy either by complete resection or open biopsy in case of unresectable tumours and/or bm aspiration . no preoperative us or ct guided biopsy from the mass was done to avoid dissemination and seeding of the tumour cells as hepatoblastoma was considered in the differential diagnosis of our case . the standard lines of treatment in management of neuroblastoma include chemotherapy , radiotherapy and/or surgical resection . many new promising therapies were introduced based on molecular mechanisms for differentiation , cell survival and apoptosis . the surgical resection offers definitive therapy with excellent outcome for management of locoregional low - risk neuroblastoma , while high - risk patients are treated with intensive multimodality therapies . in our case , the decision was surgical resection from the start if the tumour was resectable for final pathological diagnosis as the provisional diagnosis was whether hepatoblastoma or mesenchymal hamartoma . we represent a rare case of right adrenal gland neuroblastoma infiltrated the adjacent liver segments mimicking mesenchymal hamartoma of the liver . neuroblastoma should be considered in differential diagnosis of abdominal mass in all infants and children . all authors have made substantial contributions to all of the following : ( 1 ) acquisition of data , analysis and interpretation of data , ( 2 ) revising it critically for important intellectual content and ( 3 ) final approval of the version to be submitted . | highlightsthe right adrenal gland neuroblastoma and infiltrated the adjacent liver substance mimicking mesenchymal hamartoma of the liver.the presentation of fever of unknown origin in a case of right adrenal gland neuroblastoma is rare.neuroblastoma should be considered in differential diagnosis of abdominal mass in all infants and children . |
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legionnaires disease ( ld ) is usually acquired through inhalation of aerosolized water contaminated with legionella spp . , which is found ubiquitously in natural and man - made aqueous environments such as cooling towers , hot water systems or whirlpools . legionella pneumophila , especially serogroup 1 ( sg1 ) , is the major cause of ld , which remains a serious disease . several host - related risk factors have been clearly identified [ 24 ] , including older age , male sex , smoking , diabetes , chronic heart or lung disease , cancer or hematologic malignancy and steroid use or any immunosuppressive therapy . the characterization of clinical isolates by molecular typing methods is essential for epidemiologic investigations , particularly for clusters and outbreaks . thus , l. pneumophila sg1 isolates can be characterized by sequence - based typing ( sbt ) using isolates or by nested - pcr - based sbt using clinical samples . the typing method for l. pneumophila sg1 was developed by the european working group for legionella infections ( ewgli was renamed the european study group for legionella infections , esgli , in september 2012 ) . both local and worldwide sequence types ( sts ) , such as st1 , st23 , st37 , st47 and st62 , have been identified [ 712 ] . in france , st1- and st47-associated ld represented 9% and 11% of culture - proven cases in 2008 , respectively . the epidemiologic characteristics of these two sts have previously been published : st1 strains with the paris pulsotype were more frequently identified among elderly patients , immunocompromised patients or women and had a high lethality rate ( 38% ) compared to sporadic strains . in contrast , st47 strains were significantly more frequently associated with smoking , but the case fatality rate was lower than with sporadic strains . the present french epidemiologic report shows that the st most frequently associated with ld is st23 . it accounted for approximately 20% of the strains isolated in france in 2008 , but no specific host factors associated with this genotype have been reported thus far . we thus characterized the epidemiologic and clinical features of 236 patients infected with st23 and compared them to those of patients infected by two other predominant sts ( st1 , st47 ) and other nonpredominant sts . in france , all cases of ld are reported to the national health authority ( institut de veille sanitaire ) , which collects epidemiologic information through anonymous mandatory notification , including various risk factors and exposure routes for ld . the notification form allows the collection of information on current illness , the patient s personal characteristics ( sex , age and postcode of residence ) , disease outcome and factors known to be associated with ld . moreover , exposure to any of the following settings during the 10-day period preceding symptom onset ( i.e. the incubation period ) is also recorded : hospital , elderly setting , thermal centre , whirlpool spa or travel site ( campsite , hotel ) . additionally , all corresponding legionella isolates are sent to the nrcl ( national reference centre for legionella ) , where they are characterized by sbt . in our study , cases presenting clinical and radiologic signs of pneumonia combined with l. pneumophila sg1 isolation from a culture of sputum or bronchopulmonary secretions ( bronchoalveolar lavage fluid or bronchial aspirate ) were included . cases of ld due to l. pneumophila sg1 reported from 2008 through 2012 were considered for the study . the geographic distribution of cases ( based on the postcode of residence ) was used as a proxy for the geographic distribution of strains , and the patients were then classified into five regions according to their telephone area code ( fig . 1 ) . finally , on the basis of genotype distribution , for the purposes of the study , the population was divided into four groups : st1 , st23 , st47 and other st culture - confirmed cases . we constructed multivariable modified poisson regression models to estimate the incidence rate ratio ( irr ) and to identify characteristics potentially associated with st23 compared to the three other groups , considering a p value of 0.05 to be significant . analyses were performed by stata 11.2 ( statacorp , college station , tx , usa ) software . from 2008 to 2012 , a total of 6458 cases of ld were reported , and legionella isolates were recovered in 1192 cases ( 18.5% ) , which were included in the study . st23 accounted for the largest number of isolates ( n = 236 ) , representing 19.8% of all clinical strains . st1 and st47 l. pneumophila represented 9.1% and 10.3% of all isolates , respectively . st23 isolates were found ubiquitously across france , but mainly in regions 3 and 4 ( 76.4% ) ; this strain was also found in ld cases from other st groups with only minor differences . on the other hand , st1 cases occurred mostly in regions 1 and 4 , and st47 cases were mainly found in region 3 ( fig . 1 ; table 1 ) . patients infected with l. pneumophila sg1 st23 had a mean age of 59.1 years and were mostly men ( table 1 ) . of these patients , this percentage was not significantly different from the corresponding percentages among patients infected by st1 , st47 or other sts of l. pneumophila sg1 . the rarest risk factors associated with st23 ld were steroid therapy ( 4.2% ) or cancer ( 8.1% ) . conversely , smoking was the most frequently reported risk factor ( n = 124 ; 52.5% ) . the percentage of cases with this risk factor was highest in the st23 group compared to the other three groups . moreover , st23 cases were rarely hospital acquired ( 3.0% ) or acquired in elderly settings ( 3.8% ) . multivariate analysis comparing the characteristics of st 23 cases with those of other groups revealed five significant differences ( table 2 ) . first , the geographic distribution of st23 cases was significantly different than the geographic distribution of other sts ( table 2 ) . second , this genotype was significantly less associated with steroid therapy compared to st1 ( irr = 0.56 ; p 0.016 ) . this difference was not observed when comparing st23 to st47 or st23 to other sts . concerning exposure route , the st23 genotype was significantly less associated with hospital - acquired infections compared to st1 ( irr = 0.32 ; p 0.001 ) , but it was more associated with hospital - acquired infections as well as with infections acquired in elderly settings compared to st47 . finally , the st23 genotype was less frequently associated with travel cases than other sts . in this study , we found a high rate of st23 detection in ld ; this st represents 19.8% of cases with positive cultures in france between 2008 and 2012 . this proportion is higher than those found in other countries , e.g. 6% in belgium ( 20002010 ) , 4.7% in japan ( 19802008 ) , 4.5% in portugal ( 19872008 ) and 0 observed in england and wales ( 20002008 ) . a compilation of european data ( 3200 isolates from 36 countries ) showed that st23 accounted for only 10.7% of clinical isolates , including french strains . the results and statistical comparisons in the present work pertain to patients with culture - proven ld . the inclusion of only these patients could be a critical source of bias . throughout the study period , considering all cases of non - culture - proven ld with complete allelic profiles determined by nested - pcr - based sbt ( n = 70 ) , this proportion is consistent with the number of st23 culture - proven ld cases of our study . it also appeared that 72.9% of the st23-infected patients had at least one of the risk factors usually associated with ld acquisition . this overall percentage did not differ from those observed for cases of ld due to other st ; however , a more detailed analysis of the different risk factors showed that patients infected by st23 strains were less likely to have received corticosteroid therapy than patients with st1 ( irr = 0.56 ; p 0.016 ) . corticosteroids or other immunosuppressive therapies ( e.g. anti - tnf- ) are increasingly used to treat systemic diseases such as rheumatoid arthritis , crohn disease or cutaneous psoriasis . the use of these types of medications was shown to be associated with an increased relative risk of ld , leading to a 1.6- to 8-fold increase in the risk of ld compared to the general population . the overall standardized incidence ratio was 13.1 for patients receiving tnf- antagonists in the french population . the frequency with which st23-infected patients are receiving corticoids suggests that they are less likely to have chronic diseases requiring hospital admission . this finding is consistent with our observations , which revealed that st23 infections were more often community acquired compared to st1 infections . indeed , the observed irr was low ( irr = 0.32 ; p 0.001 ) . comparing st23 vs. st1 cases regarding hospital exposure and corticosteroid therapy confirmed specific features related to st1 strains that have already been reported . in this study , 22% of st1 patients received corticosteroids , and a high proportion of these patients were hospitalized ( 31.2% ) . of course , we verified that there was no regional effect associated with these nosocomial infections ( data not shown ) . the combination of environmental factors , strain virulence and host susceptibility may explain the observed differences among sts . thus , concerning st1 strains , we could hypothesize that they are either very well adapted to hospital water networks or are widely present in the environment . european data showed that st1 accounted for 21.4% of environmental isolates , which is a relatively high percentage compared to other sts . other studies have also shown that st1 was the most dominant st in analysed strains from the environment in china ( 81/164 , 49.4% ) and in the united states ( 73/149 , 49% ) . moreover , in the chinese study , st1 accounted for 92.3% and 53.1% of the environmental sg1 isolates from potable water systems and cooling towers , respectively . this finding emphasises that st1 strains are widely present in artificial water sources , but to our knowledge , these strains were never reported specifically in hospital water networks . therefore , designing a st1-specific pcr for rapid detection and identification in hospital environmental samples would be helpful . we could then hypothesize that this clone would be more ubiquitous and have lower virulence , infecting mainly immunocompromised patients . finally , another approach would be for l. pneumophila st1 to contain specific virulence determinants adapted for immunocompromised patients , who may also present specific host characteristics . the epidemiologic characteristics of st1 cases could explain the higher case fatality rate observed with this clone ( table 1 ) . by contrast , both the st47 and st23 strains were rarely recovered from environmental samples . european data indicated that st47 and st23 accounted for 0.4% and 1.5% of environmental isolates , respectively . these rates suggest that these strains could have a peculiar virulence pattern because they are less disseminated in the environment and are less associated with patients receiving corticosteroids . nevertheless , the st23 genotype was more frequently associated with hospital - acquired infections and with infections acquired in elderly settings compared to st47 . we might therefore hypothesize that the source of infection could be different for these two clones . finally , although st23 isolates could have developed a peculiar virulence pattern adapted to a larger population , we should have observed at least as many travel - associated cases in the st23 group as we did in the other st groups . in our study , this genotype was less frequently associated with travel cases than other sts . all of these observations suggest that st23 could be a dominant infective clone in the global population of legionella strains , as could st1 and st47 . nevertheless , there are still several unresolved issues , and further analysis will be necessary to determine the key host characteristics and virulence factors associated with susceptibility to these clones . finally , this study reminds us that it is important to maintain clinician awareness of the need to collect sputum samples in order to document ld cases and to better understand the association between host characteristics and legionella strains . | in france , approximately 1200 cases of legionnaires disease ( ld ) are reported annually , and isolates are available for approximately 20% of cases identified since 2000 .
all legionella pneumophila serogroup 1 ( sg1 ) isolates are characterized by sequence - based typing at the national reference centre .
ld cases caused by l. pneumophila sg1 reported from 2008 through 2012 were considered for the study .
our study objective was to describe cases according to their sequence type ( st ) .
we also constructed multivariable modified poisson regression models to estimate the incidence rate ratio ( irr ) and to identify characteristics potentially associated with st23 clones compared to st1 and st47 clones .
we studied 1192 patients infected by st1 ( n = 109 ) , st23 ( n = 236 ) , st47 ( n = 123 ) or other sts ( n = 724 ) .
the geographic distribution of the st23 cases across the country was significantly different compared to other st groups .
this genotype was significantly associated with the absence of corticosteroid therapy compared to st1 ( irr = 0.56 ; p 0.016 ) .
concerning exposure , the st23 genotype was significantly less associated with hospital - acquired infections compared to st1 ( irr = 0.32 ; p 0.001 ) , but it was more associated with infections acquired in hospitals and elderly settings compared with st47 .
finally , the st23 genotype was less frequently associated with travel than other sts . despite the large number of cases of st23 infection , we did not identify any characteristics specific to this st .
however , we identified independent associations between st1 and nosocomial transmission and steroid therapy .
these findings should encourage further exploration , especially in terms of environmental diffusion , strain virulence and host factors . |
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familial mediterranean fever ( fmf ) is an autosomal recessive disease characterized by recurrent episodes of fever accompanied by peritonitis , pleuritis , arthritis , or erysipelas - like erythema ( 1 ) . it is known to occur mainly among mediterranean and middle eastern populations such as non - ashkenazi jews , arabs , turks , and armenians . fmf is not familiar to clinicians beyond this area and diagnosing fmf can be challenging . we report a case of fmf associated with multiple venous thrombosis and elevated immunoglobulin d ( igd ) level diagnosed by mefv mutation analysis . a 22-yr old man was referred to our hospital on february 17 , 2012 due to fever and arthalgia which started 2 days prior to admission . he had a history of recurrent episodes of fever associated with arthalgia lasting 5 days with 5 - 6 month intervals which started at the age of 2 . fever would subside spontaneously or with the use of antipyretics . on admission he showed a fever of 40 with abdominal pain and arthalgia in both knees . laboratory findings on admission showed mild leukopenia and thrombocytopenia ( white blood cell count of 2,650/l and platelet count of 102,000/l ) . erythrocyte sedimentation rate was normal but c - reactive protein was highly elevated at 27.88 mg / dl . chest and abdomen computed tomography ( ct ) was performed as part of the diagnostic work up for fever of unknown origin . chest ct showed no specific findings while abdomen ct showed thrombosis in portal vein ( pv ) and superior mesenteric vein ( smv ) with splenomegaly . on the 10th hospital day , the patient complained of left upper quadrant pain and 3d angio ct showed total thrombosis of splenic vein with partial thrombosis of proximal smv , main pv and intrahepatic both pv with small amount of ascites ( fig . considering the patient 's history of periodic fever with no familial history , autosomal recessive periodic fever syndromes such as fmf and hyperimmunoglobulin - d syndrome ( hids ) were suspected . igg , iga , and igm were all in the normal range while igd was elevated at 39.2 mg / dl . the patient also satisfied the three major criteria ( fever , abdominal pain , joint pain ) of the tel - hashomer criteria . to verify the diagnosis , dna analysis of the mvk and mefv gene mvk for hids was negative while 3 mutations ( p.glu148gln , p.pro369ser , p.arg408gln ) were identified in the mefv gene for fmf . after diagnosis of fmf , colchine was prescribed and the patient is currently without recurrent fever . in recent years , more and more cases of fmf have been reported outside the mediterranean or the middle eastern countries , such as the us and japan . in japan , in korea , however , only one case of fmf has been reported , in a 8 yr - old boy associated with renal amyloidosis ( 4 ) . the 4 main mutations found in the studies that identified the mefv gene responsible for fmf were p.m680i , p.m694v , p.m694i , and pv726a on exon 10 ( 5 , 6 ) . several studies have pointed out that these 5 mutations are responsible for more than 85% of fmf patients in the middle eastern area ( 1 ) . our patient showed complex mutations ( p.glu148gln , p. pro369ser , p.arg408gln ) in the mefv gene . a significant number of patients diagnosed as fmf have only a single mutation despite sequencing of the entire mefv genome region or other autoinflammatory genes , and this has led to a reconsideration of the simple loss of function of the recessive model of fmf inheritance ( 8 , 9 ) . in some cases , fmf can be viewed as a dominant condition with low penetrance and variable disease expression , presenting not only in homozygous subjects , but also in heterozygous subjects ( 10 ) . over 200 sequence variants have been recorded in the database of the registry of hereditary auto - inflammatory disorders mutations ( http://fmf.igh.cnrs.fr/infevers ) and the same complex mutation as our patient could be found in an armenian patient . it remains controversial whether the e148q mutation is a disease - causing mutation or a simple polymorphism because of high allele frequency in healthy controls ( 11 ) . ( 12 ) reported that both homozygous and compound heterozygous patients for e148q were symptomatic and e148q mutation was described as producing a milder fmf phenotype with low penetrance ( 13 ) . mefv mutation analysis on korean patients is yet to be studied , as our case is the first adult case to be reported in korea . hids was considered as the diagnosis due to the high level of serum igd . however elevation in serum igd has been described in other autoinflammatory diseases ( 14 ) and although different from our mutation , medlej - hashim et al . ( 15 ) reported elevated igd plasma levels associated with homozygotic status for m694v , and to a lesser extent v726a . hypercoagulability has been recently described in patients with fmf , with major thrombotic events occurring with amyloidosis ( 16 ) . fmf presenting with pulmonary embolism ( 17 ) and fmf complicated by budd - chiari syndrome ( 18 ) have been reported but this is the first case presenting with thrombosis in pv , smv and splenic vein . it has been reported that decreased protein s activity is related to thrombotic disorder in systemic inflammatory disease such as behcet 's disease patients ( 19 ) and protein s activity was decreased in our patient . there are no reports of hids associated with thrombosis . in conclusion , we report a 22-yr old male diagnosed with fmf despite high level of serum igd . the diagnosis of fmf does not require a mefv mutation analysis but clinical diagnosis of fmf is not easy and analysis of mefv mutation can be helpful , especially in patients of non - mediterranean origin . | familial mediterranean fever ( fmf ) is an autosomal recessive disease characterized by recurrent episodes of fever accompanied by peritonitis , pleuritis , arthritis , or erysipelas - like erythema .
it is known to occur mainly among mediterranean and middle eastern populations such as non - ashkenazi jews , arabs , turks , and armenians .
fmf is not familiar to clinicians beyond this area and diagnosing fmf can be challenging .
we report a 22-yr old boy who presented with fever , arthalgia and abdominal pain .
he had a history of recurrent episodes of fever associated with arthalgia which would subside spontaneously or by antipyretics .
autosomal recessive periodic fever syndromes were suspected .
immunoglobulin d ( igd ) level in the serum was elevated and dna analysis showed complex mutations ( p.glu148gln , p.pro369ser , p.arg408gln ) in the mefv gene .
3d angio computed tomography showed total thrombosis of splenic vein with partial thrombosis of proximal superior mesenteric vein , main portal vein and intrahepatic both portal vein .
this is a case of fmf associated with multiple venous thrombosis and elevated igd level .
when thrombosis is associated with elevated igd , fmf should be suspected .
this is the first adult case reported in korea . |
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pseudohypoparathyroidism ( php ) type ia is an autosomal dominant condition characterized by
target organ unresponsiveness to a number of hormones that share the same signaling
mechanism , i.e. , a gs protein - coupled receptor ( 1 ) . heterozygous loss of function mutations in the stimulatory gs protein
-subunit ( gs ) gene inherited from the mother lead to the development of php type ia ( 2 ) . this disease is associated with albright s hereditary
osteodystrophy ( aho ) and resistance to pth and some other hormones whose receptors are
coupled to gs protein ( 1 ) . aho includes short stature ,
obesity , a round face , brachydactyly , subcutaneous ossification , and mild to moderate mental
retardation ( 3 ) . considerable variability occurs in
the clinical and biochemical manifestations even among affected members of a single family ,
and all of these features may not be present in every case ( 4 ) . we report two cases of php type ia in duozygotic twins with different
phenotypes . the proband , a 10-yr - old girl , was referred to our department with subcutaneous
ossification and brachydactyly . she was born as the first born of twins at term with
routine discharge from the hospital . a small hard tumor was noticed on her left
ankle at the age of 6 mo , but no definite diagnosis was made at that time . she began
gaining weight excessively from early infancy , and she was observed from 6 yr old as
simple obesity by a local pediatrician . she did not show hypocalcemia in the local
hospital . at the age of 10 yr , a tumor of 20 mm in diameter was noticed on her back , and a
biopsy was performed by a dermatologist . she was referred to our department by the dermatologist because she had
brachydactyly as well as osteoma cutis . her height and weight at that time were 130.6 cm
( 1.27 sd ) and 38.3 kg , respectively . her bone
age was 12 yr and 4 mo , and her height sd score for bone age was 3.28 sd . she had typical
aho with a round face , short neck , brachymetaphalangism ( fig . 1fig . 1 panel a ( case 1 ) : radiograph of the left hand showing shortening of the 4th and
5th metacarpals . panel b ( case 2 ) : no brachymetaphalangism was found . ) and obesity . examination of the eyes , ears , nose , throat , chest ,
heart , and abdomen disclosed no other abnormalities . biochemical and hormonal profiles are shown in table 1table 1 biochemical and hormonal characteristics of the familiy members . she had normocalcemia , high - normal phosphate and increased serum pth and
tsh levels . an ellsworth - howard test showed the absence of the phosphaturic response and
the absence of the expected rise in urinary and plasma cyclic amp ( data not shown ) . we
diagnosed her as having a php type ia on the basis of clinical and laboratory findings ,
and the ellsworth - howard test . hatched symbols show the presence of pth resistance .. panel a ( case 1 ) : radiograph of the left hand showing shortening of the 4th and
5th metacarpals . he began gaining weight excessively from early infancy and
received lifestyle advice from a doctor . at the age of 10 his height and weight were 134.1 cm ( 0.69
sd ) and 37.7 kg , respectively . his bone age
was 12 yr and 3 mo , and his height sd score for bone age was 2.0 sd . an
ellsworth - howard test showed the absence of the phosphaturic response and the absence of
the expected rise in urinary and plasma cyclic amp ( data not shown ) . we also diagnosed him
as having a php type ia on the basis of clinical and laboratory findings , the
ellsworth - howard test and family history . clinical and biochemical features of the mother and father are shown in fig . 2 and table 1 . cases 1 and 2 underwent the following endocrinological tests ( table 2table 2 endocrinological provocation tests ) . human crh , trh , lhrh and ghrh were administered simultaneously , and gh
stimulation tests , i.e. , insulin and clonidine stimulation tests were
performed on separate days after an overnight fast . case 1 had increased peak levels of
acth and tsh , delayed fsh peak , and low response of gh , whereas case 2 had increased peak
levels of acth and lh , and delayed fsh and lh peaks . total genomic dna was isolated from peripheral blood leukocytes of case 1 , and the 13
coding exons and intron - exon boundaries of gnas1 were analyzed as
previously described ( 8) . no mutation was identified in coding exons and
intron - exon boundaries of gnas1 . the proband , a 10-yr - old girl , was referred to our department with subcutaneous
ossification and brachydactyly . she was born as the first born of twins at term with
routine discharge from the hospital . a small hard tumor was noticed on her left
ankle at the age of 6 mo , but no definite diagnosis was made at that time . she began
gaining weight excessively from early infancy , and she was observed from 6 yr old as
simple obesity by a local pediatrician . she did not show hypocalcemia in the local
hospital . at the age of 10 yr , a tumor of 20 mm in diameter was noticed on her back , and a
biopsy was performed by a dermatologist . she was referred to our department by the dermatologist because she had
brachydactyly as well as osteoma cutis . her height and weight at that time were 130.6 cm
( 1.27 sd ) and 38.3 kg , respectively . her bone
age was 12 yr and 4 mo , and her height sd score for bone age was 3.28 sd . she had typical
aho with a round face , short neck , brachymetaphalangism ( fig . 1fig . 1 panel a ( case 1 ) : radiograph of the left hand showing shortening of the 4th and
5th metacarpals . panel b ( case 2 ) : no brachymetaphalangism was found . ) and obesity . examination of the eyes , ears , nose , throat , chest ,
heart , and abdomen disclosed no other abnormalities . biochemical and hormonal profiles are shown in table 1table 1 biochemical and hormonal characteristics of the familiy members . she had normocalcemia , high - normal phosphate and increased serum pth and
tsh levels . an ellsworth - howard test showed the absence of the phosphaturic response and
the absence of the expected rise in urinary and plasma cyclic amp ( data not shown ) . we
diagnosed her as having a php type ia on the basis of clinical and laboratory findings ,
and the ellsworth - howard test . hatched symbols show the presence of pth resistance .. panel a ( case 1 ) : radiograph of the left hand showing shortening of the 4th and
5th metacarpals . he began gaining weight excessively from early infancy and
received lifestyle advice from a doctor . at the age of 10 his height and weight were 134.1 cm ( 0.69
sd ) and 37.7 kg , respectively . his bone age
was 12 yr and 3 mo , and his height sd score for bone age was 2.0 sd . an
ellsworth - howard test showed the absence of the phosphaturic response and the absence of
the expected rise in urinary and plasma cyclic amp ( data not shown ) . we also diagnosed him
as having a php type ia on the basis of clinical and laboratory findings , the
ellsworth - howard test and family history . clinical and biochemical features of the mother and father are shown in fig . 2 and table 1 . cases 1 and 2 underwent the following endocrinological tests ( table 2table 2 endocrinological provocation tests ) . human crh , trh , lhrh and ghrh were administered simultaneously , and gh
stimulation tests , i.e. , insulin and clonidine stimulation tests were
performed on separate days after an overnight fast . case 1 had increased peak levels of
acth and tsh , delayed fsh peak , and low response of gh , whereas case 2 had increased peak
levels of acth and lh , and delayed fsh and lh peaks . total genomic dna was isolated from peripheral blood leukocytes of case 1 , and the 13
coding exons and intron - exon boundaries of gnas1 were analyzed as
previously described ( 8) . no mutation was identified in coding exons and
intron - exon boundaries of gnas1 . these two cases are particularly interesting because they demonstrate a variety of clinical
and hormonal features . case 1 had typical features of aho except for mental retardation but
showed normocalcemia and high - normal phosphate . on the other hand , case 2 had atypical aho
with only subcutaneous ossification and mild obesity but showed hypocalcemia and
hyperphosphatemia . the mother had an aho phenotype , but calcium metabolism and serum pth
levels were normal , and it was suggestive of pseudo - php . the maternal grandfather had short
stature ( body height 153 cm ) and obesity , however he had died before the referral of the
present cases , and the details are uncertain . the maternal grandmother showed neither short
stature nor obesity . if the maternal grandfather had aho , it is supposed that the mother is
pseudo - php . analysis of the gs activity or the gnas1 gene is required for
definitive diagnosis . the gnas1 mutation was not identified in case 1 by
our approach . in another study , the mutation detection rate was 72% in php patients with
reduced activity of gs ( 9 ) . the genetic defect may be
located outside the coding region in the promoter region of gnas1 or in
another regulatory region leading to gs deficiency . considerable variability occurs in the clinical expression of aho even among affected
members of a single family , and all of the features may not be present in every case ( 4 ) . on rare occasions , it may impossible to detect any
features of aho in an individual with gs deficiency ( 10 ) . our cases are duozygotic twins , whose intrauterine environment and growth
history resemble each other very much . however , the phenotypes of both were completely
different . the reason for the phenotypic difference is unknown , but we suggest that it is
not caused by the environment . tissue- or cell - specific imprinting of gs has been suggested
as an explanation of phenotypic variation ( 11 , 12 ) . the pth / pthrp receptor not only mediates pth - dependent regulation of calcium and phosphate
homeostasis but also plays an important role in chondrocyte proliferation and
differentiation and thus in bone growth and elongation ( 13 ) . gs mutations in the proliferative layer of growth plate chondrocytes result
in an insufficient pthrp - dependent inhibition of chondrocyte maturation . mantovani
et al . reported that the clinical findings of osteodystrophy and obesity
in php type ia patients despite the presence of one normal gs allele might be due to the
presence of haploinsufficiency of this gene in bone and adipose tissue ( 12 ) . case 1 showed typical shortening of both 4th and 5th
metacarpals and moderate obesity , whereas case 2 had no brachydactyly and only mild obesity . the reason for this difference is not clear , though the degree of haploinsufficiency of
gnas1 might explain the difference . php type ia is associated with resistance to multiple hormones , including pth , tsh ,
gonadotropins and glucagons , whose effects are mediated by gs - coupled pathways ( 14 ) . reported the
presence of imprinting of gs paternal allele in selective tissues , such as the thyroid ,
pituitary and gonad , which , besides the kidney , are affected in php type ia ( 15 ) . on the other hand , both the paternal and maternal
alleles were equally expressed in the adrenal gland ( 15 ) . patients with php type ia usually show a normal
hypothalamic - pituitary - adrenal axis . both of our cases showed high basal tsh levels and
delayed exaggerated response to lhrh , besides hyperresponsiveness of acth to the crh test . we suppose that the presence of
50% activity of the gs protein in their adrenal gland is not sufficient in our cases . there is considerable variability in clinical and biochemical features of php type ia even
among affected duozygotic twins . the differences of intrauterine environment and growth
history can not account for the variable phenotype of php type ia . even if a patient shows no
aho features , examination of all family members should be undertaken . | pseudohypoparathyroidism ( php ) type ia is characterized by hypocalcemia due to
pth resistance and by features of albright s hereditary osteodystrophy , including short
stature , obesity , subcutaneous calcification and brachydactyly .
a wide variety of clinical
and biochemical manifestations have been reported .
we report two cases of php type ia in
duozygotic twins with different phenotypes .
the proband was a 10-yr - old girl .
she showed
subcutaneous ossification , shortening of the metacarpal bone , short stature , obesity and
round face .
she had normocalcemia ( 8.9 mg / dl ) , high - normal phosphate ( 5.0 mg / dl ) and
increased levels of serum intact pth ( 152 pg / ml ) and tsh ( 9.17 iu / ml )
levels .
her twin younger brother had atypical albright s hereditary osteodystrophy with
only mild obesity and subcutaneous calcifications , but he showed a low level of serum
calcium ( 7.0 mg / dl ) and high levels of serum phosphate ( 7.6 mg / dl ) , intact pth ( 377 pg / ml )
and tsh ( 6.9 iu / ml ) .
we diagnosed them as having php type ia on the
basis of clinical and biochemical findings , ellsworth - howard test and family history .
there is considerable variability in clinical and biochemical features of php type ia even
among affected duozygotic twins .
the differences of intrauterine environment and growth
history can not account for the variable phenotypes of php type ia .
even if a patient shows
no aho features , examination of all family members should be undertaken . |
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paragonimiasis is a parasitic disease , which is caused by the lung flukes of the trematode genus paragonimus . human infection occurs by ingestion of raw or incompletely cooked freshwater crab or crayfish infected with the metacercariae . as ranges of the freshwater crab and crayfish have been rapidly reduced , due to pollution of the water bodies with environmental waste , and the infection rate of fresh water crab and crayfish with metacercariae of paragonimus westermani have been continuously decreased in korea [ 1 - 3 ] , cases of human paragonimiasis have been found in rare occasions . the lung flukes may be found in various organs , such as the brain , peritoneum , subcutaneous tissues , and retroperitoneum , other than the lungs . abdominal paragonimiasis raises a considerable diagnostic challenge to clinicians , due to its uncommon nature and it may be confused with other abdominopelvic inflammatory diseases , particularly , peritoneal tuberculosis and peritoneal carcinomatosis . moreover , subcutaneous paragonimiasis does not easily bring up clinical suspicion , due to its rarity . here , we report 2 cases of abdominal paragonimiasis and 1 case of subcutaneous paragonimiasis , which we have experienced during the past 7 years . a 48-year - old man was referred to our emergency room for evaluation and treatment of 8-hr history of periumbilical pain and abdominal mass on local abdominal ultrasonograms . he had previously been healthy with no prior medical problems . at the time of admission ( june , lived in kunsan , jeollabuk - do ) , body temperature was 37.8 , and physical examination showed direct tenderness on the periumbilical area with no other specific findings . his white blood cell ( wbc ) count was 16,100/mm with no other abnormal laboratory findings . chest x - ray was normal and abdominal computed tomography ( ct ) showed a ring - shaped mass lesion , with focal irregular enhancement in the omentum ( fig . we suspected non - specific intra - abdominal abscess and performed exploratory laparotomy . during the surgery , microscopic pathologic examinations revealed the eggs of p. westermani scattered with acute suppurative inflammation and foreign body - type giant cells ( fig . a retrospective history taking showed that he had frequently consumed ' kejang ' ( drunken crab ) , which was prepared using freshwater crabs . the treatment decided for the patient was praziquantel ( 25 mg / kg , 3 times daily for 2 days ) , and the patient recovered uneventfully . a 57-year - old woman visited our hospital to seek treatment of transverse colon cancer ( october , lived in gunsan , jeollabuk - do ) . her vital sign was stable and laboratory findings revealed no abnormalities with normal eosinophil count and normal carcinoembryonic antigen , except anemia ( hemoglobin ; 9.6 g / dl ) . the preoperative abdominal ct showed circumferential wall thickening of the proximal transverse colon with adjacent fatty infiltrations . intraoperative findings showed a small whitish nodule on the omentum , which mimicked an omental seeding nodule . pathologic reports confirmed that there were numerous eggs of p. westermani in the fibrotic and collagenous background on the omentum . she could not recall any history of consuming freshwater crabs or crayfish in the past . she was treated with praziquantel ( 25 mg / kg , 3 times daily for 2 days ) , and had recovered uneventfully . a 58-year - old woman visited our hospital for evaluation and treatment of the right flank pain over 4 days ( august , lived in namwon , jeollabuk - do ) . abdominal ct revealed localized abscess in the subcutaneous layer of the right back ( fig . fine needle aspiration cytology ( fnac ) showed the eggs of p. westermani with eosinophil - dominant inflammatory cells ( fig . elisa ( performed at seoul medical institute ) was positive for antibodies against p. westermani in the serum ( 0.69 ) . retrospective history results revealed that she had frequently consumed ' kejang ' , which was prepared using freshwater crabs . on the basis of these findings , she was treated with praziquantel ( 25 mg / kg , 3 times daily for 2 days ) and her right flank pain was resolved after treatment . a 48-year - old man was referred to our emergency room for evaluation and treatment of 8-hr history of periumbilical pain and abdominal mass on local abdominal ultrasonograms . he had previously been healthy with no prior medical problems . at the time of admission ( june , lived in kunsan , jeollabuk - do ) , body temperature was 37.8 , and physical examination showed direct tenderness on the periumbilical area with no other specific findings . his white blood cell ( wbc ) count was 16,100/mm with no other abnormal laboratory findings . chest x - ray was normal and abdominal computed tomography ( ct ) showed a ring - shaped mass lesion , with focal irregular enhancement in the omentum ( fig . we suspected non - specific intra - abdominal abscess and performed exploratory laparotomy . during the surgery , microscopic pathologic examinations revealed the eggs of p. westermani scattered with acute suppurative inflammation and foreign body - type giant cells ( fig . a retrospective history taking showed that he had frequently consumed ' kejang ' ( drunken crab ) , which was prepared using freshwater crabs . the treatment decided for the patient was praziquantel ( 25 mg / kg , 3 times daily for 2 days ) , and the patient recovered uneventfully . a 57-year - old woman visited our hospital to seek treatment of transverse colon cancer ( october , lived in gunsan , jeollabuk - do ) . her vital sign was stable and laboratory findings revealed no abnormalities with normal eosinophil count and normal carcinoembryonic antigen , except anemia ( hemoglobin ; 9.6 g / dl ) . the preoperative abdominal ct showed circumferential wall thickening of the proximal transverse colon with adjacent fatty infiltrations . intraoperative findings showed a small whitish nodule on the omentum , which mimicked an omental seeding nodule . pathologic reports confirmed that there were numerous eggs of p. westermani in the fibrotic and collagenous background on the omentum . she could not recall any history of consuming freshwater crabs or crayfish in the past . she was treated with praziquantel ( 25 mg / kg , 3 times daily for 2 days ) , and had recovered uneventfully . a 58-year - old woman visited our hospital for evaluation and treatment of the right flank pain over 4 days ( august , lived in namwon , jeollabuk - do ) . abdominal ct revealed localized abscess in the subcutaneous layer of the right back ( fig . fine needle aspiration cytology ( fnac ) showed the eggs of p. westermani with eosinophil - dominant inflammatory cells ( fig . elisa ( performed at seoul medical institute ) was positive for antibodies against p. westermani in the serum ( 0.69 ) . retrospective history results revealed that she had frequently consumed ' kejang ' , which was prepared using freshwater crabs . on the basis of these findings , she was treated with praziquantel ( 25 mg / kg , 3 times daily for 2 days ) and her right flank pain was resolved after treatment . when p. westermani metacercariae infect man , they excyst in the duodenum , and then pass through the intestinal wall , peritoneal cavity , diaphragm , and pleural cavity to the lung parenchyma , where they mature to adult flukes . due to this migratory route , from the intestine to the lungs , ectopic infections can occur at unexpected locations , such as the brain , muscle , omentum , retroperitoneum , and subcutaneous tissues . the prototype species , p. westermani , is distributed in korea , japan , china , southeast asia , and the indian subcontinent . the major source of infection has been presumed to be soy - sauced freshwater crabs ( kejang ) , in south korea . however , according to a recent study in south korea , freshwater crabs may transmit the metacercariae only in rare occasions , and p. westermani metacerariae may be transmitted mainly by crayfish . in our cases , 2 patients had a history of consumption of ' kejang ' , but 1 patient could not recall any history of consuming freshwater crabs or crayfish . the diagnosis of paragonimiasis can be made based on detection of characteristic eggs in the stool , tissues , sputum , or pleural fluid , like our cases . however , the detection procedures in the tissues may be invasive , especially in the abdominal cavity . thus , elisa is commonly used for the immunodiagnosis of paragonimiasis , as the overall sensitivity and specificity of the assay were reported to be 90.2% and 100% , respectively . however , elisa can be used only based on clinical suspicion , which should be made in cases of eosinophilia , with the history of consumption of freshwater crabs or crayfish . abdominal paragonimiasis may be confused with other abdominopelvic inflammatory diseases , particularly peritoneal tuberculosis ( mainly granuloma formation ) , and peritoneal carcinomatosis . in this case , laparoscopic exploration and biopsy can be a useful diagnostic tool to distinguish the difference between abdominal paragonimiasis and peritoneal tuberculosis . in case of a peritoneal nodule , which is combined with cancer , just like our patient ( case 2 ) , frozen and permanent biopsy must be done to rule out other inflammatory diseases before decision making and treatment . also , fnac can show the eggs of p. westermani at the abscess in the subcutaneous tissue , like our patient ( case 3 ) , and can be used as a diagnostic tool . | paragonimiasis is a parasitic disease caused by the lung fluke , paragonimus spp .
lung flukes may be found in various organs , such as the brain , peritoneum , subcutaneous tissues , and retroperitoneum , other than the lungs .
abdominal paragonimiasis raises a considerable diagnostic challenge to clinicians , because it is uncommon and may be confused with other abdominopelvic inflammatory diseases , particularly peritoneal tuberculosis , and peritoneal carcinomatosis .
also , subcutaneous paragonimiasis does not easily bring up clinical suspicion , due to its rarity .
we herein report 2 cases of abdominal paragonimiasis and 1 case of subcutaneous paragonimiasis in korea . |
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kabuki syndrome ( ks ) , first reported by japanese researchers kuroki et al ( 1 ) and niikawa et al ( 2 ) , is a rare genetic disorder characterized by intellectual disability and multiple congenital anomalies ( 3 ) . the cardinal diagnostic manifestations of ks include characteristic facial features , mild - to - moderate mental retardation , dermatoglyphic abnormalities , skeletal anomalies , and postnatal growth deficiencies ( 5 ) . in addition , speech and language deficits are commonly present in patients with ks ( 6 ) . etiologically , ks was attributed originally to loss - of - function mutations in the gene encoding lysine methyltransferase 2d ( kmt2d ) , namely kmt2d ( formerly mll2 ) , with an autosomal dominant inheritance pattern ( 7 ) . more recently , the ks phenotype was identified in boys with a loss - of - function mutation in kdma6 on the x chromosome , the product of which is important for kmt2d function ( 8) . currently , no effective treatments for ks are available ( 9 ) . treatments are required that are beneficial for ks patients with developmental disability or cognitive limitations ; group therapy may be an option ( 10 ) . the present study reports the case of a young chinese girl with unclear speech ( nasal and muffled pronunciation ) and possible mental retardation , who was diagnosed with ks based on dna sequencing analysis of kmt2d . a 4-year - old chinese girl was admitted to the central south university xiangya school of medicine affiliated haikou hospital ( haikou , china ) on march 8th , 2015 with unclear speech ( nasal and muffled pronunciation ) , which her parents indicated had been present for > 2 years , and possible mental retardation . mental retardation was suspected due to the weak expressive language ability of the patient compared with healthy peers . the patient was a first - born child with no siblings , born full - term via uncomplicated vaginal delivery when her mother was 24 years old . there was no history of consanguineous marriage in the family , and no family history of genetic disorders . the patient 's father was employed as a road construction engineer and her mother had previously worked with cosmetics . during the first trimester , prior to awareness of the pregnancy , the patient 's mother reportedly had a febrile illness and received oral cold medication at a local clinic ( no records available ) . the patient 's mother did not consume alcohol , smoke , or take any other drugs or medications during the pregnancy . at 1 year of age , the patient was diagnosed with right congenital hip dysplasia , and was treated successfully with external bracing . the patient 's speech was delayed ; she was only able to say one word by 1 year of age . upon admission to the hospital at 4 years of age , the patient was able to speak in 5- and 6-word sentences . in addition , no problems with hearing or difficulties with understanding and following instructions were observed . the patient was able to recognize simple colors and the numbers 15 , and could color with a crayon . a physical examination revealed that the patient was of normal height ( ~100 cm ) and weight ( ~16 kg ) . however , she presented with abnormal facial features , including slanted eyes and sparse eyebrows ( fig . 1b ) , and eversion of the lateral parts of the lower eyelids ( fig . 1c ) . the results of heart and respiratory system examinations were normal , and there was no hepatosplenomegaly . neurologically , the patient exhibited normal physiological reflexes and did not exhibit any pathological reflexes . blood analysis revealed normal complete blood count , electrolyte measurements , blood lipid levels and renal and liver functional markers , but high blood lactic acid levels ( 2.8 mmol / l , august 2013 ; 3.4 mmol / l , january 2015 ; normal range , 0.72.1 in addition , the patient 's blood ammonia levels were normal and a urine mucopolysaccharide test was weakly positive [ 5 l ( + ) , 15 l ( + + + ) and 25 l ( + + + ) ] . gas chromatography - mass spectrometry analysis of the patient 's urine and blood revealed no evidence of any amino acid or aliphatic acid metabolic disorders . furthermore , g - banding karyotyping demonstrated that the patient had macroscopically normal chromosomes ( 46 , xx ) . chest x - rays , retinal examinations , and brain magnetic resonance imaging results were also normal . notably , x - rays of the fingers revealed the presence of three phalanges in the short fifth digits of the two hands . due to the clinical manifestations and urine mucopolysaccharide test results , it was suspected initially that the patient may be suffering from mucopolysaccharidosis type iii ( mps iii ) . mps iii and ks are characterized by atypical facial features together with developmental and/or speech delays . specifically , mps iii is characterized by an early - onset developmental and/or speech delay subsequent to an initial period of normal development ( 11 ) . the developmental delays evolve into a progressive cognitive decline , behavioral abnormalities and severe hyperactivity that does not respond to treatment with stimulants ( 11 ) . the somatic features of mps iii , however , are relatively mild , including a dolichocephalic skull shape with a short forehead , prominent eyebrows , an everted and thick lower lip , and an upturned upper lip with a protruding philtrum ( 11 ) . the patient in the present study exhibited abnormal facial features and delayed speech , but displayed no overt behavioral abnormalities , hyperactivity , or mps iii - characteristic facial features , and her urine mps test was only slightly abnormal . autism is a neurodevelopmental disorder that is characterized by abnormalities in reciprocal social and communicative behaviors , and an inflexible adherence to routinized patterns of thought and behavior ( 12 ) . the patient did not display any difficulties in understanding or following instructions given by her parents or preschool teacher . in addition , the patient gave reliable responses and maintained eye contact when spoken to . furthermore , sensory processing difficulties are ubiquitous , albeit highly variable , among people with autism . the patient of the present study presented no evidence of sensory hyper- or hypo - sensitivity , or any motor coordination delays , further indicating that she did not have autism . following the exclusion of mps iii and autism , ks was considered as a possible diagnosis . various clinical characteristics of the patient supported a diagnosis of ks , including abnormal facial features ( fig . dna sequence analysis of kmt2d , the gene primarily associated with ks , was performed . a peripheral blood sample was first obtained from the sample , and dna was extracted from blood with a qiaamp blood dna mini kit ( catalog no . high - throughput sequencing of kmt2d was performed using the trusight one sequencing panel ( illumina , san diego , ca , usa ) . the sequence was analyzed in annovar software ( annovar.openbioinformatics.org/en/latest/ ) ( 13 ) , and various databases , including 1,000 genomes ( www.1000genomes.org/ ) , exome sequencing project 6500 ( evs.gs.washington.edu/evs/ ) , single nucleotide polymorphisms ( www.ncbi.nlm.nih.gov/snp/ ) and human gene mutation data ( www.hgmd.cf.ac.uk/ac/index.php ) , were used for screening and annotation of gene variants in accordance with the american college of medical genetics and genomics guidelines ( 14 ) . 2 ) , which confirmed the diagnosis of ks . subsequently , the patient 's parents underwent polymerase chain reaction ( pcr ) genetic testing together with their daughter with the following kmt2d - exon 16 primers , designed using primer premier software version 5.0 ( premier biosoft international , palo alto , ca , usa ) : forward , 5-tatgatgttgagtatgtgacagagg-3 and reverse , 5-aatcctagcagtgaagagaccat-3. the primers were used to amplify the kmt2d gene using an abi 9700 pcr instrument ( applied biosystems ; thermo fisher scientific , inc . , waltham , ma , usa ) . exon 16 was amplified in a reaction solution containing 1 l 10x pcr buffer , 0.35 l dntps ( 10 mmol / l ) , 0.07 l faststart taq dna polymerase ( 5 u/l ; roche diagnostics , basel , switzerland ) , 1 l genomic dna ( 100 ng/l ) , 1 l each primer ( 3.2 mol / l ) and 5.6 l ddh2o . the cycling conditions were as follows : 94c for 12 min , followed by 35 cycles of 94c for 30 sec , 55c for 30 sec and 72c for 30 sec , and a final elongation step at 72c for 10 min , as previously described ( 5,15 ) . was then used to sequence the pcr products by the sanger method , as previously described ( 15 ) , and the resultant sequences were compared with known sequences of ks - associated genes ( ncbi sequences , nm_003482.3 and ng_027827.1 ) . the patient was found to harbor a nonsense mutation in exon 16 of the kmt2d gene ( c.4485c > a , tyr1495ter ; fig . 3b and c ) . based on the clinical manifestations of the patient and the identification of a de novo nonsense mutation of kmt2d , there is currently no cure for ks , and the only treatments available are supportive and psychological therapies ( 9 ) . there are several features that should be monitored , including the patient 's height , weight , head circumference , vision and hearing ( 9 ) . the patient described in the present report was referred immediately for speech therapy with the aim of improving her speech quality . the patient 's prognosis is unclear ; therefore , a long - term follow - up plan was implemented with the intention of providing potentially beneficial supportive and psychological therapies as required . at a follow - up in may 2015 , following 10 months of speech training , the patient 's speech remained unclear . following her diagnosis in our hospital , she has not been subjected to any further tests , has not exhibited any new manifestations of pathology and has not been administered any other treatments beyond speech therapy . the current case underscores the importance of genetic testing in enabling a clear differentiated diagnosis of ks . the present study reports a new case of the rare genetic syndrome ks , which is characterized primarily by intellectual deficiencies , multiple congenital malformations , and specific craniofacial abnormalities ( 16 ) . more specifically , ks is characterized by the following five , mainly external , structural features : i ) a dysmorphic face , including eversion of the lower lateral eyelids and arched eyebrows with sparse lateral eyebrows ; ii ) dermatoglyphic abnormalities , with increased digital ulnar loops and hypothenar loop patterns , absence of digital triradius , and the presence of fingertip pads in 93% of patients ; iii ) skeletal anomalies , with brachydactyly and spinal deformities with or without sagittal cleft vertebrae in 92% of patients ; iv ) mild - to - moderate mental retardation in 92% of patients ; and v ) postnatal growth deficiencies in 83% of patients ( 17 ) . patients with ks frequently exhibit internal malformations of the heart , kidneys , gastrointestinal system , skeletal system and/or eyes ( 18 ) . speech and language deficits are common in ks , with the most prominent deficit being dysarthria , characterized by imprecise pronunciation of consonants , a harsh vocal quality , hypernasality , reduced speech rate and stress , and a distorted pitch ( 6 ) . ks has an incidence rate of 1 in 32,000 individuals ; however , this figure is likely to be underestimated due to missed diagnosis and misdiagnosis ( 4 ) . diagnosis of ks can be challenging due to the spectrum of clinical , radiological and biological factors associated with ks , various complications , and incomplete penetrance in certain cases ( 16 ) . additionally , numerous other conditions present with features similar to those observed in ks , including global developmental delays , fetal alcohol syndrome , autism spectrum disorder and down syndrome ( 19 ) . the majority of patients diagnosed with ks are diagnosed based primarily on the presentation of archetypical features . currently , although genetic testing of families is important for genetic counseling , confirmatory genetic testing is performed only in a minority of cases . mutations in kmt2d are present in 5580% of ks - diagnosed individuals subjected to genetic testing ( 9 ) ; kmd6a mutations have been observed in the remaining ks cases in which genetic testing was performed , although there may be other as yet unidentified genetic mutations that lead to ks ( 20 ) . a number of different kmt2d mutations have been reported to date , including missense mutations , nonsense mutations , frameshift mutations , splice site mutations , and indel mutations ( 21 ) . the majority of these result in a truncated protein product . in the present study , a novel nonsense mutation in exon 16 of kmt2d ( c.4485c > a , tyr1495ter ) was identified . this mutation is a loss - of - function mutation in which codon 1495 is replaced with a termination codon , resulting in truncation of the encoded kmt2d protein product . kmt2d is a histone methyltransferase , comprised of 5,537 amino acid residues , that serves an important role in regulating gene transcription . kmt2d methylates lysine 4 of histone 3 ; this methylation serves as a marker of gene activation ( 22 ) . numerous de novo kmt2d mutations have been identified in sporadic ks cases ( 20 ) . among ks cases that are not sporadic , autosomal dominant inheritance of ks due to parent - to - child transmission of kmt2d mutations has been observed ( 23 ) . in the present study , the patient 's parents were not found to carry kmt2d mutations , which indicates that the patient harbored a de novo genetic mutation . although the molecular mechanisms underlying the ks phenotype development remain to be elucidated , the clinical manifestations of ks are generally presumed to be due to the loss of kmt2d functionality . this view is supported by the fact that the other gene in which mutations have been demonstrated to cause ks , kdm6a , encodes a histone demethylase that interacts with kmt2d ( 24 ) . the kmt2d mutation found in the present case ( c.4485c > a ) appears to be novel in that it was not listed among the hundreds of previously identified ks - causing mutations ( 5 ) represented in the leiden open variation database ( www.lovd.nl/ ) , the pubmed database of the national center for biotechnology information ( www.ncbi.nlm.nih.gov/ ) , the european molecular biology laboratory ( www.embl.org/ ) , or the human gene mutation database ( www.hgmd.org/ ) . in conclusion , clinicians should be aware that there are a variety of clinical manifestations of ks . in the present study , the patient had abnormal facial features and unclear speech ; however , her overall presentation was not clearly indicative of ks . | kabuki syndrome ( ks ) is a rare genetic syndrome characterized by multiple congenital anomalies and varying degrees of mental retardation .
patients with ks often present with facial , skeletal , visceral and dermatoglyphic abnormalities , cardiac anomalies and immunological defects .
mutation of the lysine methyltransferase 2d ( kmt2d ) gene ( formerly known as mll2 ) is the primary cause of ks .
the present study reported the case of a 4-year - old chinese girl who presented with atypical ks , including atypical facial features , unclear speech and suspected mental retardation .
a diagnosis of ks was confirmed by genetic testing , which revealed a nonsense mutation in exon 16 of kmt2d ( c.4485c > a , tyr1495ter ) . to the best of our knowledge ,
this is a novel mutation that has not been reported previously .
the present case underscores the importance of genetic testing in ks diagnosis . |
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impaction of teeth can result firstly from local biomechanical impediments , and secondarily from childhood maxillofacial or dentoalveolar trauma , reconstructive surgery of the facial skeleton , malpositioning of an adjacent tooth , thickened overlying osseous or mucosal tissues , insufficient maxillofacial skeletal development or a low correlation between maxillofacial skeletal development and tooth maturation , eruption disturbances and owing to direct or indirect effects of cysts or neoplasm.1 a host of systemic prenatal and postnatal disorders , diseases and syndromes ( table 1)1,2 can also cause tooth impaction . therefore , it is prudent to perform a thorough clinical examination and obtain adequate radiographs when teeth do not appear according to the usual eruption schedule.1 the purpose of this article is to present 3 cases with multiple impacted teeth in which no syndrome or systemic conditions have been detected involving both jaws . in 2000 , 21 year old male patient referred to our clinic with respect to the failure of eruption of the upper and lower anterior teeth . in his medical history , it was detected that he was born at 39 weeks gestation to a 29 year - old mother and a 33 year - old father . the pregnancy and delivery were normal and his birth weight was approximately 3.1 kg . during the clinical examination at the age of 21 , a height of 152 cm and a weight of 55 kg were registered . his ears were thick and floppy . despite the fact that the examination of the hands showed normal findings , the examination of the feet showed that the nails of his toes were hypoplastic bilaterally and the third toes were rudimentary . in addition , congenital curly ( or varus ) toe was noted that on left foot s fourth and fifth toes and right foot s fifth toe and was also noted that hammer toe on both of feet s toes were apart from first toes . no other family members were known to have similar abnormalities of the hands and feet . radiological evaluations of the clavicles , vertebral skeleton , skull and chest proved to be normal . the skin was dry , soft and velvety . head hair and body hair were clinically normal but his eyebrows were thick and bushy . results of routine hematology tests and karyotyping ( 46 , xy ) were normal . during the intraoral examination of the patient , the oral soft tissues were unremarkable and histopathological evaluations of gingivae were normal . the patient was found to be in the permanent dentition phase with the following teeth being fully erupted ( figure 1 ) : maxillary right permanent primary molar , mandibular right permanent second molar and mandibular left permanent second molar were extracted because of caries . mandibular right and left permanent central incisor , mandibular right and left second premolar were partially erupted . the following teeth were unerupted : radiologically , five unerupted supernumerary teeth were also noted . one of them was in the maxillary right posterior region and others were in the maxillary anterior region ( figure 2 ) . his impacted primary teeth , some of the impacted permanent and supernumerary teeth were removed and it was planned to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . but he did nt continue to the treatment after the end of his military service . in 2001 , 20-year - old male patient referred to our clinic with respect to the failure of eruption of some upper and all lower teeth . in his medical history , it was detected that he was born at 38 weeks gestation to a 41-year - old mother and a 45 year - old father . the pregnancy and delivery were normal and his birth weight was approximately 3.150 kg . during the general examination , radiologically , clavicles , vertebral skeleton , chest , skull , hands and feet were normal . ct scan of the paranasal sinus evaluations showed frontal sinus agenesis and maxillary sinus hypoplasia ( figure 3 ) . intraoral examination revealed that the patient s maxilla was partially edentulous with the following erupted teeth : the patient s mandible was clinically edentulous ( figure 4 ) . the panoramic radiograph of the patient demonstrated multiple impacted teeth in maxilla and mandible . some dental crown - root abnormalities and ectopic teeth the following teeth were impacted : the patient stated that , during his childhood , his mandibular right deciduous primary molar tooth erupted but soon decayed and came out . it was planned to remove the impacted primary teeth , some of the impacted permanent teeth and to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . it was also planned to insert six implants in the mandible and to apply implant supported prosthesis . in the initial phase of the treatment , the patient was taken to the operating room and following induction of general anesthesia , all of the twenty three of impacted teeth in the mandible were removed . after two weeks the patient was taken to the operating room for the secondary phase of the treatment . maxillary right and left permanent second premolars , right and left third molars were removed . the treatments of maxillary right and left permanent central incisors and right and left second molars by surgical exposure and orthodontic traction were started . but he did nt continue to the treatment after the end of his military service . in 2001 , 21-year - old male patient referred to our clinic with respect to the failure of eruption of some upper and all lower teeth . in his medical history , it was detected that his pregnancy and delivery were normal . during the general examination , despite none of these measurements deviate from the norm , malar hypoplasia was noted facially . the radiological evaluations of his hands and feet showed that minimal clinodactyly was on bilaterally fourth and fifth fingers and first and fifth toes . during the radiological examination of the lower extremities intraoral examination revealed that the patient s maxilla and mandible were partially edentulous with the following erupted teeth ( figure 6 ) : the panoramic radiograph of the patient demonstrated multiple impacted teeth in maxilla and mandible ( figure 7 ) . the following teeth were impacted : radiologically , four unerupted supernumerary teeth were also noted . two of them were in the maxillary and mandibulary left posterior region and others were in the maxillary right posterior region . it was planned to remove the persisted primary teeth , some of the impacted permanent and supernumerary teeth and to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . but he did nt continue to the treatment after the end of his military service . in 2000 , 21 year old male patient referred to our clinic with respect to the failure of eruption of the upper and lower anterior teeth . in his medical history , it was detected that he was born at 39 weeks gestation to a 29 year - old mother and a 33 year - old father . the pregnancy and delivery were normal and his birth weight was approximately 3.1 kg . during the clinical examination at the age of 21 , a height of 152 cm and a weight of 55 kg were registered . his ears were thick and floppy . despite the fact that the examination of the hands showed normal findings , the examination of the feet showed that the nails of his toes were hypoplastic bilaterally and the third toes were rudimentary . in addition , congenital curly ( or varus ) toe was noted that on left foot s fourth and fifth toes and right foot s fifth toe and was also noted that hammer toe on both of feet s toes were apart from first toes . no other family members were known to have similar abnormalities of the hands and feet . radiological evaluations of the clavicles , vertebral skeleton , skull and chest proved to be normal . the skin was dry , soft and velvety . head hair and body hair were clinically normal but his eyebrows were thick and bushy . results of routine hematology tests and karyotyping ( 46 , xy ) were normal . during the intraoral examination of the patient , the oral soft tissues were unremarkable and histopathological evaluations of gingivae were normal . the patient was found to be in the permanent dentition phase with the following teeth being fully erupted ( figure 1 ) : maxillary right permanent primary molar , mandibular right permanent second molar and mandibular left permanent second molar were extracted because of caries . mandibular right and left permanent central incisor , mandibular right and left second premolar were partially erupted . the following teeth were unerupted : radiologically , five unerupted supernumerary teeth were also noted . one of them was in the maxillary right posterior region and others were in the maxillary anterior region ( figure 2 ) . his impacted primary teeth , some of the impacted permanent and supernumerary teeth were removed and it was planned to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . but he did nt continue to the treatment after the end of his military service . in 2001 , 20-year - old male patient referred to our clinic with respect to the failure of eruption of some upper and all lower teeth . in his medical history , it was detected that he was born at 38 weeks gestation to a 41-year - old mother and a 45 year - old father . the pregnancy and delivery were normal and his birth weight was approximately 3.150 kg . during the general examination , radiologically , clavicles , vertebral skeleton , chest , skull , hands and feet were normal . ct scan of the paranasal sinus evaluations showed frontal sinus agenesis and maxillary sinus hypoplasia ( figure 3 ) . intraoral examination revealed that the patient s maxilla was partially edentulous with the following erupted teeth : the patient s mandible was clinically edentulous ( figure 4 ) . the panoramic radiograph of the patient demonstrated multiple impacted teeth in maxilla and mandible . some dental crown - root abnormalities and ectopic teeth the following teeth were impacted : the patient stated that , during his childhood , his mandibular right deciduous primary molar tooth erupted but soon decayed and came out . it was planned to remove the impacted primary teeth , some of the impacted permanent teeth and to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . it was also planned to insert six implants in the mandible and to apply implant supported prosthesis . in the initial phase of the treatment , the patient was taken to the operating room and following induction of general anesthesia , all of the twenty three of impacted teeth in the mandible were removed . after two weeks the patient was taken to the operating room for the secondary phase of the treatment . maxillary right and left permanent second premolars , right and left third molars were removed . the treatments of maxillary right and left permanent central incisors and right and left second molars by surgical exposure and orthodontic traction were started . but he did nt continue to the treatment after the end of his military service . in 2001 , 21-year - old male patient referred to our clinic with respect to the failure of eruption of some upper and all lower teeth . in his medical history , it was detected that his pregnancy and delivery were normal . during the general examination , the circumference of the head was measured as 52 cm . despite none of these measurements the radiological evaluations of his hands and feet showed that minimal clinodactyly was on bilaterally fourth and fifth fingers and first and fifth toes . during the radiological examination of the lower extremities intraoral examination revealed that the patient s maxilla and mandible were partially edentulous with the following erupted teeth ( figure 6 ) : the panoramic radiograph of the patient demonstrated multiple impacted teeth in maxilla and mandible ( figure 7 ) . the following teeth were impacted : radiologically , four unerupted supernumerary teeth were also noted . two of them were in the maxillary and mandibulary left posterior region and others were in the maxillary right posterior region . it was planned to remove the persisted primary teeth , some of the impacted permanent and supernumerary teeth and to assist the eruption of other permanent teeth by surgical exposure and orthodontic traction with intermittent monitoring periods . but he did nt continue to the treatment after the end of his military service . multiple impacted teeth by itself is a rare condition and often found in association with syndromes such as cleidocranial dysplasia,24 gardner s syndrome,2,57 down syndrome,2,7 aarskog syndrome,2,8 zimmerman - laband syndrome2,9,10 and noonan s syndrome.2,7 although some other features were discovered on our cases beside multiple impacted teeth , all features of any syndrome could nt be diagnosed exactly . only a few cases of non - syndrome multiple impacted teeth were reported.3,7,1113 impacted teeth are those which are prevented from eruption by some physical barrier in the eruption path . the oral soft tissues of our cases were unremarkable and histopathological evaluations of gingivae were normal . the clinical and radiographic examinations of our cases revealed relatively normal jaws and teeth despite the fact that some dental crown - root abnormalities were detected on case 2 . abnormalities of tooth morphology are related to inadequate space and arrested eruption . delayed or arrested eruption is probably caused by diminished resorption of bone and of primary teeth and to the presence of multiple supernumerary teeth.3 conditions which cause lacking of eruptive force in such cases could be due to either general , endocrinal , neurogenic or mucosal and bone disorder . especially , at the case 2 operation dense compact bone in the mandible was observed . conditions where we find retained teeth are : hemifacial atrophy , hypopitutarism , hypothyroidism , cherubism , gingival fibromatosis , cleft palate and preceding syndromes.5,7 on account of the fact that the medical history of our cases was non - contributory ; above conditions were excluded . babu et al3 stated that the exact cause and the significance of multiple impacted supernumerary teeth remain an enigma to us . multiple supernumerary teeth without any associated systemic conditions or syndromes are not common and often associated with various syndromes . review of the literature revealed only a few reported cases . leslie14 reported of a 25-year old black male with non - syndrome multiple impacted supernumerary teeth that resembled regular mandibulary premolar teeth . yucel15 and yusof16 reviewed most of the cases reported in the english language literature from 1969 to 1990 and found that there was a predilection of non - syndrome multiple supernumerary teeth that occur in the mandible . however , in our patients ( case 1 and 3 ) nine unerupted supernumerary teeth were noted , eight of them were in the maxillary and the other was in the mandible . the frontal sinus is the only sinus that can not be recognized radiologically in the neonate . the frontal diverticulum is present , but the frontal bone is not pneumatized until between the first and second years of life . the frontal sinus may assume its definite shape as early as the third year of life , but continued growth and development are carried well into puberty . agenesis and massive pneumatization are both seen with regularity.17,18 maxillary sinus hypoplasia is rare , with an estimated prevalence of 15%.19,20 occasionally the lumen of the maxillary sinus may be small and its bony walls thick as a result of incomplete pneumatization and development . complete failure of development of the antral lumen and total absence of the maxillary sinus are rare.17,19 rhinootolaryngologycal examination of our patient ( case 2 ) revealed no pathological symptom . but ct scan of the paranasal sinus evaluations showed frontal sinus agenesis and maxillary sinus hypoplasia . besides , an instance of physical trauma to the left frontal region during childhood and recurrent bronchitis were detected . to achieve optimum function and aesthetics , an interdisciplinary cooperation between the oral surgeon , orthodontist , prosthodontist and pedodontist should be based for the management of the case.6 the concept of treatment of these cases contains serial examination of primary teeth , removal of the supernumerary teeth , assisting the eruption of the permanent teeth by orthodontic traction or surgical exposure and any impacted tooth which could nt erupt after surgical or orthodontic methods needs extraction and replacement.3,21 | while impaction of tooth is widespread , multiple impacted teeth by itself is a rare condition and often found in association with syndromes such as cleidocranial dysplasia or gardner s syndrome .
a light of radiographic examination , we describe three turkish young males with multiple impacted teeth who did nt possess any systemic conditions or syndromes involving both jaws .
the first patient , a 21-year - old young male , had 16 unerupted teeth and 5 unerupted supernumerary teeth . the second patient with totally edentulous mandible , a 20-year - old young male , had 31 unerupted teeth .
the third patient , a 21-year - old young male , had 22 unerupted teeth and 4 unerupted supernumerary teeth .
based on the clinical presentation , radiographic examination and histopathological studies , this paper discusses the differential diagnosis and management of such cases . |
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at present , implants have become a standard means of replacing missing teeth since they have the advantage of allowing preservation of the integrity of sound teeth adjacent to the edentulous area . the most widely used material for dental implants is titanium owing to its superior properties such as biocompatibility and corrosion resistant . after placement and osseointegration of implants , it is the responsibility of the dentist to maintain properly designed and fitted prosthesis . clinical decisions are not limited to the selection of the type of implant but are also dependent on the material of prosthesis . regardless of the implant system used , a restoration is selected to emerge from the tissues to imitate a natural tooth . the quest for predictable long - term results has raised several questions concerning the materials used as well as the techniques followed in clinical practice . implant supported restorations can be fabricated by nonprecious alloys or all ceramic materials . in the recent scenario of advanced technology , recent progress in casting techniques as well as the introduction of computer aided design / computer aided machined ( cad / cam ) has made it possible to make the prostheses using titanium also . cad / cam technology , all ceramic systems , and high strength ceramic materials have become integral parts of modern dentistry . optimal esthetics and characteristics such as color stability , high wear resistance , and low thermal conductivity make all ceramic materials ideal for the fabrication of dental prostheses . the main advantage of all ceramic systems lies in the absence of metal , especially in cases with very thin tissue thickness and shallow implant depths allowing a more esthetically pleasing restoration . dental implant restorations can be cement retained , screw retained or a combination of both . because it is difficult to achieve passive fit with screw - retained implant restorations , some dentists prefer cemented prosthesis . on the other hand , cement retained prosthesis has the disadvantage of compromised stability in case of less interocclusal distance , as the abutment lacks the height and taper for cement retention . retrievability also serves as the safety mechanism to allow the prosthesis to be removed without causing harm to underlying implant superstructures . retention certainly influences the lack of complications as well as the longevity of implant prostheses . over the years , those engaged in implant supported restorations have debated as to which type of mechanism is preferable for increase bonding of cemented restorations . the success of implant - supported restorations depends on the success of bonding between prosthesis and metal interface . retention of crown depends on cement used , surface preparation of the implant abutment , and other variables such as internal surface characteristics of coping and the height and taper of implant abutment . for better simulation of clinical conditions , investigation of the bonding or retentive strength numerous studies investigating the bonding strength of different luting agents on retention of cement - retained prosthesis and also effect of airborne - particle abrasion on zirconia copings are available , but there is a paucity of studies having direct comparison of different copings to the best of our knowledge . due to the above concerns , there is a need for comparing the shear bond strength of abutment copings of different materials , made by different techniques as well as effect of surface conditioning on bonding . the null hypothesis was that there was no difference in bond strength of abutment copings made of different materials and also no effect of surface conditioning on bonding . the alternate hypothesis states that there was difference in bond strength of abutment copings , and also surface conditioning affected the bonding strength . the present research is a cross - sectional in vitro study aimed to compare the shear bond strength of different copings and effect of surface conditioning on shear bond strength of three different types of abutment copings made on titanium implant abutment ( indident dental implant system , drdo inmas , new delhi , india ) . in the present study , three different types of abutment copings were fabricated on titanium implant abutment ( indident dental implant system , drdo inmas , new delhi , india ) . the sample size was estimated to be 30 in each group based on previous studies . for group a , 30 cad / cam zirconia copings were fabricated on one implant abutment [ figure 1a ] using cercon cad / cam system ( dentsply , germany ) . optical scanning was done with cercon eye . once the scanning was completed with cercon eye , cement gap , wall thickness , and occlusal geometry were adjusted for the coping with the help of cercon art program . a luting gap of 30 m and wall thickness of 0.8 mm were chosen for the coping fabrication . presintered zirconia blank of 38 size was inserted in cercon brain to complete the milling . after milling was completed , the frameworks of the coping were separated from the blank with the help of micromotor and sintered in furnace , according to manufacturer 's instructions . dimensions of cad / cam coping was measured with digital caliper ( shenzhen yks technology ltd . , ( a - c ) abutment copings made of computer aided design / computer aided machined ( left ) , pressable ceramic ( middle ) , and milled metal ( right ) for group b , 30 pressable ceramic copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [ figure 1b ] . implant abutment was screwed on implant analog , and two coatings of die spacer ( durolan , dfs ) were applied on implant abutment to attain the same cement space . to attain the similar dimension as cad / cam coping , silicon index was fabricated of cad / cam coping extending on the predetermined groove on implant analog . molten inlay wax ( geo - dip , renfert , germany ) was poured in silicon index , and mounted abutment was inserted in that index to the level of predetermined groove . investment of wax pattern was done in silicone ring with the phosphate bonded investment material ( ips pressvest speed ) . investment ring was inserted in the center of hot press furnace ( multimat 2 touch + press ) using investment tongs ; selected program was started . after cooling of the ring , the sprue and reaction layer on the copings were removed , and copings were retrieved . dimensions of pressable ceramic coping were verified with a digital caliper . for group c , 30 milled metal copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [ figure 1c ] . the cad volume data was transferred to the multi - axis metal milling machine ( tds cutter , turbodent system , taiwan ) . metal milling of titanium blank for abutment copings copings of cad / cam , pressable ceramic and metal millings were divided into two halves . to maintain standardization , one - half ( 15 out of 30 samples in each group ) was surface conditioned by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s at a distance of 10 mm in the sandblaster [ figure 3 ] , and other half were left nonconditioned . thus , three groups with two subgroups of each of the copings were prepared : surface conditioning done by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s
group a ( n = 30 ) : copings made by computer - aided technique
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditionedsubgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned
group b ( n = 30 ) : copings made by pressable ceramic technique subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned
group c ( n = 30 ) : copings made by metal milling
group a ( n = 30 ) : copings made by computer - aided technique
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditionedsubgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditioned subgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned group b ( n = 30 ) : copings made by pressable ceramic technique subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned
subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditioned subgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned group c ( n = 30 ) : copings made by metal milling
subgroup c1 ( n = 15 ) : milled metal copings that were surface conditionedsubgroup c2 ( n = 15 ) : milled metal copings that were nonconditioned . subgroup c1 ( n = 15 ) : milled metal copings that were surface conditioned subgroup c2 ( n = 15 ) : milled metal copings that were nonconditioned . all specimens were cemented to titanium abutment using glass ionomer cement ( gic ) ( meron , voco gmbh , germany ) to make standardization , which was mixed according to manufacturers recommendations . an alignment apparatus [ figure 4 ] was used that applied a weight of 750 g to the bonded specimens . extra cement was scraped out , and after initial set ( 8 min ) , specimens were stored in distilled water for 24 h to simulate the moist oral environment . alignment apparatus applying a weight of 750 g to the bonded specimens in the present study , shear bond strength was evaluated by universal testing machine [ figure 5 ] ( 22.5 k instron , model 4482 ) . forces were applied axially on coping cemented on abutment at a cross - head speed of 1.0 mm / min . a computer connected with the instron machine recorded the result of each test . the nonadhesive chemical bond strength ( ) values ( expressed in effects of mpa ) were calculated using the formula : = l / a . where l is load ( in n ) and a is the adhesive area ( in m ) . pull - off test for determination of bonding strength the shear bond strength was evaluated by pull - off test for all the abutment copings . all calculations were performed using the spss ( version 16 ) for windows ( spss inc . , the present research is a cross - sectional in vitro study aimed to compare the shear bond strength of different copings and effect of surface conditioning on shear bond strength of three different types of abutment copings made on titanium implant abutment ( indident dental implant system , drdo inmas , new delhi , india ) . in the present study , three different types of abutment copings were fabricated on titanium implant abutment ( indident dental implant system , drdo inmas , new delhi , india ) . the sample size was estimated to be 30 in each group based on previous studies . for group a , 30 cad / cam zirconia copings were fabricated on one implant abutment [ figure 1a ] using cercon cad / cam system ( dentsply , germany ) . optical scanning was done with cercon eye . once the scanning was completed with cercon eye , cement gap , wall thickness , and occlusal geometry were adjusted for the coping with the help of cercon art program . a luting gap of 30 m and wall thickness of 0.8 mm were chosen for the coping fabrication . presintered zirconia blank of 38 size was inserted in cercon brain to complete the milling . after milling was completed , the frameworks of the coping were separated from the blank with the help of micromotor and sintered in furnace , according to manufacturer 's instructions . dimensions of cad / cam coping was measured with digital caliper ( shenzhen yks technology ltd . , ( a - c ) abutment copings made of computer aided design / computer aided machined ( left ) , pressable ceramic ( middle ) , and milled metal ( right ) for group b , 30 pressable ceramic copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [ figure 1b ] . implant abutment was screwed on implant analog , and two coatings of die spacer ( durolan , dfs ) were applied on implant abutment to attain the same cement space . to attain the similar dimension as cad / cam coping , silicon index was fabricated of cad / cam coping extending on the predetermined groove on implant analog . molten inlay wax ( geo - dip , renfert , germany ) was poured in silicon index , and mounted abutment was inserted in that index to the level of predetermined groove . investment of wax pattern was done in silicone ring with the phosphate bonded investment material ( ips pressvest speed ) . investment ring was inserted in the center of hot press furnace ( multimat 2 touch + press ) using investment tongs ; selected program was started . after cooling of the ring , the sprue and reaction layer on the copings were removed , and copings were retrieved . dimensions of pressable ceramic coping were verified with a digital caliper . for group c , 30 milled metal copings of similar dimensions as that of cad / cam copings were fabricated on implant abutment [ figure 1c ] . the cad volume data was transferred to the multi - axis metal milling machine ( tds cutter , turbodent system , taiwan ) . copings of cad / cam , pressable ceramic and metal millings were divided into two halves . to maintain standardization , one - half ( 15 out of 30 samples in each group ) was surface conditioned by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s at a distance of 10 mm in the sandblaster [ figure 3 ] , and other half were left nonconditioned . thus , three groups with two subgroups of each of the copings were prepared : surface conditioning done by air - abrasion with 50 m al2o3 at 2.5 bars pressure for 20 s
group a ( n = 30 ) : copings made by computer - aided technique
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditionedsubgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned
group b ( n = 30 ) : copings made by pressable ceramic technique subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned
group c ( n = 30 ) : copings made by metal milling
group a ( n = 30 ) : copings made by computer - aided technique
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditionedsubgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned
subgroup a1 ( n = 15 ) : copings made by computer - aided technique that were surface conditioned subgroup a2 ( n = 15 ) : copings made by computer - aided technique that were nonconditioned group b ( n = 30 ) : copings made by pressable ceramic technique subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditionedsubgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned
subgroup b1 ( n = 15 ) : copings made by pressable ceramic technique that were surface conditioned subgroup b2 ( n = 15 ) : copings made by pressable ceramic technique that were nonconditioned group c ( n = 30 ) : copings made by metal milling
subgroup c1 ( n = 15 ) : milled metal copings that were surface conditionedsubgroup c2 ( n = 15 ) : milled metal copings that were nonconditioned . subgroup c1 ( n = 15 ) : milled metal copings that were surface conditioned subgroup c2 ( n = 15 ) : milled metal copings that were nonconditioned . all specimens were cemented to titanium abutment using glass ionomer cement ( gic ) ( meron , voco gmbh , germany ) to make standardization , which was mixed according to manufacturers recommendations . an alignment apparatus [ figure 4 ] was used that applied a weight of 750 g to the bonded specimens . extra cement was scraped out , and after initial set ( 8 min ) , specimens were stored in distilled water for 24 h to simulate the moist oral environment . in the present study , shear bond strength was evaluated by universal testing machine [ figure 5 ] ( 22.5 k instron , model 4482 ) . forces were applied axially on coping cemented on abutment at a cross - head speed of 1.0 mm / min . a computer connected with the instron machine recorded the result of each test . the nonadhesive chemical bond strength ( ) values ( expressed in effects of mpa ) were calculated using the formula : = l / a . where l is load ( in n ) and a is the adhesive area ( in m ) . the shear bond strength was evaluated by pull - off test for all the abutment copings . all calculations were performed using the spss ( version 16 ) for windows ( spss inc . , chicago , il , usa ) . a one - way analysis of variance was computed for statistical significance at p = 0.05 for individual groups with post - hoc ( tukey 's honest significant difference ) and intragroup comparison was done by unpaired t - test . the mean difference for cad / cam surface the mean difference for pressable surface conditioned group was 1.18 0.04 , and for nonconditioned group was 0.75 0.28 . the mean difference for milled metal surface conditioned group was 2.57 0.58 , and for nonconditioned group was 1.49 0.15 [ graph 1 and table 1 ] . mean and standard deviation of bonding strength ( in mpa ) between surface conditioned ( c ) and nonconditioned specimens ( nc ) mean bonding strength ( in mpa ) of different specimens on intragroup comparison , results showed that pressable ceramic and milled metal group had the statistically significant difference ( p = 0.05 ) in conditioned and nonconditioned specimens [ table 2 ] . on intergroup comparisons , cad / cam , pressable , and milled metal surface conditioned specimens had statistically significant difference , and similar results were found for nonconditioned specimens [ table 3 ] . mean comparison and standard deviation of bonding strength(in mpa ) between different abutment copings mean comparison of bonding strength ( in mpa ) between surface conditioned and nonconditioned specimens this study assessed the bonding strength of copings made out of different materials and also evaluated the effect of surface conditioning on bonding strength . the results of this study led to the rejection of null hypothesis that there was no significant difference in the bond strength of tested groups . metal copings fabricated by milling technique provided much stronger retention as compared to other materials and also surface conditioning increases the bonding strength . two all ceramic materials and titanium metal were used in this study , and titanium metal was not casted because milling of titanium metal overcomes the problem of production of reactive surface layer on its surface when cast in thin and fragile sections . over the years , questions have been put on bonding of implant supported restorations . in order to improve bonding , the internal surface of restoration chemical surface treatments include the use of various reactive agents while mechanical procedures include airborne - particle abrasion with alumina particles and abrasion with a mounted stone . investigated the factors which determine the retentiveness of copings made out of co - cr alloy and zirconia luted with permanent and provisional luting cements . the conclusion of this study was that copings made of metal alloy and zirconia showed no different level of retentiveness when set onto titanium abutments fixed with permanent or provisional cements . as concluded in this study , surface conditioning with airborne particle abrasion enhances the bonding of metallic as well as ceramic - based restorations . tashkandi evaluated the effect of surface treatment on micro - shear bond strength of zirconia frameworks . ebert et al . in 2007 found similar results when evaluating the effect of two surface conditioning methods and two luting gap sizes on the bonding of zirconia copings . airborne - particle abrasion has also been recommended as the best method of pretreatment in previous studies to improve the bond strength to oxide ceramics . sadig and al harbi compared the effect of different surface conditions on the retentiveness of titanium crowns cemented abutments using two types of cement . shahin and kern in 2010 also concluded that air - abrasion significantly increases crown retention . in the present study , although resin cements has got the maximum retention for all ceramic restorations , previous studies assessed that gic has also got the adequate retention . compared the retentive strength of metal copings on prefabricated abutments with five different luting agents . gic was used for luting all the specimens to make standardization , and moreover different resin cements require different pretreatment which could alter results . gic show high water solubility and require sufficient time for complete setting reaction to maximize their retention . manufacturer of gic states that the material will reach its full set in 47 min so 8 min setting period that was allowed in the previous study should prove satisfactory . the limitations of this study include that all copings were produced and tested under ideal conditions , which may not reflect conditions in daily clinical practice . thermal cycling and long - term water storage are the factors that can have effects on the durability of bond strength , and they are important parameters to simulate the oral conditions . in terms of in vivo loading , the masticatory cycle consists of combination of vertical and lateral forces , subjecting the restoration to a variety of off - axis loading . in the current investigation , hence , future studies should attempt to simulate intraoral conditions , as well as utilize additional mechanical tests , which can provide a more reliable assessment of retention . within the conditions and limitations of this in vitro study , milled metal copings had the highest bond strength as compared to cad / cam and pressable ceramic copings . surface conditioning with air - abrasion achieved better bond strength of all type of copings . further studies are needed to evaluate optimal surface conditioning in order to enhance the longevity of restorations clinically . | background / purpose : the aim of this study was to compare the shear bond strength of computer aided design / computer aided machined ceramic ( cad / cam ) , pressable ceramic , and milled metal implant copings on abutment and the effect of surface conditioning on bonding strength.materials and methods : a total of 90 test samples were fabricated on three titanium abutments . among 90 test samples , 30 copings were fabricated by cad / cam , 30 by pressable , and 30 by milling of titanium metal .
these 30 test samples in each group were further subdivided equally for surface treatment .
fifteen out of 30 test samples in each group were surface conditioned with airborne particle abrasion .
all the 90 test samples were luted on abutment with glass ionomer cement .
bonding strength was evaluated for all the samples using universal testing machine at a crosshead speed of 5 mm / min .
the results obtained were compared and evaluated using one - way anova with post - hoc and unpaired t - test at a significance level of 0.05.results:the mean difference for cad / cam surface conditioned subgroup was 1.28 0.12 , for nonconditioned subgroup was 1.20 0.11 . the mean difference for pressable surface
conditioned subgroup was 1.18 0.04 , and for nonconditioned subgroup was 0.75 0.28 .
the mean difference for milled metal surface conditioned subgroup was 2.57 0.58 , and for nonconditioned subgroup was 1.49 0.15.conclusions:on comparison of bonding strength , milled metal copings had an edge over the other two materials , and surface conditioning increased the bond strength . |
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individually - habituated resting head posture is believed to be determined by a dynamic
combination of factors , including body build , muscle performance , age - related structural
changes , occupation , and cultural factors1,2,3 . forward head posture ( fhp ) is one of the most common postural problems described as a
posture disorder , and in fhp the cervical spine is offset in the anterior direction4 , 5 . fhp is commonly adopted by computer workers , and it involves a combination of lower cervical
flexion , upper cervical extension and rounded shoulders ; also , it has been anecdotally
linked to musculoskeletal dysfunctions , such as upper crossed syndrome6 . recently , several studies have reported that fhp contributes to the onset and perpetuation
of neck and back pain syndromes7 , 8 . this causes a shortening of the posterior
cervical and suboccipital muscles , lengthening and weakness of the anterior neck muscles ,
weakness of the scapular retractor muscles , and increased stress on the ligaments . a study
showed that fhp reduces the average length of muscle fibers , which would contribute to an
extensor torque about the atlanto - occipital joint , and it is possible that this shortening
reduces the tension - generating capabilities of the muscles7 . proper posture is considered to be a state of musculoskeletal balance that involves
minimizing stresses and strains acting on the body9 . in clinical settings , assessment of posture is a common treatment
approach for individuals with neck , shoulder , or back pain10,11,12 . in particular , head posture assessment is recommended as part of
the examination of patients with neck pain to aid with diagnosis , determine treatment
strategies and monitor the progress of the patient10 ,
12,13,14 . major therapeutic tasks performed in
physical therapy involve teaching the ideal posture to patients in order to prevent postural
problems , such as fhp , and the correction of faulty postures . however , clinical evaluation and head posture is
generally based on the clinician s subjective visual impression . besides , it is difficult to
compare patients with each other and to quantify the improvements . therefore , the aim of
this study was to investigate the intra - rater and inter - rater reliabilities of the
assessment of fhp using a digital camera to capture images of the sagittal plane of the
upper body . forty - five participants ( 20 males , 25 females , meansd age of 21.331.7 years ) were
recruited . the subject inclusion criteria were as follows : free of neck and back pain for at
least 12 months , and no shoulder or cervical spine pathologies , or rheumatoid or
neurological condition . all subjects understood the purpose of this study and provided their
written informed consent prior to participation . the eligibility criteria were having at least 3 years of clinical
experience and a self - report that they routinely assessed fhp by observation in their
clinical practice . images of each subject were captured using a digital camera to capture the sagittal plane
profile of the upper body in the sitting position . rand markers were placed over the right
tragus ( ear ) , acromion , and c7 spinous process . forward shoulder angle was measured as the
angle from the vertical to a line connecting the c7 marker and the acromial marker . forward
head posture was defined as a forward a shoulder angle of 54 or less13 . according to norkin and white15 rotation of the head was not assessed because the angular
measurements of head rotation are easily distorted by trunk rotation . measurement of the
forward shoulder angle was made of blind of the qualitative assessment of fhp made by the
raters and the digitisation procedure was found to be highly reliable ( iccs _ 0.98 ) . a
standard distance of 3 m , between the camera and the subjects , was used . in the instructions
sheet , raters were asked to rate fhp and extension from the lateral image using the position
asked by physical therapists . this scale was chosen because it was found to be used in
clinical practice to characterize fhp14 . raters were told to view each image only once , to not change their assessment after moving
to the next image , and not to give any information related to their assessment to the
measurement of the forward shoulder angle . raters were then shown the images and their
assessments were recorded on the answer sheet . at the end of the assessment , they placed the
answer sheet in the envelope , and gave it to the researcher . the envelope was kept sealed
until assessments had been completed by the two raters . no external references were used to
aid the assessment , because in a previous study physical therapists reported not to use
external aids to inform the assessment of fhp by an observation14 . the order of appearance of the images
was counterbalanced for assessment 1 and again for assessment 2 to minimize any learning or
order effects . percentage of agreement , confidence intervals and the standard error of the mean were
calculated for the intra- and inter - rater reliabilities . angular values were grouped
according to the rating attributed by all raters in the first assessment and analyzed using
descriptive statistics . angular values and the ratings attributed by each rater were also
correlated using kendall s taub correlation coefficient16 . fhp : forward head posture ; icc : intraclass correlation coefficient sem : standard error of measurement ; ci : confidence interval participants meansd height was 166.339.09 cm , weight was 58.3810.86 kg and body mass
index was 20.932.33 kg / m . the results of intra - rater and inter - rater reliability are shown in table 1 . for the determination of intra - rater reliability , the
first measurement had a meansd of 59.642.79 , and the second measurement had a meansd of
59.212.58 for forward head posture . the measurements of tester 1 had a meansd of
59.093.84 , and the measurements of tester 2 had a meansd of 60.143.06. intra - class
correlation of intra - rater measurements indicated a very strong relationship ( 0.91 ) , and
intra - class correlation of inter - rater measurements showed a strong relationship ( 0.75 )
( p<0.05 ) . in the current study , we investigated the intra - rater and inter - rater reliabilities of the
assessment of fhp . photographic images were chosen to standardize fhp so that the same
posture was assessed by all raters in the first and second assessments . two physical
therapists with five years of clinical experience participanted in this experiment
investigating the intra- and inter - rater reliabilities of the assessment of fhp . the
participating physical therapists were recruited from local outpatient clinics where their
caseloads were mostly orthopedic . the results of this study demonstrated excellent intra - rater and inter - rater
agreement levels for the assessment of fhp by the trained physical therapists . therefore ,
the use of this method to assess fhp in clinical settings would improve the ability of the
clinician to detect and quantify posture alterations and treatment effect . forward head posture or head posture assessment is suggested for the examination of
patients with cervical joint pain to aid diagnosis , and decide treatment strategies for
patients1 , 11 , 14 , 17 . our results show that fhp measurement was strong reliability
between intra - rater and inter - rater . nancy et al.18 investigated intra - rater and inter - rater reliabilities of head
posture measurement in non - pathologic individuals aged 1834 . they reported that all raters
demonstrated significant correlations across the repeated measures , icc=0.920.94 , that
suggesting that head posture measurement has high intra - rater reliability . in addition ,
several studies have indicated that head posture measurements utilizing a flexible tape and
30 cm combination square have intra - rater and inter - rater reliability iccs ranging from 0.67
to 0.992 , 7 ,
11 . silva et al.13 investigated whether the
assessment of head posture as performed in clinical practice is reliable and valid . ten
physical therapists assessed forward head posture , head extension and side - flexion from
images of 40 individuals with and without previous experience of neck pain , using a
four - category scale . the assessment of head posture by observation and the four - category
scale showed poor reliability and validity . they suggested that when comparing the ratings
with angular measurements , some degree of overlap between adjacent categories and using
assessment of visual , and goniometer can occur error and deviation . even so , the extent of
the overlap was too big and occurred not only between adjacent categories , suggesting that
physical therapists rated similar head deviations as different , and different head posture
deviations as similar3 , 5 , 13 . therefore , we consider that the reason our results were strong
relationship in intra - rater and inter - rater reliabilities , because of the investigated
sagittal plane . the
reliability and validity of using a computer for making measurements of images is well
established . the results of our present study provide support for , computer use based on its
reliability shown in forward head posture assessment . it may use the standardized clinical
assessments in clinical implication . in this study , we have shown that it is possible to assess fhp reliably . however , the
method used relies on the assessor s judgment to identify a change in the posture and the
accuracy of making angular measurements on a digital image . it is difficult to compare the
results of most postural studies due to the lack of detailed quantitative data of postural
assessment and the difficulty of reproducing certain assessment tools in the clinical
setting . further studies will be required to abtain detailed quantitative data for accurate
measurement of the postural changes . | [ purpose ] assessment of posture is an important goal of physical therapy interventions
for preventing the progression of forward head posture ( fhp ) .
the purpose of this study
was to determine the inter- and intra - rater reliabilities of the assessment of fhp .
[ subjects and methods ] we recruited 45 participants ( 20 male subjects , 25 female subjects )
from a university student population . two physical therapists assessed fhp using images of
head extension .
fhp is characterized by the measurement of angles and distances between
anatomical landmarks .
forward shoulder angle of 54 or less was defined as fhp .
intra- and
inter - rater reliabilities were estimated using kendall s taub correlation coefficients .
[ results ] intra - class correlation of intra - rater measurements indicated an excellent level
of reliability ( 0.91 ) , and intra - class correlation of inter - rater measurements showed a
good level of reliability in the assessment of fhp ( 0.75 ) .
[ conclusion ] assessment of fhp
is an important component of evaluation and affects the design of the treatment regimen .
the assessment of fhp was reliably measured by two physical therapists .
it could therefore
become a useful method for assessing fhp in the clinical setting .
future studies will be
needed to provide more detailed quantitative data for accurate assessment of posture . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
computed tomography angiography ( cta ) is currently the standard non - invasive imaging method used for the assessment of the aorta . this is mainly due to high image quality , fast acquisition and easy post processing . indications for cta are broad , including preoperative evaluation , imaging of aortic aneurysm , aortic dissection or planning for endovascular procedures ( evar ) . several studies have been conducted concerning sufficient diagnostic luminal attenuation , , . according to the results of those studies , minimal enhancement of vascular structures of diagnostic quality varies between 250 and 350 hu . to achieve optimal enhancement various factors have to be considered : ( 1 ) scanner- and scan protocol - related factors , such as kv , tube current , pitch , gantry rotation , and timing of the scan ( scan delay ) ; especially in newest scan techniques such as high pitch scanning , . ( 2 ) patient - related factors , such as weight , body mass index ( bmi ) and cardiac output , whose influence can be taken into account and compensated for in individualized protocols ( e.g. weight - adapted cm protocols ) , , , ; ( 3 ) cm and cm administration parameters , which can be modified according to scan technique and patient characteristics . according to bae . the key parameters of cm are concentration , volume , physicochemistry ( e.g. viscosity or temperature ) , injection duration and flow rate . several studies indicate that the iodine delivery rate ( idr = the amount of iodine administered to a patient per second in gi / s ) is one of the most important parameters for intravascular enhancement , , . in recent years , cm volume reduction has consistently been in the center of research , , , , , with the goal to use cm more efficiently . apart from faster scanning techniques , new scanner- and especially the latest detector technologies with use of lower kv protocols facilitate lower cm volume . with lower kv settings , contrast to noise ratio ( cnr ) increases due to decreasing photon energy which approaches the k - edge of iodine at 33 kev resulting in higher enhancement with lower amount of iodine . thus a reduction in cm volume may be achieved without loss of diagnostic image quality , which would be an important improvement in view of potential contrast - induced nephropathy ( cin ) , , . therefore the purpose of this study was to evaluate the feasibility of a low volume cm protocol in 100 kv cta of the aorta . indications for cta were as follows : preoperative evaluation ( n = 7 ) , evar or open aortic surgery follow - up ( n = 35 ) , aortic aneurysm ( n = 21 ) , clinical suspicion of bleeding ( n = 3 ) , aortic dissection ( n = 1 ) , ischemia ( n = 5 ) , search for source of pulmonary embolism ( n = 2 ) , follow - up of lung tumor ( n = 1 ) and no specified indication ( n = 2 ) . for statistical purposes , patients were divided into 3 groups according to the aorta protocol they were referred for : thoracic aorta ( group 1 ) , abdominal aorta ( group 2 ) or combined thoracic - abdominal aorta ( group 3 ) . a waiver of informed consent was given by the local ethical committee ( metc 14 - 4 - 050 ) . in order to assess scan start delay , all patients received a test bolus ( 10 ml iopromide 300 ultravist , bayer healthcare , berlin , germany at 6 ml / s , followed by 40 ml saline flush at the same flow rate ) through an 18 g i.v . needle ( vasofix , bbraun , melsung , germany ) placed in an antecubital vein . in order to assess time to peak ( ttp ) , a region of interest ( roi ) was placed either at the level of the aortic arch for thoracic or combined thoracic - abdominal ctas , or at the level of l1l2 abdominal aorta ctas , using a dedicated software tool ( dyneva , siemens medical , forchheim , germany ) . scan start delay was calculated from test bolus delay ( 10 s ) + measured ttp + 4 s. during the latter 4 s breathing instructions were given to the patient and the scan table was moved to its starting position . the main bolus consisted of 50 ml cm followed by a 40 ml saline chaser , both injected at a flow rate of 6 ml / s ( idr 1.8 g / s , total iodine load til = 15 g ) . all injection parameters were closely monitored by a dedicated software tool ( certegra informatics platform , bayer healthcare , berlin , germany ) . patients were scanned on a multidetector - row ct ( mdct ) ( 2nd generation dual source scanner , somatom definition flash , siemens , forchheim , germany ) . scan parameters were as follows : 2 128 0.6 mm ( using z - flying focal spot techniques ) , pitch value 0.9 , 100 kv , quality ref . time 0.33 s , 1.0/0.8 mm slice reconstruction and soft tissue filter ( b30f ) . measurements were carried out at 10 different anatomic locations using a dedicated software tool ( syngo.via , siemens , forchheim , germany ) 5 thoracic and 5 abdominal : t1 ascending aorta ; t2 aortic arch ; t3 transition aortic arch descending aorta ; t4 halfway t3t5 ; t5 aorta at thoracic spine level th12 ; a1 aorta at lumbar spine level l1 ; a2 halfway a1a3 ; a3 aortic bifurcation ; a4 right common iliac artery above bifurcation ; a5 right common femoral artery ( see fig . was manually placed in the lumen of the aorta , carefully avoiding calcifications ( fig . 1 ) . mean attenuation [ hu ] and standard deviation ( sd ) ( indicator of image noise ) were recorded as well as contrast to noise ratio ( cnr ) . overall image quality was assessed by an experienced radiologist ( 10 years of experience ) for every patient on a five - point scale : grade 1 bad , no diagnosis possible ; grade 2 poor , significantly reduced diagnostic confidence ; grade 3 moderate , sufficient image quality ; grade 4 good ; and grade 5 excellent . body weight , length and age were tested for distribution among groups using a one - way anova . the same test was used in order to compare attenuation and standard deviations between groups . all values are expressed as mean sd or as a percentage with the absolute value in brackets . indications for cta were as follows : preoperative evaluation ( n = 7 ) , evar or open aortic surgery follow - up ( n = 35 ) , aortic aneurysm ( n = 21 ) , clinical suspicion of bleeding ( n = 3 ) , aortic dissection ( n = 1 ) , ischemia ( n = 5 ) , search for source of pulmonary embolism ( n = 2 ) , follow - up of lung tumor ( n = 1 ) and no specified indication ( n = 2 ) . for statistical purposes , patients were divided into 3 groups according to the aorta protocol they were referred for : thoracic aorta ( group 1 ) , abdominal aorta ( group 2 ) or combined thoracic - abdominal aorta ( group 3 ) . a waiver of informed consent was given by the local ethical committee ( metc 14 - 4 - 050 ) . in order to assess scan start delay , all patients received a test bolus ( 10 ml iopromide 300 ultravist , bayer healthcare , berlin , germany at 6 ml / s , followed by 40 ml saline flush at the same flow rate ) through an 18 g i.v . needle ( vasofix , bbraun , melsung , germany ) placed in an antecubital vein . in order to assess time to peak ( ttp ) , a region of interest ( roi ) was placed either at the level of the aortic arch for thoracic or combined thoracic - abdominal ctas , or at the level of l1l2 abdominal aorta ctas , using a dedicated software tool ( dyneva , siemens medical , forchheim , germany ) . scan start delay was calculated from test bolus delay ( 10 s ) + measured ttp + 4 s. during the latter 4 s breathing instructions were given to the patient and the scan table was moved to its starting position . the main bolus consisted of 50 ml cm followed by a 40 ml saline chaser , both injected at a flow rate of 6 ml / s ( idr 1.8 g / s , total iodine load til = 15 g ) . all injection parameters were closely monitored by a dedicated software tool ( certegra informatics platform , bayer healthcare , berlin , germany ) . patients were scanned on a multidetector - row ct ( mdct ) ( 2nd generation dual source scanner , somatom definition flash , siemens , forchheim , germany ) . scan parameters were as follows : 2 128 0.6 mm ( using z - flying focal spot techniques ) , pitch value 0.9 , 100 kv , quality ref . time 0.33 s , 1.0/0.8 mm slice reconstruction and soft tissue filter ( b30f ) . measurements were carried out at 10 different anatomic locations using a dedicated software tool ( syngo.via , siemens , forchheim , germany ) 5 thoracic and 5 abdominal : t1 ascending aorta ; t2 aortic arch ; t3 transition aortic arch descending aorta ; t4 halfway t3t5 ; t5 aorta at thoracic spine level th12 ; a1 aorta at lumbar spine level l1 ; a2 halfway a1a3 ; a3 aortic bifurcation ; a4 right common iliac artery above bifurcation ; a5 right common femoral artery ( see fig . was manually placed in the lumen of the aorta , carefully avoiding calcifications ( fig . 1 ) . mean attenuation [ hu ] and standard deviation ( sd ) ( indicator of image noise ) were recorded as well as contrast to noise ratio ( cnr ) . overall image quality was assessed by an experienced radiologist ( 10 years of experience ) for every patient on a five - point scale : grade 1 bad , no diagnosis possible ; grade 2 poor , significantly reduced diagnostic confidence ; grade 3 moderate , sufficient image quality ; grade 4 good ; and grade 5 excellent . all data were processed via ibm spss statistics ver . 20 ( spss inc . , the same test was used in order to compare attenuation and standard deviations between groups . all values are expressed as mean sd or as a percentage with the absolute value in brackets . seven patients were excluded in the course of the study : one due to incorrect scan delay timing , six due to non - adherence to the cm injection protocol the condition of the venous system of five patients meant that flow rates had to be decreased , and one patient had very low cardiac output with poor test bolus attenuation , leading to an increase in volume of the main cm bolus to 90 ml . no significant difference was found within the age , weight or length distribution between groups . for detailed patient information , the overall mean attenuation was 324 28 hu . the highest attenuation was reached in the region of the aortic arch and the thoracic descending aorta ( t3 ) , the lowest intravascular enhancement was obtained in the common femoral artery ( a5 ) . for detailed information on attenuation in different anatomic regions and groups , see table 2 . the attenuation values for each anatomic region are shown in fig . 2 , and the same results for each scan protocol can be found in fig . overall sd range was 1175 hu , with lowest sd at aortic arch ( t2 ) and highest sd at half way between l1 and aortic bifurcation ( a2 ) . except for the a4 region ( right common iliac artery above bifurcation ) , there was no significant difference among various protocols . mean cnr was 15 ( sd 3 ) . comparison of cnr and snr can be found in fig . pearson correlation of cnr to bmi did show a decrease of cnr with increasing bmi , both in thoracic and abdominal region ( fig . our results show that the use of low volume cm is feasible for aortic cta . compared to our standard cm protocol ( volume 96 ml ) , a reduction of 52% was achieved with the new protocol . however , a combination of 100 kv with optimal scan delay timing is necessary to facilitate this injection protocol . most of the studies have been performed on pulmonary cta , usually associated with a reduction of tube voltage to between 110 and 80 kv , , , , . however , aortic cta has also been the center of research : diehm et al . used 50 ml of cm in the evaluation of aortoiliac aneurysm and compared it to their standard protocol using 100 ml cm ( iodine concentration 320/350 mgi / ml , flow rate 3.5 ml / s ) . in contrast to our study , cta was only performed for the abdominal aorta , and bolus tracking technique was used with roi placed at the suprarenal aorta with a scan delay of 6 s after reaching 200 hu trigger point . the results revealed no significant difference in attenuation levels between groups , despite the use of various cm . compared a standard uniphasic injection protocol of 100 ml cm ( ioversol 350 ) to a multiphasic injection protocol , resulting in a decrease of cm volume to 89 ml in a 120 kv protocol for scanning the abdominal aorta . the multiphasic protocol was established using an exponentially decelerating rate with a decay of 0.01 ml / s , according to outcomes of previous investigations by bae et al . . results were quantitatively and qualitatively comparable but resulted only in 11% reduction in cm volume . the same authors compared their multiphasic injection protocol mentioned in a previous article , to a protocol with a 10 ml test bolus followed by a main bolus of 40 ml ( ioversol 350 ) in 30 patients ( 15 patients per group ) . they did not find any statistically significant difference between attenuations , but it must be noted that only the abdominal aorta was evaluated , and a 120 kv protocol was used . in our study , a 100 kv protocol was used which facilitates higher contrast enhancement due to the contrast to noise ratio ( cnr ) increase described earlier . lowering kv thus allows a reduction in cm volume . as was shown in our study , this reduction is functional not only for abdominal or thoracic cta , but also in combined thoracic - abdominal protocols . it is important to mention , however , that a relatively low volume bolus must be exactly timed . the chosen timing in our study was feasible , but we did experience some decrease of enhancement in peripheral vessels in some patients undergoing combined thoracic - abdominal cta . this could be due to the fact that a fixed bolus was used , which did not account for individual patient differences ( e.g. body weight ) , and which furthermore did not account for longer scan time necessary in thoracic - abdominal examinations in comparison to just thoracic or just abdominal scanning . future developments in scanner technique especially in detector technology might facilitate the use of even lower kv settings in an even broader range of patients , which may lead to further increased contrast enhancement using the same bolus . although 70 kv and 80 kv were possible at the used scanner the necessary tube current to have sufficient low image noise is not always achievable in all patients , thus lowering kv increases image noise if tube current can not be adjusted accordingly . this makes the results applicable to a broad range of scanners and not only to the newest high - end machines . furthermore iterative reconstruction allows reducing image noise also facilitating the use of lower kv settings in future protocols and in scanners which are not capable of higher tube current settings as lower kv settings . it is a single center study performed on a limited number of patients , especially as far as the thoracic aorta protocol is concerned . in addition , only one scan acquisition technique ( spiral , standard acquisition pitch ) was used . more advanced techniques such as high pitch scanning might not be suitable for reduced cm volume , due to the fast image acquisition outrunning the bolus . using low volume cm is beneficial in terms of cin prevention , especially in critically ill patients and patients with known impaired renal function . furthermore , efficient use of cm in combination with lower kv settings could decrease scanning costs . it is a single center study performed on a limited number of patients , especially as far as the thoracic aorta protocol is concerned . in addition , only one scan acquisition technique ( spiral , standard acquisition pitch ) was used . more advanced techniques such as high pitch scanning might not be suitable for reduced cm volume , due to the fast image acquisition outrunning the bolus . using low volume cm is beneficial in terms of cin prevention , especially in critically ill patients and patients with known impaired renal function . furthermore , efficient use of cm in combination with lower kv settings could decrease scanning costs . the use of a relatively low volume of cm is feasible and diagnostic image quality in 100 kv cta of the aorta can be reached . | objectivesusing smaller volumes of contrast media ( cm ) in ct angiography ( cta ) is desirable in terms of cost reduction and prevention of contrast - induced nephropathy ( cin ) .
the purpose was to evaluate the feasibility of low cm volume in cta of the aorta.methods77 patients referred for cta of the aorta were scanned using a standard mdct protocol at 100 kv .
a bolus of 50 ml cm ( iopromide 300 mg iodine / ml ) at a flow rate of 6 ml / s was applied ( iodine delivery rate idr = 1.8 g / s ; iodine load 15 g ) followed by a saline bolus of 40 ml at the same flow rate .
scan delay was determined by the test bolus method .
subjective image quality was assessed and contrast enhancement was measured at 10 anatomical levels of the aorta.resultsdiagnostic quality images were obtained for all patients , reaching a mean overall contrast enhancement of 324 28 hu .
mean attenuation was 350 60 hu at the thoracic aorta and 315 83 hu at the abdominal aorta.conclusionsa straightforward low volume cm protocol proved to be technically feasible and led to cta examinations reaching diagnostic image quality of the aorta at 100 kv .
based on these findings , the use of a relatively small cm bolus can be incorporated into routine clinical imaging . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in australia , a relatively popular strategy , aimed at improving collaboration between clinicians and research focused academics , has been the appointment of a professor of nursing and/or midwifery into a clinical chair position , jointly funded by a university and a health service or hospital ( a joint clinical chair ) . there has been much commentary , especially in australia , about the development of such positions [ 14 ] . what is not so clear from this commentary is what elements surrounding this strategy lead to successful collaborative partnerships and the development of a clinical bridge to enable nursing effectiveness science . by nursing effectiveness science we mean primary , secondary , and translational , clinically focused research activities which aim to improve patient or client outcomes . activities involved in nursing effectiveness science include systematic reviews and meta - analyses of previously conducted research studies , exploratory , correlational , and experimental clinical research studies , and translational research studies aimed at changing clinical practice . in this paper we outline the antecedents that shaped one university - health service collaboration and the change management strategies which helped construct the clinical bridge . we also explore how different types of leadership facilitated the achievement of outcomes , and finally we comment on future directions for nursing effectiveness science . joint clinical chair positions in nursing emerged , in australia , in the late 1980s . by 1996 there were 20 joint clinical chairs in australia and currently there are many more . generally , these positions are appointed at the level of professor although some have been appointed at the level of associate professor . there is also a distinction between generic joint clinical chairs ( i.e. , professor of nursing , professor of midwifery or professor of clinical nursing research ) and specialist joint clinical chairs ( i.e. , professor of critical care nursing ) . in the early days , the focus , goals , and structures associated with these positions were largely driven by individuals . at a two - day symposium , in sydney , in the late 1990s , a number of incumbents and stakeholders described the positions that were then in place and it was clear that there was no coherent model for what the joint clinical chairs would do , whether the focus should be broad or narrow and how they would work across academic and service organisations ( personal communication , m. wallis 2011 ) . qualitative research into the perspectives of a number of the early incumbents of these positions confirmed that there was a diversity of arrangements between university and health sector partners in establishing their respective roles [ 2 , page 165 ] . the main challenge that was identified early in the development of these positions was the potential for unrealistic workloads . as darbyshire expressed it .
not only will the professor be expected to be a top researcher , winning grants , establishing research programmes , publishing , supervising , teaching , leading , consulting , examining , networking , presenting and more , but in the service sector they will also be expected to be a kind of super staff development guru and contract researcher whose role is no less than to change the nursing culture of the organization , improve care and service provision quality and give the research answers that will extinguish the most troublesome clinical , professional or organizational fires of the day . after all , you can hear the hospital executive thinking , we pay half of their salary , so we may as well use them . this quote also alludes to the fact that universities and health service providers may not only have quite disparate cultures , they may also have dissimilar goals and expected outcomes . despite these problems , there continues to be university and healthcare organisational commitment to these positions . in 2000 , the faculty of nursing , at griffith university , in southeast queensland , australia , and the division of nursing in one of the local healthcare services , gold coast health service district , appointed a joint chair , clinical nursing research . if a clinical bridge is to be built to support nursing effectiveness science , the first things that have to be in place are senior people with vision and the resources to support that vision [ 3 , 4 , 6 ] . on the gold coast , in queensland , australia , in 1997 , the two people with vision were the dean of the faculty of nursing , at griffith university , and the executive director of nursing ( edon ) of the local health service . these two transformational leaders had a mutual respect for the contribution of healthcare services and academic institutions to professional nursing and to client outcomes . while the role of the edon necessarily focused on service delivery , there was a clear understanding of the value of a collaborative approach to healthcare that incorporated research and education . the dean was clear about the critical importance of clinical practice to the discipline and the need for clinically informed and clinically relevant research . together , because of their perspectives , they developed a plan to address the theory - practice gap that was evident in australia at the time . the plan that was developed , and eventually endorsed by senior management in both organisations , was to appoint a joint clinical chair . the dean and edon had carefully considered what was required within the local context and had decided that this needed to be a transitional position . the joint clinical chair was not designed to last forever and the need for change and evolution of the role were incorporated from the beginning . it would have a generic focus ( i.e. , nursing ) but would specifically include in the title the word a contract was drawn up for a joint chair , clinical nursing research , and appropriate resources were allocated not just to the funding of the position , but also to research nurse support at the clinical site . the incumbent was to be employed by the university , but half the salary was paid by the health service . office space was provided within both organisations although more time was to be spent on site at the hospital . based on their assessment of the clinical context , the dean and edon decided that the first step was to appoint a leader who could drive the development of the people , the infrastructure and the capacity required to build an ongoing program of clinical nursing research . this position was always seen by all parties as the beginning of a collaboration that would be focused on building a clinical research culture within the health service and on developing then the faculty of nursing ( now school of nursing and midwifery ) into a leading nursing research facility . as such , the second vitally important element to the successful development of the joint clinical chair was clarity and unity related to the anticipated outcomes for the position . the dean and the edon were both broadly in agreement that the goals for the position were to increase research funding from external , competitive funding bodies , increase peer - reviewed publications , and increase the number of ph.d . completions ; and they allowed the first incumbent to negotiate the key performance indicators and the timelines . when building the clinical bridge to facilitate effectiveness science on the gold coast , an approach was taken that integrated lewin 's model of unfreezing , moving , and refreezing with leadership approaches that included both servant leadership and transformational leadership . figure 1 depicts the cyclical nature of the bridge building process , outlines some of the key elements , and indicates leadership approaches that can bring success . in 2000 , when the joint chair , clinical nursing research , was appointed , a sense of urgency and excitement was palpable within both organisations . as kotter suggests establishing this sense of excitement is important as it motivates people to get outside their comfort zones and contributes to lewin 's unfreezing phase . local and regional promotion of the new position built on this excitement and an initial process of meeting and discussing potential collaborative endeavours provided the opportunity for the incumbent to commence the position with a servant leader focus . they give priority to their relationships with followers rather than their relationship with the organization and emphasise followers ' holistic needs , development , and autonomy . in contrast , transformational leaders focus on mobilizing followers to achieve performance beyond expectations , which is the ultimate priority of the organisation . if any leadership position is to function effectively , there also have to be willing followers and collaborators . both organisations had enthusiastic academics and clinicians who had already begun to establish links around specialist clinical education and clinical research projects . one of the things that made the bridge easier was the movement of specialty postgraduate nursing education from the hospital setting to the university which occurred a few years prior to the establishment of the joint clinical chair . as another element in the unfreezing of the clinical and academic environments ( see figure 1 ) , specialist master - of - nursing programs ( e.g. , critical care nursing , emergency nursing , etc . ) were offered in a university - hospital partnership model . consequently , university academics and hospital - based clinical educators had established excellent working relationships . also because the courses were at the master - of - nursing level , the students ( many of whom were clinical leaders ) received some research training , increasing their appreciation of the value of clinical research . prepared academic was already working with a clinical manager , a clinical specialist , and an educator and building a strong track record of research around icu nursing . despite this ground work , in 2000 the gold coast health service district was not a research - ready , let alone a research intensive , healthcare facility ; it was primarily focused on service provision for members of the local community . the faculty of nursing at griffith university was similarly just beginning its development as a centre for clinical research . there were many researchers beginning their careers , but there was little focus and no critical mass of researchers who were working together either in the university or the health district . as southeast queensland became the focus of internal migration within australia , and the population along the corridor from gold coast to brisbane boomed , it became clear that there would need to be a huge expansion in healthcare services and that this presented a unique opportunity to build a research centre that would not only improve the health of queenslanders , but also could develop into a centre of international repute . as discussed by ba banutu - gomez [ 15 , page 147 ] the central role of a servant leader is to establish a sustainable strategic vision for the organisation . thus at the end of 2002 , as a strategy both to complete the unfreezing stage and then to direct the moving stage of change ( see figure 1 ) , the joint clinical chair and other leaders in the nursing faculty , building on their beginning successes in collaborative clinical research , wrote a proposal for a research centre to focus on clinical innovation . the griffith university research centre for clinical and community practice innovation ( rcccpi ) was launched in 2003 . while initially rcccpi was a collaboration between the griffith university school of nursing and midwifery and gold coast health , it expanded quickly and membership now includes researchers in six major teaching hospitals in southeast queensland and has strong links to universities in australia , canada , the uk , and the usa . there were a number of iterations of the programs within the centre until eventually significant multidisciplinary teams of people coalesced around the areas of acute and critical care ; ageing and older people ; nutrition ; and maternity and family . the leadership of rcccpi maintained and grew the strong links with the joint clinical chair and the growing number of clinicians engaged in research in the health service . more clinicians enrolled in and graduated with research degrees and the joint clinical chair and senior research colleagues started to acquire competitive grants . in order to maintain the momentum and to keep the change process moving , a number of strategies were employed that were designed to build effective teams and to build team capacity ( see figure 1 ) . one of these strategies was to conduct workshops for clinicians related to evidence - based practice . university - based academics as well as the joint clinical chair and hospital - based researchers all contributed to these workshops . funding was sought to allow nurses to attend these workshops in work hours , and an evaluative research study of these workshops clearly indicated positive attitudinal change in clinicians . the health service then had a cohort of senior clinicians who were primed with a critical approach to their practice and ready to move into research . having a joint research seminar series between rcccpi and the health service meant that research conversations between academic and clinical nurses continued that movement . other early strategies that worked included setting up a visiting nursing and midwifery research fellow program and setting up a mentorship program for clinicians involved in research . the visiting nursing and midwifery research fellows were griffith university nurse researchers who would come and work in an honorary capacity with clinicians in particular specialist areas . they were appointed in areas such as icu , community care , mental health nursing , and midwifery and they joined teams of clinical nurses and midwives to help develop programs of research in specialist areas . funding for research activities , in the early days , was a challenge , as is commonly the case . establishing a funding stream for research activities is , however , vital if the clinical bridge is to be sustained and the change to a research intensive clinical culture is to be refrozen . so various strategies were employed including : always having research teams that incorporated academics and clinicians ( something that appeals strongly to the organisations that fund clinical research ) ; focusing research activity on areas of high interest to funding bodies ; approaching local health service power brokers with clearly articulated proposals for projects of mutual benefit ; accepting appointments to strategic committees , at higher levels of government ; linking with medical colleagues to establish research positions within the clinical environment ; and finally making sure that there were working relationships and strategic alliances at all levels in the health department . together all these strategies , and the work of a growing number of collaborative groups , began to bear fruit . as a way of both measuring and celebrating the successes of the first few years , the health service produced a compendium of nursing research every second year . both the health service and the university produced many reports about their total activities , but the research compendium served to celebrate , specifically , the successes of teams that involved the clinical nursing staff and served to highlight the strong connections between the two organisations . table 1 is a reproduction of the table originally published in the 2009 compendium . in the early phase of building the clinical bridge , the successes steadily accumulated but it was not until 2005 that significant funding successes began to flow through to research outputs such as publications in international journals . the joint clinical chair and visiting nursing and midwifery research fellows worked closely with clinicians and followed their suggestions for areas of research focus . by weaving this approach with a transformational leadership approach , which involves shifting the values , beliefs , and needs of followers to meet organisational goals through empowering and building capacity in the workforce , a firm foundation for building a program of nursing effectiveness science was established ( see figure 1 ) . in the initial period , while a successful track record was being established , by the joint clinical chair and teams of clinicians , there were two interlinked strategies that helped the health service appreciate the value of the joint clinical chair . these strategies were increasing the number of clinicians who successfully completed research - based masters programs and engagement in smaller projects that resulted in clinical practice changes within the local health service . examples of research studies that resulted in changes in practice are listed in table 2 . by 2005 rcccpi was expanding and the number of university researchers working in collaboration with clinicians , in a number of health services in southeast queensland , was expanding . these nurse researchers brought additional skills in effectiveness science that , in turn , led to greater grant success . one team was successful in attracting over aud 500,000 for a randomised controlled trial of routine removal of peripheral intravenous catheters compared to removal on clinical indication , from the australian national health and medical research council ( nhmrc ) ( similar to but much smaller than the us national institutes for health ) . the nhmrc traditionally allocates less than 3% of its funds to research lead by nurses , so this was a considerable breakthrough for the team . this grant success , and the ultimately successful completion of the research project , would not have been possible without the excellent collaborative working relationships , developed over these past years , between the university academics and their clinical partners , in a number of hospitals in southeast queensland . under the transformational leadership of the rcccpi director , researchers were able to formalise links with a number of international research teams , continue to strengthen their clinical collaborations , and expand their research capacity . health - service - based clinical nurse researchers were offered adjunct positions with rcccpi ( i.e. , these researchers were employed by their hospitals but were given adjunct or honorary positions in griffith university ) . one of the rcccpi adjunct professors at another hospital in southeast queensland had a strong link with the cochrane collaboration wounds group . these activities were successfully refreezing the academic and clinical environments into one where research was the norm , especially in the acute care , in - patient areas of a number of southeast queensland health services ( see figure 1 ) . the rcccpi director with assistance from other griffith university professors and the joint clinical chair then led the team in a successful application to become an nhmrc centre of research excellence . the nhmrc manages the australian government 's competitive grant process that funds health - related research . in addition to managing the competitive process for project grants , program grants , and a variety of research fellowships , it also funds a very small number of centres for research excellence . the centre for research excellence in nursing interventions for hospitalised patients ( ncren ) , established in 2011 , focuses on providing evidence to improve the nursing care of a broad range of hospitalised patients who have compromised skin integrity and/or require symptom management . these two particular areas were chosen because of ( 1 ) their high risk and high cost ; ( 2 ) the research expertise within the team ; and ( 3 ) registered nurses are largely responsible for patient care related to both skin integrity and symptom management . this is the first ever nhmrc centre of research excellence to be awarded to a centre focused on nursing . ncren now sits within rcccpi as one strand of research . both university academics ( including the joint clinical chair ) and clinicians in the local health service are named investigators within this new centre for research excellence , a clear statement of the strength of the clinical bridge . in line with the thoughts of the original leaders who envisioned the joint chair , clinical nursing research , it became clear that the success of the collaboration and the strength of the clinical bridge could support the growth and evolution of the joint clinical chair position . in 2006 griffith university then in 2011 griffith university and the health service on the gold coast were successful in securing funding for a chair in midwifery from the department of health . when the incumbent of the chair , clinical nursing research , resigned in early 2011 , the health service and griffith university decided that the groundwork had been accomplished and that it was time for the chair to evolve from a generic chair to a more focused chair in acute and complex care nursing . there are also longer term plans to appoint clinical chairs in other specialist areas such as mental health nursing , aged care nursing , and community care . ncren has been funded for five years and the team of researchers involved are back at the first element in the model depicted in figure 1 . the increased research activity demanded by ncren 's success will put stress on the clinical bridge . as clinicians struggle to maintain their standards in the face of increased clinical demand and now increased research demand , it behooves the research leadership to remain cognisant of the need for followership which is inherent within the models of servant and transformational leadership which have been manifestly successful to date . | nursing effectiveness science includes primary , secondary , and translational , clinically focused research activities which aim to improve patient or client outcomes .
it is imperative , for the successful conduct of a program of nursing effectiveness science , that a clinical bridge is established between academic and healthcare service facilities .
an australian example of the development of a robust clinical bridge through the use of jointly funded positions at the professorial level is outlined .
in addition , an analysis of the practical application of lewin 's model of change management and the contribution of both servant and transformational leadership styles to the bridge building process is provided . |
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according to the statistics of the world health organization in 2014 , the per capita medical expenditure of all countries showed a significant growth trend in the past 10 years . from 2000 to 2011 , the per capita consumption of medical consumption of the united states increased by 96.30% , the united kingdom 92.50% , japan 76.20% , australia 75.40% , germany 60.50% , and france 56.30% . as for the case of china , the per capita medical expenses increased from usd 41 dollars in the year 2000 to usd 236 dollars in 2011 , an increase of nearly five times . high medical costs have caused serious economic burdens to the society as well as individuals . thus , seeking an appropriate strategy for medical cost control to reduce the expenses of medical services and to keep the coordinated development between the health care and social economy is one of the key points of health care reforms in china . since inpatient medical expenditure is a crucial part as well as an important indicator of the overall medical cost , the research on the influence factors of medical expenses is in constant progress and development . the article aimed to analyze the distribution characteristics as well as the influence factors of high impatient expenditure cases through various aspects and multiple angles to explore the causes of high inpatient expenditure , and supply practical suggestions for strengthening inpatient expenditure management in hospitals , reducing unreasonable medical costs , regulating diagnosis and treatment behaviors , and improving medical quality . the standard for high inpatient expenditure varies greatly between different areas and periods . by analyzing current researches combined with the standard of medical service charges in china , we set rmb 100,000 yuan as the standard and included the inpatient cases in a grade - a tertiary hospital during 20132015 that met the criteria in the research . the detailed information of the patients included the admission form , diagnoses , treatments , therapeutic results , prognoses , length of hospital stay , international classification of diseases ( icd)-10 codes , complications of the disease , rescues , lists of surgeries , secondary operations , and other basic information such as age , name , gender , social characteristics , departments for treatment , and total costs ( including medicine cost , material cost , treatment cost , ward cost , blood transfusion cost , inspection cost , physical test cost , operation cost , and other cost ) ; all the information were extracted from the medical record system . according to the data available , we set the following 8 indexes as independent variables to identify the major factors that led to high inpatient expenditure : age , gender , length of hospital stay , death , surgery , complications of the disease , hospital infections , and admission form , while setting the total cost as the dependent variable to conduct multivariate analysis of average medical expense . using multiple regression analysis to quantify the index and set sub - variables for polytomous variables all the data and material collected were entered into excel 2010 software for microsoft ( microsoft corporation , washington , usa ) , and statistical analyses were performed using spss 14.0 ( spss inc . , single factor analysis of age was analyzed using nonparametric test ; admission condition , hospital infection , the occurrence of rescue , complications of diseases , occurrence of death , and surgery were analyzed using t - test ; variance of analysis ( anova ) and multivariate linear regression were used to analyze the degree of the influence factors above . the standard for high inpatient expenditure varies greatly between different areas and periods . by analyzing current researches combined with the standard of medical service charges in china , we set rmb 100,000 yuan as the standard and included the inpatient cases in a grade - a tertiary hospital during 20132015 that met the criteria in the research . the detailed information of the patients included the admission form , diagnoses , treatments , therapeutic results , prognoses , length of hospital stay , international classification of diseases ( icd)-10 codes , complications of the disease , rescues , lists of surgeries , secondary operations , and other basic information such as age , name , gender , social characteristics , departments for treatment , and total costs ( including medicine cost , material cost , treatment cost , ward cost , blood transfusion cost , inspection cost , physical test cost , operation cost , and other cost ) ; all the information were extracted from the medical record system . according to the data available , we set the following 8 indexes as independent variables to identify the major factors that led to high inpatient expenditure : age , gender , length of hospital stay , death , surgery , complications of the disease , hospital infections , and admission form , while setting the total cost as the dependent variable to conduct multivariate analysis of average medical expense . using multiple regression analysis to quantify the index and set sub - variables for polytomous variables , we analyzed the impact of the major factors to inpatient medical expenditure comprehensively . all the data and material collected were entered into excel 2010 software for microsoft ( microsoft corporation , washington , usa ) , and statistical analyses were performed using spss 14.0 ( spss inc . , single factor analysis of age was analyzed using nonparametric test ; admission condition , hospital infection , the occurrence of rescue , complications of diseases , occurrence of death , and surgery were analyzed using t - test ; variance of analysis ( anova ) and multivariate linear regression were used to analyze the degree of the influence factors above . according to the statistics , 65,520 inpatients were admitted to the hospital during 20132015 , with the average expenditure of rmb 21,556 yuan and an average length of hospital stay of 9.94 days . a number of 2041 cases ( cost more than rmb 100,000 yuan / person ) made up 3.12% of the total number of inpatients ; while the total cost of the cases was rmb 31.06 million yuan , which made up 21.99% of the total inpatient revenue of the hospital , the average cost of the cases was rmb 152,173 yuan , with the average length of hospital stay of 28.39 days / person . the total cost of the inpatient expenditure comprised medicine costs ( including cost for pharmaceutics , chinese patent drugs , and herbal medicines ) , material cost ( including coronary stents , stapling devices , and other materials of high values ) , surgery cost ( mainly referring to the value of the labor of hospital staffs for operations , and cost for drugs and anesthesia in the process were not included in this item ) , ward cost , diagnosis cost ( including cost for imaging inspections , pathological and laboratory examinations ) , treatment cost ( including dialysis and other treatment measures ) , nursing cost , and other costs . according to the statistics , the compositions of high inpatient expenditure during the 3 years are shown in table 1 . compositions of the total cost for high inpatient expenditure as shown in table 2 , the groups aged 5070 years were the majority of high inpatient expenditure cases . since the inpatient expenditure was presented as nonnormal distribution , we used nonparametric test to process the data , indicating that high inpatient expenditure was not directly related to age ( = 13.872 , p = 0.079 ) . age distributions of high inpatient expenditure cases ( n = 2,041 ) length of hospital stay as shown in table 3 , the average cost of each group varied greatly , and the average cost increased with the length of hospital stay . distributions of the length of hospital stay of high expenditure cases ( n = 2041 ) characteristics of the diseases according to the statistics from table 4 , patients of severe , emergency , and moderate conditions made up 15.33% , 42.53% , and 42.14% of 2041 cases , respectively , with the average cost of rmb 162,823 yuan , rmb 154,132 yuan , and rmb 146,880 yuan , respectively . patients with or without hospital infection were 2.11% and 97.89% of the total , and the average cost of rmb 159,191 yuan and rmb 152,071 yuan , respectively . patients who experienced rescue and those without made up 27.68% and 72.32% of the total population , with the average costs of rmb 156,376 yuan and rmb 146,512 yuan , respectively . patients with and without complications of the diseases made up 3.82% and 96.18% of the total , with the average costs of rmb 158,825 yuan and rmb 148,545 yuan , respectively . death and non - death cases made up 3.48% and 96.52% , respectively , with the average cost of rmb 178,801 yuan and rmb 150,667 yuan . cases of surgeries ( among which 50 underwent the secondary operation ) and those without comprised 82.61% and 17.39% of the population , with the average costs of rmb 152,682 yuan and rmb 150,014 yuan , respectively . single factor analysis from table 4 showed that admission condition , surgery , and hospital infection were not directly related to high inpatient medical expenditure , while complications of diseases , occurrence of death , and rescue showed a significant difference in medical expenses . characteristics of the disease related to high inpatient expenditure cases ( n = 2,041 ) * f value ; t value . the first line of each group was the control group ; p : sample group versus control group . we set the indexes of age , gender , length of hospital stay , death , surgery , complications of the disease , hospital infections , and admission conditions as independent variables and the total cost as dependent variable to conduct multivariate analysis of inpatient medical expense . results showed that complications of diseases , criticality of the disease , gender , occurrence of death , and length of hospital stay all had a great impact on inpatient medical expenditure while age , hospital infection , and surgery showed no significant impact on the inpatient medical cost . with the increased length of hospital stay by 1 day the average cost for severity and emergency cases were rmb 15,942 yuan and rmb 7864 yuan , higher than that for cases of moderate , respectively . multivariate analysis of high inpatient expenditure cases the first line of prognosis , admission condition , and gender groups were the control group ; p : sample group versus control group . as shown in table 6 , patients with diseases of skeletal and muscular system , circulatory system , and tumor comprised a large proportion of the population , which altogether accounted for 90.48% of the total , indicating that treating such diseases relatively cost higher than those of other disease categories . there were 796 cases of skeletal and muscular system diseased patients , which accounted for 39.00% of the investigated population ; 683 cases of circulatory system diseased patients , accounted for 33.46% of the total ; 368 cases of tumor , accounted for 18.03% ; and 132 cases of respiratory system diseased patients , accounted for 6.47% of the total . as for the average cost of patients , the alimentary system diseases ranked the top , with the average cost of rmb 183,715 yuan ; respiratory system ranked the next , with the average cost of rmb 179,347 yuan ; the average cost for diseases of skeletal and muscular was rmb 159,158 yuan , ranking the third . according to the statistics , 65,520 inpatients were admitted to the hospital during 20132015 , with the average expenditure of rmb 21,556 yuan and an average length of hospital stay of 9.94 days . a number of 2041 cases ( cost more than rmb 100,000 yuan / person ) made up 3.12% of the total number of inpatients ; while the total cost of the cases was rmb 31.06 million yuan , which made up 21.99% of the total inpatient revenue of the hospital , the average cost of the cases was rmb 152,173 yuan , with the average length of hospital stay of 28.39 days / person . the total cost of the inpatient expenditure comprised medicine costs ( including cost for pharmaceutics , chinese patent drugs , and herbal medicines ) , material cost ( including coronary stents , stapling devices , and other materials of high values ) , surgery cost ( mainly referring to the value of the labor of hospital staffs for operations , and cost for drugs and anesthesia in the process were not included in this item ) , ward cost , diagnosis cost ( including cost for imaging inspections , pathological and laboratory examinations ) , treatment cost ( including dialysis and other treatment measures ) , nursing cost , and other costs . according to the statistics , the compositions of high inpatient expenditure during the 3 years are shown in table 1 . as shown in table 2 , the groups aged 5070 years were the majority of high inpatient expenditure cases . since the inpatient expenditure was presented as nonnormal distribution , we used nonparametric test to process the data , indicating that high inpatient expenditure was not directly related to age ( = 13.872 , p = 0.079 ) . age distributions of high inpatient expenditure cases ( n = 2,041 ) length of hospital stay as shown in table 3 , the average cost of each group varied greatly , and the average cost increased with the length of hospital stay . distributions of the length of hospital stay of high expenditure cases ( n = 2041 ) characteristics of the diseases according to the statistics from table 4 , patients of severe , emergency , and moderate conditions made up 15.33% , 42.53% , and 42.14% of 2041 cases , respectively , with the average cost of rmb 162,823 yuan , rmb 154,132 yuan , and rmb 146,880 yuan , respectively . patients with or without hospital infection were 2.11% and 97.89% of the total , and the average cost of rmb 159,191 yuan and rmb 152,071 yuan , respectively . patients who experienced rescue and those without made up 27.68% and 72.32% of the total population , with the average costs of rmb 156,376 yuan and rmb 146,512 yuan , respectively . patients with and without complications of the diseases made up 3.82% and 96.18% of the total , with the average costs of rmb 158,825 yuan and rmb 148,545 yuan , respectively . death and non - death cases made up 3.48% and 96.52% , respectively , with the average cost of rmb 178,801 yuan and rmb 150,667 yuan . cases of surgeries ( among which 50 underwent the secondary operation ) and those without comprised 82.61% and 17.39% of the population , with the average costs of rmb 152,682 yuan and rmb 150,014 yuan , respectively . single factor analysis from table 4 showed that admission condition , surgery , and hospital infection were not directly related to high inpatient medical expenditure , while complications of diseases , occurrence of death , and rescue showed a significant difference in medical expenses . characteristics of the disease related to high inpatient expenditure cases ( n = 2,041 ) * f value ; t value . the first line of each group was the control group ; p : sample group versus control group . we set the indexes of age , gender , length of hospital stay , death , surgery , complications of the disease , hospital infections , and admission conditions as independent variables and the total cost as dependent variable to conduct multivariate analysis of inpatient medical expense . results showed that complications of diseases , criticality of the disease , gender , occurrence of death , and length of hospital stay all had a great impact on inpatient medical expenditure while age , hospital infection , and surgery showed no significant impact on the inpatient medical cost . with the increased length of hospital stay by 1 day the average cost for severity and emergency cases were rmb 15,942 yuan and rmb 7864 yuan , higher than that for cases of moderate , respectively . multivariate analysis of high inpatient expenditure cases the first line of prognosis , admission condition , and gender groups were the control group ; p : sample group versus control group . as shown in table 6 , patients with diseases of skeletal and muscular system , circulatory system , and tumor comprised a large proportion of the population , which altogether accounted for 90.48% of the total , indicating that treating such diseases relatively cost higher than those of other disease categories . there were 796 cases of skeletal and muscular system diseased patients , which accounted for 39.00% of the investigated population ; 683 cases of circulatory system diseased patients , accounted for 33.46% of the total ; 368 cases of tumor , accounted for 18.03% ; and 132 cases of respiratory system diseased patients , accounted for 6.47% of the total . as for the average cost of patients , the alimentary system diseases ranked the top , with the average cost of rmb 183,715 yuan ; respiratory system ranked the next , with the average cost of rmb 179,347 yuan ; the average cost for diseases of skeletal and muscular was rmb 159,158 yuan , ranking the third . the statistical analysis of the 2041 cases of high inpatient expenditure showed that there were a number of factors for the occurrence of high medical costs that came with regularity . first , the average length of hospital stay was relatively long of the cases . according to the study , the average length of hospital stay for the 2041 high medical expenditure patients was 28.39 days / person , while the average length of hospital stay for regular patients was 9.94 days / person . statistics showed that the average length of hospital stay in the united states is 6.9 days / person , australia is 7.4 days / person , and ireland is 9.0 days / person while the average length of hospital stay for grade - a tertiary hospital of china is 13.5 days / person . researches also showed that the excessive length of hospital stay is not entirely due to the needs of clinical diagnosis and treatment . moreover , the phenomenon might be a large extent , results from defects of the medical management control , and low work efficiency . since the length of hospital stay is directly related to inpatient expenditure in the premise of ensuring medical quality and safety , taking various measures such as shortening length of hospital stay and speeding up the bed turnover rate will not only improve the work efficiency of the hospital , but also beneficial to the limitation of high medical expenses . in addition , the research showed that fewer people consumed more medical resources . in the study , we found that 3.12% of the patients consumed 21.99% of the total inpatient medical expenses during 3 years . such high costs laid heavy burdens on individuals and the society , which also demonstrated that the distribution of medical resources was not so fair . besides , the study also revealed that the categories of diseases were concentrated among high medical costs group . patients with diseases of skeletal and muscular system , circulatory system , and tumor made up 90.48% of the population , indicating that the diagnosis as well as treatment of those disease categories cost much more higher than that of other disease categories , which meant diagnosing and treating these diseases were more difficult and called for advanced medical technologies in the process . there were 71 death cases among the 2041 high inpatient expenditure cases , accounted for 3.48% of the group , about four times of the average mortality of the hospital . moreover , the results also showed that high expenditure cases distributed in various age groups , indicating that elderliness was not a major influence factor for high medical expenses . differences in admission , death , and complications all had a great impact on medical expenses . the condition of admission , death , and complications all in some extent reflected the severity of the illnesses . cases of acute or severe illnesses consumed more medical resources and took longer to cure and , thus , spent higher medical expenses . according to the statistics , patients in severe and emergency conditions accounted for 57.91% of the high - cost groups , patients with complications accounted for 3.82% , and rescue cases accounted for 27.68% , respectively . comparing with the general , patients of the high costs group relatively suffered from more serious illnesses . most of these cases were in late stage of cerebral vascular diseases , terminal cancers , and end - stage organ failures . thus , it was not hard to understand diagnosing and treating such illnesses might cost more than regular diseases . last but not least , the costs for medicine and material took up a large proportion of the inpatient expenditure cases , which caused an irrational structure of medical expenses . according to the statistics , the average costs for medicine , materials , diagnosis , and treatment of the high inpatient expenditure cases were rmb 10,260 thousand yuan , rmb 10,040 thousand yuan , rmb 3,520 thousand yuan , and rmb 3,170 thousand yuan , accounted for 33.03% , 32.32% , 11.34% , and 10.19% of the total cost , respectively . researches showed that the average cost of medicine in developed countries was below 10.00% . while the possible reasons for high medicine and materials costs in china were high pricing , the excessive use by doctors as well as the imperfection of medical policies and so on . by randomly selecting some high expenditure cases to analyze the prescriptions in depth , we found that high - class antibiotics and auxiliary therapeutic drug of high costs were involved in the therapeutic process . the improper use of medicine would lead to the increase of bacterial resistance as well as the waste of medical resources , meanwhile , aggravating the burden on patients and the society . on the other hand , the fact that materials cost accounted for a larger proportion of hospital cost suggested that the hospital should strengthen its supervision on the consumption of high - value materials . classification analyses showed that the highest proportion of medicine cost was skeletal and muscular system diseases ( 39.00% ) . since the cost for medicine and materials took up over 60.00% of the total , it had become the primary cost for inpatient medical expenditure . by reviewing the medical records of some typical cases , we found that the application of high - level antimicrobial drugs and high adjuvant drugs in these cases was relatively common . in addition , we found that unreasonable material costs have also gradually become a serious issue in those cases , while the income for the labor of medical personnel was relatively low , and the values for the technical of medical staffs cost were hardly reflected in the process . the study was based on a single - center study due to the differences in the characteristics of the population as well as the medical situation of different areas , and the results of the study might not be applied to other medical institutions . besides , due to the accessibility of the data and information of the patients , more samples should be adopted for the generalization of the conclusion . in conclusion , according to the research , complications of diseases , criticality of the disease , gender of the patients , death , and length of hospital stay all had a great impact on inpatient medical expenditure , while age , hospital infection , and surgery were not directly related to the average cost . the main factors for high inpatient expenditure included the inadequate use of medicines and materials of high value , lacking cost control measures within the hospital , and the excessive average length of hospital stay for inpatients as well as the unnecessary treatment for the patients . thus , it is crucial for medical administration departments to establish strong supervision mechanisms and adopt various cost control measures to reduce inpatient medical expenditure . this study was supported by the grant from the beijing hospital nova project ( no . this study was supported by the grant from the beijing hospital nova project ( no . | background : the study was to explore the causes of high inpatient expenditure through analyzing the distribution characteristics as well as the influence factors of high inpatient expenditure cases during 3 years within a grade - a tertiary hospital through various aspects and multiple angles , thus identifying the major influence factors for high medical expenditure to develop further research.methods:we retrospectively studied 2041 inpatient cases which cost more than rmb 100,000 yuan per case in a grade - a tertiary hospital from 2013 to 2015 .
we analyzed the compositions of the medical cost to evaluate the major factors that cause the high inpatient expenditure .
all the data and materials were collected from medical record system , and the statistical methods included t - test , variance of analysis , and multivariate linear regression.results:the average cost of the 2,041 cases was rmb 152,173 yuan for medicines and materials of medical costs , which respectively accounted for 33.03% and 32.32% of the total cost ; and the average length of hospital stay was 28.39 days / person .
diseases of skeletal and muscular system , circulatory system , and tumor were the top three disease categories of high inpatient expenditure , which accounted for 39.00% , 33.46% , and 18.03% , respectively .
complications , criticality of the disease , gender of the patients , the occurrence of death , and the excessive length of hospital stay all had great impacts on average medical expenditure , while age , hospital infection , and surgery showed no significant impact on average medical cost.conclusions:the main factors for high inpatient expenditure included the inadequate use of high - value medicines and materials , lacking cost control measures within the hospital , the excessive length of hospital stay for inpatients , and the unnecessary treatment for the patients . |
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necrotizing soft tissue infections ( nstis ) are rare but highly lethal with a reported mortality rate of up to 76% . prior reports have identified prompt treatment ( defined by surgical debridement within 824 h after the patient arrives at the emergency department ) as leading to decreased mortality rates . the general agreement among surgeons is that the sooner one operates and debrides the necrotic tissue , the better the outcome . however , how early is early enough has not been well - defined . in our practice most of our patients with nstis undergo an operation within the first 24 h after their arrival or diagnosis . however , we could not find any large study that reported operations in the first 24 h , probably because some patients need aggressive resuscitation before the operation . we sought to examine the effect of early surgery on patients ' outcomes and , in particular , on hospital length of stay ( los ) and intensive care unit ( icu ) los . our hypothesis was that operating within the first 6 h after diagnosis of nstis would improve outcomes . the university of arizona institutional review board approved this as a single institution observational retrospective study , which we conducted in our urban academic level i trauma center . we analyzed the records of those who underwent an operation for nstis ( defined by necrotizing fasciitis , gas gangrene , and fournier gangrene ) over a 6-year period ( january 2003 through december 2008 ) . in our computer - generated search of our medical database , we used the international classification of diseases version 9 codes ( icd-9 ) discharge codes for nstis for necrotizing fasciitis ( 728.86 ) , gas gangrene ( 040.0 ) , and fournier gangrene ( 608.83 ) . the study group included only patients whose diagnosis was confirmed by operative findings revealed by analysis of the electronic medical record . patients were categorized into two groups by time of surgery ( i.e. , the interval from being diagnosed by the surgery team in the emergency department or elsewhere in the hospital to surgical intervention ) : group i , early ( < 6 h ) and group ii , late ( 6 h ) . for these two groups , we compared baseline demographic characteristics , symptoms , and outcomes . septic shock was defined as sepsis - induced hypotension in terms of systolic blood pressure < 90 mmhg or a fall in systolic blood pressure of > 40 mmhg that are persisting despite adequate fluid resuscitation . data were presented as proportion , mean ( standard deviation ) , median ( range ) , and interquartile range ( iqr ) , whenever applicable . student t - test was performed to compare continuous variables and the pearson chi - square ( ) test for categorical variables . pearson correlation coefficient was used to assess for any correlation between the time to intervention and the icu los . we considered any differences to be statistically significant when the two - tailed p < 0.05 . further , to assess for any correlation between the total hospital los and the time to intervention ( < 6 vs. 6 h ) , we used a side - to - side boxplot . to evaluate the clinical predictors of early diagnosis of nstis , we performed multivariate logistic regression analysis and determined the adjusted odds ratio and 95% confidence interval . for all of our data analysis , we used the statistical package for social sciences version 18 ( spss inc . , chicago , usa ) . data were presented as proportion , mean ( standard deviation ) , median ( range ) , and interquartile range ( iqr ) , whenever applicable . student t - test was performed to compare continuous variables and the pearson chi - square ( ) test for categorical variables . pearson correlation coefficient was used to assess for any correlation between the time to intervention and the icu los . we considered any differences to be statistically significant when the two - tailed p < 0.05 . further , to assess for any correlation between the total hospital los and the time to intervention ( < 6 vs. 6 h ) , we used a side - to - side boxplot . to evaluate the clinical predictors of early diagnosis of nstis , we performed multivariate logistic regression analysis and determined the adjusted odds ratio and 95% confidence interval . for all of our data analysis , we used the statistical package for social sciences version 18 ( spss inc . , chicago , usa ) . our study population comprised a total of 87 patients ( 62% male , 38% female ) with nstis . the mean age was 46 17 years . in almost 50% of the patients , the anatomic location of the nstis was the lower extremity , followed by the upper extremity , fournier gangrene , buttocks , and trunk . the most common systemic presentation was tachycardia ( 70% in group i vs. 57.4% in group ii ) , followed by hypotension ( 45% vs. 39% ) and tachypnea ( 33.3% vs. 30% ) . the most common wound characteristic was erythema ( 92% vs. 87% ) , followed by tenderness ( 98% vs. 95% ) , necrosis ( 50% vs. 45% ) , bullae ( 24% vs. 17.5% ) , and foul smelling ( 15% vs. 21.7% ) . the mean number of surgical intervention in each group was 3 [ table 1 ] . patient characteristics and outcomes median time to intervention in the two groups was 3 h ( range 24 , iqr 2 ) and 24 ( range 660 , iqr 18 ) , respectively . we found no statistically significant difference in morbidity and mortality between the two groups [ figure 1 ] . however , group i had a significantly shorter time to surgery ( 2.95 1.1 vs. 22.3 17.8 h ) , a significantly shorter median hospital los ( 13.5 [ 433 ] vs. 18 [ 182 ] days ) , and a significantly shorter median icu los ( 4 vs. 10 days ) than group ii [ table 1 ] . morbidity and mortality the correlation between time to surgery and the total hospital los is shown in a side - by - side boxplot in figure 2 . group i ( early operative group ) had a significantly shorter mean hospital los and a significantly shorter median icu los . moreover , there was a correlation between the time to surgery and icu los ( r = 0.27 , p = 0.01 ) . there were no significant differences between the two groups with regard to laboratory findings [ table 2 ] , comorbidities [ table 3 ] , and initial diagnosis ( such as cellulitis or abscess ) or location of that initial diagnosis [ table 4 ] . in addition , in our multivariate logistic regression analysis , we found no single clinical sign , on admission , that independently predicted early diagnosis of nstis [ table 5 ] . time to surgery and hospital length of stay associated comorbidities initial diagnosis on admission and location clinical predictors , on admission , of early diagnosis of nstis the present report demonstrates that surgical intervention within the first 6 h after diagnosis of nstis would improve hospital outcomes in terms of shortening both the hospital los and icu los . in our study , the overall mortality was 11/87 or 12.5% , which is less than that reported in many previous studies . although there was a clinically significant difference in the mortality between the groups based on the timing of surgical intervention ( 17.5% in late vs. 7.5% in early intervention group ) , this did not reach statistical significance . nstis , in particular , necrotizing fasciitis , remain potentially the most deadly surgical infections and require aggressive resuscitation and surgical debridement . early diagnosis , early antibiotic treatment , and early surgical debridement remain the cornerstone of care for these patients . early means has not been clearly defined . in the study by mchenry et al . , the mean time from admission to operation was 45 h ( 1.7312 h ) while average time from admission to operation was 90 h for nonsurvivors versus 25 h in survivors group ( p = 0.002 ) . in our study , we found that patients with nstis required an operation as soon as possible , but certainly no later than 6 h after their arrival or presentation to the emergency department . in fact , most of our group i patients underwent an operation even earlier within a mean time of 2.95 1.1 h. in patients with nstis , the most common reason for a delay in surgery is difficulty in making the correct diagnosis . the clinical presentation can be deceiving , particularly in immunocompromised patients , ranging from indolent wound infections to severe gangrene with septic shock . often , patients seem too sick to be immediately operated on , so clinicians will attempt to resuscitate them first , resulting in significantly delayed surgery or clinical presentation is deceiving in particular in immunocompromised patients . however , one has to keep in mind that source control of the infection is priority in the management of any critically ill patients . these patients should be treated just like a gunshot wound or any other major insult to the body . historically , a number of comorbidities have been associated with nstis ; however , our patients ' comorbidities showed no effect on the in - hospital outcomes . the incidence of tachycardia , hypotension , and tachypnea was significantly higher in patients with nstis , but we found that none of them is specific enough or sensitive enough to predict nstis . despite numerous scoring systems and models introduced to discriminate between nstis and nonnstis , the diagnosis predicting mortality and limb loss in nstis is still difficult , and the most important element remains early clinical recognition . so far , there is a considerable diagnostic challenge , when a surgeon is faced with bad - looking cellulitis and distinguishing it from nsts . while we do not have a set protocol , most patients would get a computed tomography scan or magnetic resonance imaging ( if available ) ; if no clear clinical indication for surgery exists . on occasion , patient may get a plain film to rule out gas in the tissue ; however , this is rare . as a rule , we may use frequent imaging techniques to confidently rule out the need for immediate surgery . laboratory test results in patients with nstis have been well studied by a number of authors . the laboratory risk indicator for necrotizing fasciitis ( lrinec ) scoring system has been advocated to be helpful in distinguishing between nstis and nonnstis , as well as in differentiating between severe and not severe nstis . however , in our study , we found that no single laboratory value independently predicted early diagnosis of nstis . furthermore , most recent study of small group of patients strongly suggested that the lrinec system is too insensitive for diagnosis of nstis . although hypoalbuminemia ( < 2 g / dl ) is a known factor for postoperative complications , in our study , the albumin level did not significantly differ between our two groups ( 2.1 0.7 vs. 1.9 0.5 ; p = 0.579 ) . our microbiologic findings were similar to those of other reported series and reflected a wide spectrum of bacteria ( data not shown ) . we believe that our rapid surgical treatment of our patients , once the diagnosis was established especially our relatively short time to surgery was the main reason for our low mortality rate . the overall reported mortality rate has been reported to be very high ( up to 72% ) if the patient does not undergo surgical debridement as soon as possible . a median time to surgery of 8.4 h had a relatively low mortality rate of 16.4% , while an interval > 14 h from diagnosis to surgery in patients with septic shock was independently associated with in - hospital death . at our institution , the in - house presence of a trauma surgeon ( acute care surgeon ) ensures that these patients are seen almost immediately . after the surgery resident is consulted by emergency medicine or internal medicine colleagues . this in turn ensures early intervention for patients with nstis , although for this study , we did not specifically assess the impact of our practice style . hyperbaric oxygen therapy for nsti , despite all commercial activities , continues to be controversial . our center does not have hyperbaric oxygen chamber , so none of our patients underwent such treatment . although we have no experience with such treatment , we believe that hyperbaric therapy may actually delay treatment of patients with ntsi . although there are a number of lower extremities nstis , those cases are managed by trauma or acute care surgeons in our institution . in summary , evidence suggests that early surgical intervention is crucial in reducing morbidity and mortality in nsti patients . however , there is still a lack of clear definition on early . therefore , together with future studies , the present study may contribute to the definition of early intervention . the mean time of surgical intervention ( interval between diagnosis and surgical treatment ) was 25 h in survivors . in their study , other risk factors previously associated with the development of nsti did not affect mortality . the authors also showed that a delay of surgical treatment of > 12 h is associated with an increased number of surgical debridement , septic shock , and acute renal failure . the present study reported outcomes of further earlier surgical treatment ( within the first 6 h ) . although there was no statistically significant difference in mortality between the study groups , higher mortality among late intervention group was clinically significant . moreover , the outcomes in terms of duration of hospital and icu stay were in favor of early intervention . notably , the mean time for surgical treatment in early intervention group was around 3 h. the comparable frequency of surgical debridement along with other comparable prognostic factors for morbidity and mortality such as age , gender , site of infection , comorbidities , and relevant physical and laboratory findings shows the significance of time of intervention . our study has several strengths . to the best of our knowledge , we believe that it is the first study to clearly show that operating early , within the first 46 h in patients with nstis , would shorten both icu los and hospital los . other studies including that of mchenry et al . and have shown that early and aggressive debridement is crucial . however , in the later study , patients undergoing early and aggressive treatment had significantly lower mortality ( p = 0.0007 ) ; however , timing from definite diagnosis to operation was not reported . this finding has significant implication , and it may set a new standard and goal to achieve . clinicians must consider patients with nstis to be similar to those with severe trauma in their need for early surgical intervention . the concept of the golden hour resuscitation with simultaneous source control should be applied in patients with nstis as well . although we could not prove a statistically significant difference in mortality between the two groups , our overall low mortality rate ( 12.5% ) , especially in earlier operative group with a mortality rate of 7.5% ; we believe that our low mortality rate was the result of early surgery and in - house acute care surgery model . as with any retrospective study , we could not account for a number of variables beyond our control , such as the number of surgeons involved in each patient 's care and the variability between surgeons . different surgeons have different approaches ; different degrees of surgical aggressiveness could account for differences in patient outcomes . furthermore , we defined time to surgery as the interval from being diagnosed by the surgery team in the emergency department to undergoing the operation ; we did not look at how long patients might have waited after first coming to the emergency department or after first being admitted to the hospital , nor did we take into account whether or not patients were transferred from other institutions . moreover , the description of physical exam depends on the examiner , and more often than not , these are usually junior residents who perform the initial examination . the small sample size could affect the power of the study to adopt solid conclusions . , we could not account for a number of variables beyond our control , such as the number of surgeons involved in each patient 's care and the variability between surgeons . different surgeons have different approaches ; different degrees of surgical aggressiveness could account for differences in patient outcomes . furthermore , we defined time to surgery as the interval from being diagnosed by the surgery team in the emergency department to undergoing the operation ; we did not look at how long patients might have waited after first coming to the emergency department or after first being admitted to the hospital , nor did we take into account whether or not patients were transferred from other institutions . moreover , the description of physical exam depends on the examiner , and more often than not , these are usually junior residents who perform the initial examination . the small sample size could affect the power of the study to adopt solid conclusions . early recognition of nstis is difficult , but their clinical presentation is of key importance . patients who are thought to possibly have nstis must be seen , as soon as feasible , by the most experienced surgeons . early surgery ( within the first 6 h after diagnosis ) improves hospital outcomes in patients with severe nstis . | objectives : early diagnosis and emergent surgical debridement of necrotizing soft tissue infections ( nstis ) remains the cornerstone of care .
we aimed to study the effect of early surgery on patients ' outcomes and , in particular , on hospital length of stay ( los ) and intensive care unit ( icu ) los.materials and methods : over a 6-year period ( january 2003 through december 2008 ) , we analyzed the records of patients with nstis .
we divided patients into two groups based on the time of surgery ( i.e. , the interval from being diagnosed and surgical intervention ) : early ( < 6 h ) and late ( 6 h ) intervention groups .
for these two groups , we compared baseline demographic characteristics , symptoms , and outcomes . for our statistical analysis , we used the student 's t - test and pearson chi - square ( 2 ) test . to evaluate the clinical predictors of early diagnosis of nstis , we performed multivariate logistic regression analysis.results:in the study population ( n = 87 ; 62% males and 38% females ) , age , gender , wound locations , and comorbidities were comparable in the two groups . except for higher proportion of crepitus , the clinical presentations showed no significant differences between the two groups .
there were significantly shorter hospital los and icu los in the early than late intervention group .
the overall mortality rate in our study patients with nstis was 12.5% , but early intervention group had a mortality of 7.5% , but this did not reach statistical significance.conclusions:our findings show that early surgery , within the first 6 h after being diagnosed , improves in - hospital outcomes in patients with nstis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the management scientists have consensus that planning , organizing , leadership and control are the main managerial tasks . in control task , the organizational functions and activities compare with targets and standards that determined in planning task and the breeding activities done with observe a deviation . therefore , the organization required to develop suitable methods and tools to monitoring , assessment and evaluation of delivered services(1 ) . accreditation is the most powerful tool to evaluation that firstly was born in health services filed and then extended to other fields such as industry and educational services . the accreditation defines as recognition of meet to pre - determined and excellent standards from a health care organization by the external peer reviewer from the same organizational level(2 , 3 ) . in a various filed , accreditationis recognized as a symbol of qualityand indicates that the organization meets certainperformance standards , therefore provide an opportunity to organizations toevaluate their operation against national orinternational standards(4 ) . the main objectives of accreditation system are - continuous quality improvement in service deli - very via determining of optimum and achievable goals and standards , making integration in health care management , making database for health care information , increase safety for patient and health providers , delivering educational and consultant services to health care organizations , increase public confidence to health services and reduce the health services cost by focusing on efficiency and effectiveness(5 , 6 ) . a glimpse to world health report in 2000 indicates that iranian health system performance in main health indicators such as equity , responsibility and etchas unacceptable situation , so that iranian health system is ranked over than 100 in the world , and this facts required to more attention of managers and policy makers and their improving interventions(7 ) . by regarding to notable impacts of accreditation on main performance indicators in health system , no doubt that one of the best intervention is developing and implementation of a national accreditation program , because the current iranian evaluation standards are not premium and comprehensive standards . furthermore , this weakness especially in primary care evaluation system is more evident and iranian health system had great neglect from this sector(4 , 8) . according to these facts , the aim of this study was developing of national accreditation model to iranian rural health centers for stimulating the continuous quality improvement and betterment in main performance indicators in health system . this is a descriptive study conducted in eastern azerbaijan in 2013 . by using ofsystematic review , the researchers determined the best accreditation models worldwide for benchmarking to develop anappropriateaccreditation model . sid , ovid medline and pubmed databases had been searched with key words of accreditation , phc , medical practice , clinic , accreditation models and health care . the inclusion and exclusion criteria was articles in english and persian language , assessing accreditation models , and considering advantages and disadvantages of various accreditation models . first of all , titles of all articles were reviewed and 826 articles were excluded due to inconsistency with the study aims and 42 articles were excluded for repetition in both medline and pubmed databases ( 6 ) . after that the abstracts of 1501 remained articles were reviewed and 747 excluded because of focus on stakeholders perception about general issues in accreditation and methodology . all 736 articles that assessed accreditation models at any setting and level of health care system were reviewed and 681 articles were excluded because of no mention the advantages and disadvantages of accreditation models . as a final point , considering all inclusion and exclusion criteria , 73 articles remained of most relevant to the topic and study aims ( fig . systematic review approach for searched articles moreover , google scholar , most relevant websites and library search have be done for electronic sources , reports ( 3 reports ) and books ( 7 books ) related to the study aim . finally , the content of the remaining 73 articles and 10 other sources were separately reviewed ( 6 ) . then , the researcher studied the iranian medical sciences universities web sitesto obtain suitable standards for evaluation of this health care centers and generally to primary health care centers . furthermore , the researchers to using of all data gathering methods and creating the valuable and acceptable accreditation model assessed the main textbooks related to primary health care and health administration in english and persian languages and making single and plural interview with health management experts , and finally the all of standards used in design of delphi questionnaire . in continue , for assessing the competency of each standard for presence in final accreditation model from the experts perspectives by regarding to importance and being practical of standardsvia the delphi technique . the experts of this study are the individuals with valuable academic and scientific experiences including the main expert in regional and provincial health centers , the academic professors in health services managements , the managers of huge research centers , the iranian health management and economy board members , the hospital managers with notable experienced and the previous and current accreditation models experiences , and evaluation systems designer in health systems ( table 1 ) . the characteristics of participated experts the comparison between accreditation models based on 25 defined indicators the delphi technique has some features such as existence of expert panel , the ability of responses repetition to questionnaires in unlimited rounds with getting feedbacks from previous rounds , statistical analysis of responses and anonymous of participants . another advantage of this technique is the ability of gathering ideas and opinions of experts that have long geographical distance with low cost ( 912 ) . also , there is no agreement on consent threshold in delphi technique , but the majority of experts have agreed that the similarity of 70 to 80 percent of responses shows consent(9 , 13 ) . by regarding that the using scale in this study has 9 degree , thus we must elect the point of 5 as neutralize point and the responses have 3 categories : the phase of no agreement with the score of 1 to 3 , the neutralized phase with the score of 4 to 6 , and the phase of agreement with the score of 7 to 9 . in this category , the standards with the scores ( median ) of low than 4 eliminate from study , the standards with high than 7 accepts , and the standards with the scores between 4 to 7 advent to next round of delphi technique(14 ) . by regarding to literature review and for sustain of study validity , the response rate must be upper than 70 percent in all round of study(15 ) , and if the change of two sequence rounds is less than 15 percent , the consent was done and the standards do nt go to next rounds(11 ) . after obtaining to final standards , the quality of designed model assessed by using of quality indicators , then the components and framework of complete accreditation model will designed to new model . in the final phase of study , the researcher acting pilot study in 3 rural health centers for determining strengthens and weaknesses of designed model and the problems of model will resolve . by regarding to defined inclusion and exclusion criteria , the result of this study was 2379 articles and by notice to study aim that was mention of these articles to advantages and disadvantages of accreditation models , the articles were studied by reading the titles , abstracts and full articles in turns and finally the number of 83 articles selected and analyzed , then the results that obtained from them enter to extraction tables . in next level , the grand accreditation models in the world compared with themselves by 25 items and final study conclusion was obtained from this comparison(6 ) . the comparative items that analysis was based on them include of accreditation purposes and some items that extract from the literature review . the accr - editation models that have most discussion and analysis in all of these articles in turns are the jcaho from usa and cchsa from canada , and the accreditation models of uk , australia and france were in next levels . the obtained results from this study are coordinate with previous studies that introduced these models as largest and most effective prevalence models in the world . in related to accreditation purposes and other items that extract from literature review and was base for comparison , the jcaho from usa and cchsa from canada had most advantages and least disadvantages respectively(6 ) . after a vast literature review , the researchers determined the jcaho ( jci ) from united states , cchsa from canada and achs from australia as the best accreditation models worldwide respectively for benchmarking to developing of new and suitable accreditation program . from this three accreditation model , the cchsa has more accreditation standards related to primary health care , but the deputies of health from iranian medical sciences universities do not have accreditation program and any suitable standards to evaluation of this health care centers and generally to primary health care centers . then , the research team obtains to valuable standards by using of health management textbooks and holding interviews with experts . finally , the number of 74 primary standards acquired by using of all collecting data methods and delivered to number of 46 experts . in the first round of delphi , from all dispensed questionnaire the number of 44 questionnaires collected ( response rate of 95.6% ) and the number of 68 standards accepted , other 6 standardsgained to next round and any standards did not eliminated from study . in continues , the second round of delphi hold with 6 remained standards with the feedback of median score of all responses and standards scores from the experts in first round to their own . in the second round , the responders review the total scores and their personal scores to each standards and revised their scores to standards if they wand , or give previous score to standards . in this round , the number of 46 questionnaires dispensed and 43 questionnaire collected from experts ( response rate : 97.7% ) . in the second round of delphi , all standards accepted from experts perspectives and recognized as suitable accreditation standards . then , the same standards merged with each other , finally , the number of remained 55 standards made the final accreditation model . the notable point in this study is high response rate in two round of delphi technique , so indicated that the research team select the best experts , with suitable and timely communication with responders and good follow up from researcher . the current model has suitable standards for assessment , documentation , amendment and implementation of service delivery , management and procurement process in four sections including context , input , process , output and impact , and attention to all main and strategic health services indicators . after determining final standards , the main section of health care accreditation model including intent statement ( the comments regarding to standard objectives ) and measurable sub standards ( the agents to objective measures of each standards ) thereafter , the designed model assessed from the 43 experts perspectives via 12 criteria that indicate the quality of standards in an accreditation model.(16 ) . from the experts perspectives , the current accreditation model indicates coordination with all criteria that indicate the quality of accreditation standards ( table 3 ) . checklist for evaluating an accreditation standard the pilot study conducted in 3 rural health centers and the designed accreditation model assessed to realize the quality of standards and practicality of aspects . in this phase , the selected rural health centers were evaluated using the developed standards by health center staff and health management specialists , and any shortages in each standards and overall model were detected . after evaluation , all of developed standards accepted in implementation level , but the number of 6 standards changed to better understanding of surveyor and health centers , and 4 items added to measurable components of standards . the aim of this study was developing of national accreditation model for evaluation of rural health centers in iran to encourage continuous quality improvement in these centers . in designing of this model , researchers used of all methods for gathering information and determining primary standards , suitable methods to managing study and best experts to holding delphi technique . thereafter , the final model is a complete model for diversity in standards , attention to all main indicators in health system , and having main section of well - established accreditation model . the joint commission for accreditation in healthcare organizations ( jcaho ) is the accreditation mother worldwide , that accredit all type of health care organizations . this accreditation program has suitable standards for assessing the health care centers in primary care services , but the scope , precise and quality of their standards are less than designed model during current study . this two accreditation model have intent statement and measurable sub standards for increase objectivity in evaluation process(17 ) . the cchsa accreditation standards is the one of pioneer accreditation model in the world that have suitable scope of standards to evaluation health centers , but its emphasis on primary care accreditation is more than jcaho , so that this program has a important part in this field as primary care services ( pcs ) with more standards . this accreditation program has best quality in their standards in primary health care , so that the current study use from its standards rather than other pioneer and grand accreditation models . the cchsa accreditation program has intent statement and measurable sub standards for all of their standards too(16 ) . other health care accreditation models such as achs of australia , anaes of france , and other accreditation model in emro region such as lebanon and egypt accreditation model emphasis on hospital accreditation and do not have any standards for primary health care evaluation . therefore , these models do nt have suitable intent statement and measurable sub standards for their standards(18 ) . regarding study results , the researcher suggests that this study implement to other health centers in primary care such as health house , urban health centers and etc and finally this research and activities lead to developing of holistic and coordinated accreditation program in all part of iranian primary health services and increasing quality of delivered cares . this study limitation is low access to majority of experts for theirs high work loud . the notable and strong points of this study is high response rate in two round of delphi technique , so indicated that the research team select the best experts , with suitable and timely communication with responders and good follow up from researcher . the current model has suitable standards for assessment , documentation , amendment and implementation of service delivery , management and procurement process in four sections including context , input , process , output and impact , and attention to all main and strategic health services indicators . other strong points of current accreditation model are having the main section of health care accreditation model including intent statement and measurable sub standards , accepted of all designed standards , and implementing of pilot study to eliminate the weaknesses point of this model and increase the implementation ability in real world . this study approved by ethic committee from tabriz university of medical sciences and all of experts participated in study voluntary and informed with written consent . the current accreditation model has most standards in primary care worldwide and researcher s hops using of this model for its unique characteristics led to continuous quality improvement in rural health centers . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | abstractbackgroundthe primary health care has notable effects on community health and accreditation is one of the appropriate evaluation methods that led to health system performance improvement , therefore , this study aims to developing of national accreditation model for rural health centers in iran health system.methodsfirstly the suitable accreditation models selected to benchmarking worldwide via systematic review , the related books and medical university s web site surveyed and some interviews hold with experts
. then the obtain standards surveyed from the experts perspectives via delphi technique .
finally , the obtainedmodel assessedvia the experts perspective and pilot study.resultsthe researchers identified jcaho and cchsa as the most excellent models . the obtained standards and their quality accepted from experts perspective and pilot study , and finally the number of 55 standards acquired.conclusionthe designed model has standards with acceptable quality and quantity , and researchers hopeful that its application in rural health centers led to continues quality improvement . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
as of january 2011 , peruvian migrants represent the largest latin american community in italy with 101,711 documented residents accounting for 2.9% of migrant population , corresponding to about 30% of latin americans in italy . the number of peruvian citizens in italy has increased 30 folds since the early nineties . the majority of them are young - adults ( 70% aged between 18 and 49 years ) and the female gender is slightly prevalent ( 61% ) . the large majority ( 70% ) are currently concentrated in few large metropolitan areas namely milan ( 33,919 documented residents ) , rome ( 15,262 ) , turin ( 10,872 ) , florence ( 7834 ) and genoa ( 3277 ) . in 2009 ( the last year with official surveillance data available ) , 48.3% ( 2053 ) of tuberculosis ( tb ) cases in italy were diagnosed in foreign born subjects , with peruvians accounting for 5% of them ( 103 cases ) . in the health district of florence ( azienda sanitaria locale , asl 10 ) , the number of reported tb cases remained almost stable in the last 10 years with around 100 - 130 cases per year and an incidence of 10 - 14 cases per 100,000 populations . nevertheless , in the same period , the number and the proportion of peruvian subjects diagnosed with tb in florence has steadily increased . starting from 2005 , peruvians represent the foreign community with highest tb burden in florence , accounting for about 20% of cases ( 22 out of 110 in 2009 and 25 out of 133 in 2010 ) . the findings of a retrospective study aiming to understand the epidemiological dynamics of tb transmission in the peruvian community of florence are reported here . main epidemiological and clinical features of tb cases notified in peruvian citizens resident in the asl 10 , in the period 2001 - 2010 were retrospectively collected from the databases of uf igiene e sanit pubblica , asl 10 zona firenze , florence , italy and sod microbiologia e virologia , centro di riferimento per i micobatteri della regione toscana , azienda ospedaliero universitaria careggi , florence , italy . incidence of tb in the peruvian community of florence was estimated on the basis of number of tb cases notified each year in the asl 10 ( used as numerator ) and number of documented peruvian residents in the province of florence ( used as denominator ) . available mycobacterium tuberculosis strains isolated were characterized by genotyping analysis with mycobacterial interspersed repetitive units - variable number tandem repeat ( miru - vntr-24 ) and spacer oligonucleotide typing ( spoligotyping ) . clonally related strains were defined as clustered and assumed to belong to a chain of recent transmission , while patients whose m. tuberculosis isolate did not present a shared pattern were assumed to originate from reactivation of a latent tuberculosis infection ( ltbi ) . as this was a retrospective study on routinely collected surveillance data and on samples collected at the regional tb reference laboratory , ethical approval was not deemed necessary . one hundred and thirty - eight tb cases were retrieved . among them , 119 ( 86% ) were microbiologically confirmed . the number of reported cases steadily increased through the years , from 11 ( 8% ) in 2001 - 2002 to 47 ( 34.1% ) in 2009 - 2010 . the estimated incidence rates varied , in the considered period , from 484 and 351 per 100,000 peruvian residents in florence . eighty - seven m. tuberculosis strains were available for genotyping analysis ( figure 1 ) . miru - vntr-24 analysis showed 48 strains ( 55.2% ) displaying a non - shared genotype , which were interpreted as originating from ltbi reactivation . thirty nine ( 44.8% ) m. tuberculosis strains showed a genotype shared at least by another strain , fulfilling the definition of clustered cases . three clusters included 2 subjects , 3 clusters included 3 subjects , 4 clusters included 4 subjects and 1 cluster included 6 subjects . in the majority of clusters ( 10 out of 12 ) the time elapsed between the diagnosis of the first and the last case was shorter than five years . spoligotyping showed that the lineages circulating in the community were haarlem ( n=31 , 35.6% , 14 clustered and 17 non - clustered ) , latin american - mediterranean ( lam , n=28 , 32.1% , 12 clustered and 16 non - clustered ) , t - specific ( n=8 , 9.2% , 2 clustered and 6 non - clustered ) beijing ( n=5 , 5.7% , 4 clustered and 1 non - clustered ) , ural ( n=3 , 3.4% , all clustered ) , s ( n=1 , 1.1% ) . eleven ( 12.6% , 4 clustered and 7 non - clustered ) previously unreported spoligotypes ( orphans ) were also present . the study shows that tb in the peruvian community in florence is a concerning problem considering its high estimated incidence . in 2009 , the last year for which final data are available at national level , the estimated incidence of tb in peruvian born in italy ( 117 per 100,000 ) was very similar to the one reported for peruvian residents in peru ( 113 per 100,000 ) and much lower if compared with that found in this study ( 381 per 100,000 ) . supported by the finding of a high rate of clustered cases , we can affirm that the higher tb incidence in peruvian residents in florence ( 3.2 time higher compared with incidence in the country of origin ) is related to an ongoing active transmission within the community . in the matter of facts , excluding one case for each cluster that should be considered the index case , the remaining clustered cases are due to recent infections occurred in florence . a limitation of this study is that we considered in the analysis only m. tuberculosis strains isolated from peruvian individuals living in florence . this greatly limit the possibility of inferring the origin of active tb ( new infection vs reactivation ) from cluster data . in other word , if a peruvian patients is not in a cluster with other peruvian patients , it is not possible to rule out that this patients was not part of a cluster in which the index case was a non - peruvian patient living in the same area , and thus to infer that tb in that patient was due to reactivation of a remotely acquired infection . thus the number of patients in cluster should be regarded as a minimum estimate of cases due to recent infection . peruvian origin has been previously identified as risk factors for tb clustering in a previous italian study carried out in milan . another study conducted in the late nineties in milan evidence a 1.6 time higher tb incidence in peruvian migrants compared with tb incidence in peru . considering data obtained in the studies carried out in milan and in florence , peruvian migrant communities of both cities emerge as a vulnerable group which may benefit from targeted intervention and tb control activities . strengthening of preventive intervention , such as early diagnosis of active tb cases and contact tracing procedures , should be prioritized for this population . an easy access to tb services for the members of this communities should be assured , regardless of legal status of subjects . collaboration between different partners such as public health services , primary care physicians , tertiary hospitals and non - for profit sector is warranted to achieve a timely diagnosis of tb and to ensure case holding of affected subjects . socially and culturally acceptable education programs on tb with a multidisciplinary approach should be reinforced . authors would also thank the cohemi project study group that includes : maurizio bonati ( a ) , chiara pandolfini ( a ) , francesca severino ( a ) , valeria confalonieri ( a ) , gianni tognoni ( a ) , zeno bisoffi ( b ) , dora buonfrate ( b ) , andrea angheben ( b ) , marco albonico ( b , c ) , alessandro bartoloni ( d ) , marianne strohmeyer ( d ) , lorenzo zammarchi ( d ) , filippo bartalesi ( e ) , jose muoz ( f ) , ana requena - mendez ( f ) , maria roura ( f ) , laia ventura ( f ) , robert pool ( g ) , christopher pell ( g ) , anita hardon ( g ) , peter chiodini ( j ) , juan moreira ( k ) , mariella anselmi ( k ) , roberto semprtegui ( k ) , eduardo gotuzzo ( l ) , maria alejandra mena ( l ) , carola liendo ( l ) , hctor h. garcia ( m ) , javier bustos ( m ) , saul santivaez ( m ) , faustino torrico ( n ) , daniel lozano ( n ) , teresa hinojosa cabrera ( o ) , javier ochoa morn ( o ) , ignacio abapori cuellar ( o ) , jaime amors suarez ( o ) , guido chumiray rojas ( o ) , alessandra nicoletti ( p ) , elisa bruno ( p ) . ( a ) department of public health , laboratory for mother and child health , mario negri pharmacological research institute , milan , italy . b ) centre for tropical diseases , sacro cuore - don calabria hospital , negrar , verona , italy . d ) infectious disease unit , department of experimental & clinical medicine , university of florence school of medicine , florence , italy . e ) sod malattie infettive e tropicali , azienda ospedaliero - universitaria careggi , florence , italy . f ) servicio de medicina tropical y salud internacional , centre de recerca en salut internacional de barcelona , hospital clnic - universitat de barcelona , barcelona , spain . g ) centre for social science and global health , university of amsterdam , the netherlands . j ) hospital for tropical diseases and london school of hygiene & tropical medicine , london , united kingdom . l ) instituto de medicina tropical alexander von humboldt , universidad cayetano heredia , lima , peru . m ) cysticercosis unit , instituto de ciencias neurologicas , department of microbiology , universidad peruana cayetano heredia , lima , peru . | tuberculosis is a leading cause of morbidity for peruvian migrants in florence , italy , where they account for about 20% of yearly diagnosed cases .
a retrospective study on cases notified in peruvian residents in florence in the period 2001 - 2010 was carried out and available mycobacterium tuberculosis strains were genotyped ( miru - vntr-24 and spoligotyping ) .
one hundred thirty eight cases were retrieved .
genotyping performed in 87 strains revealed that 39 ( 44.8% ) belonged to 12 clusters . assuming that in each cluster the transmission of tuberculosis from the index case took place in florence
, a large proportion of cases could be preventable by improving early diagnosis of contagious cases and contact tracing . |
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three different types of carbon nanotubes are experimentally observed : armchair , zigzag and chiral . these carbon structures are finished with semispheres containing pentagons and hexagons , being formally parts of fullerenes . due to their structure , the cnts are hydrophobic , strongly interact with light and possess interesting electrical and physical properties [ 13 ] . modification of hydrophobic carbon nanotubes by allowing stronger intermolecular interactions , leading to solubility is expected upon addition of selected small molecules covalently bonded to the ( a ) end , ( b ) surface , or ( c ) both the end and surface ( mixed ) of swcnts [ 2 , 413 ] . the rim structure of swcnt in case of zigzag or armchair open ended cnt shows a different pattern . zigzag carbon nanotubes show metallic or semiconductor properties and their ends shows saw - tooth like shape . for example , oh , cooh or nh2 functionalized cnt are easily transformed and could bear longer chains connecting antibodies or drugs . end - substituted swcnt are by - products of mild oxidation and carboxylic , carbonyl and hydroxyl groups are frequently formed . most previous works [ 46 ] concentrated on single functionalized swcnts and the impact of substituent on physical properties of modified versus pristine structure ( for example , density of states , homo - lumo gap ) . the pristine cnts are insoluble in water and organic solvents and this is a serious hindrance in their industrial applications , for example , as efficient nanocomposites [ 1 , 14 , 15 ] . earlier works demonstrated a possibility of transforming inert and hydrophobic cnts , into soluble forms [ 1417 ] . this was accomplished by subsequent chemical modification of water soluble cnts , containing cooh groups [ 1417 ] . unfortunately , little is known about the systematic changes of energy of zigzag and armchair swcnts upon consecutive replacement of rim hydrogen atoms by cooh groups . density functional theory ( dft ) and , in particular , the exchange - correlation b3lyp hybrid density functional is widely used in molecular modeling studies to predict structure , spectroscopic parameters and energy changes of small , middle and large size molecules [ 7 , 8 , 1820 ] . due to the large size of cnts , the dft calculations with relatively small basis sets ( 3 - 21 g or 6 - 31 g * ) , and also am1 and pm3 semiempirical methods , have been used for theoretical description of molecular structure and other parameters of finite models of cnts . in this study , as an extension of our previous works on hydroxyl substituted swcnts [ 19 , 21 , 22 ] , we would like to get a more detailed information at the atomistic level on the open - end cnt modification with cooh , up to full substitution with nine ( zigzag ) or ten ( armchair ) carboxylic groups . reliable exchange - correlational b3lyp hybrid density functional and basis sets of relatively small size ( 3 - 21 g and 6 - 31 g * ) , enabling completing fairly large scale calculations were selected . full structure optimization of unsubstituted open - ended ( with dangling bonds on carbon saturated with hydrogen atoms ) , and cooh - modified swcnts were performed . several models of swcnt were selected , including ( 9,0 ) zigzag and ( 5,5 ) armchair structures with one and three layers ( strings ) of hexagon units . ir and raman harmonic frequencies were calculated in case of one layer with one to 9/10 cooh substituents . all positive frequencies ensured ground state structure of the optimized system . for comparison purposes only , the calculations with small model molecules including methane , benzene , anthracene and phenanthrene before , and after replacing one hydrogen atom with the carboxyl group were performed at the same level of theory . in addition , to verify the basis set quality impact , these calculations were performed with a large basis set ( 6 - 311++g(3df,2pd ) . energy of one cooh group formation at nanotube terminated initially with h atoms was calculated by considering a hypothetical reaction:1\documentclass[12pt]{minimal }
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\begin{document}$$ { \text{swcnt } } - { \text{h } } + { \text { c}}{{\text{h}}_{{3}}}{\text{cooh } } \to { \text{swcn } } - { \text{cooh } } + { \text { c}}{{\text{h}}_{{4}}}. $ $ \end{document } energies of adding subsequent groups ( e in kcal mol ) were assumed as follows:2\documentclass[12pt]{minimal }
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\begin{document}$$ { \text{d}}{{\text{e}}_{\text{n } } } = { } \left [ { { \text{e}}\left ( { { \text{swcnt}}{{\left ( { \text{cooh } } \right)}_{\text{n } } } } \right ) + { \text{e}}\left ( { { \text{c}}{{\text{h}}_{{4 } } } } \right ) } \right]-\left [ { { \text{e}}\left ( { { \text{swcnt}}{{\left ( { \text{cooh } } \right)}_{{{\text{n } } - { 1 } } } } } \right ) + { \text{e}}\left ( { { \text{c}}{{\text{h}}_{{3}}}{\text{cooh } } } \right ) } \right ] , $ $ \end{document}where n = 1,2, 9 ( 10 ) . initially , the calculations were conducted at b3lyp/3 - 21 g level of theory . qualitatively , the changes in energies obtained with smaller basis set ( 3 - 21 g ) were similar to those , obtained at b3lyp/6 - 31g*. thus , the final results , obtained with the larger basis set will be only discussed . the carboxylic group can be considered as an asymmetric substituent with two different ends ( o atom vs. oh group ) and their position in respect to the rim of the cnt be positioned in a way which minimizes the interactions with the neighboring h - atoms and/or forms h - bonds with other cooh substituents . this was tested on model systems ( scheme 1 ) by rotating the cooh substituent relative to the aromatic ring plane ( changing the dihedral angle cring cring c = o).scheme 1small model molecules ( benzoic , anthracene-9-carboxylic and phenathrene-4-carboxylic acids small model molecules ( benzoic , anthracene-9-carboxylic and phenathrene-4-carboxylic acids the energy landscape of cooh rotation in case of monosubstituted benzene , phenanthrene and anthracene are shown in fig . 1 . in case of benzene , the energy minimum , corresponding to favorable carboxylic orientation , coplanar with the ring , is observed and the perpendicular position , e.g. , at both sides of the ring , are about 8 kcal mol higher . in case of anthracene , the energy minimum corresponds to about 45 degree deviation of cooh plane from rings plane and there are also two maxima of the same height ( at about 3 kcal mol ) for the perpendicular orientation . the rotation of cooh in phenanthrene at position 4 ( see scheme 1 ) leads to an asymmetric shape of energy curve . the basis set effect on the position of energy maxima upon cooh rotation in c6h5cooh is also shown in fig . thus , upon improving the basis set quality from 6 - 31 g * to 6 - 311++g * * and 6 - 311++g(3df,2pd ) the barrier height slightly decreases ( from 7.85 to 6.73 and 6.52 kcal mol ) . it is apparent that the barrier height decreases by about 1.3 kcal mol upon significantly improving the basis set quality.fig . 1energy change upon rotation of cooh substituent relative to ring plane in ( a ) benzoic acid and ( b ) anthracene-9-carboxylic acid and phenathrene-4-carboxylic acid energy change upon rotation of cooh substituent relative to ring plane in ( a ) benzoic acid and ( b ) anthracene-9-carboxylic acid and phenathrene-4-carboxylic acid in fig . 2a and b are shown optimized structures of zigzag and armchair cnt consisting of three ring layers with a single cooh substituent at the rim.fig . 2optimized structures of model ( a ) zigzag and ( b ) armchair swcnts with a single cooh substituent at the rim ( dimensions in ) optimized structures of model ( a ) zigzag and ( b ) armchair swcnts with a single cooh substituent at the rim ( dimensions in ) in fig . 3 are shown energy landscapes of single cooh group rotation attached to zigzag and armchair swcnts . in this case , the preferred geometry is observed for both -oh and = o ends of carboxylic group outside the tube ( on the circumference ) . two energy maxima are observed for cooh group oriented along the tube radius and the slightly lower one corresponds to oh being outside the tube . in case of zigzag cnt , the energy minimum corresponds to c c c = o angle of about 5 degrees , ( substituent on the circumference ) and the highest maximum corresponds to about 90 degrees ( c = o outside the tube ) . the other maximum ( with c = o inside , or oriented toward the tube center ) is slightly lower . similarly to fig . 1a , the improvement of basis set quality from 3 - 21 g to 6 - 31 g * leads to energy barrier lowering by about 4 kcal mol . in the case of carboxylic group rotation at the armchair rim , the situation is similar and the corresponding barrier heights are 9 and 7 kcal mol and the energy minimum is observed at about 10 degrees.fig . 3energy change upon rotation of cooh substituent at the rim of model ( a ) zigzag and ( b ) armchair swcnts energy change upon rotation of cooh substituent at the rim of model ( a ) zigzag and ( b ) armchair swcnts up to nine carboxylic substituents were placed consecutively at the zigzag rim ( see fig . , a kind of threefold symmetry was observed . nevertheless , some funnel shape deformation and increase of the tube - end diameter was observed.fig . 4optimized structures of model ( a ) zigzag and ( b ) armchair swcnts fully functionalized with cooh substituents at the rim . threefold symmetry is indicated for zigzag nanotube optimized structures of model ( a ) zigzag and ( b ) armchair swcnts fully functionalized with cooh substituents at the rim . threefold symmetry is indicated for zigzag nanotube the armchair model consisting of three hexagon layers with up to nine cooh groups at one rim was also stable . in addition , upon complete functionalization of one hexagon layer ( the shortest armchair nanotube model ) a stable system was also observed ( fig . however , all attempts to obtain fully functionalized one end of a longer tube , containing three layers of hexagons , failed . this was probably due to steric crowding at the relatively rigid tube skeleton end . in the next step , starting from model systems of methane and benzene , we examined the energetics of substitution process calculated according to eq . 2 ( see table 1 ) . addition of zpv correction changes the substitution energy slightly while hydroxylation is more favorable in the case of benzene.table 1comparison of carboxylation and hydroxylation energy ( kcal mol ) calculated at b3lyp/6 - 311++g(3df,2pd ) level for two model compounds according to eq . 2ch4c6h6-coohe3.8(e + zpv)4.9-oh e29.439.9(e + zpv)26.538.4 in agreement with formula ( 2 ) an opposite sign to that in ref . is given comparison of carboxylation and hydroxylation energy ( kcal mol ) calculated at b3lyp/6 - 311++g(3df,2pd ) level for two model compounds according to eq . 2 in agreement with formula ( 2 ) an opposite sign to that in ref . 2vs . the number of cooh substituents for zigzag and armchair are displaced in fig . 2 for model zigzag and ( b ) armchair swcnts functionalization at two basis set sizes . for better visualization the data points . 2 for model zigzag and ( b ) armchair swcnts functionalization at two basis set sizes . for better visualization that the carboxylation energy for the first hydrogen atom at the rim of zigzag nanotube formed from three layers is about 13 kcal mol . this differs from the calculated previously hydroxylation energy of about 35 kcal mol . significantly smaller energy increments are needed for adding three to nine carboxylic groups . in fig . 5b is shown a similar carboxylation energy dependence on replacement of consecutive hydrogen atoms at the rim of armchair cnt model . the first carboxylation energy is higher than for zigzag model ( about 8.5 vs. 13 kcal mol ) whereas for the second group this energy is about 1 vs. 3 kcal mol , for armchair and zigzag models , respectively . however , some oscillation of energy is observed , with minima located at odd numbers of cooh . this resembles the results for hydroxylation energy pattern observed previously for armchair cnt [ 21 , 22 ] . the reason of this behavior was explained earlier as a result of different h - bond ring pattern formation at the rim . the present density theory studies using b3lyp hybrid functional indicate a possibility of cooh - functionalization of one end of zigzag cnt with one to nine substituents . however , the end of armchair nanotube can not be fully functionalized with cooh groups ( one hydrogen atom remains unsubstituted ) . on the basis of the performed b3lyp/6 - 31 g * calculations it appears that the replacement of one hydrogen atom at the rim of the zigzag cnt model is a more exothermic process than for armchair model ( 13 vs 8.5 kcal mol ) . a gradual and nearly linear increase of energy is observed for subsequent carboxylation , starting from two to nine groups on a zigzag nanotube end . | structure and energy calculations of pristine and cooh - modified model single wall carbon nanotubes ( swcnts ) of different length were performed at b3lyp/6 - 31 g * level of theory . from 1 to 9 cooh groups
were added at the end of the nanotube .
the differences in structure and energetics of partially and fully functionalized swcnts at one end of the nanotube are observed . up
to nine cooh groups could be added at one end of ( 9,0 ) zigzag swcnt in case of full functionalization . however , for ( 5,5 ) armchair swcnt , the full functionalization was impossible due to steric crowding and rim deformation .
the dependence of substituent attachment energy on the number of substituents at the carbon nanotube rim was observed.figurestructure and energy calculations of pristine and cooh - modified model single wall carbon nanotubes ( swcnts ) of different length were performed at b3lyp/6 - 31 g * level of theory and from 1 to 9 cooh groups was added at the end of the nanotube |
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the institute of medicine ( iom ) 2012 workshop report entitled envisioning a transformed clinical trials enterprise in the united states , stated that the more traditional areas of mechanistic research and efficacy trials call for specialized workforces that until now have all too often depended on ad hoc , on - the - job learning as opposed to the prospective training and education that defines a mature discipline . the data from the us food and drug administration center for drug evaluation and research inspections from fiscal years 20042011 ( n=2325 ) of us clinical trial sites show the following clinical investigator deficiencies : 42% protocol violations , 30% record - keeping deficiencies , 12% informed consent deficiencies , 10% of drug accountability violations , 9% institutional review board ( irb ) communication , and 5% with problems reporting adverse events ( aes ) . moreover , despite recent requirements for good clinical practice ( gcp ) training of investigators and clinical research staff , a decrease in these deficiencies and violations have persisted ( see http://bit.ly/1mali8w ) . these data suggest that there is a need for an intervention to increase clinical investigator and coordinator competence and improve clinical trial performance metrics . we hypothesize that clinical trial ( and particularly multisite clinical trial ) performance will be significantly improved by a standard set of systematically harmonized competencies that equip pis and crcs with the necessary skills to more effectively , efficiently , and safely execute clinical trials . the iom has issued a challenge to create a clinical research workforce that can address increasing complexity in clinical and translational research and generate study results that reach the community more efficiently . this challenge has focused attention on a clinical research workforce that includes not only pis , but also crcs and staff members . to date , education and training for pis has evolved through the ctsa , but this training did not reach all investigators , nor the many clinical research professionals , and other team members who were external to the local ctsa funding mechanism . mandates for gcp training by industry sponsors and irbs have provided a minimal training activity for investigators and their clinical research teams . academic and nonacademic sites provide local training and educational links , but often these efforts are unfunded and/or institution - specific activities . a formalized education and training requirement is lacking for individuals working in this profession . over the past decade , various professional societies and institutes have supported a cbe approach for clinical research professionals . cbe identifies specific learning outcomes for knowledge and its application and is often referred to as outcomes - based education . part of an educational trend that emerged in the 1970s , this approach has been endorsed by the iom in its 2005 report . characteristics that distinguish cbe include the following:learner outcomes that are based on analysis of typical job responsibilities of practitioners.a curriculum focused on what learners need to learn to perform specific job tasks , not necessarily on traditional subject matter.hierarchically sequenced modules that allow learners to proceed at their own pace.educators employing assessment techniques that measure learner performance in settings that approximate the real environment . a curriculum focused on what learners need to learn to perform specific job tasks , not necessarily on traditional subject matter . educators employing assessment techniques that measure learner performance in settings that approximate the real environment . the evolution of cbe / competency - based training and the publication of core competencies in clinical research offer a pathway for achieving workforce development goals . cbe / competency - based training promise a skilled workforce to a variety of stakeholders by mapping core competencies to educational and training curricula . the competency - based approach ultimately defines competences and qualifications in a systematic learner - centric pathway . competencies represent not only basic knowledge , but higher levels of knowledge , skills , and attitudes ( ksas ) that embody the profession . core competencies for clinical research nurse coordinators evolved from several works generated by the royal college of nursing in the united kingdom , oncology nursing society , and a national institutes of health ( nih ) clinical research nurse working group [ 79 ] . other role delineation work for clinical research nurses have continued to study these domains and specific ksas in practice [ 1013 ] . many of the role delineations attributed to clinical research nurses can also be attributed to non - nurse crcs . in addition , core competencies for clinical and translational pis were featured on the ctsa web site and formed the basis for curriculum development for master - level courses under the ctsa , beginning with the k-30 awards . the role of pharmaceutical physicians , which is primarily a role found in europe and south america , the iom has issued a challenge to create a clinical research workforce that can address increasing complexity in clinical and translational research and generate study results that reach the community more efficiently . this challenge has focused attention on a clinical research workforce that includes not only pis , but also crcs and staff members . to date , education and training for pis has evolved through the ctsa , but this training did not reach all investigators , nor the many clinical research professionals , and other team members who were external to the local ctsa funding mechanism . mandates for gcp training by industry sponsors and irbs have provided a minimal training activity for investigators and their clinical research teams . academic and nonacademic sites provide local training and educational links , but often these efforts are unfunded and/or institution - specific activities . over the past decade , various professional societies and institutes have supported a cbe approach for clinical research professionals . cbe identifies specific learning outcomes for knowledge and its application and is often referred to as outcomes - based education . part of an educational trend that emerged in the 1970s , this approach has been endorsed by the iom in its 2005 report . characteristics that distinguish cbe include the following:learner outcomes that are based on analysis of typical job responsibilities of practitioners.a curriculum focused on what learners need to learn to perform specific job tasks , not necessarily on traditional subject matter.hierarchically sequenced modules that allow learners to proceed at their own pace.educators employing assessment techniques that measure learner performance in settings that approximate the real environment . a curriculum focused on what learners need to learn to perform specific job tasks , not necessarily on traditional subject matter . educators employing assessment techniques that measure learner performance in settings that approximate the real environment . the evolution of cbe / competency - based training and the publication of core competencies in clinical research offer a pathway for achieving workforce development goals . cbe / competency - based training promise a skilled workforce to a variety of stakeholders by mapping core competencies to educational and training curricula . the competency - based approach ultimately defines competences and qualifications in a systematic learner - centric pathway . competencies represent not only basic knowledge , but higher levels of knowledge , skills , and attitudes ( ksas ) that embody the profession . core competencies for clinical research nurse coordinators evolved from several works generated by the royal college of nursing in the united kingdom , oncology nursing society , and a national institutes of health ( nih ) clinical research nurse working group [ 79 ] . other role delineation work for clinical research nurses have continued to study these domains and specific ksas in practice [ 1013 ] . many of the role delineations attributed to clinical research nurses can also be attributed to non - nurse crcs . in addition , core competencies for clinical and translational pis were featured on the ctsa web site and formed the basis for curriculum development for master - level courses under the ctsa , beginning with the k-30 awards . the role of pharmaceutical physicians , which is primarily a role found in europe and south america , the ecrptq leadership team considered a variety of competency frameworks for this phase:the ctsa education and career development key function committee developed the ctsa master - level competencies , approved in 2011 ( http://bit.ly/21cx5n8 ) , to define the training standards for individuals functioning at the master s level in clinical and translational research . this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium . although the ecrptq leadership team felt these competencies to be highly relevant for investigators , they did not fully address the necessary qualifications and skills for other team members . the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees , including bioinformatics.the nih clinical research nursing domains of practice for the specialty of clinical research nursing ( http://cc.nih.gov/nursing/crn/dop_document.pdf ) was another framework that was reviewed and considered , but also did not necessarily address the qualifications and skills for all team members.competencies outlined by the oncology nursing society ( https://www.ons.org/sites/default/files/ctncompetencies.pdf ) were also reviewed but the group felt they were rather limited , focusing only on oncology studies.the ecrptq leadership also acknowledged the work of the national research coordinator consortium , formerly known as the ctsa research coordinator taskforce . like the other domains listed previously , the ecrptq leadership felt a framework that would be inclusive of all study team members was needed . the ctsa education and career development key function committee developed the ctsa master - level competencies , approved in 2011 ( http://bit.ly/21cx5n8 ) , to define the training standards for individuals functioning at the master s level in clinical and translational research . this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium . although the ecrptq leadership team felt these competencies to be highly relevant for investigators , they did not fully address the necessary qualifications and skills for other team members . the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees , including bioinformatics . the nih clinical research nursing domains of practice for the specialty of clinical research nursing ( http://cc.nih.gov/nursing/crn/dop_document.pdf ) was another framework that was reviewed and considered , but also did not necessarily address the qualifications and skills for all team members . competencies outlined by the oncology nursing society ( https://www.ons.org/sites/default/files/ctncompetencies.pdf ) were also reviewed but the group felt they were rather limited , focusing only on oncology studies . the ecrptq leadership also acknowledged the work of the national research coordinator consortium , formerly known as the ctsa research coordinator taskforce . like the other domains listed previously , the ecrptq leadership felt a framework that would be inclusive of all study team members was needed . ultimately , the framework proposed by the joint task force for clinical trial competency ( jtf ) was selected that identified 8 broad domains of competence:(1)scientific concepts and research design(2)ethical and participant safety considerations(3)medicines development and regulation(4)clinical trial operations(5)study and site management(6)data management and informatics(7)leadership and professionalism(8)communication and teamwork
scientific concepts and research design ethical and participant safety considerations medicines development and regulation clinical trial operations study and site management data management and informatics leadership and professionalism communication and teamwork the ecrptq leadership team selected this framework because of its comprehensive applicability and its widespread uptake by numerous other stakeholders in the clinical trial enterprise . an additional consideration was the knowledge that the jtf mapped its competency framework to those mentioned previously , along with additional competency frameworks identified by consortium of academic programs in clinical research ( coapcr ) and the uk national health service . the jtf model is displayed in table 1.table 1jtf core competenciesdomaindefinitioncompetenciesscientific concepts and research designknowledge of scientific concepts related to the design and analyses of clinical trials5ethical and participant safety considerationsknowledge of the care of patients , human subject protections , and safety in the conduct of a clinical trial8medicines development and regulationknowledge of how drugs , biologics , and devices are developed and regulated7clinical trial operationsknowledge of study management , gcp compliance ( regulatory affairs ) , safety reporting ( adverse event identification and reporting , postmarket surveillance , pharmacovigilance ) , and the handling of investigational product12study and site managementknowledge of requirements for site management ( financial , personnel , including site and study operations , not including regulatory affairs)6data management and informaticsknowledge of how data are acquired and managed during a clinical trial ( source data , data entry , queries , quality control , corrections ) and the concept of a locked database5leadership and professionalismknowledge of the principles and practice of leadership and professionalism in clinical research4communication and teamworkknowledge of all elements of communication within the site and between the site and sponsors , contract research organizations , regulators . this ctsa - wide endeavor drew upon expertise across the consortium for this important and complex project . the ecrptq leadership team invited members of the ctsa consortium with relevant expertise to participate in each of the competency domain working groups , which were created based on the 8 competency domains identified by the jtf . the expectations of the groups included a desire to focus on 2 roles : pis and crcs conducting clinical trials . the deliverables also included the charge to review and refine the jtf competency statements , identify assessment areas for each competency statement , and determine gaps in existing training . as part of the first phase of the ecrptq project , a social / behavioral research ( s / br ) working group was created in response to a recognized need to address gcp in an appropriate and meaningful way for researchers conducting clinical trial testing behavioral interventions . this work is described in a separate paper authored by murphy and her colleagues in this journal . in this phase of the ecrptq project , members of the s / br working group were invited to participate in each of the competency domain working groups , contributing their expertise . workgroups began working as soon as co - leads were identified and workgroup membership assigned . s / br working group members were embedded within the competency domain working groups to provide feedback to ensure that the competencies were inclusive of s / br . after a series of conference calls , email exchanges , and 2 working meetings in 2015 , all competency domain working groups submitted their deliverables to the leadership team . following this submission , a review team comprised of individuals from across the consortium conducted a thorough appraisal of this work to synthesize and collate the materials and provide a final draft to be reviewed by the project leadership team before being forwarded to the ctsa steering committee . the core competencies were then reviewed by the jtf , coapcr , and association of clinical research professionals ( acrp ) . the final meeting was attended by project leadership , the competency domain co - leads , jtf , and coapcr . at the final meeting , the attendees revisited the importance of focusing on drafting competency statements that represent clear and measurable expressions of performance for professionals involved in clinical trials . the attendees also discussed areas of potential overlap within the framework , with some competencies appearing in more than 1 general competency domain . the 51 competency statements written by the jtf were carefully reviewed by competency domain working groups and review teams . of the 51 jtf statements , 34 were modified to enhance meaning and to reflect a focus on clinical trials . five of the jtf competency statements were removed as stand - alone statements and were rewritten as assessments for other competencies , and 3 new ecrptq competency statements were added . in total , 48 ecrptq competency statements reflect the work of these groups ( see appendix 1 ) . several items are particularly noteworthy regarding the overall work:first , the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research.second , the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process , believing the concept of team science to be of critical importance for the ctsa consortium . because the field of team science is still emerging and that the relevant skills are still being defined , the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership , professionalism , and team science . as team science competencies emerge and are refined , it may make sense later to separate the domains.third , there are purposefully some areas of overlap across domains , as the group agreed that understanding concepts can be very related but still highly nuanced.the s / br working group suggested additional edits to 6 competency statements in 3 competency domains . these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments.echoing a sentiment expressed by the jtf at its meeting in april 2015 , the group recognized the importance of regular updates and revisions to this work , corresponding to advances in science and concomitant regulations . first , the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research . second , the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process , believing the concept of team science to be of critical importance for the ctsa consortium . because the field of team science is still emerging and that the relevant skills are still being defined , the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership , professionalism , and team science . as team science competencies emerge and are refined , it may make sense later to separate the domains . third , there are purposefully some areas of overlap across domains , as the group agreed that understanding concepts can be very related but still highly nuanced . the s / br working group suggested additional edits to 6 competency statements in 3 competency domains . these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments . echoing a sentiment expressed by the jtf at its meeting in april 2015 , the group recognized the importance of regular updates and revisions to this work , corresponding to advances in science and concomitant regulations . competency statements are broad and meant to be generally applicable to both pis and crcs ; however , in thinking of how to apply these statements to different roles within a study team , competency domain working groups identified areas of assessment specific to investigators and crcs , with a principal focus on entry - level individuals . competency domain working groups were provided with an overview of bloom s taxonomy to assist them in writing measurable and appropriately leveled assessments that are at higher level ksas . most assessments proposed by the competency domain working groups are specific and measurable , such as prepare a research question and demonstrate knowledge of appropriate control , storage , and dispensing of investigational products . groups identified a total of 429 potential assessments across all domains , with 220 identified as appropriate for investigators and 209 identified as appropriate for crcs , a list that is in no way exhaustive . a considerable number of these assessments are identical for investigators and for crcs , so the total does not represent unique assessments . in terms of methods , many competency domain working groups suggested the use of case studies and observation of behavior , indicating that simply passing a quiz or multiple - choice exam is not indicative of mastery ; some also recommended pre / post testing . it is important to acknowledge that while the creation of detailed assessment methods was not within the scope of this work , some working groups did suggest methods that might be employed . competency domain working groups were also tasked with identifying and examining existing training believed to address the competencies identified within the groups respective competency domains . they took a broad approach to this task , based on the expertise and experiential knowledge of their members . it was not possible to examine every training program that might exist , but the groups reviewed a reasonably representative sample of offerings . in general , included were training and education offered by ctsa institutions ; by professional organizations devoted to education and resources ; by industry ; and by government units ( see http://bit.ly/1iv821x , entitled training gaps by competency domains ) . collectively , competency domain working groups examined the following number of education and training offerings ( with some offerings identified as appropriate for multiple competencies ) ( see table 2).table 2summary of existing education and trainingsctsa training and educationprofessional organizations education and trainingindustry education and traininggovernment education and training132140863total=343 offerings ( not unique)ctsa , clinical and translational science awards . from table 2 , of the 343 , 219 unique education and training offerings were identified . every competency domain had associated training with a few exceptions , noted below:
leadership , professionalism , and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials . no training identified.
leadership , professionalism , and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams . no training identified.
communication : describe the component parts of a traditional scientific publication . leadership , professionalism , and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials . leadership , professionalism , and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams . appear to be available online ; not all of those offerings are readily accessible in the public domain , or free of charge , however . in addition to the trainings identified as meeting - specified competencies , groups cited 214 supplemental resources , including web sites , reports , books , and published articles . see http://bit.ly/1u3pybs for the catalog of identified existing education and trainings organized by competency domain . sample online offerings:northwestern university , clinical and translational sciences institute introduction to clinical research online modulesuniversity of washington , institute of translational health sciences ( iths ) , self - directed learning centeroffice of research integrity : the lab , the research clinic
nih : teaching the responsible conduct of research
acrp : gcp an introduction to ich gcp guidelines
collaborative institutional training initiative ( citi ) : populations in research requiring additional consideration
uc davis : strengthening provider patient communication skills in clinical trials . northwestern university , clinical and translational sciences institute introduction to clinical research online modules university of washington , institute of translational health sciences ( iths ) , self - directed learning center office of research integrity : the lab , the research clinic
nih : teaching the responsible conduct of research
acrp : gcp an introduction to ich gcp guidelines
collaborative institutional training initiative ( citi ) : populations in research requiring additional consideration
uc davis : strengthening provider patient communication skills in clinical trials . competency domain working groups were not tasked to consider cost as a factor in the groups work , but it is certainly an important consideration and some groups did so . the cost of these offerings ranges from free to a significant financial investment by the department or individual . similarly , the working groups were not tasked to consider training quality , but many did so nevertheless identifying the need for training that actively engages the learner , going beyond mere rote memorization . a number of competency domain working groups noted that existing training focuses too heavily on theoretical concepts and historical events and not enough on the application of knowledge . competency domain working groups frequently noted that online offerings are insufficient alone and must be supplemented by local institutional education and training . the education and training offerings that are listed in this report ( see http://bit.ly/1u3pybs ) are not endorsed by the ecrptq leadership team . it was outside the parameters of this project to conduct a thorough evaluation of the suggested trainings from the competency domain working groups . there was broad agreement that the assessment of their quality should be undertaken as a future phase of the project . the ecrptq leadership team considered a variety of competency frameworks for this phase:the ctsa education and career development key function committee developed the ctsa master - level competencies , approved in 2011 ( http://bit.ly/21cx5n8 ) , to define the training standards for individuals functioning at the master s level in clinical and translational research . this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium . although the ecrptq leadership team felt these competencies to be highly relevant for investigators , they did not fully address the necessary qualifications and skills for other team members . the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees , including bioinformatics.the nih clinical research nursing domains of practice for the specialty of clinical research nursing ( http://cc.nih.gov/nursing/crn/dop_document.pdf ) was another framework that was reviewed and considered , but also did not necessarily address the qualifications and skills for all team members.competencies outlined by the oncology nursing society ( https://www.ons.org/sites/default/files/ctncompetencies.pdf ) were also reviewed but the group felt they were rather limited , focusing only on oncology studies.the ecrptq leadership also acknowledged the work of the national research coordinator consortium , formerly known as the ctsa research coordinator taskforce . like the other domains listed previously , the ecrptq leadership felt a framework that would be inclusive of all study team members was needed . the ctsa education and career development key function committee developed the ctsa master - level competencies , approved in 2011 ( http://bit.ly/21cx5n8 ) , to define the training standards for individuals functioning at the master s level in clinical and translational research . this framework includes 14 thematic areas that are intended to shape the training experiences of early career investigators and it represents the foundation for many graduate programs in clinical research across the ctsa consortium . although the ecrptq leadership team felt these competencies to be highly relevant for investigators , they did not fully address the necessary qualifications and skills for other team members . the ecrptq leadership team also examined specialty competencies in a variety of areas developed by ctsa key function committees , including bioinformatics . the nih clinical research nursing domains of practice for the specialty of clinical research nursing ( http://cc.nih.gov/nursing/crn/dop_document.pdf ) was another framework that was reviewed and considered , but also did not necessarily address the qualifications and skills for all team members . competencies outlined by the oncology nursing society ( https://www.ons.org/sites/default/files/ctncompetencies.pdf ) were also reviewed but the group felt they were rather limited , focusing only on oncology studies . the ecrptq leadership also acknowledged the work of the national research coordinator consortium , formerly known as the ctsa research coordinator taskforce . like the other domains listed previously , the ecrptq leadership felt a framework that would be inclusive of all study team members was needed . ultimately , the framework proposed by the joint task force for clinical trial competency ( jtf ) was selected that identified 8 broad domains of competence:(1)scientific concepts and research design(2)ethical and participant safety considerations(3)medicines development and regulation(4)clinical trial operations(5)study and site management(6)data management and informatics(7)leadership and professionalism(8)communication and teamwork
scientific concepts and research design ethical and participant safety considerations medicines development and regulation clinical trial operations study and site management data management and informatics leadership and professionalism communication and teamwork the ecrptq leadership team selected this framework because of its comprehensive applicability and its widespread uptake by numerous other stakeholders in the clinical trial enterprise . an additional consideration was the knowledge that the jtf mapped its competency framework to those mentioned previously , along with additional competency frameworks identified by consortium of academic programs in clinical research ( coapcr ) and the uk national health service . the jtf model is displayed in table 1.table 1jtf core competenciesdomaindefinitioncompetenciesscientific concepts and research designknowledge of scientific concepts related to the design and analyses of clinical trials5ethical and participant safety considerationsknowledge of the care of patients , human subject protections , and safety in the conduct of a clinical trial8medicines development and regulationknowledge of how drugs , biologics , and devices are developed and regulated7clinical trial operationsknowledge of study management , gcp compliance ( regulatory affairs ) , safety reporting ( adverse event identification and reporting , postmarket surveillance , pharmacovigilance ) , and the handling of investigational product12study and site managementknowledge of requirements for site management ( financial , personnel , including site and study operations , not including regulatory affairs)6data management and informaticsknowledge of how data are acquired and managed during a clinical trial ( source data , data entry , queries , quality control , corrections ) and the concept of a locked database5leadership and professionalismknowledge of the principles and practice of leadership and professionalism in clinical research4communication and teamworkknowledge of all elements of communication within the site and between the site and sponsors , contract research organizations , regulators . this ctsa - wide endeavor drew upon expertise across the consortium for this important and complex project . the ecrptq leadership team invited members of the ctsa consortium with relevant expertise to participate in each of the competency domain working groups , which were created based on the 8 competency domains identified by the jtf . the expectations of the groups included a desire to focus on 2 roles : pis and crcs conducting clinical trials . the deliverables also included the charge to review and refine the jtf competency statements , identify assessment areas for each competency statement , and determine gaps in existing training . as part of the first phase of the ecrptq project , a social / behavioral research ( s / br ) working group was created in response to a recognized need to address gcp in an appropriate and meaningful way for researchers conducting clinical trial testing behavioral interventions . this work is described in a separate paper authored by murphy and her colleagues in this journal . in this phase of the ecrptq project , members of the s / br working group were invited to participate in each of the competency domain working groups , contributing their expertise . workgroups began working as soon as co - leads were identified and workgroup membership assigned . s / br working group members were embedded within the competency domain working groups to provide feedback to ensure that the competencies were inclusive of s / br . after a series of conference calls , email exchanges , and 2 working meetings in 2015 , all competency domain working groups submitted their deliverables to the leadership team . following this submission , a review team comprised of individuals from across the consortium conducted a thorough appraisal of this work to synthesize and collate the materials and provide a final draft to be reviewed by the project leadership team before being forwarded to the ctsa steering committee . the core competencies were then reviewed by the jtf , coapcr , and association of clinical research professionals ( acrp ) . the final meeting was attended by project leadership , the competency domain co - leads , jtf , and coapcr . at the final meeting , the attendees revisited the importance of focusing on drafting competency statements that represent clear and measurable expressions of performance for professionals involved in clinical trials . the attendees also discussed areas of potential overlap within the framework , with some competencies appearing in more than 1 general competency domain . the 51 competency statements written by the jtf were carefully reviewed by competency domain working groups and review teams . of the 51 jtf statements , 34 five of the jtf competency statements were removed as stand - alone statements and were rewritten as assessments for other competencies , and 3 new ecrptq competency statements were added . in total , 48 ecrptq competency statements reflect the work of these groups ( see appendix 1 ) . several items are particularly noteworthy regarding the overall work:first , the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research.second , the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process , believing the concept of team science to be of critical importance for the ctsa consortium . because the field of team science is still emerging and that the relevant skills are still being defined , the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership , professionalism , and team science . as team science competencies emerge and are refined , it may make sense later to separate the domains.third , there are purposefully some areas of overlap across domains , as the group agreed that understanding concepts can be very related but still highly nuanced.the s / br working group suggested additional edits to 6 competency statements in 3 competency domains . these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments.echoing a sentiment expressed by the jtf at its meeting in april 2015 , the group recognized the importance of regular updates and revisions to this work , corresponding to advances in science and concomitant regulations . first , the competency domain working group reviewing the medicines development and regulation domain called for a renaming of that domain to investigational products development and regulation to be more inclusive of device research . second , the domain communication and teamwork was separated into 2 domains at the request of the ecrptq leadership team early in the phase ii process , believing the concept of team science to be of critical importance for the ctsa consortium . because the field of team science is still emerging and that the relevant skills are still being defined , the decision was made that the domain should be combined with the leadership and professionalism domain to become leadership , professionalism , and team science . as team science competencies emerge and are refined , it may make sense later to separate the domains . third , there are purposefully some areas of overlap across domains , as the group agreed that understanding concepts can be very related but still highly nuanced . the s / br working group suggested additional edits to 6 competency statements in 3 competency domains . these suggestions are relevant to study teams specifically conducting clinical trials involving behavioral interventions or assessments . echoing a sentiment expressed by the jtf at its meeting in april 2015 , the group recognized the importance of regular updates and revisions to this work , corresponding to advances in science and concomitant regulations . competency statements are broad and meant to be generally applicable to both pis and crcs ; however , in thinking of how to apply these statements to different roles within a study team , competency domain working groups identified areas of assessment specific to investigators and crcs , with a principal focus on entry - level individuals . competency domain working groups were provided with an overview of bloom s taxonomy to assist them in writing measurable and appropriately leveled assessments that are at higher level ksas . most assessments proposed by the competency domain working groups are specific and measurable , such as prepare a research question and demonstrate knowledge of appropriate control , storage , and dispensing of investigational products . groups identified a total of 429 potential assessments across all domains , with 220 identified as appropriate for investigators and 209 identified as appropriate for crcs , a list that is in no way exhaustive . a considerable number of these assessments are identical for investigators and for crcs , so the total does not represent unique assessments . in terms of methods , many competency domain working groups suggested the use of case studies and observation of behavior , indicating that simply passing a quiz or multiple - choice exam is not indicative of mastery ; some also recommended pre / post testing . it is important to acknowledge that while the creation of detailed assessment methods was not within the scope of this work , some working groups did suggest methods that might be employed . competency domain working groups were also tasked with identifying and examining existing training believed to address the competencies identified within the groups respective competency domains . they took a broad approach to this task , based on the expertise and experiential knowledge of their members . it was not possible to examine every training program that might exist , but the groups reviewed a reasonably representative sample of offerings . in general , included were training and education offered by ctsa institutions ; by professional organizations devoted to education and resources ; by industry ; and by government units ( see http://bit.ly/1iv821x , entitled training gaps by competency domains ) . collectively , competency domain working groups examined the following number of education and training offerings ( with some offerings identified as appropriate for multiple competencies ) ( see table 2).table 2summary of existing education and trainingsctsa training and educationprofessional organizations education and trainingindustry education and traininggovernment education and training132140863total=343 offerings ( not unique)ctsa , clinical and translational science awards . from table 2 , of the 343 , 219 unique education and training offerings were identified . every competency domain had associated training with a few exceptions , noted below:
leadership , professionalism , and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials . no training identified.
leadership , professionalism , and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams . no training identified.
communication : describe the component parts of a traditional scientific publication . leadership , professionalism , and team science : identify and apply professional guidelines and codes of ethics as they relate to the conduct of clinical trials . leadership , professionalism , and team science : describe the methods necessary to work effectively with multidisciplinary and interprofessional research teams . appear to be available online ; not all of those offerings are readily accessible in the public domain , or free of charge , however . in addition to the trainings identified as meeting - specified competencies , groups cited 214 supplemental resources , including web sites , reports , books , and published articles . see http://bit.ly/1u3pybs for the catalog of identified existing education and trainings organized by competency domain . sample online offerings:northwestern university , clinical and translational sciences institute introduction to clinical research online modulesuniversity of washington , institute of translational health sciences ( iths ) , self - directed learning centeroffice of research integrity : the lab , the research clinic
nih : teaching the responsible conduct of research
acrp : gcp an introduction to ich gcp guidelines
collaborative institutional training initiative ( citi ) : populations in research requiring additional consideration
uc davis : strengthening provider patient communication skills in clinical trials . northwestern university , clinical and translational sciences institute introduction to clinical research online modules university of washington , institute of translational health sciences ( iths ) , self - directed learning center office of research integrity : the lab , the research clinic
nih : teaching the responsible conduct of research
acrp : gcp an introduction to ich gcp guidelines
collaborative institutional training initiative ( citi ) : populations in research requiring additional consideration
uc davis : strengthening provider patient communication skills in clinical trials . competency domain working groups were not tasked to consider cost as a factor in the groups work , but it is certainly an important consideration and some groups did so . the cost of these offerings ranges from free to a significant financial investment by the department or individual . similarly , the working groups were not tasked to consider training quality , but many did so nevertheless identifying the need for training that actively engages the learner , going beyond mere rote memorization . a number of competency domain working groups noted that existing training focuses too heavily on theoretical concepts and historical events and not enough on the application of knowledge . competency domain working groups frequently noted that online offerings are insufficient alone and must be supplemented by local institutional education and training . the education and training offerings that are listed in this report ( see http://bit.ly/1u3pybs ) are not endorsed by the ecrptq leadership team . it was outside the parameters of this project to conduct a thorough evaluation of the suggested trainings from the competency domain working groups . there was broad agreement that the assessment of their quality should be undertaken as a future phase of the project . competency domain working groups identified 115 specific training gaps across the 8 reviewed domains . in the review of identified trainings , 5 broad categories of gaps were identified : ( 1 ) training that is needed but does not currently exist ; ( 2 ) existing training that is not adequate ; ( 3 ) certification , documentation of skill , or formal assessment is needed ; ( 4 ) a core training curriculum needs to be defined and/or developed ; and ( 5 ) there is inadequate training at a level for crcs . overwhelmingly , groups concluded that some training exists for most ecrptq competency domains , but that this training is not adequate to fully meet the needs of the investigators and crcs . this finding is consistent with the data collected in the jtf core competency survey ; participants were asked whether they felt a need for training in these domains , with the majority of respondents indicating they felt training was needed . overall , the education and training identified is primarily investigator focused , particularly offerings provided by ctsa institutions . lack of training is keenly felt in the adoption of new technologies and in areas such as data management . training is not generally organized by level of expertise , and often does not distinguish roles and responsibilities between investigators and crcs . some competency domain working groups , such as leadership , professionalism , and team science , and communication were forced to draw from offerings completely outside the context of clinical research , because they were unable to find relevant training within clinical research . as noted previously , many of the trainings cited in this report do not provide opportunities to apply knowledge and do not incorporate learning strategies known to be effective in promoting the development of competence . the s / br working group reviewed all the competency domain working groups deliverables and offered suggested edits to the ecrptq competencies in some domains to promote greater inclusion of research teams carrying out clinical trials involving behavioral interventions and assessments . additional comments made by the group regarding trainings and assessments for select domains were made ( reference to http://bit.ly/1raesik ) . the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework . the survey asked individuals to self - assess their own level of competence in the framework s domains , as well as the relevance of those domains and their perceived needs for additional training . this organization is also currently exploring mechanisms to revise and update its competency statements , particularly as new scientific fields and technologies emerge.building upon the identification of assessment areas by competency domain working groups , specific assessments must be developed to assess competence . such assessments should focus not only on different study team member roles , but levels of mastery as well.an evaluation of the quality of existing training should be undertaken , as well as an expansion of the catalog of training that emerged from the working groups . this catalog does not include all available educational opportunities and should be expanded to include additional relevant training.a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed . if so , the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development , built upon the principles of adult learning.examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment , based on the competency framework developed in this work may be warranted.an eportfolio system would be identified to allow pis and crcs to collect , organize , and share their completed trainings , demonstrate learning , and have a portable record of their achievements . such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions , sponsors , and other regulatory bodies.because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts ( investigators and crcs ) , these continued activities should be undertaken in partnership with individuals from the jtf , coapcr , acrp , clinical trials transformation initiative ( ctti ) and other organizations invested in clinical research professional training . these organizations are committed to study team education and training , and the jtf framework is gaining significant traction nationally and internationally through their efforts.recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training , with step - wise approaches to training personnel.job descriptions should be modified to reflect specific competencies by levels.inter-institutional training courses should be developed and shared as cost - free , easily accessible , web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education.academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals . if an institution does not offer a clinical research program of study , then clinical research staff should be able to transfer educational benefits to other institutions . the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework . the survey asked individuals to self - assess their own level of competence in the framework s domains , as well as the relevance of those domains and their perceived needs for additional training . this organization is also currently exploring mechanisms to revise and update its competency statements , particularly as new scientific fields and technologies emerge . building upon the identification of assessment areas by competency domain working groups such assessments should focus not only on different study team member roles , but levels of mastery as well . an evaluation of the quality of existing training should be undertaken , as well as an expansion of the catalog of training that emerged from the working groups . this catalog does not include all available educational opportunities and should be expanded to include additional relevant training . a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed . if so , the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development , built upon the principles of adult learning . examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment , based on the competency framework developed in this work may be warranted . an eportfolio system would be identified to allow pis and crcs to collect , organize , and share their completed trainings , demonstrate learning , and have a portable record of their achievements . such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions , sponsors , and other regulatory bodies . because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts ( investigators and crcs ) , these continued activities should be undertaken in partnership with individuals from the jtf , coapcr , acrp , clinical trials transformation initiative ( ctti ) and other organizations invested in clinical research professional training . these organizations are committed to study team education and training , and the jtf framework is gaining significant traction nationally and internationally through their efforts . recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training , with step - wise approaches to training personnel . inter - institutional training courses should be developed and shared as cost - free , easily accessible , web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education . academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals . if an institution does not offer a clinical research program of study , then clinical research staff should be able to transfer educational benefits to other institutions . competency domain working groups identified 115 specific training gaps across the 8 reviewed domains . in the review of identified trainings , 5 broad categories of gaps were identified : ( 1 ) training that is needed but does not currently exist ; ( 2 ) existing training that is not adequate ; ( 3 ) certification , documentation of skill , or formal assessment is needed ; ( 4 ) a core training curriculum needs to be defined and/or developed ; and ( 5 ) there is inadequate training at a level for crcs . overwhelmingly , groups concluded that some training exists for most ecrptq competency domains , but that this training is not adequate to fully meet the needs of the investigators and crcs . this finding is consistent with the data collected in the jtf core competency survey ; participants were asked whether they felt a need for training in these domains , with the majority of respondents indicating they felt training was needed . overall , the education and training identified is primarily investigator focused , particularly offerings provided by ctsa institutions . lack of training is keenly felt in the adoption of new technologies and in areas such as data management . training is not generally organized by level of expertise , and often does not distinguish roles and responsibilities between investigators and crcs . some competency domain working groups , such as leadership , professionalism , and team science , and communication were forced to draw from offerings completely outside the context of clinical research , because they were unable to find relevant training within clinical research . as noted previously , many of the trainings cited in this report do not provide opportunities to apply knowledge and do not incorporate learning strategies known to be effective in promoting the development of competence . the s / br working group reviewed all the competency domain working groups deliverables and offered suggested edits to the ecrptq competencies in some domains to promote greater inclusion of research teams carrying out clinical trials involving behavioral interventions and assessments . additional comments made by the group regarding trainings and assessments for select domains were made ( reference to http://bit.ly/1raesik ) . the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework . the survey asked individuals to self - assess their own level of competence in the framework s domains , as well as the relevance of those domains and their perceived needs for additional training . this organization is also currently exploring mechanisms to revise and update its competency statements , particularly as new scientific fields and technologies emerge.building upon the identification of assessment areas by competency domain working groups , specific assessments must be developed to assess competence . such assessments should focus not only on different study team member roles , but levels of mastery as well.an evaluation of the quality of existing training should be undertaken , as well as an expansion of the catalog of training that emerged from the working groups . this catalog does not include all available educational opportunities and should be expanded to include additional relevant training.a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed . if so , the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development , built upon the principles of adult learning.examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment , based on the competency framework developed in this work may be warranted.an eportfolio system would be identified to allow pis and crcs to collect , organize , and share their completed trainings , demonstrate learning , and have a portable record of their achievements . such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions , sponsors , and other regulatory bodies.because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts ( investigators and crcs ) , these continued activities should be undertaken in partnership with individuals from the jtf , coapcr , acrp , clinical trials transformation initiative ( ctti ) and other organizations invested in clinical research professional training . these organizations are committed to study team education and training , and the jtf framework is gaining significant traction nationally and internationally through their efforts.recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training , with step - wise approaches to training personnel.job descriptions should be modified to reflect specific competencies by levels.inter-institutional training courses should be developed and shared as cost - free , easily accessible , web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education.academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals . if an institution does not offer a clinical research program of study , then clinical research staff should be able to transfer educational benefits to other institutions .
the recently completed jtf core competency survey that is currently being analyzed will provide important data that will help validate the jtf framework . the survey asked individuals to self - assess their own level of competence in the framework s domains , as well as the relevance of those domains and their perceived needs for additional training . this organization is also currently exploring mechanisms to revise and update its competency statements , particularly as new scientific fields and technologies emerge . building upon the identification of assessment areas by competency domain working groups such assessments should focus not only on different study team member roles , but levels of mastery as well . an evaluation of the quality of existing training should be undertaken , as well as an expansion of the catalog of training that emerged from the working groups . this catalog does not include all available educational opportunities and should be expanded to include additional relevant training . a deeper exploration of training gaps should be undertaken to determine whether new training modules are needed . if so , the development of this education and training should be undertaken by individuals skilled in instructional design and curriculum development , built upon the principles of adult learning . examination of a cloud - based learning management platform to support individuals seeking and tracking their cbe and assessment , based on the competency framework developed in this work may be warranted . an eportfolio system would be identified to allow pis and crcs to collect , organize , and share their completed trainings , demonstrate learning , and have a portable record of their achievements . such a system would allow individuals to upload artifacts of competence that could be made available to relevant institutions , sponsors , and other regulatory bodies . because of the extraordinary opportunity of working with external stakeholder organizations to identify standard competencies for these 2 cohorts ( investigators and crcs ) , these continued activities should be undertaken in partnership with individuals from the jtf , coapcr , acrp , clinical trials transformation initiative ( ctti ) and other organizations invested in clinical research professional training . these organizations are committed to study team education and training , and the jtf framework is gaining significant traction nationally and internationally through their efforts . recommendations for institutional policies on clinical research training should be expanded beyond basic irb and gcp training , with step - wise approaches to training personnel . inter - institutional training courses should be developed and shared as cost - free , easily accessible , web - based formats with additional train - the - trainer mechanisms for onsite training and continuing education . academic pathways for baccalaureate and graduate degrees in clinical research should be endorsed and more highly accessible to clinical research professionals working in academic medical centers and hospitals . if an institution does not offer a clinical research program of study , then clinical research staff should be able to transfer educational benefits to other institutions . this document is the result of the ctsa consortium ecrptq supplement and includes discussion and consensus documents with iterative revisions . the competencies and assessments generated in phase ii of the ecrptq supplement have been approved by the ctsa consortium and have the support of jtf , coapcr , and acrp . the competencies and assessments have been submitted to national center for advancing translational sciences ( ncats ) for their consideration . although these documents provide a framework for investigator and crc training it does not provide direction on how to implement a training program . the purpose of this document is to provide a standard set of core clinical trial competencies and ksa assessments that equip investigators and crcs with the necessary skills to more effectively , efficiently , and safely execute clinical trials . education and training should be patterned to these competencies and ultimately lead to specific formative and summative evaluations of learning . | introductiontraining for the clinical research workforce does not sufficiently prepare workers for today s scientific complexity ; deficiencies may be ameliorated with training .
the enhancing clinical research professionals training and qualifications developed competency standards for principal investigators and clinical research coordinators.methodsclinical and translational science awards representatives refined competency statements .
working groups developed assessments , identified training , and highlighted gaps.resultsforty-eight competency statements in 8 domains were developed.conclusionstraining is primarily investigator focused with few programs for clinical research coordinators .
lack of training is felt in new technologies and data management .
there are no standardized assessments of competence . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
gastric carcinomas arise from the mucus secreting cells of gastric crypts or from areas of intestinal metaplsia . histologically , almost all gastric carcinomas are adenocarcinomas , but adenosquamous and squamous varieties comprise a very small portion ( less than 1% ) . adenocarcinomas can be classified into four histologic types : the diffuse type ( synonymous with mucous or signet ring cell type ) , the intestinal type , the mixed type and the pylorocardiac gland cell type . among the subtypes , pure signet ring cell type is very rare and has a poor prognosis . in this type , numerous mucin vacuoles distend the cells and press the nucleus flat against the plasma membrane , resulting in the typical signet ring appearance . herein is presented a case of pure signet ring type early gastric carcinoma with extensive lymph node metastases . he was well until four months prior to admission , when he began to experience indigestion and left upper quadrant pain . an ugis was performed , revealing a round 1.5 1.3 cm depression , suggestive of a chronic gastric ulcer or early gastric cancer ( fig . 1 ) . on gastrofiberscopic examination , a depressed , discolored lesion with converging irregular radiating folds in the antral portion like that seen in early gastric cancer ( type iic ) , was found and microscopic examination revealed signet ring cell carcinoma ( fig . the patient was admitted for further evaluation and treatment , on admission his vital signs were temperature 36.4c , pulse 75 , respiration 20 and blood pressure 110/80 mmhg . on examination , the patient appeared to be slender and chronically ill . no lymphadenopathy was found . laboratory results included normal urinalysis , hemoglobin 14.8 g / dl ; hematocrit , 43% ; wbc , 5,800/mm with 67% neutrophils , 27% lymphocytes and 6% monocytes , and a platelet count of 204,000/mm . blood chemistries were nitrogen 14.7 mg / dl , creatinine 1 . 4 mg / dl , total protein 6.4 g / dl , albumin 4.2 g / dl , globulin 2.2 g / dl , cholesterol 215 mg / dl , total bilirubin 0.6 mg / dl , direct bilirubin 0.2 mg / dl and indirect 0.4 mg / dl . the chest x - ray was normal and a computerized tomographic scan of the abdomen showed a no evidence of metastatic lesions . on the 4th hospital day , the patient was transferred to the general surgery department . surgery was performed on the 7th hospital day , revealing a shallow depression in the anteior wall of the greater curvature side in the antrum and multiple perigastric lymph node enlargement . a billroth type ii subtotal gastrectomy was performed and lymph nodes along the portal hepatis , celiac axis branch were removed . mapping of the resectioned subtotal stomach was completed ( stage iii ; t1 n1 m0 ) ( fig . grossly , the subtotal stomach , 25 cm along the greater curvature and 11 cm along the lesser curvature , presented a pinkish brown , smooth serosal surface . an irregular , superficial depressed lesion , 31.5 cm , was noted on the anterior wall of the greater curvature in the antrum , 3 cm from the distal pyloric ring , giving it a reddened and granular appearance with a relatively well defined margin . microscopically , a pure signet ring cell carcinoma , confined by the muscularis mucosa but invading the lamina propria was observed . areas of the carcinoma were infiltrated by isolated signet ring cells while others were composed of compactly aggregated signet ring cells . lymph node involvement ( metastases to 7 of 11 superior gastric nodes and to 10 of 14 inferior gastric nodes ) was extensive , though the carcinoma was confined to the mucosa ( fig . 7 ) . on the 14th postoperative day , chemotherapy ( 5-fu , adriamycin and mitomycin ) was administered to the patient and on the twenty third hospital day , he was discharged in an improved condition . on the 45th postoperative day , the patient received a second dose of kur chemotherapy and was followed in the out - patient clinic . early gastric cancer has been defined as a carcinoma which is confined to the mucosa or submucosa , regardless of the presence of lymph node metastasis . in this case , a pure signet ring cell carcinoma located in the anterior wall of the greater curvature side in the antrum and restricted to the mucosa with no invasion of the muscularis mucosa was observed . also widespread lymph node metastases were seen . histologically , diffuse type adenocarcinoma accounts for 33 to 40% of the cases of gastric carcinoma , the intestinal type varies from 22 to 53 % and pylorocardiac gland cell type , 28% , but in korea , shim et al . , reported in a review of 108 cases of gastric adenocarcinoma , that mucous cell carcinoma accounted for 62% of the cases ; pylorocardiac gland cell carcinoma , 23% ; intestinal cell carcinoma , 8% and unclassified type , 7% . in a review of 574 gastric carcinomas , brander , et al . classified mucous carcinomas into three main types : pure signet ring cell type , well differentiated type and mixed type . recently , the incidence of signet ring cell in gastric carcinoma biopsy and resection has risen sharply . mulligan and rember reported that mucous cell carcinomas composed of signet ring cells are more frequent in females than in males , occur at a comparatively younger age ( less than 40 years old ) and have a poor prognosis , while intestinal cell type and pylorocardiac gland cell type occur moe frequently after the age of 40 years and have a better prognosis than mucous cell type . stemmermann and brown pointed out that the 5 year survival rates were about 27.4% for intestinal type and 9.9% for diffuse type , but according to the miwa report , only the early diffuse type had a better prognosis than early intestinal type ( 5 year survival rates of 90% and 85% respectively ) . the majority of the diffuse type shows mixed patterns , in which extracellular mucus and gland formation are present with signet ring cells , while there are only a few cases of pure signet ring cell type . the survival for pure signet ring cell type and mixed type is about 5 and 21 months respectively . in a study of 167 early gastric carcinomas , kodama , et al . reported that in the so - called small mucosal type a lesion of 4 cm . or less with only mucosal invasion , only one case exhibited lymph node metastasis . therefore , this case with widespread lymph node metastases although confined to the mucosa , is thought to be rare and highly malignant , although the pathogenesis is not yet known . | a 42-year old man was admitted with indigestion and left upper quadrant pain . a gastrofiberscopic examination revealed a depressed , discolored lesion ( 1.5 1.5 cm ) with converging irregular radiating folds in the antral portion , suggestive of early gastric cancer ( type iic ) .
histologically , signet ring cell carcinoma was seen .
a subtotal gastrectomy was performed and the opeartive findings showed a shallow depression in the anterior wall of the greater curvature side in the antrum and multiple perigastric lymph node enlargment.on microscopic examination , the authors observed pure signet ring cell carcinoma confined to the mucosa but with widespread lymph node metastases . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
we used the arizona and california state inpatient databases ( sid ) from the us agency for healthcare research and quality ( ahrq ) to describe clinical and demographic characteristics of persons hospitalized for disseminated coccidioidomycosis who did not have known primary immune conditions , hiv infection , or aids . the sid contains state - level data , including information from 90% of community hospital in - patient stays , and is maintained as part of the healthcare utilization project at ahrq . we extracted records from the available annual datasets for arizona ( 20002009 ) and california ( 20032008 ) that listed any diagnosis of disseminated coccidioidomycosis ( code 114.3 from the international classification of diseases , 9th revision , clinical modification [ icd-9-cm ] ) . we excluded records that listed any diagnosis of hiv infection or aids ( icd-9-cm code 042 ) or primary immune deficiency ( icd-9-cm code 279.xx ) . this study was not considered human subject research by the national institutes of health office of human subjects protection . we calculated the average annual incidence of hospitalizations as the average annual number of disseminated coccidioidomycosis hospitalizations divided by the midyear population , as determined by us census estimates ( 9 ) . by using the revisit files available from ahrq , we assessed differences in readmissions by race by comparing the proportion of persons with only 1 disseminated coccidioidomycosis infection among race groups . we also calculated all - cause hospitalization rates for blacks and whites in our dataset by using total hospitalizations from hcupnet ( http://hcupnet.ahrq.gov ) . in years for which patient state of residency was available , we assessed the potential bias from hospitalizations of persons with out - of - state residency by determining the percentage of total admissions from out - of - state patients . we used the mann - whitney test for nonparametric comparisons ; the significance level used for all statistical tests was = 0.05 . all analyses were completed in sas version 9.2 software ( sas institute , cary , nc , usa ) . the average annual incidence of hospitalizations/100,000 persons / year was 4.8 ( 95% ci 4.25.3 ) for arizona and 0.89 ( 95% ci 0.790.99 ) for california . overall , the rate of hospitalization for blacks in arizona was 12.0-fold higher than the rate of hospitalization for whites and 20.8-fold higher than for hispanics ( figure 1 ) . the rate of hospitalization in california was 8.8-fold higher for blacks than for whites and 5.6-fold higher for blacks than for hispanics ( figure 2 ) . average annual incidence ( per 100,000 population ) of disseminated coccidioidomycosis associated hospitalizations , by race / ethnicity , arizona , usa . average annual incidence ( per 100,000 population ) of disseminated coccidioidomycosis hospitalizations , by race / ethnicity , california , usa . ( note : average annual incidence not reported for native americans because of low numbers . ) the median age of patients with disseminated coccidioidomycosis associated hospitalizations was lower than the median age of all hospitalizations for both blacks and whites in arizona and california ( p<0.001 for all comparisons ) . the median age at hospitalization for hispanics was higher than the median age for all hospitalizations in arizona ( p<0.001 ) and california ( p = 0.01 ) ( table ) . * boldface indicates a significant difference ( p<0.05 ) compared with all - cause hospitalizations . nr , information not reported because cell size is < 10 or to prevent calculation of a cell size < 10 . the proportion of persons with > 1 hospital admission for disseminated coccidioidomycosis among blacks was higher than for whites in california in 2008 ( 29.8% vs. 17.3% ) and in arizona in 2007 ( 19.3% vs. 17.0% ) . among all disseminated coccidioidomycosis associated hospitalizations , 98% of hospitalizations in arizona and 99% of hospitalizations in california were from in - state residents , indicating minimal bias from nonresidents . all - cause hospitalization rates did not differ greatly between whites and blacks : 121 hospitalizations/1,000 persons for whites and 104 hospitalizations/1,000 persons for blacks in arizona ; 125 hospitalizations/1,000 persons for whites and 143 hospitalizations/1,000 persons for blacks in california . we identified a higher incidence and lower patient age at hospitalization for disseminated coccidioidomycosis associated hospitalizations among blacks in california and arizona . we were unable to determine if the higher incidence of hospitalization was a result of environmental , host , or behavioral factors . however , this study suggests that hiv infection , aids , and primary immune conditions are not the main reason for the racial / ethnic disparity for hospitalizations associated with disseminated coccidioidomycosis . because most coccidioidomycosis cases in california occur in the san joaquin valley region ( 6 ) and 90% of the population of california lives outside this region ( 10 ) , the california - specific incidence rate is likely to be an underrepresentation of the extent of the disease in the san joaquin valley region . in addition , because of this disparity , we are unable to make comparisons between rates for california and arizona . the state - specific incidence is a better description of the extent of disease in arizona because the 3 counties with 93% of the coccidioidomycosis cases in arizona represent 79% of the state s population ( 11 ) . the classification of race / ethnicity in this dataset may not completely describe the true distribution of disease among the diverse groups comprising these populations . these categories include a wide range of racial / ethnic backgrounds , representing multiple potential environmental , social , cultural , behavioral , or genetic susceptibilities . however , the higher incidence among blacks suggests that unknown factors uniquely affect a high proportion of this population . our analysis is likely to be specific for accurately detecting the number of cases of disseminated coccidioidomycosis because most cases require hospitalization of the patient . furthermore , in arizona and southern california , where the infection is common , awareness of coccidioidomycosis and disseminated coccidioidomycosis is high , and disseminated coccidioidomycosis cases are likely to be recognized . this analysis method is also likely to be specific because results of cultures , biopsies , histologic testing , and serologic testing provide strong evidence of infection . however , our use of administrative data , such as the sid , is limited by the use of icd-9-cm codes , and the sensitivity and specificity of those codes for disseminated coccidioidomycosis has not been evaluated . an additional limitation of our study was that we could not determine if a single person was hospitalized multiple times during the study years . although we identified higher rates of readmission among blacks , consistent with previous studies for coccidioidomycosis ( 12 ) , this does not completely explain the large relative rates that we identified . this finding could indicate different disease pathology with more serious or long - term infection . potential bias from out - of - state residents was minimal because most disseminated coccidioidomycosis hospitalizations occurred within the state of residence . differences in all - cause hospitalization rates were not likely to account for the differences in rates observed for our study condition . a better understanding of the progression of disease , including the number of previous hospitalizations , information on all coexisting conditions , and the severity of disease , could help explain the differences in incidence of hospitalization . overall , we found a higher incidence of disseminated coccidioidomycosis associated hospitalizations for blacks compared with whites and other racial / ethnic groups living in these coccidioidomycosis - endemic areas , a finding that is consistent with previous studies ( 6,7 ) . however , our study identifies this difference specifically in the absence of hiv / aids and primary immune conditions among a large cohort , which suggests other , unknown reasons for this disparity among races / ethnicities . | we analyzed hospitalization databases from arizona and california for disseminated coccidioidomycosis associated hospitalizations among immunocompetent persons .
racial / ethnic disease ratios were characterized by a higher incidence of hospitalization among blacks compared with other groups .
this finding suggests that hiv infection , aids , and primary immune conditions are not a major factor in this disparity . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
while opportunistic infections continue to be enameled care of patients infected with acquired immunodeficiency , cancer remains overlooked in the care of these patients in developing countries . according to center disease control ( cdc ) , the revised definition of aids criteria includes the biological criteria ( cd4 below 200 ) and/or clinical criteria ( opportunistic infections , cancers of the cervix , kaposi sarcoma , and non - hodgkin lymphoma ) associated with infection by human immunodeficiency virus ( hiv ) . the occurrence of malignant tumors in patients with hiv infection currently represents for years to come one of the most difficult and controversial challenges of therapeutic management of these patients . as of 2011 africa was the epicenter of viral related cancers even prior to the hiv epidemic ( e.g. , kaposi 's sarcoma ( ks ) , burkitt 's lymphoma , cervical cancer , and hepatocellular carcinoma ) . indeed , 30 to 40% of patients infected with hiv are likely to develop cancer about their disease ; the incidence of certain cancers such as kaposi 's sarcoma has decreased since the introduction of potent antiretroviral therapies ; other malignancies associated with aids do not seem to be affected by the establishment of this new therapeutic strategy . the prevalence coinfection tb / hiv ( 25.5% ) is well known while no study on cancers associated with hiv has been performed in our country . the widespread use of antiretroviral therapy ( arv ) in industrialized countries since the mid-1990s led to a dramatic decrease in mortality of patients with hiv infection and incidence of opportunistic infections . however , antiretroviral treatment types haart also had a significant impact on the natural history of hiv - associated malignancies . a us study found an increase in median survival of aids patients from 11.3 months before 1985 to 12.5 months in 1986 to 20.8 months in 1987 for aids cases diagnosed in washington . according to a recent british study , 40% of aids patients have cancer during the course of their disease . in france , a study showed that , out of a total of 964 recorded deaths , 149 were attributed to the type of aids defining cancers . according to the regional office for africa of who sub - saharan african countries have experienced nearly 600,000 deaths in 2008 related to tumors . if this epidemiological trend continues due to longer life expectancy and increased exposure to tobacco , africa will have by 2020 from 800,000 to 1,000,000 new cases of cancer each year [ 10 , 11 ] . the cancer incidence rate is estimated at 95.5 cases per 10 inhabitants in west africa and 90.0 per 10 in guinea . as in most sub - saharan african countries , there is very little data available on the prevalence of hiv infection in cancer patients in west africa , especially in our country . this study aims to determine the prevalence of hiv infection among patients seen at the surgical oncology unit of donka . data from this study were obtained from the hospital and came from surgical oncology unit of donka national hospital , teaching hospital of conakry ( guinea ) . the study involved the records of cancer patients followed with hiv infection from may 2007 to december 2012 . this checkup has become routine for any cancer patient received in our unit since january 2009 . the prevalence has been determined according to the number of patients and pathology subject of consultation or follow - up . pathologies associated with hiv have been classified into malignant , benign , and inflammatory diseases . social characteristics ( age , gender , marital status , tobacco and alcohol consumption , education , and body mass index ) and immune status ( hiv type , cd4 count , and antiretroviral treatment ) were reviewed according malignant tumor pathologies , divided into defining aids and nondefining aids cancers . gynecological history ( age at menarche , first full pregnancy ) was determined in women with breast cancer . out of 3143 patients received from may 2007 to december 2012 , 69 ( 2.2% ) tested positive for hiv in our unit . this proportion was 12 ( 1.9% ) of 625 patients from 2007 to 2008 and 57 ( 2.3% ) of 2518 patients from 2009 to 2012 . of the 64 hiv - infected patients , 54 ( 78.3% ) had a malignant tumor , 8 ( 11.6% ) of benign and 7 ( 10.1% ) of inflammatory tumors ( table 1 ) . the prevalence of hiv in cancer patients was 54 on 2598 cases ( 2.1% ) . of the 54 cases of cancers associated with hiv , there were 11 ( 20.4% ) defining aids and 43 ( 79.6% ) nondefining aids . cancer patients had a median age of 39 ( iqr 3148 ) against 32 ( iqr 2538 ) for noncancer patients . this median age was different about defining aids status : 33 ( 25.040.0 ) versus 39 ( iqr 35.750.0 ) ( p = 0.007 ) . their body mass index was 20,4 kg / m ( iqr 17.721.8 ) . they were unschooled in 40 ( 74.1% ) and married in 35 ( 64.8% ) . tobacco use was found in 13 ( 24.1% ) and alcohol consumption in 14 ( 20.2% ) . cd4 count , performed in 22 patients , showed a median of 317 cells / ml ( iqr 246.2534.2 ) . antiretroviral treatment was performed in 40 ( 74.1% ) patients of which it was performed in 39 only after the cancer diagnosis . table 2 shows the distribution of different types of cancers depending on the aids definition . among the cancers defining aids , non - hodgkin 's lymphoma ( 6 ) ( 11.1% ) and cervical cancer ( 5 ) ( 9.3% ) were the most common . breast cancer ( 6 ) ( 11.1% ) , followed by cancer of the liver ( 6 ) ( 11.1% ) , and eye and annexes ( 6 ) ( 11.1% ) were leading nondefining aids cancers . patients with breast cancer had a median age of 14.0 years ( iqr 12.515.0 ) at menarche and 22.0 years ( iqr 17.025.0 ) in the first full term pregnancy ; and they had 1.0 full pregnancy in term ( iqr 0.53.0 ) . this preliminary study shows a high prevalence ( 2.1% ) of the hiv infection in patients with cancer in our surgical oncology unit . this prevalence appeared to be higher than in the general population in guinea ( 1.5% ) but lower than in the tertiary hospital ( 6.0% ) in nigeria . risk of cancer occurrence in hiv - infected people is 6.39 times higher compared to uninfected hiv people . in this study , non - aids defining cancers were more frequent than defining aids cancers ( 77.8% versus 22.2% ) . this could be explained partly by the decrease in the number of cancer cases defining aids since the beginning of antiretroviral therapy as supported by yanik et al . in the us . also , kaposi 's sarcoma ( ks ) and cervical cancer are mainly managed , respectively , by the dermatology and gynecology departments of teaching hospital . kaposi 's sarcoma is very common in dermatology and the guinean first data were published by cisse et al . . in nigeria , ks and cervical cancer are the most common cancers among non - aids defining cancers . cervical cancer is the most common cancer in guinea with a high prevalence of human papillomavirus infection . in this study , nhl was leading defining aids cancer . this predominance of breast cancer among nondefining aids cancers is reported by studies in sub - saharan africa [ 21 , 22 ] . the hiv infection is very prevalent , about 35.4% among women with breast cancer in uganda and 19.7% in south africa . in developed countries , lung cancer is the most common of nondefining aids cancers [ 16 , 23 ] . conversely , those with aids classifying cancer were younger than those who had a non - aids defining illness cancer . the female gender and the young age of patients are already reported by several authors [ 14 , 21 ] . this could be related to the irregular availability of reagents for cd4 and antiretroviral despite their free access in our country . cd4 count , carried out for 22 cases out of 54 , showed no difference in the depth of immunosuppression that cancer is either aids defining or not . for defining aids cancers , there is no doubt that the risk is higher in the case of lower cd4 count ( < 200 cells / ml ) [ 24 , 25 ] . however , there are controversies with regard to the depth of immunosuppression in nondefining aids cancer [ 22 , 23 , 25 ] . we consider that the art coverage rate ( 74.1% ) is low in this category of the population infected with hiv , although this rate is higher than the rate of coverage in guinea ( 50.0% ) . these patients are faced with two problems : hiv / aids with the supported opportunistic infections and cancer that is not supported by the national program against aids . that is why a national prospective study is needed to determine the true prevalence of cancer among people living with hiv . it will also be important to determine whether hiv changes the risk factors , clinicopathological futures , and prognosis of these cancers . breast cancer is the most common in this association . a national survey of a large sample is needed to determine the true prevalence and impact of hiv on cancer prognosis . | aim . to determine the prevalence of hiv infection among patients seen at the surgical oncology unit of donka ( conakry , guinea )
. method .
we conducted a retrospective and descriptive study of hiv infection in cancer patients from may 2007 to december 2012 .
social characteristics ( age , gender , marital status , and education ) and immune status ( hiv type , cd4 count ) were reviewed .
results . out of 2598 cancer patients ,
54 ( 2.1% ) tested positive for hiv .
there were 11 ( 20.4% ) defining aids and 43 ( 79.6% ) nondefining aids cancers .
the most frequent cancers were breast ( 14 ) ( 26.0% ) , non - hodgkin lymphoma ( 6 ) ( 11.1% ) , liver ( 6 ) ( 11.1% ) , eye and annexes ( 6 ) ( 11.1% ) , and cervical cancer ( 5 ) ( 9.3% ) .
these patients were female in 34 ( 63.0% ) and had a median age of 39 years and body mass index was 20,3 kg / m2 .
they were unschooled in 40 ( 74.1% ) and married in 35 ( 64.8% ) .
cd4 count showed a median of 317 cells / ml .
antiretroviral treatment was performed in 40 ( 74.1% ) .
conclusion .
hiv prevalence is higher in patients in our unit of surgical oncology .
breast cancer was the most common in this association .
a national survey of a large sample is needed to determine the true prevalence and impact of hiv on cancer prognosis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in the last years , sophisticated intraocular lens ( iol ) designs , such as the multifocal and accommodating iols , with the ability of restoring distance , intermediate and near vision have been introduced in clinical practice . the designs of multifocal iols are based on a number of optical concepts that are used to create multiple foci and therefore to generate simultaneous vision . multizonal refractive , refractive rotationally asymmetric , diffractive and hybrid iols ( the combination of refractive and diffractive elements ) are the currently available options . the latest models of these types of iols have been shown to provide good distance and near functional vision without the patient having to wear corrective lenses [ 2 , 3 ] . however , some optical side effects have been reported with some models of multifocal iols such as decreased contrast sensitivity , glare disability or halos [ 4 , 5 ] . multifocal diffractive technology uses a platform based on concentric rings in the posterior surface that form 2 primary focal points independent of pupil size , allowing a functional distance and near vision , respectively . it has been shown that this multifocal design results in some optical contrast reduction that may explain some of the photic phenomena reported with some diffractive multifocal iols , but this situation can be avoided by minimizing the postoperative level of primary spherical aberration . for this reason , aspheric diffractive multifocal iols were developed , which have been shown to provide excellent visual and refractive outcomes as well as a good postoperative optical and visual quality [ 2 , 8 ] . we have recently conducted a study aimed at evaluating the visual and refractive benefit after cataract surgery with the implantation of one of the latest models of aspheric diffractive multifocal iol , the tecnis zmb00 ( abbott medical optics ) . the aspheric multifocal iol tecnis zmb00 ( abbott medical optics ) is a single - piece iol with a 6.0-mm full - aperture diffractive optic and an overall length of 13.0 mm . it has an anterior aspheric surface designed according to the average cornea eye model to compensate for the spherical aberration of the cornea with 0.27 m . the iol also incorporates a posterior diffractive multifocal optic pattern designed to provide both near and distance vision and thereby reduce spectacle dependency , with a near power representing a + 4 d addition . the tecnis zmb00 iol is available in powers ranging from + 5.0 to + 34.0 d in 0.5 d increments . the manufacturer 's a - constant of this iol is 118.8 , and the surgeon factor 1.68 . the optimized srk / t constant is currently 119.5 , the optimized surgeon factor is 2.15 ( ulib ) . the tecnis zmb00 iol is made of a soft foldable hydrophobic acrylic material , similar to the sensar ar40 material ( abbott medical optics ) with a covalently bound ultraviolet ( uv ) absorber . uv - blocking protection with full transmission of the entire visible light spectrum is achieved , which promotes a minimization of the contrast sensitivity loss . the haptics of this last - generation iol have a modified c shape with trifix design , which allows 3 points of capsular bag fixation to provide long - term stability and centration . it should be considered that minimal decentrations of the optic of a diffractive multifocal iol can have a significant negative impact on the patients visual outcome . the posterior edge of the optic has a 360-squared design to provide uninterrupted contact at the haptic - optic junction and limit lens epithelial cell migration . furthermore , a frosted design has been added to the edge of the optic to reduce potential edge glare effects . we conducted a prospective consecutive study including a total of 31 eyes of 19 patients with ages ranging between 40 and 81 years , and undergoing phacoemulsification surgery with the implantation of the aspheric multifocal iol tecnis zmb00 . patients 40 years or older with a visually significant cataract or presbyopia demanding surgical correction , corneal astigmatism of less than 1.50 d and without history of glaucoma or retinal detachment , corneal disease , irregular corneal astigmatism , abnormal iris , macular degeneration or retinopathy , neurophthalmic disease , or history of ocular inflammation were included . during the preoperative consultation patients were informed about potential side effects of multifocal lenses , such as reduced contrast vision , halos and glare disability . preoperatively , all patients had a complete ophthalmologic examination including the evaluation of the refractive status , far and near logmar uncorrected visual acuity ( ucva ) and best - corrected va ( bcva ) testing , slit lamp examination , goldmann applanation tonometry and funduscopy . postoperatively , patients were evaluated during the follow - up at 1 day , and 1 , 3 and 6 months after surgery . at 1 day after surgery , only far and near ucva , iop and the integrity of the anterior segment was evaluated . the postoperative examination protocol at 1 all surgeries were performed by the same experienced surgeon using a standard mini - incision sutureless microcoaxial phacoemulsification technique . in all cases , a corneal incision of 2.4 mm at 90 was planned . after standard capsulorhexis creation and phacoemulsification , the iol was inserted successfully into the capsular bag using a platinum injector ( abbott medical optics ) . postoperatively , the same prophylactic treatment was administered to all patients consisting of corticosteroid - antibiotic combination eye drops . the mean age of patients was 65.0 10.0 years in the analyzed sample , which included a total of 8 males ( 42.1% ) and 11 females ( 57.9% ) . mean iol power implanted was 22.6 3.6 d , ranging from 12 to 27 d. table 1 summarizes the far and near visual and refractive outcomes . as shown , a statistically significant improvement was found in far bcva ( p < 0.01 ) , with 96.8% ( 30 eyes ) of eyes having a 6-month monocular postoperative far logmar bcva of 0.1 or better . the replacement of the optically degraded crystalline lens with the optimized optics of the aspheric diffractive multifocal iol evaluated played the main role in this finding . in addition , postoperative far logmar ucva was 0.1 or better in a great percentage of cases monocularly ( 24 eyes , 77.4% ) ( fig . this was in relation to an excellent predictability of the spherocylindrical correction , as shown in fig . almost all eyes ( 30 eyes , 96.8% ) had a postoperative spherical equivalent within 1.00 d. similar visual and refractive results were obtained by bautista et al . using the same modality of diffractive iol and by other authors [ 2 , 3 , 4 ] using other types of diffractive multifocal iols . specifically , bautista et al . found in a sample of 70 eyes of 70 patients a mean far logmar ucva of 0.194 0.054 , 0.119 0.026 , and 0.076 0.014 at 15 , 30 , and 60 days after surgery , respectively . in addition , these authors found that 74.3% of eyes had a far ucva of 0.1 logmar or better at 60 days after surgery . obtained in a sample of 40 eyes of 20 patients implanted with the aspheric diffractive iol acri.lisa 366d ( zeiss ) similar distance visual outcomes compared to ours . however , these same authors reported a slightly poorer visual result with a diffractive apodized multifocal iol , the acrysof restor sn6ad3 ( alcon ) , in an additional sample of 40 eyes of 20 patients . it should be considered that these 2 iols , the acri.lisa 366d and acrysof restor sn6ad3 , are not designed to compensate for the average corneal spherical aberration . all this previous scientific evidence on the visual and in vivo intraocular optical performance with different multifocal iol designs and the tecnis platform is consistent and further supported by the outcomes of objective optical - bench experiences , which has been very useful for checking the real working principle of the currently available multifocal iols . regarding the near visual outcome obtained in our sample , 67.7% of eyes ( 21 eyes ) and 93.55% of eyes ( 29 eyes ) achieved a postoperative near logmar ucva of 0.00 ( equivalent to j1 + in jaeger notation ) and 0.10 ( equivalent to j1 ) or better , respectively . this was comparable to the levels of near va provided by other aspheric diffractive iols and better than those provided by refractive multifocal iols [ 4 , 13 ] . series , most eyes ( 78.57% ) could read jaeger j1 ( 0.1 logmar ) or smaller letters without correction at 6 months postoperatively and none of the eyes presented a near ucva of j4 ( 0.4 logmar ) or worse . this confirms the excellent ability of the tecnis zmb00 iol to provide a near functional vision , and therefore its excellent performance as a presbyopia - correcting iol . finally , we evaluated the level of correlation between the iol power implanted and the postoperative far and near va . specifically , the iol power was found to be very poorly correlated with the postoperative far logmar ( r = 0.13 , fig . this confirms that the predictability of the refractive correction achieved with the implant was not limited by its optical power . furthermore , a poor correlation was found between iol power and far logmar bcva ( r = 0.25 , fig . 3c ) , which shows the minimal impact of higher order aberrations on va in those eyes implanted with the tecnis zmb00 iol . this is in agreement with the excellent contrast sensitivity outcomes obtained by bautista et al . for this same iol . it should be remembered that the tecnis zmb00 iol provides compensation for the corneal spherical aberration with 0.27 m , potentially minimizing the global ocular magnitude of this high - order aberration . this result contrasts with the limitation imposed on the final visual outcome and optical quality by the iol power of apodized diffractive multifocal iols , potentially attributable to the aberrometric properties of the iol as well as to the possibility of foci overlapping . in conclusion , the aspheric diffractive multifocal iol tecnis zmb00 provides a restoration of the far and near visual function after phacoemulsification surgery for cataract removal or presbyopia correction . the correction provided by the iol is predictable and independent from the optical power of the iol implanted . studies currently in progress are confirming the agreement between this level of visual restoration with patient satisfaction , a minimal perception of photic phenomena , the presence of a postoperative optimized intraocular optics , and an excellent contrast sensitivity outcome , as reported preliminarily by bautista et al . , and sood and woodward . the combination of diffractive multifocality and compensation for the corneal spherical aberration is an effective option for cataract surgery and presbyopia correction , allowing an optimized distance and near visual function . the authors have no proprietary or commercial interest in the medical devices that are involved in this work . this research received no specific grant from any funding agency in the public , commercial or not - for - profit sectors . | the outcomes of a prospective consecutive study aimed at evaluating the visual and refractive benefit after cataract surgery with the implantation of the aspheric diffractive multifocal intraocular lens ( iol ) tecnis zmb00 ( abbott medical optics ) are reported .
a total of 31 eyes of 19 patients ( age range , 40 to 81 years ) underwent phacoemulsification surgery with implantation of this aspheric multifocal iol . at 6 months after surgery ,
postoperative spherical equivalent was within 1.00 d in 96.8% of eyes , with 94.7% of patients presenting a postoperative binocular far logmar uncorrected visual acuity ( ucva ) of 0.1 or better .
far best - corrected distance va improved significantly ( p < 0.01 ) , with postoperative values of 0.1 or better in 96.8% of eyes .
postoperative near ucva was 0.10 ( equivalent to j1 ) or better in 93.55% of eyes .
furthermore , the iol power was found to be very poorly correlated with the postoperative far logmar ( r = 0.13 ) and near ucva ( r = 0.13 ) . in conclusion ,
the aspheric diffractive multifocal iol tecnis zmb00 provides a restoration of the far and near visual function after phacoemulsification surgery for cataract removal or presbyopia correction , which is predictable and independent from the optical power of the implanted iol . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
it
refers to the force of the hands enabling diverse actions in ordinary life such as writing
with a ballpoint pen , using chopsticks to eat food , grasping a cup to drink water , and
holding exercise equipment during exercise1 . flexion , extension , and supination of the wrists have important
influences on grip and muscle strength1 . the wrist joints rotate in the long axis of the
forearm and may hold freely and maintain objects . the ranges of wrist flexion and extension
for a healthy person are 0 to 90 and 0 to 70 , respectively1 . taping is being used as an important means of prevention of
injuries and management of acute damage in athletes during training and competition2 . such taping affects proprioceptive senses
and therefore is thought to have an effect on prevention of injuries2 . proprioceptive senses refer to senses modifying and
delivering information on one s physical location , posture , and movements from receptors of
the skin , muscles , tendons , and joints to the central nervous system2 . when tape is applied to an area of pain by extending the
muscles as maximally as possible , the area where the tape is applied exhibits flexion when
the muscles return to their normal locations2 , 3 . when this occurs , the skin is raised
upward , and the space between the skin and the muscles increases , improving circulation of
blood , lymph fluid , and tissue fluid in the space and enhancing motor functions of the
body2 . taping is one of the treatment
techniques developed in 1985 by arikawa in japan to treat patients3 . the tapes used have a rate of expansion and contraction
similar to that of the human body and constant adhesive durability . there have been diverse studies aimed at relieving pain of the
ankle , shoulder , elbow , and wrist joints , but research on the effect of forearm elastic
taping on the muscle strength of the wrists has been lacking4 . accordingly , the aim of this study was to examine the effects of
forearm elastic taping on the grip strength and muscle strength of the wrist joints . this study selected 40 healthy subjects with no history of orthopedic disorders and was
conducted after consent to participate was obtained . written informed consent according to
the ethical standards of the declaration of helsinki was provided by all subjects prior to
participation , and all agreed to participate in the project . their average age , height , and
weight were 21.21 1.63 years old , 173.51 8.54 cm , and 71.18 8.47 kg , respectively . guidelines for taping of the forearm were consistent with the protocol for medial
epicondylitis of the elbow as suggested by kase et al3 . tape was applied to the wrist flexor muscle of the dominant hand .
to verify the effects of taping , the two tapes used in the study were the standard kinesio tex classic-2
( 5 cm ) blue tape ( kinesio holding company , albuquerque , nm , usa ) ( elastic group ) and a
nonelastic tape ( leukotape nonelastic , bsn medical gmbh & co. kg , hamburg , germany )
( noneleastic group ) . before applying the tape , the length of tape was measured from 2 cm inferior to the medial
epicondyle of the humerus to the line of the wrist joint . a strip was cut from a roll of
tape and then cut down the middle to produce 2 tails or a the y - strip was applied
to the common wrist flexor muscle from its insertion to origin with 1520% stretch tension . the first tail of the y strip was applied to the middle of the forearm with the wrist in a
hyperextended position , the elbow in full extension , and the forearm in full supination . the
second tail of the y - strip , also applied from insertion to origin with 1520% stretch
tension , was applied along the medial edge of the forearm to wrap the common wrist flexor
muscles3 . the nonelastic tape was applied with the same method as used for the elastic taping
group . the outcome measures for this study consisted of maximal grip and wrist flexor muscle
strength . it was
measured by using a jamar hydraulic hand dynamometer ( 5030 kit , preston , usa ) . to measure
wrist flexor muscle strength , a commander muscle tester ( for measuring grip strength , the subjects were asked to adduct their shoulder
joints while sitting in a chair such that their arms were resting againist their trunk , flex
their elbow joints to 90 degrees , and to place their forearms and wrist in a neutral
position according to the method applied by shimose et al6 . wrist flexor muscle strength was measured in a neutral position
( 0 ) after measuring grip strength . during the measurement of wrist flexor muscle strength , the measurement pad was applied to the second to fifth middle phalanges area while the
subjects clenched their first strongly . the maximal isometric muscle strength was measured
by applying force with a wrist joint flexion motion . ,
differences in the general characteristics of the experimental and control groups were
compared using independent t - tests . comparisons of variables before and after taping within
each group were made using paired sample t - tests . after application of forearm elastic taping , grip and wrist flexor muscle strength
significantly increased compared to the nonelastic taping ( p<0.05 ) ( table 1table 1.comparison of changes in characteristics of the forearm nonelastic taping group
and elastic taping groupnonelastic taping elastic tapinggrip strength ( kg ) 42.5 6.746.8 8.7wrist flexor muscle strength ( n ) 26.6 6.429.8 7.0values are expressed as the mean sd . the factors determining muscle strength include the strength of the muscles themselves , the
angles of joints during muscle strength measurement , and the distance of action of the force
from the joint axis5 . accordingly , this
study was conducted to examine the effects of forearm taping on grip and wrist flexor muscle
strength . after application of forearm elastic taping , grip and wrist flexor muscle strength
significantly increased . diverse methods aimed at muscle strength improvement have been presented . among the
methods , a lot of research has been conducted on taping techniques prove that the methods
also improve muscle functions6 . the
results of applying taping to the knee joints of patella femoral dysfunction patients
revealed that it was effective for improving their muscle strength7 . in the present study , the reason for the increase in muscle
strength after application of forearm elastic taping may be explained by the following
mechanism . according to the cutaneous fusimotor reflex theory , a fusimotor reflex triggers
contraction of subcutaneous muscles stimulated through the gamma motor reflex when different
types of stimulations , such as contact and vibration , are applied to the skin8 . in other words , stimulation of the skin
through application of taping may trigger contraction of muscles and improve muscle
strength9 . taping will be effective for
improving wrist flexor muscle strength and preventing wrist joint damage if it is applied to
patients who have difficulty with wrist flexion and extension , athletes , or those in the
industrial sites who use their hands often . future studies should suggest standardized
application methods for nerve mobilization techniques and continue to research nerve
mobilization techniques combined with interventions for various musculoskeletal
diseases . | [ purpose ] the purpose of this study was to examine the effects of forearm elastic taping
on grip and wrist flexor muscle strength . [ subjects and methods ]
this was a single - blind ,
crossover study .
this study selected 40 healthy subjects with no history of orthopedic
disorders and was conducted after consent to participate was obtained .
grip and wrist
flexor muscle strength of subjects were assessed by a handheld dynamometer and a commander
muscle tester , respectively , with forearm elastic taping or nonelastic taping .
[ results ]
after application of forearm elastic taping , grip strength and wrist flexor muscle
strength significantly increased compared with the nonelastic taping group . [ conclusion ]
application of forearm elastic taping is considered to have positive effects on improving
wrist and grip strength . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
central venous catheter ( cvc ) insertion is part of daily routine in modern medicine . of approximately 16 million cvc insertions per year globally , 66% are inserted by seldinger technique ( market data b. braun : www.cvc-partner.com ) . the literature is rife with statements about guide wire failings , inappropriate handling , and particularly difficult clinical situations . the reported complication rate is approximately 5 - 19% and is related to the experience of the operator and different clinical situations . known complications related to the guide wire often occur , when force is applied to the wire . known guide wire associated complications are knotting of the straight part , kinking with an acute angle , separation of the spiral wire and the core wire even with fragmentation of the core wire , fragmentation , and embolization . given the extent to which the seldinger technique is used , it is extremely beneficial to understand the root cause of the problems and to search for solutions . in our clinic , we decided to collect all used guide wires over a period of 4 weeks , and examine them for bending and kinking . of the 330 used in that period , 39 displayed significant bending , and 5 had kinked [ figure 1 ] . a sample of kinked guide wires there had also been recent fatalities after perforation of the vessel wall . on an inspection of the guide wires used in these cases once the wire kinked , the dilator could not follow the course of the vessel and its forward movement perforated the vessel wall . the failures noted above ( in the literature and our clinic ) are mechanical failures , yet these guide wires met the requirements of en iso 110701 , the guideline for testing guide wire flexing performance and tensile strength ( www.iso.org ) . unfortunately , en iso 11070 guidelines do not meet the clinical requirements , and we continue to see mechanical failures and their associated complications . therefore , we designed equipment and tests to mimic clinical flexing requirements . because we are unable to measure the actual tensile force exerted in clinical situations , we designed a wire - to - wire comparison of tensile strength . the logic is that all things being equal , the higher the tensile strength , the less likely the wire would fail because of excess force as excess force on one wire may not be excess force on the next . we studied seven different types of guide wires usually used for cvc insertions , all with core and wire construction and one monofil wire , radifocus , usually used in angiography . the radifocus has a nitinol alloy construction with a polyurethane polyamide elastomeric coating and is known for its extraordinary mechanical properties . types of guide wires tested tensile strength in newton : means of the three subgroups after tukey 's hsd the coils do not hold their original spacing , which causes the core wire to lose its mechanical integrity . the wire does not kink , but it does exhibit a permanent alteration or bend . testing for tensile strength was carried out according to the method described by en iso 11070 annex h ( 1999 ) . guide wires with a diameter between 0.750 and 1.150 mm have to withstand a tractive force of 5 newton ( n ) . one end of the guide wire is secured to a split - tapered clamp , which is attached to the moving crosshead of the tensile splitting apparatus . pneumatic grips attached to the fixed head should grip the guide wire approximately at its central point and at least 150 mm from the split - tapered clamp [ figure 2 ] . then , a tensile force at a rate of 10 mm / min is applied in the direction of the main axis of the guide wire . testing for tensile strength : one end of the guide wire is secured to a split - tapered clamp attached to the moving crosshead of the tensile splitting apparatus . pneumatic grips attached to the fixed head should grip the guide wire approximately at its central point and at least 150 mm from the split - tapered clamp [ figure 3 ] the en iso test is stopped at 5 n or at disruption of the union of core wire and coil , whichever occurs first . we continued the test procedure until disruption was audible or maximum tractive force was applied . tractive force ( n ) and changes in guide wire length ( mm ) were continuously recorded . the construction of the testing apparatus [ figure 3 ] consists of a fixed aluminum plate and a movable journal bearing which allows vertical movement . the dilator ( taken from certofix trio sb 730 set , fa b. braun ag , 34209 melsungen , germany , diameter : 7 french ) was fixed to the journal bearing as shown in figure 3 . a force transducer , measuring the vertical movement , was also connected to this bearing . on the aluminum plate , a metal tube ( diameter 1.3 mm , length 5 cm ) was installed at right angle to the vertically movable journal bearing . the testing machine for kinking consists of a movable journal bearing which allows a vertical movement . a = kinking test 1 ; b = kinking test 2 . for kinking test 2 , the dilator was fixed 2 cm closer to the distal end of the guide wire . movement in a vertical direction simulates the advancement of the dilator into the tissue . on the aluminum plate , a metal tube ( diameter 1.3 mm , length 5 cm ) was installed at a right angle position to the vertically movable journal bearing . in the start position of the bearing , the tip of the dilator was situated 3 cm above the level of the horizontal tube on the aluminum plate . for the measurement of the kinking behavior , the journal bearing was moved downward 3 cm with a defined velocity of 1.3 cm / s the flexing tests were performed at two dilator positions on the journal bearing [ tables 3 and 4 ] . for flexing test 2 , the dilator was fixed 2 cm closer to the distal end of the guide wire than in test 1 , resulting in a greater flexing angle [ table 4 ] . screws , 3 cm from the j - tip , fixed the wire to the aluminum plate and onto the test apparatus . kinking test 1 : means in the four subgroups after tukey 's hsd kinking test 2 : means in the four subgroups after tukey 's hsd in the start position , the tip of the dilator was situated 3 cm above the level of the horizontal tube on the aluminum plate . for measurement , the journal bearing thereafter , the screws were released , and the journal bearing was slowly lifted until the guide wire was released . we measured tensile strength in n. we measured flexing angles by first scanning the wires with canoscan 5000 f , and then analyzing the results with the assistance of the adobe illustrator 10 ( adobe systems incorporated , 345 park avenue san jose , ca 95110 - 2704 ) . within each wire type , standard deviation and mean were determined . concerning flexing behavior , we compared variables using nonparametric mann - whitney u - test for independent samples . analysis of variance ( anova ) was used to test the type of the guide wire for significant differences . significant differences between the types were analyzed by a post - hoc - analysis ( tukey 's honestly significant difference [ tukey 's hsd ] ) . the coils do not hold their original spacing , which causes the core wire to lose its mechanical integrity . because of this definition , a monofil can never fall into this category . the wire does not kink , but it does exhibit a permanent alteration or bend . the coils do not hold their original spacing , which causes the core wire to lose its mechanical integrity . because of this definition , a monofil can never fall into this category . the wire does not kink , but it does exhibit a permanent alteration or bend . testing for tensile strength was carried out according to the method described by en iso 11070 annex h ( 1999 ) . guide wires with a diameter between 0.750 and 1.150 mm have to withstand a tractive force of 5 newton ( n ) . one end of the guide wire is secured to a split - tapered clamp , which is attached to the moving crosshead of the tensile splitting apparatus . pneumatic grips attached to the fixed head should grip the guide wire approximately at its central point and at least 150 mm from the split - tapered clamp [ figure 2 ] . then , a tensile force at a rate of 10 mm / min is applied in the direction of the main axis of the guide wire . testing for tensile strength : one end of the guide wire is secured to a split - tapered clamp attached to the moving crosshead of the tensile splitting apparatus . pneumatic grips attached to the fixed head should grip the guide wire approximately at its central point and at least 150 mm from the split - tapered clamp [ figure 3 ] the en iso test is stopped at 5 n or at disruption of the union of core wire and coil , whichever occurs first . we continued the test procedure until disruption was audible or maximum tractive force was applied . tractive force ( n ) and changes in guide wire length ( mm ) were continuously recorded . the construction of the testing apparatus [ figure 3 ] consists of a fixed aluminum plate and a movable journal bearing which allows vertical movement . the dilator ( taken from certofix trio sb 730 set , fa b. braun ag , 34209 melsungen , germany , diameter : 7 french ) was fixed to the journal bearing as shown in figure 3 . a force transducer , measuring the vertical movement , was also connected to this bearing . on the aluminum plate , a metal tube ( diameter 1.3 mm , length 5 cm ) was installed at right angle to the vertically movable journal bearing . the testing machine for kinking consists of a movable journal bearing which allows a vertical movement . a = kinking test 1 ; b = kinking test 2 . for kinking test 2 , the dilator was fixed 2 cm closer to the distal end of the guide wire . movement in a vertical direction simulates the advancement of the dilator into the tissue . on the aluminum plate , a metal tube ( diameter 1.3 mm , length 5 cm ) was installed at a right angle position to the vertically movable journal bearing . in the start position of the bearing , the tip of the dilator was situated 3 cm above the level of the horizontal tube on the aluminum plate . for the measurement of the kinking behavior , the journal bearing was moved downward 3 cm with a defined velocity of 1.3 cm / s the flexing tests were performed at two dilator positions on the journal bearing [ tables 3 and 4 ] . for flexing test 2 , the dilator was fixed 2 cm closer to the distal end of the guide wire than in test 1 , resulting in a greater flexing angle [ table 4 ] . screws , 3 cm from the j - tip , fixed the wire to the aluminum plate and onto the test apparatus . kinking test 1 : means in the four subgroups after tukey 's hsd kinking test 2 : means in the four subgroups after tukey 's hsd in the start position , the tip of the dilator was situated 3 cm above the level of the horizontal tube on the aluminum plate . for measurement , the journal bearing thereafter , the screws were released , and the journal bearing was slowly lifted until the guide wire was released . we measured tensile strength in n. we measured flexing angles by first scanning the wires with canoscan 5000 f , and then analyzing the results with the assistance of the adobe illustrator 10 ( adobe systems incorporated , 345 park avenue san jose , ca 95110 - 2704 ) . within each wire type , standard deviation and mean were determined . concerning flexing behavior , we compared variables using nonparametric mann - whitney u - test for independent samples . analysis of variance ( anova ) was used to test the type of the guide wire for significant differences . significant differences between the types were analyzed by a post - hoc - analysis ( tukey 's honestly significant difference [ tukey 's hsd ] ) . all guide wires , except one of the six braun nk1 prototypes , met en iso 11070 requirements for tensile force of 5 n. one - way anova showed statistically significant differences . after post - hoc analysis of means by tukey 's hsd , three subgroups could be defined [ table 2 ] . the mean of the wire types tested ranged from 15.06 to 257.76 n. none of the wires kinked . two core / coil wires displayed minor bending , ( angle 1.5 ) and all other wires displayed bending angles between 22.5 and 43.0. the applied insertion angle ( test 1 : 20 , test 2 : 50 ) had an influence on the resulting bending angle of the guide wire . this was significant in the guide wires of accoat , braun n , braun s , and arrow [ table 5 ] . all guide wires , except one of the six braun nk1 prototypes , met en iso 11070 requirements for tensile force of 5 n. one - way anova showed statistically significant differences . after post - hoc analysis of means by tukey 's hsd , three subgroups could be defined [ table 2 ] . two core / coil wires displayed minor bending , ( angle 1.5 ) and all other wires displayed bending angles between 22.5 and 43.0. the applied insertion angle ( test 1 : 20 , test 2 : 50 ) had an influence on the resulting bending angle of the guide wire . this was significant in the guide wires of accoat , braun n , braun s , and arrow [ table 5 ] . two core / coil wires displayed minor bending , ( angle 1.5 ) and all other wires displayed bending angles between 22.5 and 43.0. the applied insertion angle ( test 1 : 20 , test 2 : 50 ) had an influence on the resulting bending angle of the guide wire . this was significant in the guide wires of accoat , braun n , braun s , and arrow [ table 5 ] . central line insertion is a daily routine in anesthesiology and the management of critically ill patients . even though materials and insertion technique have improved dramatically over the last few years , serious complications are still being regularly reported in the medical literature . we do have it in our power to produce and use high - end guide wires which can actually neutralize many of the human errors associated with cvc and other procedures . the primary purpose of this study was to test and compare the mechanical properties of core and coil seldinger wires used in commercially available cvc kits . the intent of its inclusion was to give clinicians comparative information necessary to make informed decisions when faced with certain procedures and body types known to be extremely problematic , for example :
patient is of small stature , especially if corpulent , and the procedure involves a large - bore catheter;patient is of small stature , especially if corpulent , and the procedure involves the right subclavian or the left internal jugular vein;patient has undergone or is believed to have undergone previous / numerous cannulations . patient is of small stature , especially if corpulent , and the procedure involves a large - bore catheter ; patient is of small stature , especially if corpulent , and the procedure involves the right subclavian or the left internal jugular vein ; patient has undergone or is believed to have undergone previous / numerous cannulations . to avoid serious complications , even the instructions for use included with the cvc kit read : do not apply excessive force in removing the guide wire . excessive force is an extremely nebulous measurement , and in any event , the actual n rating of the guide wire in the kit is not provided . this means clinicians often do not know if they are working with an extremely robust guide wire or a marginally adequate guide wire . hence , the human force applied successfully with unknown guide wire x , may be too much force for unknown guide wire z. since there is no way to measure the tensile force used in an actual clinical situation , the clinician must perform a great deal of this delicate procedure through learned behavior , through feel which may be extremely difficult to achieve if the mechanical properties of the wires are different from kit to kit . this leads to one obvious conclusion : in order to avoid the complication caused by excessive force use a kit which consistently contains a guide wire with the highest possible tensile strength . the second conclusion is that the 5 n en iso requirement is simply not adequate to meet clinical requirements or would not continue to see the high level of mechanical failures . in our study of tensile strength , we found three significantly different groups . in subgroup ii , the mean ranged from 35 to 25 n , and overlapped with subgroup i which ranged from 24 to 26 n. we would recommend guide wires of group ii for seldinger technique , especially those which are not in both subgroups ( > 27 n ) . it consists of an elastic alloy core coated with a polyurethane jacket and a hydrophilic coating . the result is an extremely pliable tip , a smooth outer coating and excellent torque control . because of the monofil construction of this wire , the tensile strength is only influenced by the stability of the metal wire itself . this kind of the guide wire is routinely used in radiology but not for cvc procedures . one critical step during the insertion of cvc via the seldinger technique is the dilation of the puncture canal . in order to dilate the perivascular tissue , a dilator a forward movement of the guide wire is prevented by immobilizing the wire with a firm grip . when this is the case , the force intended for tissue dilation often diverts to the guide wire , potentially leading to patient complications . clinicians can avoid this result using nonkinking wires , such as the braun nk2 or the radifocus monofil tested in this study . but , medical devices now exist that would allow us to make an immediate improvement in patient results . not using these devices would appear difficult to justify . to simulate this cvc insertion process , a method of testing flexing characteristics , mimicking clinical demands , was developed . main features influencing the insertion procedure were taken into consideration for developing the test design , that is , resistances of tissue or friction by fixing the distal end of the wire , muscle force of the physician and dilation velocity by defining the sliding velocity , as well as the angle between the dilator and guide wire by testing at two different angles . even the instructions for use included with the cvc kit read : do not apply excessive force in removing the guide wire . excessive force is an extremely nebulous measurement , and in any event , the actual n rating of the guide wire in the kit is not provided . this means clinicians often do not know if they are working with an extremely robust guide wire or a marginally adequate guide wire . hence , the human force applied successfully with unknown guide wire x , may be too much force for unknown guide wire z. since there is no way to measure the tensile force used in an actual clinical situation , the clinician must perform a great deal of this delicate procedure through learned behavior , through feel which may be extremely difficult to achieve if the mechanical properties of the wires are different from kit to kit . this leads to one obvious conclusion : in order to avoid the complication caused by excessive force use a kit which consistently contains a guide wire with the highest possible tensile strength . the second conclusion is that the 5 n en iso requirement is simply not adequate to meet clinical requirements or would not continue to see the high level of mechanical failures . in our study of tensile strength , we found three significantly different groups . in subgroup ii , the mean ranged from 35 to 25 n , and overlapped with subgroup i which ranged from 24 to 26 n. we would recommend guide wires of group ii for seldinger technique , especially those which are not in both subgroups ( > 27 n ) . it consists of an elastic alloy core coated with a polyurethane jacket and a hydrophilic coating . the result is an extremely pliable tip , a smooth outer coating and excellent torque control . because of the monofil construction of this wire , the tensile strength is only influenced by the stability of the metal wire itself . this kind of the guide wire is routinely used in radiology but not for cvc procedures . one critical step during the insertion of cvc via the seldinger technique is the dilation of the puncture canal . in order to dilate the perivascular tissue , a dilator a forward movement of the guide wire is prevented by immobilizing the wire with a firm grip . when this is the case , the force intended for tissue dilation often diverts to the guide wire , potentially leading to patient complications . clinicians can avoid this result using nonkinking wires , such as the braun nk2 or the radifocus monofil tested in this study . but , medical devices now exist that would allow us to make an immediate improvement in patient results . not using these devices would appear difficult to justify . to simulate this cvc insertion process , a method of testing flexing characteristics , mimicking clinical demands , was developed . main features influencing the insertion procedure were taken into consideration for developing the test design , that is , resistances of tissue or friction by fixing the distal end of the wire , muscle force of the physician and dilation velocity by defining the sliding velocity , as well as the angle between the dilator and guide wire by testing at two different angles . clinicians use guide wires with high - end mechanical properties , ( nonkinking , high tensile strength),en iso 11070 incorporate our flexing test into their testing method , raise the flexing requirement to kink - proof , and raise the tensile strength requirement to a minimum of 30 n , andall manufacturers and suppliers be required to display mechanical properties of all guide wires and cvc kits sold . clinicians use guide wires with high - end mechanical properties , ( nonkinking , high tensile strength ) , en iso 11070 incorporate our flexing test into their testing method , raise the flexing requirement to kink - proof , and raise the tensile strength requirement to a minimum of 30 n , and all manufacturers and suppliers be required to display mechanical properties of all guide wires and cvc kits sold . we recommend that clinicians use the least amount of force necessary during cvc insertion / retraction , and remain alert to specific patient profiles . | background and aims : most central venous catheters are placed using seldinger guide wires .
en iso 11070 is the guideline for testing guide wire flexing performance and tensile strength , and we can safely assume that guide wires in use meet these requirements . unfortunately , en iso 11070 guidelines do not reflect the clinical requirements and we continue to see mechanical failures and their associated complications.material and methods : this in vitro study was performed in an accredited laboratory . with regard to flexing , we : ( 1 ) established the minimum flexing performance needed to meet clinical requirements , ( 2 ) developed flexing performance tests which mimic clinical requirement , and ( 3 ) evaluated the mechanical properties of various guide wires relative to these requirements . with regard to tensile strength , we used the testing method prescribed in iso 11070 , but did not end the test at 5 newton ( n ) .
we continued until the guide wire was damaged , or we reached maximum tractive force .
we then did a wire - to - wire comparison .
we examined two basic wire constructions , monofil and core and coil.results:tensile strength : all wires tested , except one , met en iso 11070 requirements for 5 n tensile strength .
the mean of the wire types tested ranged from 15.06 n to 257.76 n. flexing performance : none of the wires kinked .
the monofil had no evidence of bending .
two core / coil wires displayed minor bending ( angle 1.5 ) . all other wires
displayed bending angles between 22.5 and 43.0.conclusion:we recommend that : ( 1 ) clinicians use guide wires with high - end mechanical properties , ( 2 ) en iso 11070 incorporate our flexing test into their testing method , raise the flexing requirement to kink - proof , ( 3 ) and raise the tensile strength requirement to a minimum of 30 n , and ( 3 ) all manufacturers and suppliers be required to display mechanical properties of all guide wire , and guide wire kits sold . |
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crossed renal ectopia ( re ) is a very rare anomaly with an incidence of 1 in 7500 autopsies or 1 in 14,000 pediatric admissions . solitary crossed re ( scre ) occurs due to a combination of unilateral agenesis and re . we report a case of scre associated with vesicoureteric junction ( vuj ) obstruction in a 7-year - old male child . ultrasonography abdomen revealed hydroureteronephrosis with a solitary left kidney with a nonvisualization of the right kidney . intravenous urogram ( ivu ) findings demonstrated that the ureter from the left kidney was inserted into the bladder on the right side after crossing the midline [ figure 1 ] . renal radionuclide 99m - technetium ( 99mtc ) -scan was again suggestive of absent right kidney and ectopic left kidney with vuj obstruction ( delayed drainage of whole ureter and kidney ) [ figure 2 ] . cystoscopy showed one ureteric orifice on the right side which was normal in caliber and could not be cannulated . upon exploration , diagnosis of vuj obstruction intravenous urogram showing solitary right to left ectopia with grossly dilated ureter crossing the midline renal radionuclide scan showing crossed ectopia with obstruction to drainage at vesicoureteric junction and proximal dilated ureter and pelvis crossed re exists when a kidney is located on the opposite side of the midline from where ureters enter the bladder . scre results due to renal agenesis of one side and contralateral displacement of the ureter to the opposite side . the crossed re is a rare disorder , furthermore the incidence of scre is exceedingly low , and till now only 35 cases have been reported in the literature . scre can be diagnosed by ultrasonography and radionuclide scintigraphy using 99mtc - dimercaptosuccinic acid ( dmsa ) in most of the asymptomatic cases . excretory urography and multi - detector three - dimensional computed tomography urography are excellent for delineating re . in our case , 99mtc - dmsa scintigraphy is useful in the diagnosis of simple re , renal cortical lesions , and function of cortical mass . hydronephrosis is the most significant finding in re , and the associated vur is present in 25 - 70% of cases . dilatation of the ectopic renal pelvis due to the ureteropelvic junction obstruction is present in 37% of patients . other abnormalities associated with re include abnormalities of spinal growth and genital abnormalities such as anorectal malformation , hypospadias , and sacral agenesis . in our case , scre ( right to left ) was associated with vuj obstruction which was the cause of hydroureteronephrosis . to our best knowledge , this is the first case report in the world literature although vuj obstruction with crossed re has been reported in one case which was bilateral . in this case , the ectopic kidney was poorly functioning , while the contra lateral orthotopic kidney was malrotated , with fetal appearance . ectopic kidneys are more disease prone than normal kidney , and they are often hypoplastic or dysplastic . some authors encourage a vcug in boys and cystoultrasonography in girls affected by crossed re for the high risk of associated vur as postnatal us is a poor predictor of vur . crossed renal ectopic kidneys are smaller than simple renal ectopic kidneys and have inherently reduced function . a solitary crossed ectopic renal unit will have further worsening of function if associated with an obstructive or refluxing lesion . furthermore , it is important to distinguish the vur from vuj obstruction as association of vuj obstruction causes a more rapid deterioration of renal function particularly in a solitary renal unit . most patients with scre do not require treatment for this condition , but because of associated vuj obstruction reimplantation was unavoidable in our case . we recommend that the urological evaluation should be sought due to the high proportion of these cases have associated urological anomalies . intravenous urography , magnetic resonance urography , and 99mtc - dmsa are the investigations used for the diagnosis of such unusual cases . an associated vuj obstruction once documented should be treated urgently to prevent deterioration of renal function . | solitary crossed renal ectopia ( scre ) is a very rare anomaly of urinary tract .
most cases are diagnosed incidentally .
we report a case of scre associated with vesicoureteric junction obstruction in a 7-year - old child which has not been reported in the literature till date . |
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mitochondrial dysfunction is implicated in the etiology of various diseases , such as neurodegenerative diseases , diabetes , cardiovascular disease , various forms of hepatic disorders , skeletal muscle disorders , sepsis , and psychiatric disorders [ 110 ] . abnormalities in mitochondrial functions such as defects in the electron transport chain ( etc)/oxidative phosphorylation ( oxphos ) system , krebs 's cycle enzymes , and atp production have all been suggested as the primary causative factors in the pathogenesis of neurodegenerative disorders and sepsis . enhanced production of reactive oxygen species ( ros ) and possibly accumulation of mitochondrial ( mt ) dna mutations in postmitotic cells are considered to be contributory factors to age - related degeneration . mitochondria not only generate ros / reactive nitrogen species ( rns ) but are also the main target of their actions . as a result of this action , damage occurs in the mitochondrial respiratory chain , thus producing further increases in free radical generation , ultimately self - inducing a vicious cycle . during the last decade , a number of studies have demonstrated that melatonin plays an effective role in regulating mitochondrial homeostasis . in addition to being a free radical scavenger , melatonin reduces nitric oxide ( no ) generation within mitochondria . it maintains the electron flow , efficiency of oxidative phosphorylation , atp production and bioenergetic function of the cell by regulating respiratory complex activities , ca influx , and mitochondrial permeability transition pore opening [ 1418 ] . in this article , the several mechanisms through which melatonin exerts neuroprotective actions in neurodegenerative disorders such as parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and huntington 's disease ( hd ) and in a number of mitochondrial dysfunction related conditions such as aging , ischemia / reperfusion ( i / r ) , or septic shock , are reviewed . mitochondria contain multiple copies of a circular genome ( chromosome ) known as mtdna as it has been characterized in humans . although the majority of mitochondrial proteins essential for normal bioenergetic function are encoded by nuclear dna , some proteins needed for etc / oxphos are encoded by mtdna . human mitochondrial genome encodes for 13 peptides of subunits of complexes i , iii , and iv , and atp synthase complex , 22 transfer rnas and 2 ribosomal nucleic acids , while nuclear dna encodes for at least 1000 mitochondrial proteins . the primary function of mitochondria is to generate atp within the cell through the etc resulting in oxphos . the etc , which is present in the inner mitochondrial membrane , comprises a series of electron carriers grouped into four enzyme complexes , namely , complex i ( nadh ubiquinone reductase ) , complex ii ( succinate ubiquinone reductase ) , complex iii ( ubiquinol cytochrome - c - reductase ) , and complex iv ( cytochrome c oxidase ) . the main function of the etc is to convert redox energy into an electrochemical gradient of protons that subsequently causes atp formation from adp and phosphate by atp synthase . the end product of the respiratory chain is water that is generated in a four - electron reduction of molecular oxygen ( o2 ) by complex iv . during this process ( electron leakage especially at complex i and iii ) , a small percentage of o2 is converted into ros , such as superoxide anion radical ( o2 ) and its secondary products hydrogen peroxide ( h2o2 ) and reactive hydroxyl radical ( oh ) [ 23 , 24 ] . under normal conditions , the iron - sulfur cluster n2 of complex i appears to be the primary source of free radicals in the brain [ 23 , 25 ] . mitochondrial no synthase ( mtnos ) localized in the inner mitochondrial membrane is responsible for generating no radical ( no ) from l - arginine . localization of mtnos refers only to myristoylated nnos splice variant alpha , which is also relevant because of its interaction with complex iv . high rates of no synthesis , which typically occur in the calcium - dependent excited state of neurons , can contribute to oxidative and nitrosative stress . the availability of no determines the rates at which the adduct peroxynitrite ( onoo ) and the decomposition products are generated . the free radical no is produced by several forms of nos . in the mitochondria , two nos isoforms , namely constitutive and inducible have been reported ( c - mtnos and i - mtnos ) . since no is an uncharged gaseous compound , it crosses membranes with ease and can enter mitochondria regardless of their neuronal , glial , or vascular origin . no strongly interferes with components of the respiratory chain , in particular cytochrome c oxidase [ 2729 ] . other nitrosation processes , like transnitrosation or reversible nitrosation and nitration as well as irreversible protein and lipid oxidation , can occur . upon entering neuronal mitochondria , no , in combination with onoo ( formed there by combination with o2 from electron leakage ) , not only interferes with respiratory chain complexes but , when it reaches elevated levels , can trigger free radical - mediated chain reactions that in turn destroy protein , lipid , and dna molecules [ 3032 ] . as it has been stated above , damage to the mitochondrial respiratory chain can cause a breakdown of the proton potential , apoptosis , or lead to further generation of free radicals , thus maintaining a vicious cycle that ultimately results in cell death [ 23 , 33 ] . in the mitochondrial - mediated cell death pathway , a nonspecific increase in the permeability of the inner mitochondrial this is known as mitochondrial permeability transition ( mtpt ) , a process associated with the opening of channels in the inner mitochondrial membrane , which in turn causes a flux of molecules of < 1500 daltons . the inner mitochondrial membrane possesses a uniporter to transport ca into the matrix . with ca overload , there is complete uniporter inhibition , mitochondrial swelling , loss of respiratory control , and a release of matrix calcium caused by mtpt pore opening [ 35 , 36 ] . under these conditions , atp is hydrolyzed by mitochondria , the mitochondria undergo swelling , and mitochondrial - mediated apoptosis occurs . a number of mechanisms take part in the control of ros / rns production . among these is the action of the enzyme superoxide dismutase ( sod ) , which occurs in the inner side of the inner mitochondrial membrane ( mn - sod ) , that remove o2 . oh generated from h2o2 in the presence of reduced transition metals are scavenged by the enzyme glutathione peroxidase ( gpx ) , during the process of metabolism of reduced glutathione ( gsh ) to its disulfide ( gssg ) , which in turn is reduced back to gsh by the enzyme glutathione reductase ( grd ) . these enzymes form part of the endogenous antioxidant defense system and suppress ros levels within the cell as well as within the mitochondria . antioxidants such as ascorbate , ubiquinone , or -tocopherol can participate in the mitochondrial antioxidative defense system , but none of them can convert o2 to o2 . it is gsh that participates in scavenging o2 , as well as in several redox reactions and maintains the mttp pore closed . the redox cycling in the mitochondria is very active and serves to prevent significant loss of gsh . this is important because the mitochondria contain gpx and grd activities and depend only on gsh uptake from the cytoplasm to keep adequate gsh levels . since melatonin promotes de novo synthesis of gsh by stimulating the activity of the enzyme -glutamyl - cysteine synthetase and also through its effects on gene expression of gpx , grd , sod , and cat [ 4145 ] helping in the recycling of gsh and in maintaining high gsh / gssg ratio , the role it plays in mitochondrial physiology is important . the synthesis of melatonin that occurs in a number of other tissues and cells does not contribute significantly to the circulating melatonin levels but rather exerts an autocrine or paracrine role . within this category , melatonin synthesis by lymphocytes , skin , the gastrointestinal tract , thymus , serotonin is then acetylated to form n - acetylserotonin through the action of arylalkylamine n - acetyltransferase , one of the key enzymes in melatonin synthesis . n - acetylserotonin is then converted to melatonin by hydroxyindole - o - methyltransferase ( hiomt ) , which has been identified as a rate - limiting enzyme in the biosynthesis of pineal melatonin [ 55 , 56 ] . once formed , melatonin is not stored within the pineal gland but diffuses into the capillary blood and cerebrospinal fluid . in a recent study conducted in humans , csf melatonin levels were found to be higher in the third ventricle compared to the lateral one , thus indicating that melatonin enters the csf through the pineal recess , even during daytime . the brain has much higher concentrations of melatonin than any other tissue in the body . in the circulation circulating melatonin is metabolized mainly in the liver where it is first hydroxylated by cytochrome p450 mono - oxygenases ( isoenzymes cyp1b1 , cyp1a2 , cyp1a1 ) and , thereafter , conjugated with sulphate to be excreted as 6-sulfatoxymelatonin . under certain circumstances these metabolites of melatonin which are formed in the brain , namely , n -acetyl - n -formyl-5-methoxy kynuramine ( afmk ) and n -acetyl-5-methoxykynuramine ( amk ) , also share the antioxidant and anti - inflammatory properties of melatonin [ 64 , 65 ] . melatonin is involved in the control of various physiological functions of the body such as seasonal control of reproductive processes [ 66 , 67 ] , sleep regulation , immune mechanisms [ 69 , 70 ] , and regulation of circadian [ 71 , 72 ] and sleep - wake rhythms [ 73 , 74 ] . in addition to the above - mentioned physiological actions , melatonin in pharmacological doses inhibits tumor growth and may have a potential therapeutic value in treating breast cancer , prostate cancer , melanoma , and cancer of gi tract [ 75 , 76 ] . as the prototype of the chronobiotic class of drugs [ 74 , 7880 ] , melatonin regulates the phase and amplitude of circadian rhythmicity by interaction with mt1 and mt2 receptors expressed in the hypothalamic suprachiasmatic nuclei ( scn ) and other brain areas . melatonin exerts many of its actions via membrane receptors , namely , mt1 and mt2 receptors that are expressed both singly and together in various tissues of the body [ 8183 ] . a third melatonin binding site that was isolated and purified from hamster kidney has been characterized as quinone reductase type 2 . this enzyme belongs to a group of reductases that participate in the protection against oxidative stress by preventing electron transfer reactions of quinones . the melatonin mt1 receptor is coupled to different g proteins that mediate adenylyl cyclase inhibition and phospholipase c activation . the mt2 receptor is coupled to a number of signal transduction mechanisms among them phosphoinositide production , inhibition of adenylyl cyclase , and inhibition of guanylyl cyclase . melatonin gets access freely to all compartments of the cell , and can be especially concentrated in the nucleus and mitochondria [ 15 , 47 , 88 ] . the discovery that melatonin was a remarkably potent scavenger of the particularly reactive , mutagenic , and carcinogenic oh was the finding that initiated numerous studies on melatonin 's role as a protector against free radicals . melatonin was shown to be much more specific than its structural analogs in undergoing reactions which lead to the termination of the radical reaction chain and in avoiding pro - oxidant , c- or o - centered intermediates [ 8991 ] . moreover , melatonin scavenged numerous different free radical species and other oxidants , among which the carbonate radical is important because of its presumed role in mitochondrial damage . although direct radical scavenging has been effective under numerous experimental conditions at clearly supraphysiological melatonin concentrations , its relevance at physiological levels has been questioned for reasons of stoichiometry . even though a single melatonin molecule may generate products in a scavenger cascade which may collectively eliminate up to ten free radicals , such findings from chemical systems a possible indirect action as mediated by upregulation of antioxidant enzymes by melatonin was proposed ( reviewed in [ 96 , 97 ] ) . an alternate concept has been put forth to explain the protective effect at the level of radical generation rather than detoxification of already formed radicals [ 98100 ] . if melatonin is capable of decreasing the processes leading to enhanced radical formation , this might be achieved by low , physiological , concentrations of the methoxyindole . apart from oxidants released by leukocytes , the isoforms of nad(p)h oxidases ( nox ) and mitochondria should be mentioned as main sources of free radicals in the cell . a recent study showed that melatonin inhibits free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway thus giving support to the hypothesis that melatonin has a protective effect at the level of radical generation . melatonin 's ability to influence mitochondrial function has been tested both in vivo and in vitro . in initial in vivo studies conducted on rats , etc complexes from the mitochondria of brain and liver tissues melatonin was found to increase the activity of c - i and c - iv of mitochondrial etc in a time - dependent manner , c - ii and c - iii not being affected . ruthenium red was found to impair mitochondrial metabolism by reducing etc and atp synthesis through its cellular oxidative stress action . inhibition of both c - i and c - iv of the etc were noted . injections of melatonin were found to counteract the inhibitory effect of ruthenium red on c - i , c - iv , and gpx enzyme . in an in vitro study , the effect of melatonin on t - butyl hydroperoxide- ( t - bhp- ) induced mitochondrial oxidative stress was evaluated . t - bhp depletes mitochondrial gsh and inhibits gpx and grd activities . in mitochondrial preparations , 100 nm melatonin was found to prevent the oxidation of gsh to gssg induced by t - bhp and also restored the normal activities of both gpx and grd . melatonin increased c - i and c - iv in a dose - dependent manner , the effect being significant at 1 nm . melatonin also counteracted cyanide - induced inhibition of c - iv showing thereby that melatonin can increase the activity of etc coupled to oxphos and increase atp synthesis in normal mitochondria as well as in mitochondria depleted of atp by cyanide . the effects of melatonin in regulating complexes i and iv presumably do not reflect its antioxidant role but indicates an interaction with etc complexes by donating and accepting electrons , thereby increasing electron flow , an effect not shared by other antioxidants . the major consequence of melatonin 's action on mitochondria may be avoidance of damage and dysfunction thus contributing to increase atp production [ 16 , 107 ] . melatonin increases the efficiency of etc thereby limiting electron leakage and free radical generation , and consequently promoting protein synthesis [ 17 , 47 ] . the possible mechanism by which melatonin controls mitochondrial respiration in the liver was examined in two groups of rats . in one group , melatonin ( 16 to 50 g / ml ) or vehicle was administered for a period of 45 days . in another study , rats received melatonin in drinking water ( 50 g / ml ) for 45 days or the same amount for 30 days , followed by a withdrawal period of 15 days . at sacrifice , the liver mitochondrial fraction was prepared and oxygen consumption was measured in the presence of excess concentration dl-3 -hydroxybutyrate or l - succinate . melatonin treatment decreased krebs 's cycle substrate - induced respiration significantly at both examined doses . the stimulation of mitochondrial respiration , caused by excess concentration of substrate , recovered after melatonin withdrawal . this study shows that melatonin can protect mitochondria from oxidative damage resulting from overstimulation of cellular respiration caused by excess krebs ' cycle substrate . a similar study on melatonin 's mechanism of action on mitochondrial respiration was carried out by another group of investigators . in this study , mitochondria from mouse liver cells was incubated in vitro with melatonin at concentrations ranging from 1 nm to 1 mm . melatonin decreased oxygen consumption , inhibited the increase in oxygen flux in the presence of excess of adp , reduced membrane potential and inhibited the production of o2 and h2o2 . melatonin was also able to maintain the efficiency of oxidative phosphorylation and atp synthesis by increasing the activity of the respiratory complexes i , iii , and iv . these effects were attributed to the intramitochondrial presence of melatonin , thus showing melatonin 's participation in the physiological regulation of mitochondrial homeostasis . melatonin 's action in preventing the opening of the mtpt pore given by oxidative stress caused by t - bhp was shown in another study on primary skeletal muscle cultures . using isolated mitochondria , melatonin ( 1100 m ) fully prevented myotube death induced by t - bhp . melatonin desensitized the mtpt pore to ca and prevented t - bhp - induced mitochondrial swelling and gsh oxidation . the inhibition of the mtpt pore opening by melatonin was suggested as an explanation for the protective action of melatonin against oxidative stress in myotubes . recently , the role of melatonin on cardiolipin and mitochondrial bioenergetics was explored [ 110 , 111 ] . cardiolipin , a phospholipid located at the level of inner mitochondrial membrane , is required for several mitochondrial bioenergetic processes as well as in mitochondrial - dependent steps of apoptosis . alterations in cardiolipin structure , content , and acyl chain composition have been associated with mitochondrial dysfunction in various tissues under a variety of pathophysiological conditions . melatonin was reported to protect the mitochondria from oxidative damage by preventing cardiolipin oxidation and this may explain , at least in part , the beneficial effect of this molecule in mitochondrial physiology [ 110 , 111 ] . the enhanced production of ros and accumulation of mtdna mutations in mitochondria may be contributory factors to human aging . many studies have established that the respiratory function of mitochondria declines with age [ 113115 ] . the increased production of free radicals such as o2 and h2o2 in mitochondria along with advancing age has been demonstrated [ 116 , 117 ] . accumulation of mtdna mutations can cause defective respiratory function resulting in enhanced production of ros . many of these mtdna mutations begin after adults reach their the mid thirties and accumulate with age in postmitotic tissues . overproliferation of abnormal mitochondria has been shown to occur in the muscle of aged individuals and in patients with mitochondrial myopathies [ 115 , 118 ] . the presence of these defective mitochondria is one of the factors involved in the decline in respiratory function during the aging process . enhanced activation of the mtpt pore in the brain and liver of aging mice has also been demonstrated . this in turn causes the release of proapoptotic factors from the intermembrane space of mitochondria . hence , an increased mitochondrial ros production , oxidative stress , respiratory functional decline , and susceptibility to apoptosis constitute central events in the aging process . the mechanism of the aging process can be studied in experimental models like the senescence accelerated mouse ( samp8 ) and senescence resistant mouse ( samr1 ) . the accelerated aging seen in this mouse strain is due to oxidative stress , which occurs with greater intensity as compared to the samr1 . at an age of 12 months , reductions in the activities of respiratory complexes i and iv have been demonstrated in liver mitochondria of samp8 mice , but not in samr1 mice . greater concentrations of lipid peroxidation products in the liver and brain homogenates were found in samp8 mice as compared to samr1 mice . in contrast to this , the concentration of the antioxidant enzyme gpx from samp8 mice at 12 months of age was found to be significantly lower than in samr1 mice . these studies support the conclusion that excess free radical generation coupled with less effective defense against the oxidative stress is responsible for alteration of mitochondrial function seen in samp 8 mice [ 122124 ] . since melatonin can readily reach the mitochondria due to its high lipophilicity , it seems feasible that , upon its entry into the cell , it could become concentrated at a superficial position in lipid layers near the polar heads of membrane phospholipids , a key place to function as a free radical scavenger . the effect of melatonin on age - dependent changes in the redox status of mitochondria in the heart and diaphragm was thus evaluated . melatonin , administered in the drinking water at a dose of 10 mg / kg for 9 months , was shown ( i ) to counteract the age - dependent increase in lipoperoxidation level , ( ii ) to increase gsh content in muscle mitochondria of both samp8 and samr1 mice , ( iii ) to counteract the reduction of gsh / gssg ratio in diaphragmatic mitochondria of samr1 and samp8 mice , ( iv ) to increase the activity of the antioxidant enzymes gpx and grd in the mitochondria of samp8 mice with no effect on samr 1 mice , and ( v ) to increase the activity of grd in samr1 mice . therefore , long - term melatonin administration prevented the age - dependent mitochondrial stress in both senescence - accelerated and senescence - resistant mice . as a continuation of the above - mentioned study , the effect of melatonin at earlier stages of the life span was evaluated at the 5th and 10th months of age in samp8 and samr1 mice . mitochondrial oxidative stress was determined by measuring the levels of lipid peroxidation , gsh and gssg , and the activities of gpx and grd in diaphragmatic mitochondria . age did not affect diaphragmatic mitochondrial levels of lipid peroxidation in samr1 mice but increased them in samp8 animals . when melatonin was administered in the drinking water at a daily dose of 10 mg / kg , the level of lipid peroxidation in 10-month - old samp8 mice was reduced to that found at 5 months of age . the decrease of gpx seen with age in both strains of mice was counteracted by melatonin administration with a higher effect in samp8 mice . as far as grd , although age caused significant reductions in both strains of mice , treatment with melatonin partially restored grd activity in samr1 mice only . the chronic administration of melatonin significantly increased complex ii and complex iii activity in samr1 and samp 8 mice and complex iv activity in samp 8 mice . measurement of atp levels and atp / adp ratio showed that treatment with melatonin counteracted the reduction in atp levels and atp / adp ratio found in samp8 mice . as far as longevity , melatonin treatment increased the half - life of samp8 mice from 16 to 22 months while longevity increased from 23 to 27 months . melatonin 's beneficial effects on longevity were significantly higher in samp8 mice than in samr1 mice . the study thus showed that melatonin administration counteracted age - dependent oxidative damage and mitochondrial dysfunction in senescence accelerated mice by improving mitochondrial function as reflected by the increase in atp production and a prolonged longevity . another study using rat brain mitochondria was designed to evaluate the beneficial effects of melatonin on age - associated reductions in mitochondrial bioenergetic function . mitochondria from control and aged rats treated or not with melatonin were obtained , and various bioenergetic parameters such as complex i activity , rates of state 3 respiration , mitochondrial h2o2 production , and membrane potential were evaluated . melatonin was found to prevent the significant age - related changes that occurred in all of these parameters in untreated animals . the ability to prevent complex i dysfunction and cardiolipin peroxidation was melatonin 's principal mechanism of action for achieving its effects . age - associated impairments in mitochondrial oxphos found in the brain of samp8 mice did not exhibit any major gender differences . however , a higher reduction in the gsh / gssg ratio at 10 months of age in female than in male samp8 mice has been reported in one study . chronic melatonin treatment completely prevented age - dependent oxidative stress as assessed by the recovery of the gsh / gssg in mitochondria of brain samples of both male and female mice . the ability of melatonin to prevent gsh loss with age probably reflects its influence on the activities of the gsh redox cycle enzymes . this is not due to reduction of brain mitochondria with aging but has been demonstrated to be due to diminished activities of respiratory complexes i , ii , and iii . mitochondrial dysfunction with aging is not an irreversible process as shown by studies using melatonin to prevent age - dependent declines in bioenergetic impairment of brain mitochondria in mice . i / r lesions are seen in many clinical conditions and are triggered by multiple factors including overproduction of ros [ 131133 ] . for example , ros produced at the level of complex i and iii of the respiratory chain are responsible for injury seen in cardiac i / r [ 134 , 135 ] as well as in stroke . the available evidence indicates the opening of the mtpt pore is responsible for the cardiomyocyte death occurring during i / r . while these pores remain closed during the ischemic period , at reperfusion the influx of ca into the mitochondria and an associated burst of ros production caused the opening of mtpt channels . this leads to mitochondrial depolarization , swelling , and rupture of the external mitochondrial membrane , with uncoupling of the respiratory chain and efflux of cytochrome c and other proapoptotic factors , all of which lead to either cell death by either apoptosis or necrosis . melatonin has also been shown to be effective in protecting the cardiac musculature against i / r [ 137139 ] . melatonin 's protective effect during i / r has been attributed to its action in inhibiting the mtpt pore , and in preserving the content and integrity of cardiolipin molecules . the fact that melatonin treatment inhibits both mtpt pore opening and cardiolipin peroxidation following i / r suggests a possible link between these two processes . it has been suggested that that increased levels of peroxidized cardiolipin together with increased ca overload can contribute to the mtpt pore opening during reperfusion . it must be noted that a significant cytoprotective effect of melatonin was described at a very early phase of a myocardial infarction , when i / r and thus oxidative damage were minimal , indicating that not all cardiac protective effects of melatonin are attributable to its antioxidant activity . septic shock is a lethal condition caused by a complex chain of pathogen - induced events involving immune cells , the epithelium , the endothelium , and the neuroendocrine system . the lethal effects of septic shock are associated with the production and release of numerous proinflammatory mediators as well as no and ros , thus inducing massive apoptosis . since many years ago , research interest was focused on the hypothesis that mitochondrial dysfunction plays a pivotal role in septic shock . this hypothesis was indeed confirmed by the finding of decreased respiratory complex i activity and low levels of atp levels in skeletal muscle biopsies obtained from critically ill patients with septic shock . increased no production and decreased levels of gsh were also found in septic shock patients . the protective effect of melatonin on the lethal effects of bacterial lipopolysaccharide ( lps ) on respiratory complex activities i and iv and a mitochondrial subform of inos ( mt inos ) activity was examined in liver and lung mitochondria of rats . lps administered at a dose of 10 mg / kg i.v . was found to increase mt inos activity and no , an effect that was greater in old rats than in young ones . melatonin administration ( 60 mg / kg , i.p . ) prevented lps toxicity by decreasing mt inos activity and no production . it also counteracted lps - induced inhibition of the activity of respiratory complexes i and iv . it is interesting to note that the effectiveness of melatonin to prevent the mitochondrial failure that occurs during endotoxemia were more pronounced in older animals than in young ones . using a long - term ( 3-day ) rat model of sepsis , the model comprises a long - term , fluid - resuscitated , fecal peritonitis model utilizing male wistar rats that closely replicates human physiological , biochemical , and histological findings with a 40% mortality . compared to sham - operated controls severely septic rats had lower ( 2022% ) hepatic and muscle complex i activities . moderate increases in nitrite / nitrate production were seen in both muscle and liver peaking at 2448 h and returning to sham - operated levels at 72 h. a fall in gsh was associated with lower complex i and increased no production was also demonstrated . a number of animal model studies and a few clinical observations have now shown that melatonin is beneficial for treating septic shock ( see , for a recent review , ) . to examine the effect of melatonin on changes in mt inos in septic skeletal muscles wild - type ( inos ) and inos knockout ( inos ) mice after sepsis , increases in mt inos and no levels , and decreases in electron chain activity were noted in inos mice but not in inos mice . in addition , an increase in oxidative stress was also found as indicated by an increase in lipid peroxidation products as well as a reduction in gsh levels and in the activities of gpx and grd . melatonin treatment counteracted the changes in mt inos activities and oxidative stress , and , further , restored the mitochondrial respiratory chain in inos mice . this study confirmed that mtnos is responsible for the mitochondrial dysfunction seen during sepsis and thus supported the conclusion that melatonin has the ability to protect against mt inos - mediated mitochondrial failure . a similar study performed in mitochondria isolated from the diaphragm of septic mice indicated that melatonin administration to inos mice counteracted mt inos induction and respiratory chain failure , and , finally , normalized the redox state after sepsis . considering the effects of melatonin and its virtual absence of toxicity , the use of melatonin along with conventional therapy to preserve mitochondrial bioenergetics as well as to limit inflammatory response and oxidative damage should be taken into account as a treatment option . pd is a neurodegenerative disorder with a multifactorial etiology , mainly characterized by the death of dopaminergic neurons in the pars compacta of substantia nigra and by the formation of lewy bodies . the initiating factor in pd is increased release of free radicals and enhanced signs of oxidative stress as demonstrated in brains of pd patients [ 150152 ] . although the molecular mechanisms responsible for the pathogenesis of ad are still under intense investigation , reduced complex i activity in the substantia nigra and loss of gsh have been reported in pd patients . the selective inhibition of complex i in the etc compromises energy availability and leads to apoptosis and death of the dopaminergic cells of substantia nigra . a commonly accepted model of pd is that achieved by the systemic or intracerebral administration of neurotoxins like 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine ( mptp ) . the loss of dopamine neurons occurring in these animal models causes severe sensory and motor impairment which in turn gives rise to tremor , rigidity and akinesia similar to those seen in pd patients [ 155 , 156 ] . the active glial metabolite of mptp , 1-methyl-4-phenylpyridinium ( mpp ) , is taken up into the dopaminergic neurons through the dopamine transporter , and then accumulates in the mitochondria of substantia nigra pars compacta . by binding to complex i , mpp increases the production of ros and enhances oxidative stress causing reduction of atp and death of cells in the substantia nigra . in the striatum , the damage caused by mpp is attributed to increased generation of o2 that reacts with no to generate the highly toxic onoo . this impairs mitochondrial function as a result of irreversible inhibition of all etc complexes leading to the death of neuronal cells . a neuroprotective effect of melatonin in isolated rat striatal synaptosomes and liver mitochondria treated with mpp has been demonstrated . melatonin prevented the inhibition of mitochondrial respiration by limiting the interaction of mpp with complex i of etc . although the role of ros generation has been demonstrated in the etiology of pd , the participation of mtnos in the mitochondrial dysfunction and nigrostriatal degeneration has only recently been examined . in a study conducted in adult male mice , mptp was administered at a dose of 15 mg / kg in four separate doses . animals also received melatonin or its metabolite amk ( 20 mg / kg ) 1 h prior to mptp injection . the administration of melatonin or amk concomitantly with mptp significantly reduced the inos activity stimulated by mptp . in the mitochondria , two nos isoforms , namely , constitutive and inducible , mptp administration significantly increased the activity of mt inos without affecting mt constitutive nos activity . interestingly , mptp administration induced i - mtnos activity in the mitochondria of substantia nigra whereas i - mtnos was only slightly induced by mptp in striatal mitochondria . treatment with either melatonin or its brain metabolite amk effectively counteracted i - mtnos induction , oxidative stress , and mitochondrial dysfunction induced by mptp . the nitrosative / oxidative stress reduction seen after therapeutic intervention with melatonin or amk in mptp treated mice was attributed to an effect in preventing damage to mitochondria . as already mentioned , mitochondria take up melatonin in a concentration- and time - dependent manner . a small number of controlled trials indicate that melatonin is useful to treat disturbed sleep in pd [ 162 , 163 ] , particularly rapid eye movement - associated sleep behavior disorder [ 164169 ] . whether melatonin or the recently introduced melatonergic agents ( ramelteon , agomelatine ) have the potential for treating insomnia in pd patients and , more generally , for arresting the progression of pd merits further investigation . several recent studies have confirmed the involvement of mitochondrial ros production and abnormal mitochondrial function in the pathophysiology of ad [ 170177 ] . ad is characterized by extracellular senile plaques of aggregated -amyloid ( a ) and intracellular neurofibrillary tangles that contain hyperphosphorylated tau protein . the resulting clinical effect is a progressive loss of memory and deterioration of cognition . there is substantial evidence to prove that mitochondrial toxicity is linked to the progressive accumulation of mitochondrial a . in the early phase of ad , inhibitors of and -secretase can be therapeutically effective to halt ad disease progression by inhibition of the protein misfolding of a into neurotoxic oligomeric aggregates . an increased expression of mitochondrial antioxidant enzyme sod-2 has been shown to prevent memory deficits and amyloid plaque deposition associated with ad . although a hypothetical occurrence of mutations in mtdna could cause increased oxidative stress and energy failure , no causative mutations in mtdna have been detected in ad so far . several actions of melatonin have been described which antagonize the deleterious effects of a. the effects of melatonin can be grouped as ( i ) antioxidant , including influences on mitochondrial metabolism ; ( ii ) antifibrillogenic , blocking a synthesis ; ( iii ) cytoskeletal , including suppression of tau protein hyperphosphorylation ( for a recent review see ) . the antifibrillogenic effects of melatonin were observed not only in vitro but also in vivo in transgenic mouse models [ 182184 ] . one such pathway is the bcl-2 pathway , which stabilizes mitochondrial function by antiapoptotic bcl-2 family modulators . melatonin inhibited free radical formation in microglia exposed to amyloid-142 by preventing the phosphorylation of the p47 nox subunit via the pi3k / akt pathway . in view of the consequences of excitation - dependent calcium overload on mitochondrial membrane potential and mtpt pore sensitivity towards excitotoxins like a , the actions of melatonin at the level of this important cellular compartment deserve particular attention . modulation of mitochondrial ca handling has been suggested as the potential pharmacological target for ad . in a recent study , a possible melatonin prevention of damage induced by a was evaluated in young and senescent hippocampal neurons . rat hippocampal neurons were incubated with a2535 and cell viability , mitochondrial membrane potential , atp , and the activity of the respiratory chain complexes were measured . cells exposed to a2535 showed decreased mitochondrial membrane potential , inhibited activity of respiratory chain complexes , and a depletion of atp levels . molecular studies undertaken with mitochondrial preparations suggest that melatonin has a therapeutic value in treating ad through its antiapoptotic activities . as outlined , melatonin acts at different levels relevant to the development and manifestation of ad . the antioxidant , mitochondrial , and antiamyloidogenic effects may be seen as a possibility of interfering with the onset of the disease . mild cognitive impairment ( mci ) is an etiologically heterogeneous syndrome characterized by cognitive impairment shown by objective measures adjusted for age and education in advance of dementia . a small number of controlled trials indicate that melatonin is useful to treat mci and to prevent progression to ad [ 181 , 191194 ] . a huntington 's chorea animal model was developed by using 3-nitropropionic acid , an inhibitor of mitochondrial complex ii . in this model , that replicates the neurochemical , histological , and clinical features of hd current evidence from genetic models of hd including mutation of the huntingtin gene ( mhtt ) , supports the mitochondrial dysfunction as major cause of the disease , with respiratory chain impairment relegated to a late secondary event . upstream events include defective mitochondrial calcium handling and impaired atp production . also , transcription abnormalities affecting mitochondria composition , reduced mitochondria trafficking to synapses , and direct interference with mitochondrial structures enriched in striatal neurons , are possible mechanisms by which mhtt amplifies striatal vulnerability . evidence is lacking on whether melatonin 's action on mitochondria could affect evolution in the genetic model of hd . at least on the accumulation of insoluble protein aggregates in intra- and perinuclear inclusions in hd melatonin had little or no inhibitory effect on huntingtin aggregation . mitochondrial dysfunction is implicated as the major causative factor in a variety of conditions such as the aging process , i / r , and septic shock . in addition , abnormal mitochondrial function , decreased respiratory enzyme complex activities , increased electron leakage , opening of the mtpt pore , and increased ca entry have all been shown to play a role in the pathophysiology of neurodegenerative disorders such as pd , ad , and hd . in addition to aging as a factor for low melatonin levels , it is well documented that there is a huge interindividual variation in circulating levels of melatonin which is stable within individuals and , which has been hypothesized to be genetic in origin [ 198201 ] . there is now evidence that there are polymorphisms in the gene for hiomt , the rate limiting enzyme in melatonin synthesis , and that the hiomt transcript level depends significantly on the genotype distributions . thus , there may be a genetically determined low melatonin syndrome that causes a predisposition to a variety of diseases . among the number of substances involved in maintaining mitochondrial bioenergetics a number of in vivo and in vitro studies in animals ( table 1 ) indicate that melatonin may emerge as a major therapeutic candidate to preserve the bioenergetic function of mitochondria . double - blind placebo controlled studies are needed to assess to what extent melatonin has therapeutic value in the treatment of the several disorders associated with mitochondrial dysfunction . s. r. pandi - perumal is a stockholder and the president and chief executive officer of somnogen inc . , a new york corporation . he declared no competing interests that might be perceived to influence the content of this paper . all remaining authors declare that they have no proprietary , financial , professional , nor any other personal interest of any kind in any product or services and/or company that could be construed or considered to be a potential conflict of interest that might have influenced the views expressed in this paper . | mitochondrial dysfunction is considered one of the major causative factors in the aging process , ischemia / reperfusion ( i / r ) , septic shock , and neurodegenerative disorders like parkinson 's disease ( pd ) , alzheimer 's disease ( ad ) , and huntington 's disease ( hd ) . increased free radical generation , enhanced mitochondrial inducible nitric oxide ( no ) synthase activity , enhanced no production , decreased respiratory complex activity , impaired electron transport system , and opening of mitochondrial permeability transition pore all have been suggested as factors responsible for impaired mitochondrial function .
melatonin , the major hormone of the pineal gland , also acts as an antioxidant and as a regulator of mitochondrial bioenergetic function . both in vitro and in vivo , melatonin was effective for preventing oxidative stress / nitrosative stress - induced mitochondrial dysfunction seen in experimental models of pd , ad , and hd .
in addition , melatonin is known to retard aging and to inhibit the lethal effects of septic shock or i / r lesions by maintaining respiratory complex activities , electron transport chain , and atp production in mitochondria .
melatonin is selectively taken up by mitochondrial membranes , a function not shared by other antioxidants .
melatonin has thus emerged as a major potential therapeutic tool for treating neurodegenerative disorders such as pd or ad , and for preventing the lethal effects of septic shock or i / r . |
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numerous common human diseases and phenotypic traits are believed to arise from a combination of genetic and environmental factors . the unravelling of the genetic predisposition to complex traits is a major challenge , and it could lead to better prevention , diagnosis and treatment of disease . recently , advances in genotyping technologies , reduction in genotyping costs and the availability of data regarding genome - wide sequence variation through the international hapmap project and 1000 genomes project have made genome - wide association studies ( gwas ) possible . gwas have emerged as a powerful tool for identifying genetic variants associated with complex traits . in the past few years , more than 500 loci have been found to be associated with human common diseases and traits ( 1 ) . gwas have proven to be much more successful than linkage studies , which were underpowered to detect variants of modest effect ( 2 ) , and candidate gene studies , which are non - systematic and biased due to our limited knowledge of the biological pathways implicated in disease pathogenesis ( 3 ) . gwas are based on the common disease common variant ( cdcv ) hypothesis ( 4 ) , which states that relatively common genetic variants ( maf > 5% ) of relatively low penetrance are important contributors to the genetic susceptibility to common diseases . well - powered gwas , which capture a substantial majority of common variation in the genome , have been now conducted for many common diseases . however , for the majority of these diseases , common variants explain only a small proportion of heritability ( 5 ) , due to small individual effect sizes . it has been estimated that only 13% of all identified susceptibility loci have odds ratios ( or ) above 2 , and only 1% have or above 10 ( 6 ) . for example , if we consider a total estimated sibling recurrence risk ratio ( s ) of 510 for rheumatoid arthritis ( ra ) ( 7 ) , 15 for type 1 diabetes ( t1d ) ( 8) , 1735 for crohn 's disease ( cd ) ( 9 ) and 3 for type 2 diabetes ( t2d ) ( 10 ) , their established susceptibility loci would contribute 3347% , 55.6% , 1012.6% and 11.9% of the total heritability , respectively ( table 1 ) . table 1.established susceptibility loci for ra , t1d , cd and t2dchromosomesnppositionregion / generaforsreferencerheumatoid arthritis 1p36rs38907452553624tnfsfr140.681.121.003(16 ) 1p13rs2476601114377568ptpn220.101.941.068(16 ) 1p13rs11586238117263138cd2 , cd580.241.131.003(16 ) 1q23rs12746613161467042fcgr2a0.121.131.002(16 ) 1q31rs10919563198700442ptprc0.871.141.002(16 ) 2p16rs1303123761136129rel0.371.131.004(16 ) 2p14rs93473465595586spred20.491.131.004(16 ) 2q11rs10865035100835734aff30.471.121.003(16 ) 2q32rs7574865191964633stat40.221.161.004(16 ) 2q33rs1980422204610396cd280.241.121.002(16 ) 2q33rs3087243204738919ctla40.561.151.005(16 ) 3p14rs1331559158556841pxk0.091.291.007(16 ) 4p15rs87404026108197rbpj0.301.141.004(16 ) 4q27rs6822844123509421il2 , il210.821.111.002(16 ) 5q11rs685921955438580ankrd55 , il6st0.791.281.009(16 ) 5q21rs26232102596720c5orf130.681.141.004(16 ) 6p21hla1.800(70,71 ) 6q21rs548234106568034prdm10.331.101.002(16 ) 6q23rs10499194138002637tnfaip30.731.101.002(16 ) 6q23rs6920220138006504tnfaip30.221.221.008(16 ) 6q23rs5029937138195151tnfaip30.041.401.006(16 ) 6q25rs394581159482521tagap0.701.101.002(16 ) 6q27rs3093023167534290ccr60.431.131.004(16 ) 7q32rs10488631128594183irf50.111.191.003(16 ) 8p23rs273634011343973blk0.251.121.003(16 ) 9p13rs281237834710260ccl210.341.101.002(16 ) 9q33rs3761847123690239traf1 , c50.431.131.004(16 ) 10p15rs21042866099045il2ra0.731.091.001(16 ) 10p15rs47503166393260prkcq0.811.151.003(16 ) 11p12rs54038636525293traf60.861.141.002(16 ) 12q13rs167854257968715kif5a0.621.101.002(16 ) 20q13rs481048544747947cd400.751.181.005(16 ) 22q12rs321825337544810il2rb0.261.091.001(16)type 1 diabetes 1p31rs226924164108771pgm10.191.101.001(15 ) 1p13rs2476601114377568ptpn220.142.051.104(15 ) 1q31rs2816316192536813rgs10.821.121.002(15 ) 1q32rs3024505206939904il100.831.191.004(15 ) 2p25rs1534422126407412p250.461.081.001(15 ) 2q12rs917997103070568il18rap0.781.201.005(15 ) 2q24rs1990760163124051ifih10.601.161.005(15 ) 2q33rs3087243204738919ctla40.551.141.004(15 ) 3p21rs33346345611ccr50.881.181.003(15 ) 4p15rs10517086260855114p150.301.091.002(15 ) 4q27rs17388568123132492il20.261.261.011(15 ) 5p13rs689793235874575il7r0.731.121.002(15 ) 6p21mhc3.058(72 ) 6q15rs1175552790958231bach20.471.131.004(15 ) 6q22rs9388489126698719c6orf1730.451.171.006(15 ) 6q23rs6920220137973068tnfaip30.221.091.001(15 ) 6q25rs1738074159465977tagap0.561.091.002(15 ) 7p15rs7804356268916657p150.761.141.003(15 ) 7p12rs494808851027194cobl0.951.301.002(15 ) 9p24rs70206734291747glis30.501.141.004(15 ) 10p15rs115946566,62015il2ra0.751.191.005(15 ) 10p15rs127224956137289il2ra0.891.591.015(15 ) 10p15rs9474746430456prkcq0.811.101.001(15 ) 10q23rs1050954090023033c10orf590.721.331.015(15 ) 11p15rs6892138800ins0.712.301.096(15 ) 12p13rs47638799910164cd690.371.091.002(15 ) 12q13rs229223956482180erbb30.341.311.018(15 ) 12q13rs167853657979190multiple0.721.121.002(15 ) 12q24rs3184504111884608sh2b30.491.281.015(15 ) 14q24rs14657886926359914q240.711.161.004(15 ) 14q32rs49003849849895114q320.291.091.002(15 ) 15q25rs382593279235446ctsh0.681.161.005(15 ) 16p13rs1270871611179873clec16a0.651.231.009(15 ) 16p12rs124442682034257216p120.301.101.002(15 ) 16p11rs478808428539848il270.581.161.005(15 ) 16q23rs72028777524724516q230.101.131.001(15 ) 17p13rs16956936763369217p130.871.091.001(15 ) 17q12rs229040038066240ormdl30.511.151.005(15 ) 17q21rs72211093877028617q210.651.051.001(15 ) 18p11rs189321712809340ptpn20.171.131.002(15 ) 18q22rs76336167531642cd2260.471.161.006(15 ) 19q13rs4251054720848119q130.841.161.003(15 ) 20p13rs2281808161055120p130.641.111.002(15 ) 21q22rs1120320343836186ubash3a0.431.131.004(15 ) 22q12rs57530373058172222q120.391.101.002(15 ) 22q13rs22954137591318c1qtnf60.431.111.003(15 ) xq28rs2664170153945602xq280.321.161.005(15)crohn 's disease 1p31rs1146580467475114il23r0.932.501.025(14 ) 1p13rs2476601114179091ptpn220.901.311.005(14 ) 1q23rs2274910159118670itln10.681.141.004(14 ) 1q24rs92868791711288571q240.241.191.006(14 ) 1q32rs115843831992024891q320.701.181.005(14 ) 2q27rs3828309233845149atg16l10.531.281.015(14 ) 3p21rs319799949696536mst10.271.201.007(14 ) 5p13rs461376340428485ptger40.131.321.010(14 ) 5q31rs21889621317987045q310.431.251.013(14 ) 5q33rs11747270150239060irgm0.091.331.008(14 ) 5q33rs10045431158747111il12b0.711.111.002(14 ) 6p22rs690842520836710cdkal10.781.211.006(14 ) 6q21rs77460821065419626q210.291.171.005(14 ) 6q27rs2301436167357978ccr60.461.211.009(14 ) 7p12rs1456893502402187p120.681.201.007(14 ) 8q24rs15513981266092338q240.621.081.001(14 ) 9p24rs107586694971602jak20.351.121.003(14 ) 9q32rs4263839116606261tnfsf150.681.221.008(14 ) 10p11rs175824163532765610p110.351.161.005(14 ) 10q21rs1099527164108492znf3650.391.251.012(14 ) 10q24rs11190140101281583nkx2 - 30.481.201.008(14 ) 11q13rs792789475978964c11orf300.391.161.005(14 ) 12q12rs1117559338888207lrrk2 , muc190.021.541.005(14 ) 13q14rs37641474335592513q140.221.251.010(14 ) 16q12rs206684749321280nod20.023.991.147(14 ) 17q21rs287250735294289ormdl30.471.121.003(14 ) 17q21rs74416637767727stat30.571.181.007(14 ) 18p11rs254215112769947ptpn20.151.351.014(14 ) 21q21rs17361351572709121q210.571.181.007(14 ) 21q22rs76242144439989icoslg0.391.131.004(14)type 2 diabetes 1p13p11rs10923931120319482notch20.111.091.001(73 ) 1q32rs340874212225879prox10.561.071.001(18 ) 2p23rs78009427594741gckr0.611.061.001(18 ) 2p21rs757859743586327thada0.901.151.002(73 ) 2p16rs24302160438323bcl11a0.461.081.001(74 ) 2q26rs7578326226728897irs10.641.111.002(74 ) 3p25rs180128212368125pparg0.851.231.005(73 ) 3p14rs460710364686944adamts90.761.101.002(73 ) 3q13q21rs11708067124548468adcy50.771.121.002(18 ) 3q27rs4402960186994381igf2bp20.311.111.002(73 ) 4p16rs100101316343816wfs10.591.141.004(73 ) 5q13rs445705376460705zbed30.261.081.001(74 ) 6p22rs1094639820769013cdkal10.331.091.002(73 ) 7p21rs219134915030834dgkb / tmem1950.471.061.001(18 ) 7p15rs86474528147081jazf10.501.081.001(73 ) 7p15rs460751744202193gck0.21.071.001(18 ) 7q32rs972283130117394klf140.551.071.001(74 ) 8q22rs89685496029687tp53inp10.481.061.001(74 ) 8q24rs13266634118253964slc30a80.681.121.003(73 ) 9p21rs1081166122124094cdkn2a / b0.841.171.003(73 ) 9q21rs1329213681141948chchd90.931.111.001(74 ) 10p13rs1277979012368016cdc123/camk1d0.181.111.002(73 ) 10q23rs501548094455539hhex / ide0.591.101.002(73 ) 11p15rs23344991653428dusp80.411.081.001(32 ) 11p15rs2313622648047kcnq10.521.081.001(74 ) 11p15rs22378922796327kcnq10.341.421.031(73 ) 11p15rs521917366148kcnj110.391.151.005(73 ) 11q13rs155222472110746centd20.881.141.002(74 ) 11q21rs1083096392348358mtnr1b0.301.091.001(73 ) 12q14rs153134364461161hmga20.11.11.001(74 ) 12q14q21rs796158169949369tspan8/lgr50.271.061.001(73 ) 12q24rs7957197119945069hnf1a0.851.071.001(74 ) 15q25rs1163439778219277zfand60.61.061.001(74 ) 15q26rs804268089322341prc10.221.071.001(74 ) 16q12rs805013652373776fto0.381.211.009(73 ) 17cen q21.3rs75721033170628hnf1b ( tcf2)0.381.101.002(73 ) xq28rs5945326152553116dusp90.211.271.011(74)ra , rheumatoid arthritis ; t1d , type 1 diabetes ; cd , crohn 's disease ; t2d , type 2 diabetes ; raf , risk allele frequency in controls ; or , odds ratio.sibling recurrence risk ratio ( s ) was calculated using the formula :
where q is the risk allele frequency , p = 1 q , and is the genotype relative risk under the additive model . established susceptibility loci for ra , t1d , cd and t2d ra , rheumatoid arthritis ; t1d , type 1 diabetes ; cd , crohn 's disease ; t2d , type 2 diabetes ; raf , risk allele frequency in controls ; or , odds ratio . sibling recurrence risk ratio ( s ) was calculated using the formula :
where q is the risk allele frequency , p = 1 q , and is the genotype relative risk under the additive model . explaining this missing heritability ' of complex diseases ( 1113 ) is an area of active research , and there are likely to be multiple contributing factors . part of the explanation is likely to be an underestimate of the contribution made by the types of variants targeted by gwas . for instance , it might be that there are large numbers of variants of very small effect , which early gwas were underpowered to detect , yet to be found . this idea is supported by the observation that meta - analyses of published gwas are discovering a substantial number of new susceptibility loci ( 1425 ) . in addition , for most loci , causal variants and potential independent additional markers within the region have not been identified yet . new ways of analysing the genetic architecture of complex traits using gwas data are suggesting that indeed a large proportion of heritability can be explained by common variants and that larger gwas will yield many more validated loci for complex traits ( 26,27 ) . of course , gwas only interrogate a portion of the types of variation that could underlie disease risk . analysis of gwas data has been mainly focused on single nucleotide polymorphisms ( snps ) , but there are other types of genetic variation , such as structural variants , that have not been studied in depth . however , recent studies of common ( maf > 5% ) copy number variants ( cnvs ) have shown that they seem unlikely to account for a substantial proportion of the missing heritability ( 28 ) . similarly , the analysis of gene environment and gene gene interactions ( epistasis ) might improve the fraction of heritability explained by loci documented thus far . several epistatic interactions have been indentified in humans [ e.g. between the ret protooncogene and endothelin receptor type b genes in hirschsprung disease ( 29 ) , the interleukin 4 receptor variants and interleukin 13 promoter variants in asthma ( 30 ) and the alpha- and beta - adrenergic receptors in congestive heart failure ( 31 ) ] , although they have not been replicated . however , this phenomenon has not been thoroughly explored through large - scale analysis of genome - wide snp interactions , first due to the fact that current sample sizes are underpowered to detect modest interaction effects and secondly due to the paucity of sample collections with genetic and detailed environmental exposure data . complex patterns of inheritance , such as parent of origin effects ( 32 ) , as well as inherited epigenetic modifications of the genome , the presence of phenotype heterogeneity in the cohorts used in the first wave of gwas , or even an initial over - estimation of the heritability of complex traits ( 33 ) can also contribute to the missing heritability . while the above - mentioned plausible contributors seem unlikely to play a substantial role in explaining missing heritability , rare variants are increasingly thought to account for a large proportion of it ( 3436 ) . contrary to the cdcv hypothesis , the multiple rare variant ( mrv ) hypothesis argues that the summation of the effects of low - frequency polymorphisms , each conferring an intermediate increase in risk ( i.e. incompletely penetrant , but greater than those observed for common variants ) , can explain a significant proportion of the genetic susceptibility to common diseases and traits . some studies analysing rare variants using gwas data have been carried out , but these have proven to be underpowered to detect robust associations . re - sequencing approaches are more suitable for rare variant analysis , and , as these are becoming more cost - effective and new analysis methods are being developed ( 37,38 ) , they will soon be applied to large - scale studies of rare variants . indeed , several targeted sequencing studies have already proven successful for the identification of associations between rare variants and some human diseases and disease - related phenotypes ( 3943 ) . the same argument can also be extended to other forms of genetic variation , and it has been recently proposed that rare cnvs may be responsible for some fraction of the missing heritability of complex traits ( 44,45 ) . it has been recently proposed that gwas signals that have been credited to common variants could instead reflect the effect of mrvs . synthetic association ' signals in gwas , by occurring more often in association with one of the alleles of a common tag snp ( fig . 1 ) , which would thus synthetically confer an increased risk for disease this might also mean that the causal variants could be megabases away from the common variants detected in gwas , and that the real effect size could be much stronger than that implied by the common tag snp . if true , the synthetic association hypothesis would suggest that follow - up studies from gwas hits should encompass a much larger region than the linkage disequilibrium region surrounding the detected common variant ( 6 ) . figure 1.simplified view of genetic variation at the nod2 locus , a well - documented example of a synthetic association . the left - hand side shows a genealogical tree representing six snps in this region after discarding rare recombinant events . the right - hand side shows the resulting haplotypes and their population frequencies ( 48 ) , with coloured circles representing common gwas snps , and starbursts representing previously identified low - frequency coding variants responsible for association between nod2 and cd . while none of the gwas snps is strongly correlated with any individual causal allele , the three coding variants create a synthetic association because they cluster by chance on the side of the tree marked by the green gwas snp ( rs2076756 ) . simplified view of genetic variation at the nod2 locus , a well - documented example of a synthetic association . the left - hand side shows a genealogical tree representing six snps in this region after discarding rare recombinant events . the right - hand side shows the resulting haplotypes and their population frequencies ( 48 ) , with coloured circles representing common gwas snps , and starbursts representing previously identified low - frequency coding variants responsible for association between nod2 and cd . while none of the gwas snps is strongly correlated with any individual causal allele , the three coding variants create a synthetic association because they cluster by chance on the side of the tree marked by the green gwas snp ( rs2076756 ) . there are very few documented examples showing that mrvs may be responsible for a common variant gwas signal ( 47 ) . it therefore seems sensible to evaluate this hypothesis in the broader context of human disease genetics , including historical study designs , functional annotations of gwas regions and experiments in human populations with diverse ancestry . while sequencing experiments currently underway or in planning will ultimately resolve the role of synthetic association , the balance of evidence available today is already illuminating . one line of evidence that suggests that synthetic associations do not underlie many reported gwas associations is provided by linkage scans that have been conducted in the past . the genetic model that underpins synthetic association ( allelic heterogeneity caused by several low - frequency variants with larger effects than commonly seen in gwas ) is highly tractable by linkage analysis , which combines information from all causal variants at a particular locus . this relationship is highlighted by the widely replicated linkage between the nod2 gene and cd , which is driven by three independent , low - frequency causal variants ( 4850 ) which cause a synthetic association signal in gwas of cd ( fig . nod2 is the exception that proves the rule that , despite many attempts , very few replicable linkages to complex diseases have been discovered ( 51 ) . this dearth of findings is informative when considering the likelihood of synthetic associations because it rules out a class of genetic models from playing a substantial role in complex disease . power calculations comparing a large - scale linkage scan ( 52 ) with the largest gwas considered by dickson et al . ( 46 ) show that only a small fraction of the genetic models which can give rise to synthetic associations would not be detected by linkage . furthermore , the scenario where synthetic associations could have escaped linkage comprises models with a small number of causal variants with genotype relative risk < 2.5 ( 53 ) . while these observations do not entirely rule out synthetic associations , they seriously confine the parameter space in which they might exist . in addition , comparisons of even modest linkage signals with gwas regions have shown only a few overlaps , and even these are largely driven by atypically large effects like the mhc in autoimmunity . in addition , attempts to explicitly use linkage information to boost the power of gwas ( 54 ) have not been successful . this contrast between largely overlapping genetic models that linkage and synthetic association are well powered to detect and almost completely non - overlapping results from linkage and gwas strongly suggests that synthetic associations do not underlie many gwas signals . another prediction made by the synthetic association hypothesis is that the most significantly associated common variant identified by gwas might be located several megabases away from the underlying low - frequency functional variants . the empirical properties of linkage disequilibrium between low - frequency and common variants are not fully understood , although the complete 1000 genomes project ( http://www.1000genomes.org/ ) will soon provide information necessary to evaluate this question directly . nevertheless , two indirect pieces of evidence suggest that most gwas hit snps are within a few hundred kilobases ( and many within tens of kilobases ) of their tagged functional alleles . first , a large number of gwas signals across a variety of traits are nearby to genes previously established to cause mendelian forms of the same trait ( 55 ) . for example , 8 of 10 proteins involved in the th17-differentiation signalling pathway have been associated with one or more auto - inflammatory diseases ( 56 ) . as with many aspects relating to the evaluation of the prevalence of synthetic associations , deeper sequence data sets will be needed to fully answer the question of the distance between gwas tag snps and causal variants , but these early patterns imply that the tag snp often resides in the proximity of the relevant functional genomic element . under the synthetic associations model , common variant signals reflecting single or multiple rare alleles are unlikely to be consistent across populations of different ancestry . this is based on the fact that many of these rare variants would have arisen recently and will therefore not be shared across diverged populations . for example , a study from early 2010 clearly demonstrated that common variant signals for t2d are reproducible and have similar effect sizes across east asian populations including chinese , malays and asian - indians in singapore ( 57 ) . in fact , t2d - associated variants have been found to be associated with disease in diverse populations ( ranging from african - americans to chinese ) by several studies ( 5862 ) . similarly , in ra , the stat4 locus , as an example , has shown reproducible association with disease in the usa ( 63 ) , uk ( 64 ) , spanish , swedish , dutch ( 65 ) , korean ( 66 ) , colombian ( 67 ) , japanese ( 68 ) and greek ( 69 ) populations . although synthetic associations explaining common gwas signals for complex polygenic traits are certainly plausible and can occur under specific circumstances ( e.g. nod2 in cd ) , results from studies thus far suggest that these scenarios are actually a rarity . the idea that mrvs at a particular locus may be associated with complex traits of interest has been around for over a decade . we are now starting to accrue a growing body of empirical evidence in support of this hypothesis . the field of complex trait genetics has over the last few months engaged in discussions on the controversial topic of synthetic associations , but it transpires that there is little evidence to support this as a widespread scenario . empowered by advances in sequencing technologies , attention is currently shifting towards the comprehensive study of low - frequency and rare variants . resources such as the 1000 genomes project and emerging large - scale studies like the uk10k project will undoubtedly facilitate the examination of variants at this end of the allele frequency spectrum . in parallel , improved strategies for accurate imputation and powerful analysis of low - frequency and rare variants in aggregate are being further developed and fine - tuned to the needs of these next generation truly genome - wide scans for association . g.o . is funded by the european union ( marie curie ief fellowship pief - ga-2009 - 235662 ) . are supported by the wellcome trust ( wt088885/z/09/z , wt089120/z/09/z ) . funding to pay the open access charge | genome - wide association studies ( gwas ) have successfully identified a large number of genetic variants associated with complex traits , but these only explain a small proportion of the total heritability .
it has been recently proposed that rare variants can create
synthetic association ' signals in gwas , by occurring more often in association with one of the alleles of a common tag single nucleotide polymorphism . while the ultimate evaluation of this hypothesis will require the completion of large - scale sequencing studies , it is informative to place it in the broader context of what is known about the genetic architecture of complex disease . in this review ,
we draw from empirical and theoretical data to summarize evidence showing that synthetic associations do not underlie many reported gwas associations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
molecular
self - assembly is a ubiquitous process in nature , and is also believed
to play an essential role in the emergence , maintenance , and advancement
of life . while the primary focus of the research on molecular
self - assembly centers on the biomacromolecules ( proteins , nucleic
acids , and polysaccharides ) or their mimics , the self - assembly of
small molecules in water ( or an organic solvent ) also has profound
implications from fundamental science to practical applications . because
one usual consequence of the self - assembly of the small molecules
is the formation of a gel ( or gelation ) , a subset of these small molecules
is called gelators . depending on the solvents in which they form gels ,
these small molecules are further classified as hydrogelators ( using water as the liquid phase ) and organogelators ( using an organic solvent as
the liquid phase ) . more precisely , hydrogelators ( i.e. , the molecules )
self - assemble in water to form three - dimensional supramolecular networks
that encapsulate a large amount of water to afford an aqueous mixture . the aqueous mixture is a supramolecular hydrogel because it exhibits
viscoelastic behavior of a gel ( e.g. , unable to flow without shear
force ) . unlike the conventional polymeric hydrogels that are mainly
based on covalently cross - linked networks of polymers ( i.e. , gellant ) ,
the networks in supramolecular hydrogels are formed due to noncovalent
interactions between the hydrogelators ( figure 1a ) . considering
that water is the unique solvent to maintain life forms on earth , it is important and necessary to distinguish water from organic solvents .
because supramolecular hydrogels are a type of relatively simple heterogeneous
system that consists of a large amount of water , it is not surprising
that the applications of hydrogels and hydrogelators in life science
have advanced most significantly thus , in this review we mainly focus
on the works that study the properties and explore the applications
of supramolecular hydrogels and hydrogelators in biomedical science .
because of the rapid advancement of the field , it is unavoidable that
some works are inadvertently absent from this review . here we offer
our sincere apology in advance and hope readers will let us know those
deserving works so we can include them in future reviews . ( a ) illustration
of the process for creating polymeric hydrogels
via cross - linking ( left ) , or formation of supramolecular hydrogels
via a chemical or physical perturbation initiated self - assembly ( right ) .
adapted with permission from ref ( 6 ) . ( d ) optical
image and negatively stained tem image of the hydrogel of 3 . adapted from ref ( 14 ) . according
to the report by hoffman in 1921 , the first small molecule hydrogelator
was dibenzoyl - l - cystine ( 1 ) ( figure 1 ) , which was able to form a
gel of 0.1% concentration [ that ] was rigid enough to hold its shape
for a minute or more when the beaker containing the gel was inverted . use modern
physical methods in chemistry ( e.g. , x - ray crystallography , light
and electron microscopy , rheology , and calorimetry ) to examine the
hydrogel of 1 again and provide invaluable molecular
details that reveal many fundamental design principles for creating
effective hydrogelators made of small molecules . impressively , among
the 14 aroyl - l - cystine derivatives studied by menger in the
seminal work in 2000 , the best hydrogelator
( 2 ) is able to self - assemble and to rigidify aqueous
solutions at 0.25 mm , ca . 0.01 wt % , in less than 30 s , which probably
still holds the record in terms of the lowest concentration of hydrogelators
and the fastest rate for gelation . one
of the most revealing design principles in the study of 1 is that aromatic moieties are highly effective for enhancing intermolecular
interactions in water . this principle is largely responsible for the
successful use of aromatic aromatic interactions to design
hydrogelators of small peptides . these facts imply that the
use of aromatic aromatic interactions is an effective and biomimetic
way to enhance hydrogen bonds and other interactions in water for molecular self - assembly in water that usually lead to supramolecular
hydrogels . a notable example of this
principle is that a small dipeptide derivative ( 3 ) is
capable of enabling many other molecular motifs to self - assemble in
water to form supramolecular hydrogels ( figure 1 ) . despite
the above seemingly obvious general principle of supramolecular hydrogelation ,
one common theme mentioned by the researchers who study gelators ,
intriguingly , is that their work on gelators started from an accidental
discovery of a particular molecule that forms a gel in a solvent . for example , the first small molecule hydrogelator reported by hoffman
unlikely was the intended goal . the research of organogelators also
began with a surprise . in 1987 , weiss et al . , while investigating
the photochemistry of small organic molecules , serendipitously observed
that small amounts ( usually ca . 1% by weight ) of cholesteryl 4-(2-anthryloxy)butyrate
( cab ) are able to cause reversible gelation in a wide variety of organic
liquids , the different types of functional groups of which in different
positions can interact with cab to affect gelation . similarly , shinkai et al . , in the process of developing
a new synthetic route , have made a surprising finding that the recrystallization
of a calixarene derivative from certain solvents ( e.g. , n - hexane , 1-butanol , and carbon disulfide ) results in gels ,
which is the first example of a gelator derived from macrocycles . in fact , in the research of oligomeric peptides ,
it is rather common to obtain serendipitous hydrogels of the peptides
even if the intended goals are something else . it is quite intriguing that serendipity is a common
occurrence throughout the encounter of small molecule gelators . these fortuitous happenstances , paradoxically ,
imply that the formation of supramolecular hydrogels via the self - assembly
of small molecules in water is , undeniably , a general phenomenon and
a rather common process . therefore , the
exploration of supramolecular hydrogels unlikely will be fruitless ,
and the applications of supramolecular hydrogelators will have a more
profound impact than a mere serendipitous observation , as we intend
to illustrate in this review . the development
of supramolecular hydrogels in the past two decades not only has underscored
the above implication , but also has provided a fundamentally new approach
for chemists to control the properties of soft materials via the molecular
engineering of a diverse set of substrates for a wide range of applications . to further realize the far - reaching impact of supramolecular hydrogels
in many fields of science and technology , from chemistry to physics
and to biology , and from materials to pharmacy to health , it would
be helpful to review the progress made so far and to consider possible
new directions . to contribute to this objective , in this review we intend to provide
a relatively comprehensive summary of the work of supramolecular hydrogelators
after 2004 and to put emphasis particularly on the applications of
supramolecular hydrogels / hydrogelators as molecular biomaterials . we make this rather arbitrary choice for several reasons : ( i ) the
excellent review by hamilton in 2004 has
covered the works on hydrogelators prior to 2004 . ( ii ) while the early
research of gelators has made significant progress in the elucidation
of the physiochemical properties of hydrogelators and the corresponding
small molecule hydrogels , the past decade has witnessed significant and exciting advances
in the exploration of the applications of hydrogelators and hydrogels
in biomedicine . ( iii ) the recent excellent reviews on gelators ( including hydrogelators ) have already provided considerable
insights into the structure property relationships of supramolecular
hydrogelators ; thus , the emphasis on the use of supramolecular interactions
and hydrogelators for various applications will complement other review
papers and provide a broader perspective at the interface of chemistry
and other fields of molecular science . we hope that , by summarizing
the development of hydrogelators within 10 years or so and with the
emphasis on the design of molecular biomaterials and the relevant
applications , in this review we will provide a potential starting
point not only for expanding the knowledge base of supramolecular
hydrogels as soft molecular biomaterials , but also for attempting
to address fundamental questions , perhaps providing a venue for chemists
to address the holy grail question in chemistry , that is , the origin
of life . we have arranged the review
in the following order . after the brief introduction of the methods
for generating supramolecular hydrogels , we discuss the supramolecular
hydrogelators on the basis of their categories , such as small organic
molecules , coordination complexes , peptides , nucleobases , and saccharides . after the introduction of the molecular building blocks for supramolecular
hydrogels , we focus on the various potential applications of the supramolecular
hydrogels as molecular biomaterials , classified by their applications
in cell cultures , tissue engineering , cell behavior , imaging , immunology ,
and unique applications of hydrogelators . particularly , we also discuss
the applications of supramolecular hydrogelators after they form supramolecular
assemblies but prior to reaching the critical gelation concentration
( cgc ) because this subject is less explored but may hold equally great
promise for helping to address fundamental questions about the mechanisms
or the consequences of the self - assembly of small molecules . finally ,
we provide our ( probably biased ) perspectives on supramolecular hydrogelators . we hope that this review will serve as an updated introduction and
reference for researchers who are interested in exploring the potentials
of supramolecular hydrogelators for discovering , inventing , and creating
innovative molecular assemblies , including soft matter and molecular
biomaterials . we believe such molecular biomaterials will contribute
to addressing the societal needs at various frontiers . despite the fact that they
share a prominent appearance and properties
( e.g. , soft and wet ) with polymeric hydrogels , supramolecular hydrogels
differ from polymeric hydrogels in many subtle ways . one essential
difference is that supramolecular hydrogels , unlike the polymeric
hydrogels that originate from a randomly cross - linked network made
of strong covalent bonds , are the consequence of molecular self - assembly
driven by weak , noncovalent interactions among hydrogelators in water . this subtle yet fundamental difference not only renders more ordered
molecular arrangement in the supramolecular hydrogels , but also manifests
itself in the process of hydrogelation . while simple swelling usually
confers a polymeric hydrogel , a stimulus or a triggering force is
necessary to bias thermodynamic equilibrium for initiating the self - assembly
process or phase transition to obtain a supramolecular hydrogel . therefore ,
there are many forms of stimuli or triggers for manipulating the weak
interactions . for the transition from a nongel state to a hydrogel
to occur thus , the overall impact
of the stimuli or triggers usually is negative h or positive s or both , which can be achieved
by either physical methods ( e.g. , changing the temperature , applying
ultrasound , or modulating the ionic strength ) or chemical methods
( e.g. , ph change , chemical or photochemical reactions , redox , and
catalysis ) . one of
the key features of a supramolecular hydrogel , especially when compared
to most cases of gels formed by polymers , is the apt thermal reversibility
of the self - assembly process , during which the strengths of hydrophobic
interactions and/or hydrogen bonding are temperature dependent . in
this kind of hydrogel , multiple methods
are able to determine tgel , including
the dropping ball the data collected via these methods are useful
for comparing structurally diverse hydrogelators and evaluating the
potential practical applications according to the thermodynamic features
of given hydrogels . for example , miravet et al . found that
the aggregation process is enthalpy driven in an organic solvent but
entropy driven in water when studying the molecular hydrogels from
bolaform amino acid derivatives 4 ( scheme 1 ) on the basis of the thermodynamics of gel
solubilization . this observation , also reported
by others , underscores the fundamental thermodynamic differences
between supramolecular organogels and hydrogels , which deserve the
attention of researchers . though cooling from an elevated temperature is a common
approach
for making supramolecular hydrogels , an increase of the temperature
of a supramolecular hydrogel can give quite opposite consequences :
the usual one is formation of a well - dissolved solution , but it is
also possible that precipitation will occur at higher temperature . for example , nandi et al . have applied a range of techniques to demonstrate
the effects of temperature and elucidated the activation barriers
for the assembly of riboflavin melamine hydrogels , the formation of which is triggered by cooling a homogeneous solution
of the mixtures from 80 or 120 to 30 c . reported another two - component hydrogel comprising fatty acids and
amines , the spacer length of which in the di / oligomeric amine dictates
the gel melting temperature . however ,
xu et al . observed that the increase of temperature induces a hydrogel ,
formed by a dipeptide derivative ( fmoc - d - ala - d - ala ;
fmoc = ( fluoren-9-ylmethoxy)carbonyl ) , starting syneresis and finally
collapsing into a precipitate . this behavior
is similar to that of lower critical solution temperature ( lcst ) polymers , indicating that the increase of the entropy
drives the self - assembly of the hydrogelator to a kinetically stable
state . a change of temperature also results in many other hydrogels
explored for various kinds of applications . during the past decade , many thermally reversible supramolecular
hydrogels have emerged for potential applications in various fields , such as drug delivery . regardless of a particular
molecular system , gaining a more comprehensive understanding of the
thermodynamic properties of supramolecular hydrogels by a change of
temperature is always beneficial for the optimization and applications
of supramolecular hydrogels . in chemical laboratories or in
industry , ultrasound commonly serves
as a convenient physical stimulus to speed dissolution or dispersion
or clean up the surface by disrupting weak intermolecular interactions . in fact , it is quite common to use ultrasound to assist the formation
of supramolecular hydrogels , but the systematic study of ultrasound
to control the properties of soft materials is a rather recent event . in essence , the force of ultrasound readily rearranges the aggregation
of molecules by cleaving self - locked intramolecular hydrogen bonds
or stacking to form interlocked structures through intermolecular
interactions , usually involving the participation of water molecules . the interest in using ultrasound for gelation has apparently received
more attentions in generating organogels . reported an
association - inert binuclear pd complex which , being stabilized by
intramolecular stacking interactions , can instantly
form gels in a variety of organic solvents upon a brief irradiation
with ultrasound . later , naota et al .
assumed that ultrasound could destroy intramolecular h - bonding of
metal - containing peptides and consequently initiated polymerization
under the intermolecular h - bonding in the semistable system . recently , ratcliffe et al . found that ultrasound
may reshape sheetlike dipeptide particles into elongated molecular
assemblies , due to the sonocrystallization effect , as the origin of
gelation . usually , with the treatment
of ultrasound , it is easier for the gelators to induce fibril formation . reported that ultrasound
can promote cyclo(l - tyr - l - lys ) ( 5 )
to form a hydrogel when its aqueous solution is cooled ( scheme 1 ) , although it normally precipitates
in water and gels a number of polar organic solvents , including n , n - dimethylformamide ( dmf ) and dimethyl
sulfoxide ( dmso ) . in addition , gu et
al . concluded that ultrasound not only accelerates the gelation process
and recovers the properties of an l - lysine - based hydrogelator ,
but also induces the self - assembly of fibrils to entangle and to form
3d networks . reported a metastable bicomponent hydrogel of thymine and 6-methyl-1,3,5-triazine-2,4-diamine
that slowly converts into a crystalline precipitate depending on the
method of its preparation ( e.g. , sonication induced ) . the similar rich phase behavior seems to be common when
ultrasound , combined with a change of temperature , is applied to supramolecular
hydrogels . a change of ph probably is the
most effective and the simplest chemical method to trigger supramolecular
hydrogelation because a small amount of acid or base easily and rapidly
can lead to a large ph shift via a diffusion - limited process . as an
attractive chemical method , a change of ph is particularly useful
for generating hydrogels since the ph of an aqueous solution not only
is well - defined , but also can be determined easily by a ph paper and
precisely by a ph meter . although ph - triggered hydrogelation largely
relies on reversible protonation / deprotonation of basic or acidic
group(s ) in a particular hydrogelator , a change of ph also may affect
the intensity and strength of hydrogen bonding between the hydrogelator
and water molecules . moreover , ph may affect the conformation of molecules
to favor hydrogelators to grow from a homogeneous solution to a fibrillar
structure in water via noncovalent forces , including aromatic normally ,
the self - assembly of small molecules , especially molecules possessing
acidic or basic groups , requires altering the ph to dissolve in the
aqueous phase before adjusting the ph of the solution for screening
the charge repulsion to result in hydrogels . among a variety
of supramolecular hydrogelators , peptide - based hydrogelators are the
most common ones that form supramolecular hydrogels on the basis of
a change of ph . specifically , according to the molecular structures
of the peptides or peptide derivatives , a change of ph , by affecting
the state of charges on the peptidic hydrogelators , usually results
in three kinds of hydrogels . ( i ) hydrogels that form at low ph : most
of the n - terminal - blocked peptides or peptide derivatives result in
this type of hydrogel . ( ii ) hydrogels
that are stable at physiological ph : many small amphiphilic molecules
self - assemble to form hydrogels of this category , and they usually
are suitable for certain biological applications or share certain
features with natural biomaterials . ( iii ) hydrogels that exist at
a high ph : the hydrogelators serving as building blocks of this type
of hydrogel likely have a very hydrophobic group or primary amine
groups . one intriguing and often overlooked fact of supramolecular
hydrogelators is that the pka of the monomeric
hydrogelator may differ from the pka of
the assemblies of the hydrogelators . for example , ulijn et al . recently
reported that a decrease of the solution ph of fmoc - diphenylalanine ( fmoc - ff , 6 ; scheme 1 ) induces the self - assembly
of 6 to form an entangled network of flexible fibrils
or flat rigid ribbons , only the former of which results in a weak
hydrogel . according to the authors , the self - assembly of 6 to form fibrils consisting of antiparallel -sheets results
in two apparent pka shifts , which are
6.4 and 2.2 ph units above the theoretical pka ( 3.5 ) of the monomeric 6 . although protons or hydroxide anions diffuse
fast , the self - assembly
of the hydrogelators during hydrogelation introduces inhomogeneity . thus , it is rather necessary and common to combine acid or base titration
with mechanical mixing ( including ultrasound ) to achieve a homogeneous
ph change . recently , adams and donald et al . utilized the hydrolysis
of glucono--lactone ( gdl ) to gluconic acid as a means of adjusting
the ph gradually in a solution of small molecule hydrogelators , which
allows the specific targeting of a certain final ph . this method achieves
a uniform ph change of the solution by slowing the release of protons ,
which appears to be particularly useful for the hydrogelation of the
hydrogelators that are soluble at high ph and gel at a lower ph . one
notable advantage of this approach is reproducibility of self - assembly
and hydrogelation , which may be particularly important in the study of the biological
functions of the assemblies of small molecules . the same principle
should be applicable to the slow release of hydroxide anion for the
hydrogelation of amine - containing hydrogelators , which remains to
be demonstrated . it is noteworthy that a change of ph usually influences
other physiochemical properties of the hydrogelators ( e.g. , fluorescence of the hydrogels or the morphology of the matrixes of the hydrogels ) . chemical reactions ,
which often yield products with properties different from the reactants ,
have become important tools in the production of soft materials , such
as hydrogels . particularly , the incorporation of chemical functional
groups into biological molecules can create unique sites of addressable
reactivity in even large and complex targets . although many kinds
of chemical reactions have found applications to generate polymeric
hydrogels , such as click chemistry , redox reactions , michael addition , ligation reactions , acid base reactions , and ring - opening metathesis polymerization ( romp ) , the use of similar approaches to produce supramolecular hydrogels
has received much less attention . recently , xu et al . reported that
a simple chemical modification of a small molecule ( 8) could generate another molecule ( 7 ) with excellent
solubility at physiological ph . the solution of 7 turns
into a hydrogel upon the addition of a strong base ( naoh ) for the
hydrolysis of the carboxylic ester bond of 7 to produce 8 ( scheme 2 ) . the unusual property of the hydrogel of 8 is that
it is kinetically stable over a wide ph range . this result illustrates a simple method to produce supramolecular
soft materials and may be particularly useful in designing a robust
system of prodrugs that can maintain a constant release rate against
abrupt changes in the environment . demonstrated
the use of a retro - diels alder reaction to convert a bolaamphiphile
to a hydrogelator . simple heating triggers the reaction and results
in a morphological transformation ( from 2d nanosheets to a network
of 1d nanofibers , as proved by means of transmission electron microscopy
( tem ) , atomic force microscopy ( afm ) , and small - angle x - ray scattering
( saxs ) ) to give a new heat - set supramolecular hydrogel . besides hydrolysis , redox reaction provides
another useful method for controlling the self - assembly of small molecules . for example , nilsson et al . demonstrated that reduction of a disulfide
bond in a cyclic peptide is a viable strategy for controlling peptide
self - assembly to form a hydrogel . as
shown in scheme 3 ,
xu et al . reported that a tripeptide derivative ( nap - ffk , 9 ) , a versatile self - assembly motif , could be integrated with a ruthenium(ii )
tris(bipyridine ) complex to afford the first supramolecular metallohydrogelator
( 10 ) . as a hydrogelator , not only do the molecules of 10 self - assemble in water to form a hydrogel , but also the
hydrogel exhibits a gel developed
a convenient and portable triacetone triperoxide ( tatp ) sensor by
utilizing a thiol - to - disulfide oxidation to trigger a solution - to - gel
phase transition . introduced
a cysteine - containing small peptide , ac - i3cgk - nh2 ( 11 ) . under an oxidative environment , not only do the
molecules of 11 form hydrogels at low concentrations ,
but also the hydrogels exhibit a tunable strength according to the
degree of oxidation . used native ligation to generate a peptide that forms a dimer
upon the oxidation of o2 in air , and the dimer acts as
a gelator in a mixed solvent of methanol / water . these arbitrarily selected examples illustrate that there
is hardly a limitation of using chemical reactions for generating
supramolecular hydrogels . although any aqueous chemical reaction potentially
can generate hydrogels , it is likely that atom - economy reactions will be particularly more suitable for creating
supramolecular hydrogels . for chemical
reactions , light is a simple stimulus for activating the reactants
and starting a chemical transformation . particularly , the invention
of coherent light sources ( e.g. , laser ) has greatly advanced photochemistry , which has laid a perfect foundation for the application of photochemical
reactions in the creation of materials . besides being extensively
used in the fabrication of a diverse array of materials that include
industrial membranes and coatings , dental adhesives , and optical and
electronic materials , photochemical reactions have already found
application in producing polymeric hydrogels . because photochemical reactions
allow the hydrogels to be defined with both temporal and spatial resolution , it is not surprising that light - derived hydrogels have received increasing
attention for broad biomedical applications that include drug delivery ,
wound healing , tissue engineering , and construction of high - density
cell arrays . recently , researchers have explored the use of light to initiate
self - assembly for generating supramolecular hydrogels . for example ,
yamamoto et al . described the formation and biodegradation of cross - linked
natural and related polymer hydrogels , fibers , and capsules with photoinduced
methods . the irradiation of the aqueous solutions of copoly[lyslys(cou ) ]
containing 510 mol % -[(7-coumaryloxy)acetyl]-l - lysine [ lys(cou ) ] residues causes a photo - cross - linking reaction
between coumarin moieties in the side chains and turns the solutions
to transparent hydrogels . as shown in scheme 4 , schneider et al . developed a photocaged peptide , max7cnb ( 12 ) , which remains
unfolded and unable to self - assemble when being dissolved in an aqueous
medium . the irradiation ( 260 nm < < 360 nm ) of the solution
releases the photocage , -carboxy-2-nitrobenzyl protection ,
and triggers peptide folding to produce amphiphilic -hairpins
that self - assemble to generate viscoelastic hydrogels . besides initiating folding and self - assembly
and regenerating rigid , nontoxic soft materials , this photocage chemical
method is also used for other applications ( e.g. , photorelease of
functional compounds ) since light can be easily controlled in intensity ,
direction , and duration in both space and time , as illustrated by
the widely used calcium photocages . wang et al .
presents a photo - cross - linking strategy , based on the ruthenium - complex - catalyzed
conversion of tyrosine to dityrosine upon light irradiation , to enhance
the mechanical stability of a peptide - based hydrogel by 10-fold with a storage modulus of around 100 kpa , which , according
to the authors , is one of the highest reported so far for hydrogels
made of small peptide molecules at a concentration of 0.5 wt % . considering the well - established photochemistry
of [ ru(bpy)3 ] , this method is convenient
and versatile for enhancing the mechanical stability of tyrosine - containing
peptide - based photo - cross - linked supramolecular hydrogels . khan et
al . used ultraviolet ( uv ) light to cross - link alginate hydrogels modified
with methacrylate groups . by using a
rheometer to monitor the hydrogelation during uv exposure , they illustrated
a potentially powerful tool to elucidate the dynamics of gelation
and predict the mechanical properties of the hydrogels . obviously ,
it is impossible to numerate all the cases of photochemically generated
hydrogels in this section . while these examples illustrate the use
of photochemical reactions for generating hydrogels , designing new
hydrogelators , and predicting the properties of new molecules , we
shall mention specific cases when we discuss the building blocks and
applications of supramolecular hydrogels in the subsequent sections . catalysis ,
especially enzymatic reactions , undoubtedly is a prominent dynamic
feature of life . considering that self - assembly is the molecular foundation
of life , and soft and wet are another two obvious characteristics
of most types of cells , it is not surprising that catalysis and enzymes
are attracting increased attention and are achieving many unexpected
successes in the generation and applications of supramolecular hydrogelators
and hydrogels . to this day , the reports of catalytic control
over self - assembly processes mostly deal with biocatalytic formation
hydrogels ( e.g. , enzyme - instructed hydrogelation ) , and much room remains for achieving directed self - assembly by catalytic
action in fully synthetic systems . recently , van esch et al . reported
the procedure for preparation of low - molecular - weight hydrogels in
the presence of an acid or aniline , which acts as the catalyst for
the in situ formation of a hydrogelator . the concentration of the
catalyst controls the conjugation of two water - soluble precursors ,
an oligoethylene - functionalized benzaldehyde and a cyclohexane - derived
trishydrazide , thus tuning the gelation time and mechanical properties
of the final gels ( also see figure 3 ) . recognizing catalyst - assisted
self - assembly as a common process in nature to achieve spatial control
over structure formation , they developed an ingenious way to generate
a spatially controlled supramolecular hydrogel using a micropatterned
catalyst on a surface . according to
their design , the precursors ( cyclohexane-1,3,5-tricarbohydrazide
and 3,4-bis[2-(2-methoxyethoxy)ethoxy]benzaldehyde , 3:1 ) of a gelator
( trishydrazone derivative ) react on
micropatterned catalytic sites on a surface to form building blocks
of self - assembled nanofibers that act as the matrixes of the hydrogels .
unlike homogeneous catalysis , this method apparently can achieve multilevel
organization among the nanofibers , which is uniquely promising for
further development . liu et al . introduced cu into a
glutamic acid - based bolaamphiphilic lipid ( n , n-hexadecanedioyldi - l - glutamic acid , l - hdga , 13 ; scheme 5 ) to form nanotubes with multilayer walls . providing a high density of catalytic sites
( cu ) , such nanotubes showed enhanced - asymmetry catalytic
behavior and accelerated the asymmetric diels alder cycloaddition
between cyclopentadiene and azachalcone . while any aqueous catalytic
reaction may find applications for generating supramolecular hydrogels ,
the development of catalytic supramolecular hydrogelation for targeted
applications likely will be most useful . the pivotal importance of enzymes in a variety of cellular
processes ,
including self - assembly and self - organization , justifies the exploration
of enzymatic supramolecular hydrogelation . although the application
of enzymes to cross - link covalent polymers is an effective process
for generating hydrogels , the use of
enzymes to prepare supramolecular hydrogels has several distinct advantages ,
such as the opportunity to achieve sophisticated secondary structure ,
adaptability to structural modification , and , most importantly , excellent
accessibility to enzymes both in vitro and in vivo due to the fast
diffusion of small molecules . despite the huge diversity of enzymes ,
so far only a handful of enzymes these enzymes are phosphatase , -lactamase , esterase , matrix metalloproteinase-9 ( mmp-9 ) , -chymotrypsin , thrombin , chymotrypsin , and -galactosidase ( for catalyzing
bond cleavage reaction ) , lipase , microbial
transglutaminase ( mtgase ) , and thermolysin ( for catalyzing bond - forming
reactions ) , and some other enzymes such as glucose oxidase , peroxidase , and tyrosinase . regardless of the types of reactions or enzymes ,
the essential feature for enzymatic hydrogelation of small molecules
involves the enzymatic conversion of a precursor into a hydrogelator
( normally via bond cleavage or bond formation , but not limited to
these two ) . the self - assembly of the hydrogelators to form supramolecular
nanostructures ( usually nanofibers ) , and the entanglement or alignment
of the nanofibers , affords the matrixes of the hydrogel . the first
case of enzymatic formation of supramolecular hydrogels is the use
of an alkaline phosphatase to dephosphorylate
a precursor , fmoc - tyrosine phosphate ( 14 ) , under slightly
basic conditions to form a hydrogelator ( 15 ) , which self - assembles
in water to form a supramolecular hydrogel ( scheme 6 ) . besides
promising a new methodology for the creation of hydrogels in situ ,
this process also builds up a platform for screening enzyme inhibitors and detecting the presence of an enzyme . instead of catalytically breaking bonds for supramolecular hydrogelation ,
ulijn et al . took a different approach by triggering the self - assembly
of peptide hydrogels via reverse hydrolysis using thermolysin . utilizing the fact that certain proteases can
thermodynamically favor the formation of peptide bonds , they used
thermolysin to catalyze the coupling of two amino acid precursors
to form a hydrogelator , which then self - assembled to form a hydrogel . a major advantage of employing reverse hydrolysis is that no byproducts
except water are formed , although the use of hydrophobic precursors
may be problematic in water . the demonstration of enzymatic supramolecular
hydrogelation has sparked relatively active research of bioresponsive
materials . for example , yang et al . reported the use of an enzymatic
dephosphorylation process to assist the formation of supramolecular
hydrogels . they designed and developed a precursor ( 16 ; scheme 6 ) which
is unable to form a hydrogel under most conditions , but turned into
a translucent gel upon the treatment of a protease ( i.e. , thrombin ) . the past decade has witnessed a considerable success in preparation
of supramolecular hydrogels using enzymatic transformation . we hope that the more detailed discussion of the applications of the
specific cases of enzymatic hydrogelation in the later sections will
provide stimulation for further development . the increased number of hydrogelators and the requirement of more
information on supramolecular hydrogels at both the nanoscale and
molecular levels require more accurate analysis and characterization
of the hydrogels . in this review , we first give a brief introduction
of various techniques generally used to characterize supramolecular
hydrogels before discussing the classifications and potential applications
of hydrogelators . especially , we focus on the high - resolution techniques
that elucidate the molecular self - assembly processes leading to gelation . unavoidably , some sample preparation methods may affect the native
nanostructures of the hydrogels . thus , among all the characterization
techniques , those methods that preserve the native properties of hydrogels
should be preferred over the ones that need to dry or / and to stain
the samples . generally speaking , the analysis and characterization
of supramolecular hydrogels aim to help scientists better understand
how the small molecules are arranged and organized in the matrixes
of hydrogels , which may lead to new approaches not only for rational
design of supramolecular hydrogelators but also for the development
of various functional molecular biomaterials . the classic inverting - vial visual inspection of the material
just by flipping the vial upside down , which acts as a zeroth - order
characterization technique , provides an intuitive impression for researchers
on the shape and strength of the hydrogels . thus , according to the
visual inspection , one can easily classify the material as a solution ,
viscous liquid , half - gel , or solidlike gel , which may contribute to
selection of more suitable techniques for further characterizing the
hydrogels . despite its simplicity , this assay by the naked
eye , in fact , provides important information whether
a small molecule self - assembles in water . with the rapid development
and easier operation of microscopic instruments , researchers rely
more and more on the microscopy techniques to study the morphology
of micro- and nanostructures that act as the matrixes of supramolecular
hydrogels . among all the microscopy techniques , atomic force microscopy
( afm ) or scanning force microscopy ( sfm ) , as high - resolution scanning
probe microscopies , can achieve a resolution on the order of fractions
of a nanometer , which is more than 1000 times better than the optical
diffraction limit . using afm , one may analyze hydrated samples in
situ under high humidity conditions or even without dehydration . in addition , it is possible to measure the roughness of a sample
surface at a high resolution , which helps classify a sample on the
basis of its mechanical properties ( e.g. , hardness and roughness )
and offers new capabilities for the microfabrication of a sample ( e.g. ,
an atomic manipulation ) . however , scanning force microscopy can be
misleading due to multiple factors , and
it is imperative to use multiple techniques to verify uncommon observations
obtained by afm or sfm . electron microscopy techniques , including
transmission ( tem ) and scanning ( sem ) microscopies , utilize a beam of accelerated
electrons as a source of illumination . since the wavelength of an
electron is rather short , electron microscopy has the capacity to
reveal the structures of small objects with resolution up to a nanometer . for example , tem can achieve better than 50 pm resolution . on the basis of these properties , electron
microscopy can provide valuable information about the morphology of
the molecular aggregates / nanofibrils leading to hydrogelation . however ,
the requirement of completely dried samples under the operating conditions
( e.g. , high vacuum ) makes tem and sem less reliable for inferring
the native molecular arrangement in the hydrogel state since the dehydration
process may result in artifacts that are difficult to explain . furthermore ,
staining agents ( e.g. , urnayl acetate , phosphotungstate , or osmium
tetroxide ) , which are used to increase the electron density of tem
samples to improve the quality / contrast of the images , may interact
with hydrogelators to change the self - assembly morphology and to induce
artifacts . cryogenic techniques have already been used in tem for
studying the self - assembled structures . for cryo - electron microscopy
( cryo - em ) or electron cryomicroscopy , the samples are studied at cryogenic
temperatures ( generally liquid nitrogen temperatures ) through creation
of thin vitrified ice films . cryo - em
has the advantage of reducing or eliminating the artifacts by making
nanometer resolution images of the native gel state feasible . indeed ,
several research groups have already successfully determined the structures
of supramolecular hydrogels and their related fibrous assemblies with
cryo - em . particularly , a seminal work
of the use of cryo - em to solve the structures of peptide nanofibers has demonstrated the capability of em for studying
the self - assembly of small molecules in water , which is emerging as
a new frontier of supramolecular chemistry at the intersection of
chemistry and cell biology . in addition ,
the development of another technique , environmental scanning electron
microscopy ( esem ) , also provides a useful
approach to characterize supramolecular hydrogels under a certain
humidity . oscillatory rheometry , as a comprehensive technique
to characterize viscoelastic materials , is becoming a routine measurement
of supramolecular hydrogels . rheology , which studies the flow of supramolecular
hydrogels , can provide tertiary structural information about the type ,
number , and strength of networks responsible for the observed hydrogelation . several types of setups , such as parallel plates ,
concentric cylinders , and cone - and - plate systems , are suitable for
the measurement of the rheological properties of hydrogels . all the
setups contribute to making a thin layer of supramolecular hydrogels
between a stationary and a movable component . the basic principle
of oscillatory rheometry is to measure the response of supramolecular
hydrogels to an applied oscillatory stress , which is quantified by
the elastic properties , such as g * ( complex modulus ) , g ( storage or elastic modulus ) , and g ( loss modulus or viscosity ) . meanwhile , the relationship
between these variables and the oscillatory frequency , imposed stress ,
temperature , time , or hydrogelator concentration usually contributes
to the studies of certain key characteristics ( e.g. , critical strains ,
thermodynamic nature of gelation ) of hydrogels . the temperature of
gelation ( tgel ) is one of the most often
studied characteristics of a gel , which is determined by the point
that noncovalent cross - links or global molecular rearrangements are
broken by thermal energy . differential scanning calorimetry ( dsc ) is a well - established characterization method
for the test of tgel , especially when
there is a sharp phase transition associated with hydrogelation . although
the rheological , elastic , and thermodynamic properties of supramolecular
hydrogels provide limited insight into the atomistic molecular arrangement
and understanding of how small molecules self - assemble to form hydrogels ,
the combination of physical characterization with a systematic structural
modification of the hydrogelators would contribute to establishing
the structure property relationships of supramolecular hydrogels . clearly , this approach requires the synthesis of molecules , which
is a core activity of chemistry . moreover , the synthesis of new molecular
entities provides opportunities for discovering new materials , especially
supramolecular hydrogels . this kind of approach , together with additional
techniques and greater correlation of various techniques , ultimately
should help infer the nanostructures of supramolecular hydrogels in
great molecular detail . small - angle x - ray
scattering ( saxs ) is another technique for characterizing supramolecular
hydrogels with a resolution close to that of tem . different from tem , which focuses on the local morphology of gel
matrixes , saxs mainly provides averaged information on the matrixes
of supramolecular hydrogels by measuring the spatiality of the matrixes . in addition , wide - angle x - ray powder diffraction ( xrd ) contributes
to the elucidation of the molecular organization and nanostructures
of supramolecular hydrogels , especially when microcrystals are formed
in the hydrogels . the long d spacing obtained from xrd represents the longest repeat distance
in the ordered structures by molecular self - assembly , which may provide
insight into the packing of small molecules in either an extended
or a bent conformation . a related technique for the characterization
of gels is small - angle neutron scattering ( sans ) . although it is a rather specialized technique to which researchers
have limited access , sans is able to provide useful information about
the average sizes and shapes of the nanostructures in a supramolecular
hydrogel . besides the
techniques referred to above , some other physical methods , such as
circular dichroism ( cd ) , uv / vis , infrared ( ir ) spectroscopy , fluorescence ,
or nmr , may also provide certain information about the molecular arrangement
in supramolecular hydrogels through detection of the molecule , cd has a wide range of applications in many different
fields , such as the study of the secondary structures of proteins ,
or the investigation of charge - transfer transitions . in the cases
of soft materials such as hydrogels , cd is able to study the self - assembled
superstructures in the gel phase or at the gel - to - sol transition . however , it always remains a challenge to make any precise conclusion
from the cd spectra alone , which means that it is better to combine
cd with other techniques for studying secondary structures of supramolecular
hydrogels . uv / vis is the technique used for investigating
stacking ( or aromatic aromatic interactions ) or metal coordination
in the process of hydrogelation . uv / vis , in combination with cd , may provide information for certain
molecular arrangements in hydrogels . ir spectroscopy , dealing with
the infrared region of the electromagnetic spectrum , is suitable for
confirming the presence of hydrogen bonding and determining the protonation
state of carboxylic acids . fluorescence is a useful tool for the investigation
of the aggregation between aromatic groups and the formation of hydrophobic
pockets inside hydrogels . in addition , the incorporation of fluorescent
probes into supramolecular hydrogelators usually results in large ,
flat aromatic surfaces for self - assembly , thus providing a reliable
approach for the design of an effective strategy to understand the
gelation process and to discover more biological applications of supramolecular
hydrogels . furthermore , solution - state
nmr can identify the chemical shift changes in the aggregation process
from the solution spectra to the gelled ones . solid - state magic angle spinning nmr ( mas nmr ) , being extensively
used for characterizing structures of protein or peptide aggregates ,
may be useful for elucidating the structures of supramolecular hydrogels . recent reports on the use of solid - state nmr to elucidate the packing
of a in a amyloids indicate
solid - state nmr as a powerful method for elucidating the molecular
arrangement of aggregates . however , the requirement of isotope labeling
has limited the routine use of solid - state nmr for characterization
of supramolecular hydrogels . in summary , it is always beneficial to
analyze all the data collected via multiple methods for elucidating
the nanostructures and potential applications of a given supramolecular
hydrogel since data from various techniques are usually complementary
to each other . on the
basis of the molecular
structural data collected from microscopy , xrd , sans , or rheology ,
it is possible to use modeling for proposing a plausible arrangement
of the molecular organization in supramolecular hydrogels . actually , researchers have already developed
some relevant model systems from computer simulation about the gelation
process of gelators in organic solvents . however , few modeling approaches , currently , are reliable in describing
the self - assembly of small molecules in water because of the inherent
kinetic nature of hydrogels and the
lack of an accurate description of hydrophobic interactions , which ,
interestingly , are the major driving forces for small molecules self - assembling
in water to form supramolecular hydrogels . while implying that the formation of supramolecular
hydrogels via
the self - assembly of small molecules in water is a common process ,
the serendipitous discoveries of many supramolecular hydrogelators
also paradoxically indicate that , presently , it is still impossible
to predict a hydrogelator a priori on the basis of its molecular structure . in fact , many designs of supramolecular hydrogelators only become
possible after the serendipitous discovery of a particular hydrogelator . the inability of the prediction , in our opinion , mainly originates
from the inaccurate evaluation of the interactions between water molecules
and the hydrogelators ( and their assemblies ) . despite this currently
unsolved problem , supramolecular hydrogelators , indeed , share common
features despite their different molecular structures . like certain
proteins that self - assemble , supramolecular hydrogelators possess
amphiphilicity and require noncovalent interactions (
interactions , hydrogen bonding , and charge interactions among the
molecules , among others ) that allow effective building up of three - dimensional
networks as the matrixes of hydrogels . scheme 7 shows a
few classical examples of hydrogelators that certainly are the products
of multiple weak interactions . being derived from an existing family
of low - molecular - weight organic gelators ( urea derivatives),17 , an effective hydrogelator , maintains the intermolecular
hydrogen bonds provided by its bisurea motif and allows its free carboxylic acid groups for both solubility in
water and ph control . consisting of a saccharide ( instead of using
urea ) for hydrogen bonding , 18 also relies on the long
alkyl chain to enhance intermolecular hydrophobic interaction and
to promote intermolecular hydrogen bonding among hydroxyl groups and
amide bonds , which results in a gelator that
gels a diverse range of solvents ( including water ) . incorporating l - lysine , which is easily made into
an amphiphile , 19(228 ) and 20(37 ) not only act as hydrogelators ,
but also have inspired a wide range of other hydrogelators based on l - lysine . along the notion of synthetic amphiphiles,21 consists of two alkyl tails
and self - assembles to form micelles , which result in hydrogelation . instead of relying on alkyl chain(s ) , 22(11 ) utilizes aromatic aromatic interactions
of ( fluoren-9-ylmethoxy)carbonyl ( fmoc ) to promote intermolecular
hydrogen bonding for supramolecular hydrogelation . because the fmoc
group is commonly used as a protection group for peptide synthesis ,
this convenience has led to many other fmoc - based peptide hydrogelators . aromatic
interaction as an effective hydrophobic force to enhance intermolecular
hydrogen bonding for self - assembly of small molecules in water . an intriguing and unexpected candidate as a hydrogelator is a vancomycin
derivative ( 23 ) . although
a vancomycin analogue ( ramoplanin ) is able to form nanofibers upon
binding to its receptor ( a lipid i analogue ) , it is still unusual for 23(18 ) to act as a hydrogelator . this case , indeed , reflects the
essential role and the diverse origins of multiple weak interactions
for supramolecular hydrogelation . despite the immense diversity of
the hydrogelators , an essential requirement of a supramolecular although adequate intermolecular interactions among
the hydrogelator are necessary for the self - assembly of the hydrogelators
in water , one should avoid excessive intermolecular interactions that
may result in the precipitation of the molecules in water . since several
excellent reviews have
already discussed molecular design in depth , interested readers are
recommended to consult those reviews . instead of prescribing a set
of detailed rules of the design of a hydrogelator , we simply introduce
the hydrogelators and hope the readers will formulate their own intuition
on the aspect of the molecular design of supramolecular hydrogelators . because the discovery of supramolecular hydrogels was made with
small organic molecules , we first discuss
the small organic molecules that act as the molecular building blocks
of supramolecular hydrogels ( table s1 ) . the large and diverse pool of building blocks makes categorizing these
small molecules rather subjective ; thus , we arrange the hydrogelators
according to their resemblance in molecular structure and start with
the hydrogelator having the lowest molecular weight within each type .
however , it is not necessarily that the hydrogelator having the lowest
molecular weight would be the most effective hydrogelator . the most
effective one should be the one that occupies the least volume fraction
to form a hydrogel . thus , highly effective hydrogelators should be
able to gel water at a very low weight percentage . another reason
for this arrangement is that the structural similarity of the hydrogelator
may offer a feasible starting point for the theoreticians who are
interested in supramolecular hydrogels and strive to formulate principles
for predicting supramolecular hydrogelation on the basis of molecular
structures , which is still a challenge . in the following , we first
discuss supramolecular hydrogels made of homotypic hydrogelators ,
and then introduce hydrogels consisting of a mixture of small molecules . by
attaching a pyridyl group to the urea motif , dastidar et al . synthesized
a small hydrogelator ( 24 ; scheme 8) that forms a hydrogel with the cgc of 0.8
wt % . it was found that the urea group has to
be at the para position of the pyridine to form the hydrogel . because
ethylene glycol molecules interact with both 24 and water ,
the authors were able to grow the crystals of 24 in a
mixed solvent of water / ethylene glycol . the crystal structure contains
both water and ethylene glycol and reveals valuable details about
the intermolecular interactions that involve 24 , water ,
and ethylene glycol . scanning electron microscopy ( sem ) shows the
fibrils formed by 24 in water are much thinner than the
fibrils of this hydrogelator formed in water / ethylene glycol , suggesting
the addition of ethylene glycol promotes the interfibrillar interactions . there are many other hydrogelators based on the urea motif developed
during this decade . for example , john et al . reported a urea - containing hydrogelator ,
1-[3-(decyloxy)phenyl]urea ( 25 ( n =
10 ) ) that not only forms a hydrogel in water at 0.1 wt % , but also
serves as a matrix for preparing and stabilizing gold nanoparticles
by in situ reduction . steed et al . reported
the gelation ability of a series of chiral bisurea gelators ( 26 ) . when n is an even number in 26 , the molecules act as a gelator
( 1 wt % ) in a mixed solvent ( e.g. , chcl3mecn dmso : h2o = 7:1 ) , but 26 fails to form a gel when n is an odd number . according to the crystal packing diagrams ,
the antiparallel urea tape motif appears to be necessary for the formation
of hydrogels , which consist of matrixes made of microcrystals . shimizu
et al . designed second - generation self - assembling bisurea macrocycles
( e.g. , 27 ) , which consist of more flexible building blocks
that form columnar structures in the solid state . van esch et al . reported a class of efficient hydrogelators
based on a simple attachment of hydrophilic hydroxyl or amino functionalities
to cyclohexane bisurea organogelators . they found that 28 in 1 n naoh forms a hydrogel with a cgc of 0.5 wt % . interestingly ,
after the formation of the hydrogel , the ph decreases to around 11.2 .
further lowering the ph to 10.1 results in a gel while the pure enantiomer of 28 results in a more stable
hydrogel than that made of racemic 28 , the hydrogel of
racemic 28 melts , almost being independent of the concentration . the authors observed that the racemic hydrogel became turbid upon
heating , a commonly observed phenomenon for an entropy - driven hydrogel . as shown in scheme 9 , tang et al . synthesized a small gelator , 29 , from
3-hydroxy-2-aminopyridine and glutaric anhydride . the authors
found that an increase of the power of the ultrasound from 200 to
500 w decreases the width of the self - assembled fibers from 8 to 2
m , accompanied by an increasing network density in the hydrogels . dastidar et al . synthesized a series of bisamides
derived from l-(+)-tartaric acid as potential hydrogelators .
among 14 bisamides synthesized , dipyrid-3-yltartaramide ( 30 ) displays an intriguing nanotubular morphology of its gel network
in the gel made in dmf / water . bearing a pyridyl group , 30 is able to coordinate with cu(ii)/zn(ii ) salts under suitable conditions
to afford metallogels . one unique aspect of this study is that the
authors managed to obtain a considerable amount of single - crystal
structures of those gelators . while polymorphism likely exists in
the gel phase , these structural details have provided useful insights
to understand the plausible intermolecular interactions among the
gelators . while 30 fails to form a gel at
ph below 7.0 , another
pyridine - containing amino acid - based gelator ( 31 ) forms
gels in aqueous media in the presence of hydrochloric acid . besides
the fact that it forms a transparent gel in a water / ethanol mixture
at a cgc of 0.2 wt % , the solution of the gelator successfully detects
and traps hydrogen chloride gas , likely due to the sol gel
transition when the ph is lowered . tang
et al . synthesized a hydrogelator , 2,6-bis[n-[(carboxypropyl)carbonyl]amino]pyridine
( 32 ) , from 2,6-diaminopyridine and glutaric anhydride
by a one - step procedure . 32 forms a self - supporting hydrogel
at a concentration of 4 wt % , which contains microcrystalline networks
to immobilize water . reported
a class of terpyridine derivatives ( e.g. , 33 ) , in their
bisprotonated forms , to act as versatile hydrogelators upon ultrasound
irradiation . although the terpyridine ligand chelates with metal cations ,
resulting in stable gels with tunable emissive properties , the sem
images of the hydrogels exhibit only a slight change before and after
the chelation . mcneil et al . designed
an innovative class of pyridine - based gelators
that formed gels in a mixed solvent of water and dmso . for example , 34 forms a gel at 3 wt % in 1:1 dmso / water . besides investigating
the relationship between molecular structure and gelation ability
of these pyridine - based compounds , the
authors discovered that some of the gelators are able to sense nitric
oxide . if this class of compounds can
act as hydrogelators with a reduced use of dmso , they likely will
find broader applications . reported an effective
hydrogelator ( 35 ) based on ( phenylenedivinylene)bispyridinium .
with two n - octyl chains , 35 forms a
hydrogel in water with a cgc of 0.12 wt % . this hydrogelator self - assembles
to give a morphological transition from fiber to coil to tube , depending
on the concentration of the gelator . because the emission of the chromophore
is sensitive to the environment , self - assembly of the gelator and
a change of the ionic strength lead to the aggregates fluorescing
in different colors . this type of fluorescent
colloid , recently rediscovered and termed aggregation - induced
emission by tang et al . in fact , the restriction
of bond rotation to generate fluorescent colloids had already found
applications in molecular imaging about two decades ago . the generation of a white - light emission from
a single chromophore in a single solvent ( water ) , indeed , agrees with the polymorphism of the assemblies
of the hydrogelators , which illustrates the versatility of supramolecular
hydrogels . the authors also observed a similar emission switch when
the solvent was a mixture of ethanol and water and reported that the
color of the emission depends on the temperature . designed and synthesized
a series of derivatives
of n - amidated 3- and 4-aminobenzoic acids with linear
alkyl chains ranging between 3 and 13 methylene units long , among
which the 4-(octanoylamino)benzoic acid sodium salt is able to form
supramolecular hydrogels thermoreversibly in aqueous solutions of
alkaline sodium salts at a concentration of 1 wt % 36 and 1 n naoh . moreover , a mold - casting / drying process can transfer
the supramolecular assemblies to produce self - supporting , macroscopic ,
supramolecular , nanofiber mats , which are thermally and mechanically
stable , and resistant to a large variety of organic solvents . on the
basis of sem , xrd , and cryo - tem , the authors proposed the mechanism
of the formation of the nanofibers of 36 , involving the
transformation of spherical micelles into ribbons and platelets of
multiple stacks of bilayers of the sodium salt of 36 . araki et al . reported an asymmetrically substituted
sulfamide ( 37 ) that forms a hydrogel in water at a cgc
of 1.0 wt % . sem and xrd suggest the formation of lamellar superstructures
via a hydrogen - bond - directed amphiphilic 2d sheet . one intriguing
property is that the casted film of 37 and the xerogel
of 37 , from benzene or water , result in almost identical
xrd patterns . more interestingly , this gelator is able to form homogeneous
and heterogeneous biphasic gels when the solvents are benzene and
water . on the basis of a similar concept ,
araki reported that 38 forms a hydrogel at a cgc of 0.5
wt % upon protonation of the tertiary amine groups . the authors also
observed lamellar superstructures and suggested the formation of 2d
sheetlike assemblies by the 2d hydrogen bond networks between sulfamide
moieties . the authors also reported that , with an increase of the
concentration of 38 to 2.0 wt % , the hydrogel exhibits
relatively high mechanical stability . reported that cetylpyridinium chloride
( cpc ) ( 39 ) forms a gel with a cgc of 6 wt % in a mixed
solvent of
chloroform and water . on the basis of saxs , the authors suggested
that the packing of the molecules is polymorphic , which also leads
to a lamellar organization . the authors
also reported a two - component gel resulting from 39 in
the presence of a structure - forming bolaamphiphilic additive , 6-aminocaproic
acid ( 6-aca ) , and the cgc remains at 6 wt % for the mixture of 39 and 6-aca . the authors used saxs to infer that the gelators
assemble as a lamellar organization of a loosely interdigitated bilayer
structure of 39 and 6-aca molecules predominantly due
to charge transfer , hydrogen bonding , and hydrophobic interactions . recently reported a simple hydrogelator based on bisphosphonates ( bps ) , a well - known
class of compounds used for treating osteoporosis . similar to the
incorporation of bisphosphonates in both polymeric hydrogels and supramolecular hydrogels,40 is a new supramolecular hydrogelator consisting
of bisphosphonates which forms a transparent hydrogel ( at 4 wt % )
that contains lamellar structures . baskar et al . reported that n - octadecylmaleamic
acid ( 41 ) formed hydrogels with a cgc of 0.75 wt % in
basic conditions . tiller et al . reported that a simple azo dye gels water at 5 wt
% upon cooling from hot water . using a glass slide coated with positive
charge , the authors were able to induce hydrogelation on the surface
when the solution concentration of 42 was as low as 0.10
wt % . the authors suggested that this
significant reduction of cgc might be a useful concept for the design
of drugs . this concept , indeed , is supported
by the hydrogelators that inhibit bacteria or cancer cells . in a more detailed
study , the authors found that the hydrogel prepared from 42 consisted of highly ordered and stable hierarchical structures .
on the basis of nuclear magnetic resonance , rheology , x - ray scattering ,
birefringence , and microscopy , the authors suggested that 42 forms worm micelles as the matrixes of the hydrogel of 42 . reported hydrogelators 43 and 44 , which consist of three amide moieties
and one alkyl chain . at ph 9.0 , the cgc values for 43 and 44 are 1.2 and 0.3 wt % , respectively . sol transition due to
the protonation of the ternary amine . on the basis of x - ray diffraction
and ft - ir analyses , the authors concluded that 43 and 44 form lamellar - like aggregates in the hydrogels , presumably
because the amide moieties form strong intermolecular hydrogen bonds .
despite the fact that there is a suspension phase between the ph - induced
gel sol transitions , the hydrogel of 43 or 44 exhibits high sensitivity to the ph change , which is needed
for the phase transition . while
redox chemistry is a fundamental process in nature , there
are only limited numbers of reports on the electrochemical characterization
of supramolecular hydrogels . yang et al . has proposed
an electrochemical strategy to characterize the hydrophobic microenvironment
of micellar hybridized supramolecular gels . as shown in scheme 11 , by using a gemini surfactant ( 45 ) and the classical
gelator n , n - dibenzoyl - l - cystine ( 1 ) to form a micellar hybridized hydrogel ,
the authors quantitatively characterized the net positive shifts of
the redox formal potential and the change of peak currents obtained
from the cyclic voltammograms of methylene blue ( 46 ) . according to the authors , by comparing the
apparent diffusion coefficients of 46 in these different
systems , it is feasible to characterize the hydrophobicity change
of the hybrid supramolecular hydrogel made of 1 and 45 . demonstrated
an ingenious way to combine a nonionic
surfactant and an aromatic core to generate an innovative class of
molecules that self - assemble in water . for example , the authors reported that a t - shaped aromatic amphiphile ,
consisting of tetrabranched oligo(ethylene oxide ) chains , self - assembles
to form nanofibers in water , which result in hydrogelation at a concentration
of 0.5 wt % . unlike most other hydrogelators , 48 forms a hydrogel when the temperature increases . this type
of lcst , though being common for polymeric hydrogels , is less reported
for supramolecular hydrogels . investigated hydrogels
formed by mixing alkyltrimethylammonium
bromides 49 and sodium azobenzene-4,4-dicarboxylic
acid ( 50 ) . in a typical example , 49 and 50 in a 2:1 ratio form a hydrogel at a concentration of about
4.0 wt % . the authors found that uv irradiation or the addition of
a salt and an acid results in a gel sol transition , while the
addition of a base hardly changes the hydrogel , suggesting that it
is important to maintain the ionic state of 50 for the
hydrogelation . reported
that amphiphilic guanidinium alkylbenzenesulfonates 51 exhibit lyotropic behavior in aqueous solvents . at a relatively
high concentration , 10 wt % , 51 self - assembles to form
a lamellar structure and results in hydrogels . huang et al . demonstrated that the mixture of an imidazole - type
surfactant , 1-hexadecyl-3-methylimidazolium bromide ( 52 ) , and a sodium salicylate ( 53 ) produces a thermoresponsive
hydrogel at a cgc of 2 wt % . the authors reported that , above the
critical temperature , the sample exhibits viscoelastic properties
of wormlike micelles , and the viscoelastic solution transforms into
an elastic hydrogel accompanied by a remarkable increase of the elastic
modulus . studied the
phase behavior and aggregation in the aqueous solutions of mixed 2-phenylbenzimidazole-5-sulfonic
acid sodium ( 54 ) , an anionic uv absorber , and cetyltrimethylammonium
bromide ( 55 ) , a cationic surfactant . the authors found that the morphologies ( i.e. , vesicles ,
tubules , or ribbons ) of the self - assembled structures depend on the
ratio of the two components in the mixture . for example , a hydrogel
forms at 0.6 wt % with a molar ratio of 54 and 55 of 8:2 . the authors found that the formation of very long
stiff tubules about 14 nm in diameter leads to hydrogelation , and
suggested that the stiffness of the bilayer of the vesicles and the
stiffness of the tubules originate from the rigidity of 54 . as shown in scheme 12 , shan et al . reported an interesting small molecule
( 56 ) that self - assembles in 6 m koh to form a hydrogel
at a concentration as low as 0.3 wt % . when the concentration of 56 increases to 1.4 wt % , the gel 56 is the first low - molecular - weight gel electrolyte having good electrochemical
properties while solving the problem of solution leakage , which may
find application in supercapacitors . also , using sorbitol , niu et al . 57 self - assembles
in a mixed solvent of dmso / h2o to form a gel at a cgc of
3 mm ( 0.13 wt % ) . the gel turns to a solution upon the addition of
copper(ii ) , and the solution reverses back to the gel state upon the
addition of edta to competitively coordinate away the copper(ii ) . song et al . also reported a d - sorbitol - based
hydrogelator and the effect of salt on the hydrogelation of 2,4-(3,4-dichlorobenzylidene)-d - sorbitol ( dcbs , 58 ) , which forms a hydrogel at
a concentration of 1 wt % . while sem indicates that the hydrogels
consist of globular aggregates , the addition of nacl to the aqueous
medium not only accelerates the gelation , but also results in networks
of long fibers . using uv / vis and fluorescence emission spectra to
characterize the hydrogels , the authors concluded that extensive aggregation
of the phenyl rings is responsible for the gelation . variable - temperature h nmr spectra further demonstrate that the addition of the
salt nacl enhances the interactions . wide - angle
x - ray diffraction shows that the hydrogels have a layered structure
that is independent of the addition of nacl . the authors also used
density functional theory ( dft ) calculations to support the proposed
molecular packing of the gelator in the nanofibers . griffiths et al . found that
bis-,-dihydroxyl esters
are able to gel thermoreversibly a wide range of solvents . as a gelator , 59 forms a gel in
a water - rich ( 75% ) ethanol / water mixture at a concentration of 0.18
wt % . on the basis of sans , ohsedo et al . suggested that in the gelation
mechanism the bis-,-dihydroxyl ester motif forms rodlike
structures . on the basis of a well - known
organogelator , dibenzylidenesorbitol , smith et al . developed a simple
condensation between sorbitol and 2 equiv of a benzaldehyde derivative
to form a hydrogelator ( 60 ) which is functionalized with
hydrazide ( as replacements for carboxylic acids ) . 60 not
only self - assembles to form hydrogels , at a cgc of 0.8 wt % in water
across a wide ph range , with a small amount of dmso , but also exhibits
ph - switchable dye adsorption kim et al . designed
a hydrogelator ( 61a ) derived from riboflavin ( vitamin
b2 ) . the authors found that 61a forms a hydrogel at a concentration of 1.6 wt % , but 61b and 61c are too soluble to form a hydrogel . one interesting observation reported by the authors is that the ability
of hydrogelation apparently is beneficial for the delivery of vascular
endothelial growth factor small interfering rna ( vegf - sirna ) into
human cells . russo et al . reported that
arborols , a type of dumbbell - shaped molecules acting as bolaamphiphiles ,
are able to assemble spontaneously into long fibers and to lead to
thermally reversible gels . on the basis of wide - angle x - ray scattering ,
the authors concluded that the self - assembly of 62 at
0.2 wt % results in fibrils in solution and the formation of bundles
of fibrils at 2 wt % is responsible for the hydrogelation . reported a chemical - responsive
supramolecular hydrogel based on a derivative of -cyclodextrin
( 63 ) . after the hydrogelator 63 forms a
hydrogel at a cgc of 2.9 wt % , the addition of 1-adamantanecarboxylic
acid or a large amount of urea induced a gel - to - sol transition . on the basis of a detailed nmr study , the authors
suggested that the host guest and hydrogen - bonding interactions
of cyclodextrins lead to the formation of supramolecular fibrils ,
which explains the chemoresponsiveness of the hydrogels . instead of using -cyclodextrin , osakada
et al . used -cyclodextrin and an alkylpyridinium to generate
a series of pseudorotaxanes that form hydrogels . according to the
authors , the possible mechanism is that the host guest interactions
transform the micelles of an alkylpyridinium to nanofibers of the
pseudorotaxanes and result in gelation at a concentration of about
12 wt % . as shown in scheme 13 , xu et al . developed
a supramolecular gel
in a mixture of ethanol and water ( 1:1 ) based on n , n,n-tris(3-pyridyl)trimesamide
( 64a ) . the nitrogen and
amide group in hydrogelator 64a can bind with phosphate
and carbonate ions via h - bonding and act as biomineralization active
sites for growing biominerals . the authors found that the calcium
phosphate grew into curved platelike nanostructures along the fibers .
in another work , dastidar and das et al . reported hydrogelators 64a and 64b derived from the pyridyl amide of
trimesic acid and demonstrated that 64a and 64b are able to form gels in a mixed
solvent of meoh / h2o at concentrations of 0.2 and 0.1 wt
% , respectively . on the basis of the crystal structures of 64a and 64b , the authors also proposed intermolecular interactions
among the gelators , which is reasonable if the matrixes of the gels
are microcrystalline . li and xu et al . also studied the gel of 64a ( 0.55 wt % ) in a 1:1 mixture of ethanol and water at ph
7.0 . they found that the macroscopic viscoelastic properties of the
gel of 64a depend on the microscopic hydrogen bonding
between the amide n h bond and the nitrogen on the pyridyl
group ( n hpy ) . one notable feature was the
increase of the storage modulus of the gels upon a decrease of the
ph to 5.0 . hpy leads to a highly branched
and homogeneous fibrillar network in the gel , as revealed by xrd and
field - emission scanning electron microscopy ( fesem ) images . such highly branched fibrillar networks likely
result from the reduction of the crystallinity of the fibrillar network ,
which is a useful insight for the design of supramolecular hydrogels . the same laboratory reported that another molecule of trimesic amide
( 64c ) self - assembles to form hexagonal microtubes in
a mixed solvent of h2o / thf and is able to gel h2o / thf at a concentration of 1.0 wt % . replacing the pyridyl group
in 64b by a benzoic acid
in the trisamide to produce 64d , schmidt et al . demonstrated
that 64d acts as a hydrogelator and forms a photoluminescent
hydrogel in water at a cgc of 0.2 wt % . having carried out dft calculations ,
the authors suggested that the photoluminescence originates from the
formation of a supramolecular chromophore . on the basis of the structure of 64d , researchers investigated
a series of simple benzene-1,3,5-tricarboxamide ( bta ) aromatic carboxylic acid compounds . found that the n - methylation of the amide bond in 64d results in a compound
to give a precipitate upon a decrease in ph , implying the critical
role of hydrogen bonding between the amides for hydrogelation . the
authors also introduced a hydroxyl group or a naphthyl moiety to generate
the hydrogelators 64e and 64f , respectively . compared to the hydrogel of 64d , the hydrogel of 64e exhibits a 10-fold higher yield stress , and the hydrogel
of 64f results in a 4-fold higher storage modulus . nagarajan
et al . reported that the replacement of carboxylic groups in 64d by alkyl chains also leads to gelators which largely gel
organic solvents such as dmso . bommel and van esch et al . developed a class of effective hydrogelators
based on cyclohexane-1,3,5-tricarboxylic acid . by capping the c - terminal
phenylalanine with diethylene glycol , they obtained a hydrogelator
( 65 ) with a remarkably low cgc value ( 0.033 wt % ) . the authors also obtained the crystal structure
of an analogue ( a nongelator ) of 65 and provided useful
insights for the molecular design of this type of hydrogelator . in another illuminating study , friggeri and
van esch et al . investigated the ability of several 1,3,5-cyclohexanetricarboxamide phenylalanine
derivatives 66 to form hydrogels . while they found that
enantiomerically pure homochiral 1,3,5-cyclohexanetricarboxamide l - phenylalanine crystallizes from water and fails to form gels ,
the heterochiral derivatives with either two l - phenylalanine
moieties and one d - phenylalanine ( lld ) or vice versa ( ddl )
are able to form hydrogels with a cgc value of 0.04 wt % . the authors
also found that an increase of the concentration to 0.12 wt % lld-66 or ddl-66 results in hydrogels with a remarkably
high gel the authors
also demonstrated that the attachment of a second amino acid or a
hydrophilic moiety to the c - terminal of the homochiral derivatives
of 66 produces effective hydrogelators . to align the amide bond in this type of 1,3,5-triamide
cyclohexane derivatives , van esch and samori et al . used an electrical
field to assist the alignment of the nanofibers and demonstrated that
the application of a voltage bias , indeed , helps the directional orientation
of the fibrils . using the 1,3,5-triamide
cyclohexane - based hydrogelators 67 , van esch et al . demonstrated
an elegant system that forms
well - defined nanostructures by the orthogonal self - assembly of hydrogelators
and surfactants . taking advantage of
the thermoreversibility of the hydrogels made of the 1,3,5-triamide
cyclohexane - based hydrogelators ( e.g. , 67 ) , the authors
dissolved the hydrogelators in solutions of surfactants above the
gel sol transition temperature , followed by cooling the mixture
and examining the hydrogelation . one of the most interesting results
reported by the authors was that cryo - tem studies revealed that , when
lipids , dopc , are used , the unilamellar dopc vesicles encapsulate
well - defined fibers with a diameter of 5 nm in the middle of their
aqueous compartment ( referred to as another remarkable feature was that the membrane
wall is able to restrict the growth of the fibers to a few hundred
nanometers . these observations imply that one should be able to use
the mutual interactions between both of the self - assembled structures
to design sophisticated soft materials , as recently suggested and
further demonstrated by van esch et al . van esch et al . recently reported another seminal study on the catalytic
formation of the triamide cyclohexane - based hydrogelators . specifically , either an acid or a base can
catalyze the formation of a trihydrazone hydrogelator ( 70 ) from the soluble building blocks 68 and 69 . the authors demonstrated that the concentration of the catalyst
used in the in situ formation of the hydrogelator controls the gelation
time and mechanical stiffness of the final gel ( figure 3 ) . this work may lead to an elaborate
way to form hydrogels via control of the reaction kinetics . cryo - tem images of unilamellar dioleoylphosphocholine
( dopc ) vesicles
coexisting with a network of well - defined fibers of 67 with a high aspect ratio . adapted with permission from ref ( 321 ) . copyright 2008 wiley - vch
verlag gmbh & co. kgaa . catalytic formation of trishydrazone hydrogelator 70 from soluble building blocks 68 and 69 leads to supersaturation followed by formation of fibers that eventually
cross - link to form a network that traps the surrounding solvent , leading
to gelation : blue , hydrophilic functional groups ; red , hydrophobic
functional groups . adapted with permission from ref ( 142 ) . copyright 2014 nature
america . as shown in scheme 14 , terech et al . investigated
hydrogels made of cationic bile acid
derivatives ( e.g. , 71 ) . they found that 71 ( i as the anion ) forms a robust hydrogel at 2.0
wt % to exhibit a storage modulus of 0.3 mpa . on the basis of x - ray
crystallography of the single crystals and x - ray scattering experiments ,
the authors concluded that the gel state consists of a morphology
different from that of the solid , which is supported by em investigations
of the xerogels to reveal the fibrous nature of the gel networks .
on the basis of the structural difference of the derivatives and the
morphology of the networks in the hydrogels , the authors suggested
an interesting notion that more compact structures would develop at
low concentrations . the same group of
authors used sans to study the self - assembled structures of the hydrogels
formed by the hydrogelators 7173 . they found that 71 forms thick cylindrical fibers
( r = 68 ) , the aggregates of 72 are ribbons with a bimolecular thickness of t =
37 and an anisotropy of the section of b / a 0.1 , and 73 exhibits a remarkable
transition from ribbons to thicker cylindrical fibers upon an increase
of the concentration . the authors also suggested the existence of
secondary aggregation mechanisms in the formation of bundles , differing from the behaviors of the hydrogels
formed by sodium lithocholate . also
extensively investigated the effect of the electrolyte and counterions
on the gelation of 73 . they found that the addition of
a monovalent salt ( nacl ) favors the formation of gels . at larger salt
concentrations , these studies
provide a rare case to compare the molecular arrangements of the gelators
in water and in an organic solvent . to understand the remarkable ability of hydrogelators
made of cationic
derivatives of deoxycholic acid , maitra et al . designed and synthesized
a series of that class of hydrogelators and compared them with hydrogelators
based on natural anionic bile salts . according to the authors , these cationic hydrogelators are ph independent
and start to aggregate in water at a concentration an order of magnitude
lower than those at which natural anionic bile salts aggregate . recently , maitra et al . reported tunable luminescent
gels and xerogels formed by lanthanide(iii ) cholates , which , according
to the authors , might find applications as luminescent coatings on
a glass surface . is the perfluoroalkyl
bile esters , which are efficient gelators in organic and aqueous organic
media . the requirement of an organic
solvent as the cosolvent seems a quite common feature of the hydrogelators
derived from bile acid , which occurs in several other bile acid derivatives
reported . maitra et al . also reported
a hydrogelator based on tripodal cholamide ( 74 ) . as a
supergelator , 74 forms a hydrogel with a cgc of 0.02
wt % in water containing 0.01% acetic acid . using fluorescent probes ,
8-anilinonaphthalene-1-sulfonic acid and pyrene , the authors found
two critical aggregation concentrations and suggested a progressive
increase in aggregate size and the microviscosity of the aqueous pool
encompassed by the self - assembled fibrillar network during the gelation . one noteworthy result from this work is that the microviscosity of
the aqueous phase around the network of nanofibers is far less than
the bulk viscosity of the gel . the authors
also used the tripodal cholamide - based hydrogel to synthesize semiconducting
nanostructures and obtained nanotubes and nanorods of cds , zns , and
cus . galantini and tato et al .
reported an interesting hydrogelator
of a bile salt derivative that forms a hydrogel in bicarbonate buffer
( ph 10 ) at a concentration of 0.18 wt % . the authors used a range of techniques ( static light scattering
( sls ) , cd , saxs , tem , and optical microscopies ) to establish the details
of the self - assembly of 75 , which occurs at 1.8 wt %
in the buffer and forms supramolecular nanotubes . according to the
authors , the tubule formation starts with the aggregation of the fibrils ,
followed by a slow transformation and ordering of the tubule walls
in well - spaced layers . one interesting feature is that the final elongation
of the tubules proceeds without a further aggregation of fibrils . by introducing a diamine or a dicarboxylic aromatic residue on the
lateral of a natural bile acid , galantini et al . obtained compounds 76 and 77 . while 77 forms a hydrogel
at a cgc of 0.16 wt % , a mixture of 76 and 77 results in a hydrogel at a cgc of 0.05 wt % this work , thus , illustrates
that the presence of the electrostatic interaction promotes the hydrogelation
from more dilute samples , suggesting that cationic and anionic mixtures
enhance the efficiency of the gelators . since there is greater understanding of the structures of the hydrogelators
based on bile acids , the exploration of their applications is emerging
as well . for example , shen and zhang et al . reported the use of the
hydrogels of bile acid derivatives for creating gold and silver nanoparticles
in situ . xin and xu et al . reported
that sodium deoxycholate ( 78 ) forms hydrogels at a concentration
of 2 wt % in the presence of nacl or nabr . they made an interesting observation that the addition of l - lysine or l - arginine turns the hydrogels to solutions . the authors suggested that the addition of amino acids competes with
the hydrogen bonds needed for hydrogelation , thus causing a gel sol
transition . as shown in scheme 15 , shinkai et al . reported a versatile gelator ( 79 ) that
is able to gel more than 10 different solvents . although 79 is unable to form a hydrogel ,
sierra et al . used click chemistry to connect this
type of estradiol - based gelator to form another effective gelator
( 80 ) that gels a dmso and water mixture at concentrations
as low as 0.04 wt % . using a cholesteryl
derivative , fang et al . developed another gelator ( 81 ) that gels a 1:1 mixture of acetone and water at 0.06 wt % . also employing a cholesteryl group , ji et al .
developed a series of phospholipid hydrogelators ( 82 ) .
besides exhibiting polymorphism of the networks in the hydrogels ,
this type of hydrogelator forms hydrogels at cgc values as low as
0.05 wt % ( 82 , m = 0 , n = 2 ) . reported a conjugate
of oleanolic acid with adenine ( 83 ) which forms a gel
in mixed solvents of thf and water ( 2:3 ) at a cgc of 2 wt % . one feature
of this gel is that the addition of uracil decreases the stability
of the gel due to the disruption of hydrogen bonding between the hydrogelators . recently reported
that sodium glycyrrhetinate ( 84 ) is able to form a hydrogel
with a cgc of 5.6 wt % , and the authors suggested the dipole dipole
interaction of sodium carboxylates as the main driving force for the
hydrogelation of 84 . another
related interesting work is the hydrolysis of succinated triamcinolone
acetonide which forms triamcinolone acetonide and results in hydrogelation . dastidar and shibayama
et al . took a combinatorial library approach
to generate 60 organic salts by reacting 5 bile acids ( e.g. , 85 ) with 12 secondary amines ( e.g. , 86 ) . after
the gelation test with various aqueous and organic solvents , they
found that 16 salts are supramolecular gelators , 6 of which are able
to form gels in aqueous as well as organic solvents . the salt didodecylammonium
cholate ( 85 + 86 ) is the most versatile
gelator , forming a gel in a 1:1 mixed solvent of dmso and h2o at a cgc of 1 wt % . the authors used dynamic light scattering ( dls )
and sans to infer the fibrous network formed via flexible clusters
of a few tens of nanometers in length , followed by the immobilization
of the network in the gel . bhattacharya
et al . demonstrated the formation of a supramolecular hydrogel by
simply mixing lithocholic acid ( 87 ) with dimeric or oligomeric
amines ( e.g. , 88 ) , at a total concentration of 5 wt % . however , the replacement of lithocholic acid ( lca ) by cholic acid
or deoxycholic acid results in no hydrogelation . on the basis of the
single - crystal x - ray diffraction analysis with one of the amine lca
complexes , the authors suggested that the electrostatic forces and
hydrogen bonding between the amines and the carboxylate and hydroxyl
moieties result in the formation of fibers as the matrixes of the
hydrogels . used zwitterionic
alkyldimethylamine oxide 89 to interact with litholic
acid ( 87 ) to form hydrogels . when n =
12 in 89 , the two - component system exhibits a high gelation
capability ( cgc = 0.08 wt % ) . one notable feature is that an increase
in the temperature results in a transition from helical fibrils to
vesicles with an intermediate mesophase . tian et al . also reported an interesting photoswitchable cholesterol
derivative as a gelator , though it is
too hydrophobic to act as a hydrogelator . as
shown in scheme 16 , blume et al . reported a class of symmetric long - chain bolaamphiphiles
that are efficient hydrogelators ( 90 and 91 ) . among them , dotriacontane-1,19-diylbis[2-(dimethylammonio)ethyl
phosphate ] ( 90 ) forms a clear hydrogel at 0.1 wt % and
ph 5 . tem reveals the hydrogelator to form a dense network of helically
structured nanofibrils with a diameter of 34 nm . at ph 5 , 90 self - assembles to form nanofibrils that are stable up to
at least 75 c . although there is no gelation at ph 10 , nanofibrils
form , but they become fragmented at 75 c . the authors reported that sans data support the significantly
higher stability of the hydrogel of 90 . while the formation of nanofibrils of 90 or 91 in the hydrogels agrees with these hydrogelators self - assembling
to form worm micelles , the interfibrillar interactions depend on the
kinetics of the self - assembly , thus resulting in rich polymorphism . for example , the cryo - tem of the hydrogels of 91 ( n = 17 or 18 ) reveals the formation of square lamellae . besides using the ph to control the morphology
of the assemblies of those hydrogelators , the authors also demonstrated
that the changes of the symmetry of the head groups are able to tune
the self - assembly behavior of single - chain bolaamphiphiles in an aqueous
suspension . designed and synthesized a series of polymethylene-1,-bis(phosphocholine )
( pc - cn - pc ) analogues , and found that the even - numbered ones form nanofibers composed
of stretched molecules with an all - trans - alkyl chain
conformation . meanwhile , they synthesized
the odd - numbered analogues to study a possible even odd effect
of these bolaamphiphiles during their aggregation in water . in addition
to these bolaamphiphiles with phosphocholine head groups , they designed
a series of polymethylene-1,-bis(phosphodimethylethanolamine)s
( me2pe - cn - me2pe )
with smaller sizes of the head group . these bolaamphiphiles show an
additional fiber fiber transition when the alkyl chain length
exceeds 26 carbon atoms . the behavior of the mixed bolaamphiphiles
indicates that the fiber structure allows differences in the alkyl
chains of up to six carbon atoms long . the mixing of two me2pe - cn - me2pe- or pc - cn - pc - type bolaamphiphiles with different alkyl chain
lengths offers the possibility to adjust the temperature of the gel sol
transition , at which the cross - linking of the fibers breaks and the
fibers dissociate . on the basis of this feature , the authors obtained
thermally switchable hydrogels , which may allow fine - tuning for drug
delivery applications . the comparison with dotriacontane-1,32-diylbis[2-(methylammonio)ethyl
phosphate ] ( mepe - c32-mepe , 92 ) , a bolaamphiphile
with an even smaller phosphomonomethylammonium head group , illustrates
the importance of the size of the head group for self - assembly . this
bolaamphiphile self - assembles exclusively into lamellar structures ,
a type of assembly that persists in mixtures containing the fiber - forming
molecules ( 90 ) . using
different methods , researchers already have characterized
the supramolecular behavior of the bolaamphiphilic hydrogelators dotriacontane-1,32-diylbis[2-(trimethylammonio)ethyl
phosphate ] ( pc - c32-pc , 91 ) and the ph - sensitive
dotriacontane-1,32-diylbis[2-(dimethylammonio)ethyl phosphate ] ( me2pe - c32-me2pe , 90 ) . depending on the temperature , ph , and concentration , these bolaamphiphiles
self - assemble into long nanofibers or other assemblies , such as short
rods or micelles . to obtain information about the motional dynamics
and microscopic order inside these assemblies , blume et al . carried
out a systematic electron spin resonance ( esr ) spin probe study and
reported that the spectra obtained with the spin probes 5- , 12- , and
16-doxylstearic acid ( n - dsa ) are highly sensitive
to the changes in the bolaamphiphilic arrangement . the authors obtained
rotational correlation times and order parameters from full esr line
shape simulations and found that the transition temperatures , determined
by the maximum hyperfine splitting , agree with the differential scanning
calorimetry ( dsc ) data . by comparimg 5-dsa and 12-dsa , which reside
at different positions in the alkyl chain region of the assemblies gauche isomerization predominantly
occurs in the outer region of the assemblies . for me2pe - c32-me2pe ( 90 ) at ph 10 , the authors
reported that esr data indicate the micelles to be short rods rather
than spherical in shape and that an increase of the concentration
from 1 to 10 mg / ml leads only to a one - dimensional growth of these
micelles . on the basis of the
structure of 90 , drescher and
meister et al . developed two unique bolalipids ( 93 and 94 ) which not only self - assemble at a concentration of 0.01
wt % , but also are able to modulate the viscoelastic properties of
the hydrogel made of 90 or 91 . generally , these bolaamphiphiles are much more
effective hydrogelators than the hydrogelators made of diacylphosphatidylcholine . zhang et al . developed a bolaamphiphile ( 95 ) that has two carboxylic acid ends and a diketopyrrolopyrrole
chromophore in the center . on the basi of the color change associated
with the self - assembly process , the authors concluded that the
stacking of the central parts and the hydrogen bonding between the
ends are responsible for the formation of the nanofibrils of 95 in water . although 95 self - assembles at a
concentration as low as 0.06 wt % in water , the formation of a hydrogel
has not been reported by the authors . similarly , another bolaamphiphile ( 96 ) bearing a bipyridine
moiety at the central part , though forming nanofibers at 0.15 wt %
by self - assembly , was not reported to form a hydrogel by zhang et
al . reported unsymmetrical
diacetylenic bolaamphiphiles 97 , which bear a carbohydrate
residue and a cationic glycine betaine moiety . when m = 13 , 97 forms a hydrogel at a cgc of 1.7 wt % . tem
studies by the authors revealed the polymorphism of these bolalipids
and the dense filament of the hydrogelators in the hydrogels . developed a series of two - component
hydrogels based on cetyltrimethylammonium bromide ( 55 ) and bis(decyloxy)succinic acid ( 98 ) to study the effect
of the chirality of the amphiphile on gelation . besides finding that 55 and 98 form vesicles and hydrogels that are
ph and temperature responsive , the authors concluded that molecular
chirality is responsible for the formation of supertwisted fibrils
in the hydrogels at a 98:55 molar ratio
of 1:2 with 31% water . dmf
( 20 vol % ) at a concentration of about 1 wt % . one interesting feature
is the observation of two break points in the surface tension isotherms ,
which correspond to the critical micelle concentration ( cmc ) and critical
gelation concentration . the authors also observed that the ph of the
solution decreases with an increase of the concentration of the hydrogelators . on the basis of cyclotriveratrylene ( ctv ) , jiang
et al . developed
a class of supramolecular hydrogelators having a cavity . by introducing
deprotonable cooh or protonable nh2 as the terminal groups
into the rigid and hydrophobic ctv backbones , the authors successfully
used 100 and 101 ( scheme 17 ) to form supramolecular hydrogels with
cgcs of 1.0 and 1.5 wt % , respectively . the obtained hydrogels of 100 and 101 are luminescent and exhibit ph - responsive ,
reversible gel the work also illustrates that
the skeleton of an organogelator is a promising starting point for
designing a hydrogelator . mocerino and
ogden et al . reported a proline - functionalized calixarene ( 102 ) that forms hydrogels in the presence of specific anions
such as nitrate , bromide , iodide , and perchlorate . however , it requires
a considerable amount of the hydrogelators ( over 18 wt % ) to form
the hydrogels . since acidic conditions and the presence of a lanthanide drastically reduce the amount of anions needed for hydrogelation ,
the hydrogelation likely depends on more than just the presence of
the anions . the later report by the
same group , indeed , confirmed that the lanthanum cations connect two
supramolecular helices to form a 2d network for hydrogelation . escuder , miravet , and ballester et al . developed
an aryl - extended calixpyrrole that acts as a receptor for tetramethylammonium . the authors found the formation of hydrogels in basic conditions and
at neutral ph , thus suggesting that , as a guest molecule , tetramethylammonium
interacts with the calixpyrrole to form noncovalent polymers , resulting
in hydrogelation . property relationship for another
calixarene - based hydrogelator , in which large alkali - metal cations
( k or rb ) trigger the hydrogelation . marletta and cunsolo et al .
reported a type of ph - responsive hydrogelator
based on calixarene . after capping
the lower rim of calixarene with an isopropyl group and attaching
alkyl amino groups at the upper rim , the authors obtained a series
of hydrogelators , the most effective one of which exhibits a cgc of
0.2 wt % . kim et al . reported that cucurbituril ( cb , 103 ) is able to form a hydrogel in acidic conditions at a concentration
of 3 wt % . although the optical appearance
of the hydrogel is opaque , the authors demonstrated a unique guest - induced
stimulus - responsive behavior of the gel of cb by using 4,4-diaminostilbene
dihydrochoride as a guest . besides the observation by kim et al . that
cb forms a hydrogel , tan et al . reported
a thermoresponsive supramolecular hydrogel consisting of cucurbituril
( cb ) and butan-1-aminium 4-methylbenzenesulfonate ( bamb ) . however ,
the formation of the hydrogel requires relatively high concentrations
of cb ( 30 mm , 3 wt % ) and bamb ( 2.5 m ) . reported an octaamino amide resorcinarene ( 104 ) acting as a hydrogelator
that starts to aggregate at 0.1 wt % and forms a hydrogel at 1.2 wt
% . sem reveals a unique cell - like micrometer size feature when the
concentration of 104 reaches 5% . the authors suggested
that the fusion of aggregates ( micelles ) leads to the network for
gelation , and found that the walls of the cell - like using
surfactants ( e.g. , 10% n , n , n - trimethylhexadecan-1-ammonium bromide and 5% n-(3-((3-(3-((3-(3-(hexadecyldimethylammonio)-2-hydroxypropoxy)-3-oxopropanoyl)oxy)propoxy)-3-oxopropanoyl)oxy)-2-hydroxypropyl)-n , n - dimethylhexadecan-1-ammonium chloride )
as the cogelators , tian et al . reported the photoisomerization of
two pseudorotaxanes ( consisting of cucurbituril or being composed
of -cyclodextrin and cucurbituril ) in the hydrogels . recently , the same laboratory and co - workers successfully achieved
photoactivated sol gel conversion using -cyclodextrin without using the surfactants . as shown in scheme 18 , banerjee et al . introduced l - tyrosine into
the perylenebisimide core to generate a hydrogelator ( 105 ) that forms stable , semiconducting , photoresponsive , and ph - sensitive
hydrogels . the authors found that the cgc value of 105 is about 0.27 wt % at ph 5 . tem indicates that the self - assembly
of 105 starts at 8.8 m ( 6.3 g / ml ) , which
is exceptionally low . on the basis of the impressive photoswitching
behavior of this hydrogel , the authors suggested that such a high
photoresponse value could lead to soft photodetectors . reported an interesting case that
perylene diimide derivatives with melamine form fluorescent hydrogels . employing the concept of bolaamphiphiles , banerjee
et al . reported another hydrogelator ( 106 ) they found that 106 self - assembles in water at physiological
ph and forms a hydrogel at a cgc of about 1.3 wt % . the authors demonstrated
that the incorporation of graphene oxide or reduced graphene oxide
into the hydrogels enhances the photoresponsiveness of the hydrogel
of 106 . zang et al . also
reported that a perylenebisimide derivative ( 107 ) forms
a hydrogel via ph triggering . the authors found that the addition
of hydrochloric acid in the 4.4 mm ( about 0.2 wt % ) solution of 107 ( in the presence of 26.4 mm triethylamine ) results in
a dark red hydrogel , which agrees with
the formation of a charge transfer complex . george et al . reported an amphiphile
( 108 ) that consists
of coronenebisimide at the core of the molecule and self - assembles
in thf / water through aromatic aromatic interactions . despite
the observation of self - assembled nanotubes by tem acceptor pair ( 109/110 ) , which is able to self - assemble to form
a hydrogel with a cgc of 0.65 wt % . on the basis of uv vis ,
nmr , and xrd , the authors suggested a molecular packing to explain
the formation of the nanofibrils from cylindrical micelles . using a naphthalenediimide as the core and
ethylene glycol as the side chains , ghosh et al . developed a nonionic
bolaamphiphile ( 111 ) that starts to aggregate at 0.05
wt % and forms vesicles . as an electron - deficient core - based bolaamphiphile , 111 forms donor acceptor ( da ) charge - transfer ( ct )
interactions with pyrene , a water - insoluble electron - rich donor . this
interaction ruptures the membrane vesicles to form 1d fibers , thus
producing ct - mediated hydrogels with a cgc of 0.3 wt % . george et al . reported a two - component hydrogel
that consists of an oligo(phenylenevinylene ) derivative ( 112 ) and a perylenebisimide derivative ( 113 ) . these two
molecules form a strong donor and acceptor interaction in water to
result in a hydrogel at a concentration of 0.4 wt % . tem reveals that
aggregation starts at much lower concentration ( 0.012 wt % ) . one impressive
feature is the critical strain of the hydrogel is over 10% , which
is unusual for a supramolecular hydrogel at such a low concentration . zhang et al . developed a bolaamphiphile ( 114 ) consisting of a naphthalenediimide as the rigid core
and a viologen derivative as the hydrophilic head . the authors demonstrated
that the addition of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium
salt ( 115 ) turns the two - dimensional nanosheets of 114 into ultralong nanofibers . however , the hydrogelation
of this interesting two - component system remains to be tested . as shown in scheme 19 , the current record of the smallest hydrogelator likely is a squaric
acid derivative containing a phenyl group ( 116 ) reported
by ohsedo et al . according to the authors ,
the cgc of 116 , in 1 m hcl , is about 1 wt % . a slight
increase of the ph ( from 0 to 1.68 ) shifts the cgc to 10 wt % . sem
and xrd reveal that 116 forms microcrystals as the matrix
of the hydrogel . this microcrystalline morphology explains that it
requires 25 wt % 116 to obtain a reliable rheological
measurement because the weak interactions between the microcrystals
likely only offer a fragile hydrogel that fails to maintain integrity
during oscillatory rheometry . the authors also proposed that , at a
certain concentration , 116 forms fibrils with hydrophilic
porous cavities which have diameters and lengths on the order of micrometers
and submillimeters , respectively . it
would be very interesting to have more insight into the intermolecular
interactions within these fibrils . chi and xu et al . reported an n-(4-carboxyphenyl)trimellitimide ( 117 ) that
forms a gel in a mixed solvent of dmf and water with a cgc of 0.4
wt % . containing microcrystals as its matrix , this hydrogel exhibits
a thermoirreversible property and precipitates upon heating , which
indicates the hydrogelation likely is an entropy - driven process . on the basis of the earlier
works of bis(amino acid ) oxalamides , zinic reported
gelators composed of bis(amino alcohol ) oxalamides . for example , the authors found that 10 mg of ( s , s)-bis(tyrosinol ) oxalamide ( 118 ) forms a metastable
hydrogel in 7.5 ml upon rapid cooling . the structure property relationship study carried out by
feng et al . , in fact , suggests that the carbonyl groups in amino acids
play a critical role in the formation of hydrogels . developed
two c2-symmetric benzene - based hydrogelators
( 119 and 120).119 forms a hydrogel at ph 2 with a cgc of 0.25 wt
% . with ethylene glycol to cap the c - terminal of 119 ,
the authors obtained a more effective hydrogelator ( 120 ) which forms a hydrogel at 0.1 wt % . the authors found unique layered
porous structures in the hydrogel of 119 and fibrous
structures in the hydrogel of 120 . developed
a one - pot ugi reaction from simple starting materials for the synthesis
of tripeptoids as hydrogelators , which lead to gelation in a mixed
solvent of dmso and h2o ( 1:1 ) with cgcs of 0.5 wt % ( 121 ) and 0.2 wt % ( 122 ) . this result also reflects
that the sufficient unsubstituted amide moiety ( conh )
is crucial for the formation of supramolecular hydrogels without a
cosolvent . szymanski and feringa reported
the design of a dichromonyl compound ( 123 ) that bears
an azobenzene photoswitch and forms a hydrogel in its trans conformation
( with a cgc of 1.5 wt % ) . compared to other reported photoswitchable
hydrogelators,123 seems to exhibit much faster kinetics and is able
to form a gel within 1 min upon the cis form of 123 being
irradiated . a hydrogelator composed of two
components usually means that the
two constituents are nongelators by themselves , but together they
can act as a gelator via intermolecular interactions . the two - component
hydrogels have certain benefits over one - component small molecule
hydrogels because the tunability of the individual components allows
more versatile and dynamic reversibility , which may result in greater
diversity in morphology and greater variation in mechanical and optical
properties . moreover , the gelation process and the properties of the
gels can be easily tuned by changing the components or the compositions
of the components or by functional modifications in one of the components ,
which should be beneficial for the applications of these hydrogels . as shown in scheme 20 , tang et al . reported a supramolecular hydrogel consisting of two
types of building blocks , 1,2,4,5-benzenetetracarboxylic acid ( 124 ) and 4-hydroxypyridine ( 128 ) , at a concentration
of 2.5 wt % . on the basis of xrd of powders and other characterization
methods , the authors derived the interactions of the building blocks . one interesting feature is that the hydrogen bonding between the carboxylic
acid and pyridine units is strong enough to allow the fibers to be
drawn from the melted building blocks . the authors used m - hydroxypyridinium ( 129 ) to interact with 124 at a molar ratio of 1:2 to form
hydrogels . one striking feature of these hydrogels is the rare gel - to - crystal
transition , which agrees with microcrystalline particles constituting
the networks of the hydrogels and the hydrogels being metastable . the solved crystal structure also provides confirmation that water
mediates the hydrogen bonding between the pyromellitic acid and the
4-hydroxypyridine . the authors used 126 to interact with 128 , 129 , or 130 to form hydrogels with a cgc of about 0.7 wt % . using a branched gelator consisting of 1,2,4-benzenetricarboxylic
acid ( 125 ) and 4-hydroxypyridine ( 128 ) , the same laboratory shows a two - component hydrogel with a cgc of 4
wt % . by analyzing the single - crystal structures of the complex formed
from 128 and o - phthalic acid , and 125 , the authors suggested that the molecules assemble into
branched fibers via different hydrogen bondings . interestingly , the
melting of the gelators also allows the supramolecular fibers to be
pulled to a length of centimeters . when
the concentration of the gelator ( i.e. , the mixture of 125 and 128 ) is 2.5 wt % and below the cgc , the authors
found that the gelator self - assembles in water to form macrospheres
with diameters of millimeters . tang et al . developed another type of two - component hydrogelator
by mixing 3,3,4.4-benzophenonetetracarboxylic acid
( 127 ) with 128 or 129 at molar
ratios of 1:2 and 1:4 , respectively . the authors reported that the
self - assembled fibers act as the matrixes of the hydrogels . using 2-amino-3-hydroxypyridine ( 131 ) to interact with 1,2,4,5-benzenetetracarboxylic acid ( 124 ) , the authors obtained a hydrogel that exhibits a higher tgel than that of the hydrogel made of 124 and 129 , which likely originates from the
formation of stronger hydrogen bonding enhanced by the o - amino group of 131 . tang
et al . reported the use of ultrasound to promote the mixture of 1,3,5-benzenetricarboxylic
acid ( 126 ) and 4-hydroxypyridine ( 128 ) to
gel water at a concentration of 1.5 wt % . the authors observed that
the width of the nanofibrils in the hydrogels depends on the power
of the ultrasound , and demonstrated
that a higher power of the ultrasound results in nanofibrils with
a smaller fiber width . yang and shen et al . employed microfluidics to generate microgels
made of 1,2,4,5-benzenetetracarboxylic acid and 4-hydroxypyridine . on the basis of thermal analysis , the authors concluded that , due
to the entangled three - dimensional network structures crowded in a
small volume , the supramolecular hydrogel microspheres are more thermally
stable and can immobilize more water molecules . using a similar approach , feng et al . reported a
hydrogel made
of 2,6-pyridinedicarboxylic acid ( 132 ) and 4-hydroxypyridine
( 128 ) at a concentration of 5 wt % . on the basis of a
range of techniques of characterization , the authors suggested that
the interaction between 132 and 128 is highly
directional . reported a
two - component hydrogel of melamine ( 133 ) and gallic acid
( 134 ) . the authors mixed 133 and 134 in different ratios and demonstrated
that hydrogels form at 2 wt % . the optical appearance of the hydrogel
suggests the formation of microcrystalline networks or micrometer - sized
fibrillar bundles , agreeing with sem imaging . the authors also observed
the enhancement of photoluminescence ( pl ) at the gel state , which
is consistent with the enhanced fluorescence in the colloidal state . also used positional
isomers of hydroxybenzoic acid ( 135137 ) to interact with melamine ( 133 ) in a 1:1 molar ratio
to form two - component hydrogels . the
cgc values are 0.5 , 1.0 , and 0.1 wt % for the hydrogels containing 135 , 136 , and 137 , respectively .
on the basis of the upfield shift of the aromatic protons in the gels ,
the authors suggested that the stacking in
the gels follows the order 137 ( para ) > 135 ( ortho ) 136 ( meta ) . the authors found that
the thermal stability , the storage moduli , and the critical strain
of the two - component hydrogels follow the order 137 > 135 > 136 . reported
the thermoreversible gelling systems
consisting of melamine ( 133 ) and three positional isomers
of aminobenzoic acid ( 138140 ) at
a concentration of 3 wt % . the authors found that the gel strengths
at lower temperatures follow the order 140 > 139 > 138 . also used
melamine to interact with maleic acid ( 141 ) to form a
hydrogel at a cgc of 15 wt % , and applied this two - component hydrogel
as a template for making silver nanoparticles . reported a hydrogel formed by mixing melamine
and bis(2-ethylhexyl)phosphoric acid ( 142 ) at a cgc of 6 wt % . one notable feature of
this study is that the authors used a well - established polymer solubility
theory ( the fedors method ) to estimate flory recently , wang and liu et al . reported a series of supramolecular
nanotubes formed by combining melamine - based and l - glutamic acid - based bolaamphiphiles . the authors demonstrated that the ratio of
the components dictates the final nanostructures formed by the self - assembly
of the constituents . as shown in scheme 21 , douliez et al . reported that the ethanolamine
salt of 12-hydroxystearic
acid ( 143 ) forms tubes several tens of micrometers in
length with a temperature - tunable diameter at a concentration of 1
wt % . despite the observation of a gel sol transition via dsc
and sans measurement , it is unknown whether these salts result in
a hydrogel at 1 wt % . to pair melamine
( 133 ) with more complicated molecules , such as lumichrome
( 144 ) , nandi et al . produced thermoreversible hydrogels
that consist of 144 and 133 at a molar ratio
of 3:1 or 1:1 . the hydrogels , formed
at a concentration of 0.2 wt % , are thermoreversible and exhibit higher
intensities of photoluminescence than that of pure 144 . on the basis of the red shift of the emission peak , the authors
suggested a transformation from h - aggregates to j - aggregates . steed et al . reported hydrogelation by a mixed
system comprising two entirely rigid , insoluble , mutually complementary
small organic molecules , melamine ( 133 ) and uric acid
( 145 ) , which act as a planar multifunctional hydrogen
bond donor / acceptor and result in a hydrogel at a cgc of 0.8 wt % . combining molecular dynamic calculation and
the data from c mas nmr spectroscopy and the powder xrd
pattern of the xerogels , the authors proposed plausible intermolecular
interactions to explain the hydrogelation of these two components . zhang et al . found that the
addition of oxoanions ( e.g. , no3 , po4 , atp ,
and so4 ) to a solution of melamine
is able to trigger the formation of hydrogels . the total amount of salts and
melamine needed for gelation is from 3.1 to 10 wt % , apparently depending
on the anions . reported
a hydrogel of melamine ( 133 ) containing 6,7-dimethoxy[1h,3h]quinazoline-2,4-dione , riboflavin , and rhodamine b in a proper proportion . the
authors suggested that this type of gel might find application in
generating white light . hud et al . reported
an elegant design that uses cyanuric acid ( 146 ) and a
modified triaminopyrimidine ( 147 ) to form noncovalent
interactions that result in a hydrogel at ph 7 and a concentration
of 5 mm ( 0.18 wt % ) . the authors suggested that the formation of a
hexameric rosette can serve as a functional architecture to generate
the hydrogels . tang et al . synthesized two
isomeric building units , 4-oxo-4-(2-pyridinylamino)butanoic acid ( 148 ) and 4-oxo-4-(3-pyridinylamino)butanoic acid ( 149 ) . while 148 and 149 form fiber- and treelike
crystals in aqueous solutions , respectively , cooling the aqueous solutions
of their mixtures over a wide range of molar ratios ( 7:1 to 1:3 ) yields
a series of supramolecular hydrogels at a total concentration of 4
wt % . reported
two - component hydrogels consisting
of stearic acid ( 150 ) or eicosanoic acid with di- or
oligomeric amines ( 151 ) . the authors demonstrated that 150 and 151 at a molar ratio of 2:7 , with a total
concentration of 5 wt % , result in a hydrogel containing a three - dimensional
network formed by the self - assembling nanofibers made of 150 and 151 . since two of these hydrogelator salts are
able to crystallize , the authors obtained very useful crystal structures
which provide insights into the molecular packing in the condensed
phase ( figure 4 ) . taking advantage of the interaction of -cyclodextrin
( -cd , 155 ) with a ditopic molecule ( 152 ) having adamantane at one end and a pyromellitic diimide moiety ,
gopidas reported a two - component hydrogel that is stable even with
a 10 m concentration each of 155 and 152 . considering that isothermal titration calorimetry ( itc ) shows the association constant k between 155 and 152 is about 6 10 m , it is unusual ( and probably needs confirmation ) that the hydrogel
forms at such a surprisingly low cgc value ( about 2 mg harada used the interaction between 155 and adamantane to cross - link acrylamide to form self - healing hydrogels . . found that alkyltriphenylphosphonium
bromide 153 , an ionic liquid - based surfactant , and 155 are able to form a supramolecular complex which further
aggregates to result in vesicles in an aqueous solution . the authors
found that the addition of inorganic salts ( e.g. , kcl , nacl , cucl ,
and k2co3 ) induces the formation of sheetlike
hydrogels . using diimidazolium salts
with different alkyl chain lengths to interact with -cd and 155 , danna and noto et al . developed another series
of two - component hydrogels , with cgc values from 1.0 to 4.7 wt % ,
formed by host guest interactions based on cyclodextrins and
cationic imidazoliums 154 . they demonstrated that the nature of the cyclodextrin , the salts ,
and the host : guest ratio are effective tools for tuning the properties
of these two - component hydrogels . ortep diagrams of 150 and 151 with the
atom numbering scheme for the asymmetric unit , and the molecular packing
of 150 and 151 showing the columnar supramolecular
architectures , characterized by a lipophilic exterior and a polar
interior . adapted with permission from ref ( 55 ) . the incorporation of inorganic components
into the hydrogels constitutes
an irreplaceable way to introduce unique properties of the inorganic
components or metal complexes , such as redox , catalytic , conductive ,
photoresponsive , photochemical , and other properties related to coordination
complexes in soft materials . similar to the development of hydrogels
of small organic molecules , the development of metallogels largely begins with organogels . readers who are interested in organogels
of metal complexes are encouraged to consult several
excellent reviews . in this section although the coordination between organic and metal ions is common
in nature , the organic molecules used in these complexes for generating
hydrogelators are largely centered on several functional groups that
serve as the ligands for metal ions . therefore , we arrange these relevant
hydrogelators according to the following classifications : ( i ) carboxylic
groups as the ligands , ( ii ) ligands containing nitrogen as electron
donors to a metal ion , ( iii ) binding via phosphate groups , ( iv ) ligands
comprised of thiol groups , and ( v ) others . although some amphiphiles with carboxylic
groups themselves self - assemble in water to form nanostructures as
the matrixes of hydrogels , organic inorganic hybrid hydrogels
still have attracted considerable interest due to the specific functions
conferred by the metal ion or inorganic elements . as shown in scheme 22 , kogiso et al . reported that a dicarboxylic valylvaline bolaamphiphile
( 156 ) self - assembles in water to form nanofibers in the
presence of divalent transition - metal cations ( e.g. , cu and zn ) at a concentration of 1.6 wt % . the resulting
nanofibers have widths of 1520 nm and lengths of several micrometers . suzuki et al . designed molecules 157 and 158 containing a carboxylic group , which
form a thermally sensitive hydrogel . the addition of the inorganic
salts ( e.g. , k , ca ) to the hydrogel affords
improved mechanical strength at a cgc concentration of about 1.2 wt
% . on the bsais of this result , they also developed an l - lysine
derivative ( 159 ) and its alkali - metal salts . compound 159 is insoluble in water , but 159 with an alkali
metal is readily water soluble . surprisingly , the mixtures
of these compounds ( e.g. , the mixture of 159 and its
lithium salt ) form hydrogels ( at cgc values from 0.5 to 1.7 wt % )
via hydrogen - bonding and van der waals interactions . while 159 forms spherical micelles , the mixtures of the hydrogelators self - assemble
to form nanofibers . shen and zhang et al . reported an intriguing example in which the chirality of the ligand
controls supramolecular hydrogelation via the coordination of phenylalanine
( phe , 160 ) to cu(ii ) at a cgc of 0.35 wt % . according
to the authors , a decrease of the enantiomer excess of ligand l - phe ( or d - phe ) weakens the gelation ability of the
phe ,
the authors suggested that this hydrogelator may open up a new window
for developing promising chiral sensing and recognition platforms . the photoluminescence
of lanthanide ions has constantly attracted
research interest because of the long
lifetimes , narrow - band emission , and robust photochemical stability . demonstrated a lanthanide - based
luminescent hydrogel by mixing sodium cholate ( 85 ) and
europium acetate , at a concentration of about 0.7 wt % , with the addition
of pyrene at extremely low concentration ( e.g. , 10 m ) as a sensitizer . in addition , the authors also synthesized nanoparticles
( metal sulfides ) in a calcium cholate hydrogel using the matrixes
of the hydrogel as the template . huang
et al . used 85 as the ligand to bind
lanthanide to generate a superhydrogelator . at 0.04
wt % , 85 forms a hydrogel in which the hierarchical nanostructures
are thermoresponsive ( e.g. , twisted nanohelices and nanotubes at low
temperature ( 4 c ) and untwisted ribbons at a higher temperature
( 50 c ) ) . huang et al . also found that the color of the emission
of a lanthanide cholate hydrogel depends on different lanthanide
ions or codoping ions . utilizing the
iminodiacetic acid ( 161 ) as a ligand to coordinate with
lanthanides , alves et al . prepared a
series of hydrogels with the lowest cgc value at 0.04 wt % . banerjee
et al . designed a series of tyrosine - based
amphiphiles ( 162 ) which bind with ni ion
selectively and result in hydrogels at cgc values of 0.78 , 0.75 , and
0.63 wt % when the chain length is 10 , 12 , and 14 , respectively . yang
et al . reported a supramolecular hydrogel
based on n , n - dibenzoyl - l - cystine ( 1 ) with a cgc of 0.2 wt % . owing to the interaction
between eu(iii ) ions and the hydrogelators , immobilized eu in the
hydrogel exhibits enhanced luminescence . due to its versatile catalytic properties , palladium is of great
interest in synthetic chemistry . escuder
et al . reported a supramolecular hydrogel
( 4 ) containing pyridine which forms a hydrogel at 0.5
wt % . also acting as an organogelator , 4 binds with pd(ii )
to create a functional material to catalyze the oxidation of benzyl
alcohol , similar to the case reported by xu et al . as shown in scheme 23 , takaya and nakamura et al . designed a supramolecular gel ( 163 ) containing pd(ii )
for catalysis . the authors made the xerogel of 163 and
used it to act as a highly efficient catalyst for the intramolecular
addition cyclization of alkynoic acid in water . designed a molecule of 1,4-bis(dimethyl-4-pyridylsilyl)benzene
( 164 ) , which binds with ( tmeda)pd(no3)2 ( tmeda = n , n , n,n-tetramethylethylenediamine )
to afford a hydrogel containing 98.5% water below 2 c . according
to the authors , one interesting feature of these hydrogels is the
formation of dynamic catenated cyclotrimers in water . andrew et al . reported that the addition of water to the
mixture of 1h-5-(2-pyridyl)tetrazole ( 165 ) with lacl3 results in a hydrogel . surprised by this
result , the authors also obtained the crystal structure of the complex ,
which appears to be consistent with the microcrystalline nature of
the hydrogel . considering that the understanding of the relationship
between
molecular structure and gelation ability is still poor and proposed an approach that selects the molecules according to the
intermolecular interaction revealed by the cambridge structural database
( csd ) for the development of hydrogels . on the basis of this principle , the authors selected 166 , which specifically binds with
hg(oac)2 to afford a gel in 90:10 meoh / h2o with
a cgc value at 1.6 wt % . mal and rissanen et al . also found that the ligand of 40-[4-(4-aminophenyl)phenyl]-2,2:6,2-terpyridine
( 167 ) selectively binds with hg to afford
a hydrogel . the interaction between benzo-18-crown-6 ether and the
ammonium group on 167 is able to disrupt the hydrogelation ,
which can be partially recovered by the addition of k to
bind competitively with the crown ether . employing the ligand 5-(mercaptomethyl)tetrazole
( 168 ) as a surface coating to stabilize the cdte nanocrystal ,
voitekhovich and eychmuller et al . this work illustrated
an interesting way to use colloids directly as part of 3d networks
for generating hydrogels . utilizing n , n-bis(3-pyridyl)butylenebisurea
( 169 ) to coordinate with cu , dastidar et
al . made a gel in a dmso / h2o or dmf / h2o mixture with cgcs of 810 wt % . em
reveals that the matrixes of these gels are largely microcrystalline .
as shown in scheme 24 , macgillivray et al . reported a hydrogel
via coordination of copper with rctt-1,2-bis(3-pyridyl)-3,4-bis(4-pyridyl)cyclobutane
( 170 ) . being composed of nanoscale metal organic
particles , this hydrogel exhibits thixotropic properties with a yield
value of 8.33 pa . also prepared a thixotropic hydrogel via simple mixing of 171 and cu(ii ) at a concentration of 0.25 wt % . furthermore ,
they also developed a photoswitchable metallohydrogel by utilizing
the photoresponsive 2,2-azopyridine as the ligand . in addition , bhattacharya et al . also prepared
a thixotropic hydrogel based on the interaction of pyridylenevinylene
and copper(ii ) . , a terpyridylplatinum(ii ) complex with biphenylacetylide
ligands ( 172 ) self - assembles to form nanofibers with
diameters of 2040 nm and results in a hydrogel with a cgc
of 3.7 wt % . yu et al . designed a pair
of decapeptides , formyl - efeaeaeaew - carboxyl and formyl - ofoaoaoaow - amide
( o = ornithine ) , which are able to form a hydrogel at ph 7 . moreover ,
the peptide conjugated with a gd(iii ) chelate ( 173 ) speeds
the gelation process and increases the cross - sectional area of the
peptide fibers . further study found that the conjugates 173 are incorporated into the peptide fibers and aggregate toward the
center of the peptide fibers . one notable feature of this study is
that the authors correlated saxs and t2-weight magnetic resonance
imaging ( mri ) to provide insights into the self - assembly process . zhang and ye et al . prepared a metallogel
in water by using 4,6-bis(2-pyridyl)-1,3,5-triazin-2-ol ( 174 ) to coordinate with copper acetate at a molar ratio of 4 ( cu : l ,
5.6 wt % ) . it is interesting that the hydrogel is stable at room and
high temperature ( e.g. , 60 c ) , but flows upon cooling to 4 c . this kind of lcst behavior is uncommon for supramolecular metallohydrogels . upon mixing melamine ( 133 ) with zinc(ii ) orotate at
a 1:1 molar ratio , nandi et al . developed
a bicomponent hydrogel at a cgc of 0.7 wt % . employing
a dendritic ligand ( 175 ) to coordinate diplatinum(ii ) ,
yang et al . developed a gel in a mixed
solvent of acetone and water ( 5:3 , v / v ) with a cgc of 0.23 wt % . upon addition of
-cyclodextrin to the solution of the pd(ii ) complex with a
bipyridinium ligand ( 176 ) , osakada et al . prepared a hydrogel with a tgel of 47 c . the authors suggested that the complex
self - assembles to form polyrotaxane - like fibrous architectures and
further aggregates to afford nanoparticles with a size around 2040
nm to trap the water and to result in the hydrogel . fernandez et al . designed and synthesized a ligand ( 177 ) consisting of a pyridyl group , an oligo(phenyleneethynylene ) motif ,
and three ethylene glycol chains . 177 coordinates with
pt(ii ) metal ions to result in a hydrogel . on the basis of roesy ( rotating - frame
overhauser effect spectroscopy ) nmr hydrogen
bonding drive the complex to form a hydrogel at a concentration of
1.4 wt % . the most impressive result in this work is correlation of
uv / vis spectroscopy , nmr spectroscopy , and x - ray crystal structure
analysis to infer the molecular arrangement in the hydrogel . diaz
and banerjee et al . reported a complex
of l-3-methyl-2-[(pyridin-4-ylmethyl)amino]butanoic acid
( 178 ) and zn(ii ) to form a metallohydrogel with a cgc
value of 8.4 wt % ( scheme 25 ) . according to the authors , this hydrogel undergoes a gel sol
transition upon application of different stimuli ( e.g. , edta , ammonia
solution , trifluoroacetic acid , sodium sulfide , shaking , or heating ) ,
which likely originates from the unique amphoteric property of zinc
ions . designed
2-aminothiazole and its derivatives ( 179 ) that bind with
mercury acetate to result in hydrogels with thixotropic behavior . one interesting feature of this work is the dependency of the thixotropic
property on the substitution groups in the c-4 and c-5 positions of
the aminothiazole . have
demonstrated an intriguing case of a highly selective anion - responsive
supramolecular hydrogel , based on silver(i ) glutathione ( gsh ) ( 180 ) , with a cgc value of 0.20 wt % . compared with other anions
( f , cl , br , and h2po4 ) sol transition , likely due
to the higher affinity of iodide to silver ion than that of other
halogen anions . used
a drug , n - acetyl - l - cysteine ( nac , 181 ) , to generate the supramolecular
hydrogel ( 0.82 wt % , 50 mm ) in the presence of au(iii ) , ag(i ) , and
cu(ii ) salts . although the hydrogelation of these metal complexes
occurs at a relatively low ph ( < 4 ) , this work still provides new
insights into creating supramolecular metallogels . reported a hybrid hydrogel based on the complex
( 182 ) of l - cysteine and silver nitrate ( scheme 26 ) . via sulfur silver
bonds , complex 182 self - assembles to form nanofibers
and results in hydrogelation at a rather low concentration ( 0.01 wt
% ) . designed
two n - terminal - capped tripeptides , ac - rgc and ac - rcg , which have an
isoelectric point close to 7 . because these two peptides contain the
cysteine residue , they form hydrogels upon the introduction of au(i )
to ac - rgc and ag(i ) to ac - rcg , at final peptide concentrations of
about 3 wt % . arachchige et
al . reported a highly efficient method
to prepare a monolithic ag hydrogel by using a large amount of oxidant
c(no2)4 to remove the surface thiolate in ag
nanoshells . moreover , the resulting aerogel exhibits extremely high
surface areas ( 40160 m / g ) and interconnected mesoporous
structures . pal et al . reported a cu(i ) metallogel
at a concentration of 0.15 wt % via simple mixing of cucl2 and thiourea ( 183 ) . the coordination of cu(i)thiourea
and extensive hydrogen - bonding interactions result in a hydrogel which
is redox responsive , and the oxidation of cu(i ) to cu(ii ) by fecl3 leads to a gel moreover , the metallogel
shows high selectivity for picric acid , and the addition of picric
acid causes a color change of the gel from white to yellow . yeh et al . developed a facile method to prepare hydrogel - like
gdpo4h2o nanorods . the authors reported
that the nanorods are cell compatible . on the basis of the high affinity
between phosphate groups and certain metal ions , takahara et al . reported an imogolite - based hydrogel which
is able to immobilize an enzyme containing phosphoric groups . the
authors found the enzyme is easily recovered from the reaction system
and reused . patil and mann et al . reported
that cerium oxide nanoparticles act as a catalyst to dephosphorylate
fmoc - tyr - p ( 14 ) , thus resulting in hybrid supramolecular
hydrogels . the most interesting part of this study is the observation
of maltese cross - patterns ( spherulites ) or nematic birefringence of
the hydrogels , which suggests directional orders . as shown in scheme 27 , kasuga and co - workers have prepared a metaphosphate hydrogel ( 184 ) via hydration
of the metaphosphate glass powders . the authors have also performed
extensive studies on the conductive properties of this viscous gel . discharge capability , and shows a high electrical conductivity
of 5 ms cm at room temperature because of proton
hopping between p the authors
suggested that this hydrogel promises an alternative electrolyte . besides the electron donors
discussed above , other types of nucleophiles may coordinate with metal
centers to generate hydrogelators . as shown in scheme 28 , kulkarni et al . reported that copper(ii ) binds with inositol and 2,2-bipyridine
to form a trinuclear complex ( 185 ) . the self - assembly
of the complex affords a supramolecular hydrogel at ph 12.4 via inter-
and intramolecular stacking between the bipyridyl
groups . in addition , rodriguez et al . designed a discrete molecule [ ( pta)au(4-pyridylethynyl ) ] ( pta
= 1,3,5-triaza-7-phosphaadamantane ) ( 186 ) ; this complex
forms a hydrogel immediately in water even at a concentration as low
as 0.015 wt % . designed a
dinuclear platinum(ii ) complex ( 187 ) having a [ pt(6-phenyl-2,2-bipyridyl)((2,6-dimethylphenyl)isocyanide ) ]
motif connected by an oligo(oxyethylene ) chain which is able to form
hydrogels driven by intra- and intermolecular ptpt
and interactions . moreover , 187 acts as a supramolecular cross - linker to trigger the gelation of
[ pt(6-phenyl-2,2-bipyridyl)((2,6-dimethylphenyl)isocyanide ) ]
at a molar ratio of 1:30 . 188 is able to afford a hydrogel with a cgc of 0.6 wt % . upon the addition
of tb , the resulting hydrogel exhibits enhanced green
luminescence due to the coordination between the gelator and the metal
ion . lima and rodriguez et al . developed
a hydrogelator of [ au{7-(prop-2-yn-1-yloxy)-1-benzopyran-2-one}(dapta ) ]
( dapta = 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane )
( 189 ) which self - assembles to form long luminescent fibers
and induces a hydrogel with a cgc of 0.01 wt % . other cases in this
category include the polyoxometalate ( pom ) reported by bonchio et
al . as well as others , which forms a gel at a specific condition ( e.g. , 90:10
meoh / h2o ) . besides the small organic
and inorganic hydrogelators , the hydrogelators
derived from amino acids or peptides are of great importance due to
their inherent biocompatibility and biological activities . the utilization
of amino acids as the molecular building blocks of hydrogelators provides
unique opportunities for generating supramolecular hydrogels that
are not readily available from traditional organic / inorganic molecules . peptide - based hydrogelators self - assemble via various noncovalent
interactions , including hydrogen bonding , electrostatic interactions ,
aromatic aromatic interactions , or hydrophobic interactions . these intermolecular interactions lead to the
formation of organized supramolecular structures that entrap water
under appropriate stimuli . in addition , due to the well - established
protocol of solid - phase peptide synthesis ( spps ) , it is easy to produce and to chemically and biologically
modify peptides in large quantities and at a relatively fast turnaround .
these merits have attracted considerable research activities focusing
on the exploration of supramolecular hydrogels made of peptides . in
this section we give a summary of different types of peptide - based
hydrogelators in the past decade . we hope that the introduction of
the recent progress will contribute to the understanding of the relationship
between the peptide structures , the resulting conformation of the
hydrogelators , and the morphologies of the matrixes of the supramolecular
hydrogels . by presenting an updated report on peptidic hydrogelators , we intend to provide molecular information for researchers to uncover
the hypotheses for the rational design of supramolecular hydrogelators . we start with a brief description of the supramolecular hydrogels
formed by amino acid derivatives . strictly speaking , amino acid derivatives
are not exactly peptides , because the shortest peptides
are dipeptides . however , because the building blocks of peptides are
amino acids , amino acid - based hydrogelators share considerable similarities
with peptide - based hydrogelators , making them relevant to be discussed
in this section . we categorize the amino acid derivatives into three
groups ( i.e. , those containing an alkyl chain , those that are salt - based ,
and those containing an aryl group ( i.e. , ferrocenyl , fluorenyl , naphthyl ,
pyrenyl , etc . ) ) and discuss several representative molecules in each
group . then we introduce the hydrogels formed by native peptides ( consisting
of amino acids only and without any chemical modification ) . we summarize
most of the peptidic hydrogelators according to their molecular size
( table 1 ) . in such
an arrangement , we give an overall review of the design strategies
of peptide - based hydrogelators and highlight the notable advances
made recently . after identifying key features of the native peptides , we discuss the chemically / biologically modified peptides ( i.e. , peptide
derivatives ) that are the representative hydrogelators from six subgroups . the six subgroups are the peptides with terminal acetylation ( or formylation )
and amidation , peptide derivatives containing an alkyl chain , peptide
derivatives containing an aromatic group ( e.g. , fluorene - based , naphthalene - based ,
and pyrene - based peptide derivatives ) , peptide derivatives containing
a photosensitive group , bolaamphiphiles , and dendritic peptides . most amino acid derivatives containing alkyl chains
are conventional amphiphiles , consisting of a polar head group and
one or two hydrophobic tails . the amino
acids make the polar head , while the alkyl chains are the hydrophobic
tails with many variations in the length and flexibility . these molecules
are quite common in nature and are well - known to form a variety of
nanostructures in water . for example , bhattacharya et al . reported
the gelation of enantiomerically pure n - alkanoyl - l - alanine amphiphiles in a series of organic solvents . recently , dey et al . designed and developed
a series of amino acid - based gelators , n-(n - alkylcarbamoyl)-l - alanine ( e.g. , 190 ) ( scheme 29 ) , all
of which form stable organogels in solvents in the presence of water
at a concentration of 1% ( w / v ) . the authors concluded that water - mediated
intermolecular hydrogen bonds between amphiphiles result in the formation
of supramolecular self - assemblies . the urea linkage of this hydrogelator ,
as the simplest motif that provides two hydrogen bond donors and one
acceptor , is a common and useful hydrogen - bonding building block for
supramolecular hydrogels . similarly , n-[4-(n - alkyloxy)benzoyl]-l - histidine ( 191 ) with a hydrocarbon chain , reported
by the same group , affords thermoreversible hydrogels in a wide range
of ph at room temperature . they also reported that the cgc value of 191 at different ph values varies from 2.5% to 5.0% ( w / v ) . liu et al . reported a multiple - h - bonding amphiphile , n - stearoyl - l - glutaminc acid ( c18-glu , 192 ) , which forms disk- and fiberlike nanostructures in both
hydrophilic and hydrophobic environments , respectively , due to intra-
and intermolecular hydrogen bonds . maruyama
et al . reported that the attachment of a carbohydrate motif to the
n - terminal of the alkyl chain amino acid structure affords
another hydrogelator , 193 , which is able to harden a
broad range of solvents , in addition to gel water over a wide ph range
at gelation concentrations of 0.12 wt % . using a similar synthetic strategy , cai et al . interestingly , l - glutamic acid , through both - and
-aminoalkylation , affords a double - long - chain - terminated hydrogelator , 194 . this amphiphile disperses in various solvents ranging
from water to hexane , and forms nanofibrils or aggregates . it also
has a relatively good solubility , which results in a cgc value higher
than 20 mm . designed a zwitterionic
hydrogelator of sodium n-(n - dodecyl-2-aminoethanoyl)-l - valinate ( 195 ) that is able to afford a supramolecular
hydrogel with a cgc of 0.05 wt % in the presence of sodium dodecyl
sulfate ( sds ) . in addition , the resulting hydrogel is thermoreversible
and sensitive to a change of ph . while they augment the self - assembly ability of hydrogelators , long
alkyl chains or aromatic rings are the hydrophobic segments that decrease
the solubility of the hydrogelators in water . one of the simplest
strategies to achieve relatively better solubility required for forming
a hydrogel is to introduce charge(s ) into the hydrogelators . reported the synthesis and properties of l - lysine alkali - metal salt 159 . in water , 159 forms a supramolecular hydrogel , within which the self - assembled
nanofibers entangle to result in a 3d network.158 is another l - lysine - based hydrogelator ,
with a pyridinium bromide salt , developed by suzuki and liu et al . it forms a hydrogel at a wide range of ph
and has a cgc as low as 0.3 wt % at neutral ph . developed
two chiral l - phenylalanine - based salts ( 196 and 197(530 ) ) as hydrogelators . as shown in scheme 30 , 196 has an ammonium salt , and 197 has
a pyridinium salt . they both self - assemble in aqueous media at different
ph values to form supramolecular hydrogels with cgc values of 1.22.0
wt % ( for 196 ) and < 1.0 wt % ( for 197 ) . although ionic force and the overlap between alkyl
chains offer interfiber interactions , they are intrinsically weak
and inefficient . aromatic interaction , extensively
used by nature as a stabilizing force for generating ordered structures
in proteins , not only exceeds the van
der waals interaction between the alkyl chains , but also leads to
more predictable and efficient self - assembly of the molecules in the
aqueous phase for the formation of mechanically strong and stable
supramolecular hydrogels due to its compact volume . thus , aryl motifs
( e.g. , ferrocenyl , fluorenyl , naphthyl , and pyrenyl ) and phenylalanine ( or its derivatives ) become prevalent and effective
residues used in generating supramolecular hydrogels . as shown
in scheme 31 , zhang
et al . serendipitously discovered that ferrocenoylphenylalanine ( 198 ) aggregates in water via a rapid self - assembly to form
a stable hydrogel which exhibits a sharp phase transition in response
to multiple stimuli ( i.e. , oxidation reduction reaction , guest host
interaction , and ph changes ) . though being exceptionally simple and
small , this hydrogelator is electrochemically active , which illustrates
a remarkably facile approach to introduce an organometallic moiety
into a supramolecular hydrogel . inspired
by this work , he et al . also investigated the self - assembly properties
of ferrocenoyldiphenylalanine . since
the serendipitous observation of an effective hydrogelator made of
[ ( fluorenylmethyl)oxy]carbonyl ( fmoc)-protected dialanine , fmoc , widely used as the protecting group in
spps , has become a popular n - terminal capping motif in peptidic hydrogelators
because the fmoc - protected amino acids or peptides are commercially
available or relatively easy to make . the intensive use of fmoc in
making supramolecular hydrogelators also originates from its advantages ,
such as ease of being incorporated into peptide / amino acid derivatives
( i.e. , derived directly from spps ) , low cost , and excellent capacity
to promote self - assembly . changing the ph is a common way to make
supramolecular hydrogels from fmoc - protected amino acids . for example ,
nilsson et al . reported supramolecular hydrogels generated by fluorinated
fmoc - phenylalanine derivatives . suspensions of fmoc - pentafluorophenylalanine
( 199 ) in water undergo rapid self - assembly , which gives
rise to rigid supramolecular hydrogels even at concentrations as low
as 0.1 wt % . to better understand the electronic and steric contributions
of the benzyl side chain to the hydrophobic and aromatic aromatic
interactions during self - assembly , the authors also changed the halogen
substituents ( i.e. , f , cl , br ) or the position of substitution ( e.g. ,
ortho , meta , or para ) on the aromatic side chain of fmoc - phe ( 200 ) . the authors reported that the position of the halogen
and the size of the halogen atom significantly affect the rate of
self - assembly and the bulk rheological properties of the resulting
hydrogels . for example , the rate of self - assembly increases in the
order ortho < meta < para , and the hydrogel rigidity increases
in the order br < cl < f. xu et al . also reported
the first case of the use of fmoc - amino
acid derivatives to generate multicomponent supramolecular hydrogels . equal moles of 14 and 201 and 2 equiv of na2co3 afford a clear hydrogel after heating and addition of alkaline phosphatase . similarly , mixing and then heating 1 equiv
of 200 , 1 equiv of 201 , and 2 equiv of na2co3 in water eventually leads to the formation
of a clear solution , which turns into a hydrogel after being cooled
back to room temperature . the subsequent hydrogel exhibits exceptionally
high storage moduli and a rapid recovery of its original mechanical
strength after removal of the external forces . in another study , xu et al . prepared a supramolecular hydrogel by
mixing 200 and fmoc - lys ( 202 ) . the resulting
hydrogel acts as a reaction medium to mimick a bioluminescence environment
with a more than 10-fold enhanced quantum yield of chemiluminescence . banerjee et al . also reported that supramolecular
hydrogels were formed by coassembling two oppositely charged fmoc - amino
acids ( i.e. , 202 and fmoc - glu , 203 ) with
a cgc value of 50 mm ( 1.8 wt % ) . the
authors reported that molecular chirality is translated into the supramolecular
helicity and the handedness of these fibers because the latter depends
on the corresponding molecular chirality of the two - component system . fluorescent spectroscopy is a useful tool to study the interactions
among the fluorophore - bearing hydrogelators in the gel state . besides
providing strong and efficient aromatic aromatic interaction ,
pyrene is a suitable motif for fluorescent spectroscopy as its emission
changes due to aromatic aromatic interaction ( e.g. , the formation
of an excimer ) . thus , pyrene has become
a popular moiety to be incorporated into supramolecular hydrogelators . banerjee et al . designed and developed a hydrogelator ( 204 ) consisting of a single amino acid and a pyrene group . 204 forms hydrogels in a wide range of ph ( 7.514 ) in the aqueous
phase . with an increase of the ph , a distinct morphological change
of the nanofibers of the hydrogel occurs , and the morphology transforms
from helical to tapelike . although the
aromatic aromatic interaction between two phenyl groups is
weaker than that between two fluorenyl or two pyrenyl groups , phenylalanine
is often incorporated into peptides for increasing their ability to
self - assemble . kim et al . reported a biotin - based small molecule ( biotin - l - phe , 205 ) that displays remarkable gelation properties
in aqueous media , including buffer solutions with different ph values . although biotin - based organogelators have been
reported , the study of supramolecular
hydrogelators of biotin is rare . bile acids are among the most
investigated rigid molecular motifs
due to their ability to self - organize into many different supramolecular
structures . reported the synthesis and self - assembly
behavior of cholic acid - connected amino acid derivatives ( e.g. , 206 ) . the molecules of 206 aggregate in globular micelles at high ph , whereas they
form tubular superstructures under acidic conditions and result in
hydrogels at a concentration of 18 mm ( 1.2 wt % ) . these two representative
cases imply that the conjugation of l - phenylalanine to natural
products ( i.e. , biotin ( 205 ) and cholic acid ( 206 ) ) likely will result in a rich class of supramolecular hydrogelators . bolaamphiphiles
are a class of hydrogelators composed of two terminal
hydrophilic groups linked by a hydrophobic backbone / chain . variation
of the structures of the head group and the linker
of bolaamphiphiles has great influence on their ability to aggregate
and the properties of the hydrogels . on the basis of l - valine-
and l - proline - based peptide bolaamphiphiles , a class of efficient
organogelators , miravet et al . designed and developed one bolaform
amino acid derivative ( 4 ) which self - assembles in water
to form hydrogels . the cgcs for these small molecules range from 4 to 26 mm ( from 0.18
to 1.2 wt % ) . as shown in scheme 32 , liu et al . reported another series of bolaamphiphiles with head
groups made of l - glutamic acid and a hybrid linker composed
of two rigid benzene rings and a butyl segment ( 207 ) . for example , at ph 3 , these molecules form a hydrogel
in water at a concentration as low as 0.5 wt % ( cgc ) ; at ph 12 , these
hydrogelators then form vesicle - like aggregates . as
a consequence of evolution ,
proteins ( e.g. , insulin ) form hydrogels ,
implying that peptides , as the component of proteins , should be able
to form supramolecular hydrogels . for example , namba et al . have demonstrated that the terminal regions
of flagellin ( consisting of 25 amino acids ) form hydrogels at concentrations
> 15 mg / ml . surprisingly , much smaller
peptides , such as dipeptides , are able
to self - assemble in water to generate supramolecular hydrogels . besides
the easy synthesis of peptides , several seminal works on peptide - based
hydrogels at the beginning of the century have stimulated
the development of supramolecular hydrogels based on small peptides .
in this section on supramolecular hydrogels formed by peptides , we
put the emphasis on small peptides ( 20 or less amino acid residues )
because there are already several excellent reviews on medium - sized
oligopeptides and their derivatives . we arrange the discussion of peptide - based hydrogelators according
to the length of the peptides and start with the shortest one . the report by gazit et al . on the nanofibers formed by the self - assembly
of diphenylalanine ( ff , 208 ) has stimulated intensive investigations of the self - assembly of
small peptides , including dipeptides . ventura et al . examined the
dipeptides ile - phe ( if , 209 ) and val - phe ( vf , 210 ) and found that 209 , at 1.5 wt % , forms a hydrogel
consisting of persistent nanofibers with a diameter of about 55 nm . unlike 209 , 210 can not self - assemble . besides the fact
that 209 is probably the smallest hydrogelator , it is
worth noting that the difference of one methyl group between 210 and 209 has a profound impact on their capabilities
to self - assemble . the authors also proposed
the self - assembled structure of 209 ( figure 5 ) , which is quite similar to
the recent structure of a140 determined
by solid - state nmr . since the report
of gazit et al . on diphenylalanine , several other groups have explored
the properties of 208 in great detail . have
demonstrated that 208 self - assembles to form nanowires
and nanotubes with high thermal , chemical , and proteolytic stability . qi et al . reasoned the use of hexafluoroisopropyl
alcohol ( hfip ) to dissolve 208 would introduce a solvent
effect , so they used ch3cn they found that cooling the solution
of 208 results in microtubes and the evaporation of the
solution of 208 on a glass surface affords nanofibers . krishnan et al . also
investigated the effect of hfip and found that the amount of hfip
significantly affects the morphologies of the nanotubes formed by
the self - assembly of 208 . considering the morphology of nanoscale assemblies dictates their
interactions with proteins , this work , indeed ,
provided a useful insight for understanding the discrepancy in the reports on the cytotoxicities of a
because hfip is the most common solvent used to dissolve commercially
available a. molecular model of 209 self - assembled structures . the model is based on the crystal x - ray structure of the diphenylalanine
peptide . copyright 2007 biophysical
society . as shown in scheme 33 , feng et al . reported that
an innovative cyclodipeptide ( 211 ) , made of tyrosine
and lysine , acts as a hydrogelator and forms
a hydrogel at a cgc of 0.6 wt % . the
hydrogel , having a relatively low mechanical strength , only forms
during cooling with the assistance of ultrasound for breaking up the
intermolecular interactions . interestingly , after the attachment of
an alkyl chain at the -amino group of 211 , the
cgc in water becomes 1.5 wt % . due to the difficulty of synthesis ,
reports on the self - assembly of cyclic peptides are rather scarce . zhao and dory et al . reported a cyclic tripeptide ( 212 ) forming micrometer - sized tubes by self - assembly in a liquid crystal .
on the basis of molecular dynamics , the authors suggested a hierarchical
hexagonal assembly to generate the observed hollow macrotubes of 212 . in a similar study , kimura
et al . reported the self - assembly of a unique tri--peptide
( 213 ) that consists of two -glucosamino acids
and one trans-2-aminocyclohexanecarboxylic acid . 213 self - assembles in a mixture of formic acid and water to
form nanofibers at a concentration of about 0.006 wt % . it would be interesting to obtain more information
about the biological properties of 213 . systematically investigated
tripeptides containing
one d - amino acid residue at the n - terminal and found that
chirality plays a critical role in the morphology of the nanofiber
networks of the resulting hydrogels . the authors observed a very interesting
and unexpected result by modulating the chirality of the tripeptides :
while the tripeptides vff and ffv fail to self - assemble at physiological
ph , vff ( 214 ) and ffv ( 215 ) are able to self - assemble at a concentration of about
0.67 wt % . both 214 and 215 self - assemble
to form distinct nanostructures : the former results in nanotapes and
the latter twisted nanofibers . the authors
also observed similar behavior for lff ( 216 ) , which forms a hydrogel at 0.67 wt % . while the self - assembly of 216 results in mainly nanofiber networks , the self - assembly
of lff ( 217 ) is rather polymorphic and fails to form
a self - supporting hydrogel . cao et al .
examined the self - assembly of ffy ( 218 ) and fmoc - ffy
( 219 ) for photo - cross - linking after the self - assembly . they found 218 self - assembles to form nanoparticles which
turn into nanorods after photo - cross - linking tyrosine residues . 219 forms a weak hydrogel at 0.1 wt % which consists of nanofibers
that turn into nanoparticles after photo - cross - linking . this result
provides a useful insight into the effect of cross - linking to the
self - assembled nanostructures . moreover ,
ulijn and tuttle et al . have presented a coarse - grain molecular dynamics
( md ) protocol for screening tripeptides for their aggregation behavior
and applied this to a set of 8000 gene - encoded tripeptides . it would be highly valuable to verify if these
self - assembly tripeptides exist endogenously and at concentrations
high enough to self - assemble . miller et al . designed a tetrapeptide
( fefk , 220 )
consisting of two hydrophobic residues , one positive residue , and
one negative residue . while 220 itself hardly forms a
hydrogel , 220 can serve as a substrate for thermolysin
to undergo reverse hydrolysis for enzymatic hydrogelation by the formation
of octapeptides as the final products . although the hydrogelation
concentration of the tetrapeptide concentration is 10 wt % , this unique
reversible hydrolysis of the amide bond represents an evolutionary
approach to screen hydrogelators from a dynamic library of peptides . further studied this tetrapeptide
for enzymatic hydrogelation and reported that the initial concentration
of the tetrapeptide dictates the morphology of the peptide nanofiber
network , and the concentration of the enzyme has little effect on
the final morphology of the nanostructures formed by the self - assembly
of the peptides . reported
the self - assembly of a tetrapeptide , pwwp ( 221 ) , which
forms vesicular microstructures . the author reported that the vesicles
can entrap dye molecules and the addition of kcl ( 0.25 mm ) disrupts
the vesicles . examined the
effect of temperature on the self - assembly of rwdw ( 222 ) and found that an increase of temperature produces nanofibers with
smaller diameters . according to the
afm experiment , the authors also found that 222 self - assembles
to form a dense network of entangled nanofibers at 15 c , but
assembles into sparse globular and fibrillar structures at 35 c . used an array of techniques to investigate the self - assembly
of the pentapeptide klvff ( 223 ) , which is the core motif
of -amyloid peptide . the authors reported that 223 forms a hydrogel at 2.5 wt % in water , but at 3.0 wt % in phophate - buffered
saline ( pbs ) . the nanofiber dimensions determined by cryo - tem and
saxs confirm that fibrils ( 10 nm in diameter ) are formed in aqueous
solution , which helps to address some contradictions in the literature ,
as the authors pointed out . on the basis
of the motif aaklvff , hamley et al . replaced phenylalanine with -2-thienylalanine
( 2-thi ) to generate ( 224 starts to self - assemble at a concentration of 0.03 wt
% and forms a hydrogel in water at 2.0 wt % . although the self - assembly
of 224 occurs as designed , the conductivity of the nanofibrils
has yet to be demonstrated . doyle
et al . studied the kinetics of the hydrogelation of an octapeptide
( fkfefkfe , 225 ) and found that 225 forms a hydrogel at about 0.1 wt % . one notable feature of the hydrogelation
of 225 is that an increase of the ph from 3.5 to 4.0
speeds the formation of the nanofiber networks by almost 2 orders
of magnitude , from hours to minutes , but the gelation follows the
same mechanism . the authors proposed a simple model of self - assembly
based on derjaguin landau verwey overbeek ( dlvo )
theory . this detail kinetic study offers a useful
insight for the design of fast responsive hydrogelators . investigated a set of four octapeptides ,
aeaeakak ( 226 ) , aeakaeak ( 227 ) , fefefkfk ( 228 ) , and fefkfefk
( 229 ) , which consist of oppositely charged residues and
hydrophobic residues with slightly different positionings . the authors
found the phenylalanine in the octapeptides to promote -sheet
conformations in solution and the alanine to favor -helices .
according to the authors , 227 is unable to self - assemble
in solution , but 226 self - assembles to form thick , rigid
nanofibers ( 6 nm in diameter ) . however , 226 fails to
form a hydrogel at concentrations up to 10 wt % . on the contrary ,
both phenylalanine - based peptides 228 and 229 self - assemble in solution and form hydrogels at a cgc of 0.8 wt
% . both hydrogels contain a dense network of semiflexible nanofibers
4 nm in diameter . besides further confirming the importance of aromatic aromatic
interactions for the self - assembly of peptides in water , the detailed
sans measurement in this work has provided invaluable information
on the morphologies of the nanofibers in solutions and in hydrogels . miller et al . developed an innovative way to
stabilize enzymes by covalently attaching a self - assembling peptide
( 230 ) to pentaerythritol tetranitrate reductase , which
co - self - assemble to form hydrogels . the stability of the entrapped
enzyme increases significantly , and the activity of the enzyme is
retained even after exposure to a high temperature ( 90 c ) and
12 months of storage , which is quite
remarkable . cavalli et al . synthesized a nonapeptide , fefefkfkk
( 231 ) , which forms a hydrogel at 2.0 wt % . on the basis
of
this result , the authors conjugated 231 with a dna - recognizing
protein ( tus ) by a sortase a methodology . gpggdgpggd ( 232 ) based on the spider
flagelliform silk protein and the ca binding domain of
lipase lip a. the authors suggested that such a design can avoid redundancy
and preserve the properties of both sequences . at 1.0 wt % , 232 largely forms nanospheres which become nanofibers upon
the addition of ca . on the basis of cd data , the authors
suggested a -spiral structure as the secondary structure of
the peptide . mcpherson et al . reported
a new method to synthesize peptide qqrfewefeqq ( 233 ) based on the protein expression in escherichia coli . the recombinant peptide self - assembles
to form nanofibers and results in a hydrogel at a concentration of
1.5 wt % and ph 2.0 . mcpherson et al . used a sumo ( small ubiquitin - related
modifier)peptide
fusion approach for efficient production and purification of -structured
recombinant self - assembling peptides with native n- and c - terminals
( 233 ) to compare with the chemically synthesized peptide
that has an acetylated n - terminal ( 234 ) . the authors
reported that the chemically synthesized peptide 234 forms
a hydrogel at 1.0 wt % and ph 2.0 , which
are essentially the same conditions as those needed for sumo233 . intending to investigate
the effect of the topology of the peptides on self - assembly , lim et
al . designed and synthesized two dodecapeptides that have the same
sequence ( rrrrgswwwwsg ) but different topologies : one
( 235 ) is linear , and the other ( 236 ) is
cyclic . their study revealed that the cyclic and linear peptides exhibit
significantly different self - assembled nanostructures and thermal
stabilities , but share similar critical aggregation concentrations
and cytotoxicity profiles . this study
offers an important topological approach to tailor the nanostructures
formed by the self - assembling peptides . rapaport et al . reported four
tridecapeptides ( e.g. , pelelelelelep ( 237 ) and pefefefefefep ( 238 ) ) consisting of
negatively charged residues and hydrophobic residues . as an amphiphilic
and acidic peptide , 237 self - assembles to form -sheet
structures and results in a hydrogel at ph 7 and a concentration of
4 wt % . the addition of calcium ion can increase the storage modulus
of the hydrogel by almost 10-fold . furthermore , the authors also demonstrated
that the hydrogel of 238 could provide scaffolds for
cell adhesion and spreading of saos-2 cells . reported a tetradecapeptide ( eiaqleyeisqleq , 239 ) consisting of two heptads for adopting coiled - coil structures .
at a concentration of 1 wt % , the mixture of 239 with
another tetradecapeptide ( kiaqlkykisqlkq , 240 ) containing a positively charged residue results in the formation
of nanofibers having -sheet features the authors suggested
that these short peptides illustrate the minimum requirements necessary
to form dimeric coiled coils . developed a hexadecapeptide ( kkqlqlqlqlqlqlkk , 241 ) which fits into an aba triblock motif ( termed multidomain
peptides the authors reported that 241 , at 1 wt % and with 10 mm tris at ph 7 , self - assembles
to form nanofibers and results in a hydrogel . one impressive result
in this work is the control of the length of the nanofibers , as demonstrated
by the fact that the aging of the solution of 241 hardly
changes the maximal length of the nanofibers . on the basis of the same principle , the authors also developed
several other mdps ( kkslslslslslslkk ( 242 ) , eeslslslslslslslee ( 243 ) , kkqlqlqlqlqlqlkk
( 244 ) ) and used them to form hydrogels at 2 wt % . moreover ,
they demonstrated the use of lysyl oxidase or plasma amine oxidase
to oxidize primary amines to aldehydes so the reactive aldehydes react
further to cross - link the nanofibers in the hydrogels . kinoshita et al . designed two kinds of -peptide ,
( le)8 ( 245 ) and ( vevsvkvs)2 ( 246 ) . both peptides self - assembled to form nanofibers
and afford hydrogels upon addition of calcium ion . owing to the interaction
between the carboxylic acid in the peptide and the calcium ions , the
resulting hydrogel exhibited enhanced mechanical properties with an
increase of the concentration of the calcium ions . the authors suggested
that this mineralization approach provides a new way to prepare bone - filling
materials . after schweitzer - stenner
et al . observed that the hexadecapeptide
aakaaakaaakaaaka ( 247 ) forms a hydrogel at
low concentration ( 0.5 wt % ) , li et
al . analyzed the initial phase of the aggregation process by molecular
dynamics . in addition , the authors suggested that the formation
of trimers is very sensitive to the concentration of the peptides , which provides a useful insight to explain
the conformation transition of 247 from an -helix
to a -sheet . after zhang et al .
reported the hydrogels of radaradaradarada ( 248a ) , unsworth et al . carried out a detailed study to provide a mechanistic
understanding of the self - assembly process by designing and synthesizing
two peptides ( radaradaradaradakkkk ( 248b ) and radaradaradaradasssss ( 248c ) )
that are related to 248a . the authors observed that 248b is unable to self - assemble , suggesting the role of the
charged residues . the authors also found that self - assembly of 248a and 248c is entropy driven , with hydrophobic
force as the main factor for 248a and hydrogen bonding
as the main factor for 248c . the authors also confirmed that counterions contribute little to
self - assembly , which differs from the current conceptual understanding
of the self - assembly of 248a . muller et al . designed
a library of peptides having alternating hydrophobic and polar amino
acids by substituting phenylalanine in fefefkfkfefefkfk
( 249 ) with glycine , alanine , valine , leucine , or isoleucine . the authors found that an increase of the number of glycines eventually
prevents the hydrogelation of the peptide , and concluded that the
sequence and steric size of the nonpolar residues dictate the secondary
structure and morphology of the nanofibers of the self - assembled peptides . as supramolecular hydrogelators , one of the
most impressive classes of oligopeptides made of 20 amino acids is
the de novo -hairpin peptides ( 250264 ) pioneered by schneider and pochan et al . these synthetic peptides undergo intramolecular folding at a proper
condition , and the subsequent intermolecular self - assembly generates
-sheets in water to result in hydrogels . the authors ingeniously used d - pro - l - pro
to define a type ii turn . at physiological ph , the
peptides exist as random coils and exhibit excellent solubility in
water , but a stimulus ( e.g. , ionic strength , ph , temperature , light ,
or shear forces ) switches the peptide to a -hairpin conformation
so these -hairpin peptides self - assemble into a highly cross - linked ,
but semiflexible , network of nanofibers to afford mechanically rigid hydrogels . since
there are several excellent reviews on the -hairpin hydrogelators
and their applications , we only list some representative sequences in table 1 , and interested
readers are encouraged to read the excellent reviews written by schneider
et al . when the peptides
get longer , the intermolecular interactions certainly
can increase and favor self - assembly and physical cross - linking to
form a hydrogel . however , the synthesis
of long peptides becomes more difficult or more expensive so the peptides
should have unique functions or aims to address important and general
problems to justify the high cost . after reporting the first example
of peptide hydrogels consisting of purely helical structures , woolfson et al . recently designed a self - assembling
fiber system consisting of a dimeric coiled - coil peptide ( e.g. , kiaalkqkiaslkqeidaleyendaleq
( 265 ) and kirrlkqknarlkqeiaaleyeiaaleq
( 266 ) ) that assemble in a controlled manner . the self - assembly
of 265 and 266 results in fibers that are
tens of nanometers wide and tens of micrometers long . using cryo - tem ,
the authors also obtained an ultrastructure for interpreting the packing
of individual -helices within the fibers . on the basis of the
electron density map , the authors derived a model for elucidating
how these -helical fibrils pack into larger fibers . to understand how to use local interactions
between proteins for creating materials that have well - determined
microstructures , fairman et al . designed and synthesized an oligopeptide
( ckqledkieellskaackqledkieellsk , 267 ) that relies on hydrophobic interactions to drive self - assembly .
as pointed out by the authors , the hydrophobic effect between 267 molecules favors axial assembly and their electrostatic
forces modulate lateral assembly . at a concentration of 0.05 wt % ,
the peptide self - assembles to form a filament consisting of about
120 molecules of 267 . the authors also reported that
various environmental factors ( e.g. , ph , salt , molecular crowding
reagents , and peptides ) can
regulate the self - assembled filaments in an assembly of predictable
manner , which provides useful insights
for developing coiled coils as peptide - based materials . it would be
interesting to know the proteolytic stability of these self - assembled
filaments . besides native
peptides acting as hydrogelators , peptide derivatives
can also self - assemble in water to form hydrogels . the most common
way to modify a peptide is to cap the n - terminal by an acetyl or the
c - terminal by an amide or to cap both ends . as shown in scheme 34 , solaro et al . reported a capped tetrapeptide , ac - rwdw - nh2 ( 268 ) , containing oppositely charged residues and hydrophobic
residues , to form a soft hydrogel at 0.026 wt % in pbs buffer . they
also observed an increase of the concentration of 268 and the addition of cacl2 to result in a thick and transparent
hydrogel . moreover , the authors demonstrated the use of the hydrogels
for culturing cells ( mouse embryo fibroblasts balb/3t3 clone a31 and
human hepatoblastoma hepg2 ) . xu and
lu et al . reported a capped hexapeptide , ac - iiicgk - nh2 ( 269 , table 2 ) , which self - assembles in water to form long nanofibers and results
in a hydrogel at 0.53 wt % . it is worth noting that oxidation of the
cys residue in 269 greatly increases the storage modulus
of the hydrogels ( from 200 to 10000 pa ) . moreover , the authors
found that oxidation reduces the cgc to as low as 0.034 wt % . replacing
the cys by a met residue allows the formation of nanofibers at 1.7
mm , but fails to generate any hydrogels . nilsson et al . synthesized a series of capped octapeptides ( ac - xkxkxkxk - nh2 ; x = val ( 270a ) , ile ( 270b ) , phe
( 270c ) , phe ( 270d ) , and cyclohexylalanine
( cha ) ( 270e ) ) that are composed of alternating hydrophobic
residues ( val ) and positively charged residues ( lys ) . at 8 mm , 270a is too hydrophilic to form a hydrogel , but 270b , 270c , 270d , or 270e self - assembles
in water to form a hydrogel . on the basis of cd , the authors concluded
that these peptides adopt -sheet structures in the nanofibers
formed by self - assembly . the authors also found that 270c and 270d , containing aromatic residues , form rigid
hydrogels , but 270b and 270e form weak gels . one notable feature is that the self - assembly of 270b270e requires the addition of nacl to the solution . to understand the relative
contributions of different hydrophobic
groups ( e.g. , aromatic vs nonaromatic hydrophobic groups ) , nilsson
et al . designed another series of octapeptides ( ac - fkfefkfe - nh2 ( 271a ) , ac - akaeakae - nh2 ( 271b ) , ac - vkvevkve - nh2 ( 271c ) , and ac - lklelkle - nh2 ( 271d ) ) . they found that only 271a formed a hydrogel at a
concentration of 0.46 wt % in a nacl ( 40 mm ) solution , suggesting
that aromatic aromatic interactions , though less essential
for the self - assembly of this type of peptide ( ac - xkxexkxe - nh2 ) , are necessary for hydrogelation . moreover , the authors
also reported that the cosolvent hfip exerts a significant influence
on the stabilization of the helical morphology of the self - assembled
nanofibers . the authors also varied
the sequence of 271a to ac - fkfkfefe - nh2 ( 271e ) , ac - keffffke - nh2 ( 271f ) , ac - kffekffe - nh2 ( 271 g ) , and ac - ffkekeff - nh2 ( 271h ) . the authors found that the self - assembly
of these peptides is ph dependent . at ph 34 , 271e self - assembles to form -sheet nanoribbons and 271f forms distinct nanotapes with a width of 20 nm , but 271 g and 271h fail to self - assemble to form fibrils / tapes ;
however , 271h does form micelle - like aggregates . at neutral
ph , 271h forms 20 nm nanotapes and the other peptides
behave similarly to the way they do at ph 34 . these results underscore that the amino acid
sequence plays a key role in the self - assembly of the oligopeptides ,
which is quite reasonable . voyer et al . designed a series of octapeptides
based on milk proteins ( lactoglobulin ) . they found that both the n - terminal - capped
octapeptide ac - livtqtmk ( 272a ) and the c - terminal - capped
octapeptide livtqtmk - nh2 ( 272b ) adopt
a -sheet conformation and self - assemble to form hydrogels at
a concentration of 0.1 wt % . however , 272a forms hydrogels
instantly at neutral ph and acidic ph , and 272b forms
hydrogels only at basic ph . this study also provided useful insight
into the transition from a random coil to a -sheet conformation
upon a change of the ph . by introducing
four phenylalanine residues into a capped nonapeptide
( ac - pro - ser - phe - asn - phe - lys - phe - giu - pro - nh2 , 273 ) , zhao et al . the authors reported that the -turn and -sheet
structures result in a concentration - dependent self - assembly to afford
hierarchically arranged nanostructures . yu et al . investigated the effect of the ionic strength on the mechanical ,
structural , and transport properties of the hydrogels made of two
capped undecapeptides , ac - kwkakakakwk - nh2 ( 274 ) and ac - eweaeaeaewe - nh2 ( 275 ) . at
a peptide concentration of about 2.3 wt % , the authors changed the
ionic strength of the solution and found that an increase of the ionic
strength results in higher final storage moduli , slows the rate of
hydrogelation , decreases the cross - section of the nanofibers in the
hydrogels , and reduces the diffusion coefficients of water in the
hydrogels . used photon
correlation spectroscopy to study the internal dynamics of self - assembled
fibrils of charged peptides , such as ac - qqrfqwqfeqq - nh2 ( 276a ) , ac - qqrfqwqfqqq - nh2 ( 276b ) , ac - qqrfewefeqq - nh2 ( 234 ) , and ac - ssrfswsfess - nh2 ( 276c ) . the
authors found that these peptides form hydrogels at 0.1 wt % , but
they have different critical concentrations for self - assembly , which
are 0.05 , 0.03 , 0.01 , and 0.1 mm for 276a , 276b , 234 , and 276c , respectively . aggeli et al . studied the effect of the ionic strength
on the self - assembly , morphology , and gelation of charged peptides ,
such as 234 , ac - qqrfowofeqq - nh2 ( 276d ) , ac - ssrfewefess - nh2 ( 276e ) , and ac - ssrfowofess - nh2 ( 276f ) ( o
= ornithine ) . according to their study , at a concentration of 0.7
wt % , 234 and 276e , in water , exhibit a
sharp transition from antipaprallel -sheets at ph < 6.5
to random coils at ph > 8 . this transition results in a change
of
the properties of the solutions from a nematic solution ( ph < 8)
to an isotropic fluid ( ph > 8) . bearing charges opposite those
of 234 and 276e , 276d and 276f undergo a transition from antiparallel -sheets
( ph > 10 )
to random coils ( ph < 8) at 8 < ph < 10 . the most insightful
observation is that , in the presence of nacl ( 130 mm ) , 234 , 276d , 276e , and 276f all
form nematic hydrogels at physiological ph . nelson et al . used electrochemical impedance spectroscopy to examine
the interaction of this class of peptides with phospholipid monolayers . they found that peptides with side chains of serine and threonine
interact with dopc layers more strongly compared to peptides with
side chains of glutamine and asparagine . these insights should be useful for designing the hydrogels to interact
with cells . by introducing cysteine residues into 271a , nilsson
et al . designed a novel cyclic peptide , ac - cfkfefkfecg - nh2 ( 277 ) . upon reduction by dtt , cyclic-277 becomes a linear peptide , linear-277 , which forms a hydrogel at 0.9 wt % . this excellent strategy also has been applied by yang et al . to
design redox - trigged hydrogelation of small peptides . to improve the mechanical properties of the
self - assembled peptide hydrogels , collier et al . devised an innovative
approach to perform native chemical ligation ( ncl ) of the peptides
after the self - assembly . on the basis of the self - assembling peptide
ac - qqkfqfqfeqq - nh2 ( 278a ) , they designed
and synthesized the sequence cys - qqkfqfqffeqq - gly - thioester
( 278b ) . the self - assembling 278b forms a
hydrogel at 0.76 wt % , and ncl of the hydrogel results in a 6-fold
increase of the storage modulus . the authors reported that ligation
also leads to a significant enhancement of huvec cell proliferation
on the surface of the hydrogel . moreover , they demonstrated that ncl
is orthogonal to the inclusion of an rgd - functionalized peptide ( e.g. ,
ac - ggrgdsgggqqkfqfqffeqq - nh2 , 279 ) , which further increases the cell proliferation . schweitzer - stenner et al . investigated the self - assembly
of a hexadecapeptide ( ac - aakaaakaaakaaaka - nh2 , 280 ) and found that the presence of a salt can stabilize
the self - assembly of 280 to result in a hydrogel at a
cgc of 0.5 wt % . it was found that 280 starts self - assembling
at about 0.001 wt % and forms a network of filaments at 1 wt % , and
the filaments turn into a nanoweb after the addition of 1.0 m nacl . yokoi , arosio ,
and zhang et al . further evaluated the ac - radaradaradarada - nh2 peptide ( 281 ) by
reassembling the peptides after disassembling the hydrogel of 281 by sonication or by changing the ionic strength . after examining the kinetics of the reassembly ,
the authors proposed a sliding diffusion model for the reassembly
of 281 . further
investigated the temperature and ph effects on the self - assembly of 281 and found that the extent of -sheet conformation
decreases at low or high ph and an increase of the temperature to
70 c results in smaller globular aggregates . later , yokoi et al . mutated 281 to change
the number and position of the net charges on the peptide . for example ,
they found that , at a concentration of 0.5 wt % and ph of 1.0 , while
ac - rasaradaradarada - nh2 ( 282 )
fails to form a hydrogel , ac - rasarasarasarada - nh2 ( 283 ) does form a hydrogel . their results confirm that
the number of charges and the sequence are critical for the formation
of the antiparallel -sheets for self - assembly . on the basis of their design of mdps , hartgerink
et al . varied the sequence of the mdp motif to generate a series of
hexadecapeptides ( ac - kkqlqlqlqlqlqlkk - nh2 ( 284a ) , ac - eqlqlqlqlqlqle - nh2 ( 284b ) , ac - kkslslslslslslkk - nh2 ( 284c ) , and ac - eclslclslclsle - nh2 ( 284d ) ) for enhancing the viscoelasticity of the hydrogels
formed by these peptides at 1 wt % . the authors found that 284a284d all self - assemble to form nanofibers with
diameters of 6 nm and lengths on the order of hundreds of nanometers . the addition of mg to the solutions of 284b and 284d and po4 to
the solution of 284c increases the length of the nanofibers
to result in entangled networks and hydrogelation . the authors also
demonstrated that the addition of mg to the hydrogels
of 284b and 284d and po4 to the hydrogels of 284a and 284c or the oxidation of the hydrogel of 284d significantly
increase the storage moduli of the hydrogels , by up to about 60-fold . in another study , introduced
aromatic residues into the mdp peptides to obtain ac - kkqfqlqfqlqfqlkk - nh2 ( 284e ) , ac - kkqfqfqfqfqfqfkk - nh2 ( 284f ) , ac - kkqwqwqwqwqwqwkk - nh2 ( 284 g ) , and kkqyqyqyqyqyqykk - nh2 ( 284h ) . the authors
reported that the peptides 284e284h self - assemble to form nanofibers with intertape spaces larger than
that of 284a . at 1 wt % , 284e284h all form hydrogels at ph 7.4 , and 284e and 284f exhibit higher storage moduli than those of 284 g and 284h . in addition ,
hartgerink et al . designed a peptide amphiphile containing a tridecapeptide
( ac - gtagligqergds , 285 ) . while the sequence
rgds promotes cell adhesion , ac - gtagligq serves as a substrate
of mmp-2 . the authors demonstrated the enzymatic degradation ( by type
iv collagenase ) of the hydrogels made of the peptide amphiphile , and
suggested that the degradation of the nanofiber networks mimics a
key property of the natural extracellular matrix ( ecm ) . dexter et al . developed a 21-residue
peptide ( ac - lkelakvlhelaklvsealha - nh2 , ( 286 ) ) that forms a hydrogel at 0.1 wt % upon
a change of the ph . on the basis of tem , dls , and electronic circular
dichroism ( ecd ) studies , the authors reported that 286 , due to hydrophobic interactions , self - assembles to form hexameric
coiled coils , which promote vertical alignment for rearrangement to
fibril networks for hydrogelation . tseng
and yu et al . designed a pair of self - repulsive peptides ( ac - wkvkvkvkvk - nh2 ( 287a ) and ac - ewevevevev - nh2 ( 287b ) ) consisting of alternating charged / neutral amino acid
sequences . the authors reported that the simple mixing of the two
peptides , at a total concentration of 0.25 wt % , affords a hydrogel . it was found that the coassembled hydrogel exhibits rapid recoveries
from repeated shear - induced breakdowns . the authors also found that
the hydrophobicity of the neutral amino acids dictates the viscoelastic
properties of the hydrogels . by designing
two similar peptides , formyl - woaoaoaoao - nh2 ( 287c ) and formyl - weaeaeaeae - nh2 ( 287d ) , yu et al . found that the temperature significantly affects the
structure and mechanical properties of hydrogels formed by mixing 287c and 287d at a total concentration of 1 wt
% . the authors found that the peptide nanofibers assemble faster to
result in mechanically stronger hydrogels at 25 c than at 5
c , suggesting entropy - driven self - assembly . later , yu et al . compared 287c and 287d with
other similar peptides that have the same alternative amino acid sequences
but different lengths of the peptides ( i.e. , formyl - woaoao - nh2 ( 287e ) , formyl - weaeae - nh2 ( 287f ) , formyl - woaoaoaoaoaoao - nh2 ( 287 g ) , and formyl - weaeaeaeaeaeae - nh2 ( 287h ) ) . they found that , upon mixing , 287c + 287d and 287 g + 287h form
hydrogels at 1 wt % , but the mixture of 287d + 287e does not . in addition , the hydrogel formed by 287 g + 287h is mechanically weaker than that of 287c + 287d , likely due to the tighter packing of the amino
acid side chains in the nanofibers formed by 287c + 287d . kinoshita et al . designed
a c - terminal - capped hexadecapeptide , saslsaslsaslsasl - nh2 ( 288 ) . they found that 288 adopts
a -sheet structure and forms a hydrogel at 1 wt % . one interesting
feature of 288 is that it also forms organgels in polar
organic solvents ( e.g , dmf , dmso , n - methyl-2-pyrrolidone
( nmp ) , and 2,2,2-rifluoroethanol ( tfe ) ) . alkyl - chain - containing peptide derivatives , sometimes termed surfactant - like peptides , essentially consisting of hydrophilic and hydrophobic
domains , are some of the most representative peptide amphiphiles . the alkyl or lipid chains , usually attaching
to the n - terminal or the c - terminal of peptides , not only drive self - assembly
through van der waals force , but also allow the functional peptides
to be presented on the surface of nanofibrils at an exceedingly high
density . moreover , the modular nature
of peptide chemistry facilitates the variation of the amino acids
or chain length , thus easily tuning both the mechanical properties
and biological activities of the subsequent self - assembled nanofibrils
or hydrogels . as shown in scheme 35 , miravet and escuder
et al . reported an l - proline - based supramolecular hydrogelator
( 289 ) which affords a supramolecular hydrogel at a concentration
of 0.25 wt % . this l - proline - based supramolecular hydrogel
has a remarkable efficiency as a heterogeneous organocatalyst for
a direct aldol reaction . another dipeptide
derivative ( 290 ) , consisting of two amino acids ( i.e. , l - histidine and -alanine ) and a lipid chain , is able
to form a hydrogel in a variety of conditions ( e.g. , at both alkaline
and acidic conditions and in the presence of additives , such as nacl
or alcohol ) with a cgc value of 3.46.8 wt % .291 ( -ala - his-(eo)2-alkyl
chain ) , with one more ethylene glycol group compared with 290 , can also self - assemble to form a hydrogel at a concentration of
2.0 wt % . goto et al . reported a tetrapeptide
amphiphile ( 292 ) which has a simple molecular structure
but results in a hydrogel at a remarkably low concentration ( 0.03
wt % ) . hamley et al . designed and developed
an enzymatically cleavable peptide amphiphile ( c16-kkffvlk , 293 ) ) . the peptide part of this small molecule is cleaved
by -chymotrypsin at two sites , leading to several products :
c16-kkf , fvlk , c16-kkff , and vlk . 293 molecules form nanotubes and helical ribbons at room temperature ,
and both c16-kkf and c16-kkff self - assemble
to form spherical micelles . fvlk and vlk are unable to adopt well - defined
aggregated structures . in this way , the enzyme can modulate the self - assembly
of the systems and tune the resulting nanostructure . it would be more interesting to determine the biological
activities of 293 in a cellular environment . the addition of soluble metal ions or an adjustment
of the ph triggers the hydrogelation of this molecule at a concentration
of 20 mm ( 2.4 wt % ) , indicating the self - assembly is mediated by a
screening counterion and stabilized by van der waals and hydrophobic
forces , ionic bridging , and hydrogen bonding . to elucidate the self - assembly behavior of the peptide
amphiphiles , which tend to form cylindrical nanostructures that imply
worm micelles , the same group used a pair of compounds ( 295 and 296 ) , containing chromophores ( i.e. , tryptophan ) ,
to study the aqueous solvation within the self - assembled structures . self - assembly constrains the chromophores to a defined location within
the aggregate , which leads to different fluorescence changes of the
chormophores after the addition of aqueous acrylamide ( a quencher
of fluorescence ) . the authors found that , at lower ph , 295 and 296 have a tendency to form a cylindrical structure
with the alkyl chain inside the nanostructure as a hydrophobic core.297 represents a class of peptide
derivatives , called gemini peptides , formed by the complexation of
cationic gemini surfactants and anionic oligoglycine - aspartate . by
studying the aggregation behaviors of these molecules ( the hydrophobic
chain length ( c10 to c22 ) and the length of
the oligoglycine ( 04 ) ) , oda et al . found that the hydrogels
of 297 only form below a certain temperature ( e.g. , the
krafft temperature ) when either the hydrophobic
chains or the peptides are long enough . developed hydrogels
formed by coassembly of amphiphiles with opposite peptide polarities
( i.e. , peptide amphiphiles with free n - terminals or c - terminals ) ,
for example , 298 and 299 . the mixture of
these molecules with complementary polarities results in coassembled
structures which show unusual thermal stability compared to the assemblies
composed by only one of the constituents . peptides functionalized at the n - terminal with large
aromatic groups have recently emerged as an exciting class of small
molecular hydrogelators . the most used aromatic group is fmoc . as shown in scheme 36 , shortly after the report of an unexpected small peptidic hydrogelator
made of fmoc - l - ala - l - ala ( 300 ) and
fmoc - d - ala - d - ala ( 22 ) , ulijn et al . reported the development of fmoc - diphenylalanine
( fmoc - ff or fmoc - phe - phe ( 6 ) ) , one of the most investigated
low molecular weight hydrogelators . the molecules of 6 form hydrogels by adjusting the ph of the aqueous solution
of 6 , by applying 6 to
a silica wafer surface , or by the addition
of water to a dmso solution of 6 . despite considerable studies on 6 , it was unclear why the mechanical properties reported for the hydrogels
of 6 vary significantly , up to 4 orders of magnitude . have systematically studied
the mechanical properties of hydrogels of 6 prepared
using different protocols . they demonstrated that , independently of
the method of gel formation , the final ph of the hydrogels is the
principal determinant of the mechanical properties , which is quite
reasonable due to the c - terminal carboxylic group in 6 . besides , additional variability arises from experimental factors
such as the fraction of dmso or the nature of the buffers used in
the selected systems . in addition to
fmoc - ff ( 6 ) , a variety of fmoc - dipeptide derivatives
have been reported in the past several decades . adams and firth et al . investigated the influence of the molecular
structure on the gelation behaviors of a range of fmoc - dipeptides . in general , they found that the overall hydrophobicity of the fmoc - dipeptide
determines the ability to form a stable hydrogel . interestingly , gazit et al . designed a 3,4-dihydroxy - l - phenylalanine ( dopa)-containing fmoc - dipeptide ( 301 ) by using dopa to substitute for the phenylalanines in 6 . the resulting hydrogelator self - assembles at a concentration of
0.5 wt % and forms supramolecular nanostructures , which can be used
as multifunctional platforms for various technological applications ,
such as glass glue . in addition to fmoc - dipeptide ,
fmoc - peptide derivatives with more than two amino acids are also excellent
candidates for self - assembly to form hydrogels . further functionalized fmoc - dipeptide fmoc - kk ( 302 ) at the -amino position with a naphthalenediimide
( ndi ) chromophore to afford fmoc - kk(ndi ) ( 303 ) . the resulting
molecule forms a self - supporting hydrogel at a concentration as low
as 1.5 wt % which exhibits high thermal stability up to 75 c . like fmoc , naphthalene is another aromatic
group frequently used in peptide derivatives to achieve strong intermolecular
interaction for self - assembly . among
them , 2-(naphthalen-2-yl)acetic acid is a convenient motif for constructing
naphthalene - based peptide derivatives because of its ease of being
directly used in spps . , is
a dipeptide derivative made by conjugating 2-(naphthalen-2-yl)acetic
acid with phe - phe . in the molecule of 3 , the naphthalenyl
group provides the hydrophobic force to enhance self - assembly in an
aqueous environment , while the dipeptide backbone acts as both hydrogen
bond acceptors and hydrogen bond donors . xu et al . demonstrated 3 as an effective hydrogelator that forms a hydrogel at a
concentration of 0.8 wt % , with the gel sol transition temperature
at about 323 k. the crystal structure
of 3 ( figure 6 ) , though obtained from single
crystals grown from a mixed solvent of ethanol and water , reveals
that the aromatic aromatic and hydrogen - bonding interactions
apparently reinforce each other . xu et al . also designed and synthesized a similar small molecule
( 304 ) with an exposed n - terminal and a naphthalene - blocked
c - terminal . this n - terminated hydrogelator affords a stable hydrogel
even below a concentration of 0.8 wt % , but unlike its c - terminal
analogues 3 , 304 forms hydrogels only within
a narrow ph range ( 56 ) ( scheme 37 ) . adams et
al . used 2-naphthol to construct naphthalene - based dipeptide derivatives with an ether bond , such as 305(101 ) and 306 . they reported that the
hydrogelation of 305 and 306 could be controlled
by reducing the ph value with the hydrolysis of gdl at a concentration
of 2.2 mm . they chose gdl for ph control because its hydrolysis to
gluconic acid allows a slow , uniform ph change . also developed
a series of hydrogelators ( e.g. , 307 ) with 2-naphthol
as a terminal , but with an ester bond . molecular packing
and hydrogen bonds in the crystal of 3 ( recrystallized
from ethanol ) : views from the ( a ) a , ( b ) b , and ( c ) c axes and ( d )
view from the c axis to show the hydrogen bonding
( green dotted lines ) of one molecule with four other molecules and
some aromatic aromatic interactions ( yellow lines ) . adapted
from ref ( 14 ) . ,
the pyrene motif not only enhances intermolecular
interaction to promote self - assembly , but also exhibits fluorescence
that acts as a useful tool for studying the aggregation behaviors
of the hydrogelators . atkins et al . replaced the disulfide
bond in the oxidized disulfide form of glutathione ( -glutamylcysteinylglycine
( gsh ) ) ( gssg ) to increase the self - assembly ability of gssg in aqueous
solution , which otherwise only self - assembles to generate fibrillar
aggregates and gels in organic solvents . pyrene
( 308 ; scheme 38 ) then forms a gel in a mixed solvent ( 95% h2o
and 5% dmso ) at a concentration of 1 mm ( 0.056 wt % ) . xu et al . developed a pyrene - terminated dipeptide
( 309 ) which affords a weak hydrogel at a concentration
of 30 mm ( 1.3 wt % ) . however , the addition of vancomycin ( 310 ) at a 1:1 ratio remolds the self - assembly of hydrogelators and drastically
increases the elasticity of the hydrogel by 10-fold due
to the ligand receptor interaction between vancomycin and 309 . em also reveals the formation of highly cross - linked
networks in the hydrogels , likely contributing to the significant
increase of the storage modulus . developed several molecules
( 311 , 312 , and 313 ) with pyrene
chromophores in the backbone to investigate the aqueous solvation
within the self - assembled structure formed by these peptide amphiphiles . they found that , as the chromophore is placed closer to the exterior
of the aggregates , the stern volmer quenching constants and
the fractional accessibility of covalently bound pyrene progressively
increase . their study also demonstrated that covalently bound fluorophores
within an aggregate can interact with the external environment . in addition to the
exploration of fmoc , naphthalene , and pyrene ,
ladouceur et al . have synthesized novel self - assembling hydrogelators
that contain an electroactive aromatic group , anthraquinone . as shown
in scheme 39 , they
used the well - known redox couple of anthraquinone / anthrahydroquinone
as the hydrophobic component for a series of hydrogelators , such as 314 . the molecules of 314 undergo two separate
processes : a reversible redox reaction and a reversible self - assembly
at ta concentration of 4 wt % . dynamic
combinatorial chemistry was originally a method for developing synthetic
receptors and ligands for biomolecules by linking building blocks
together using a reversible reaction , resulting in a thermodynamically
controlled product distribution . the studies of otto et al . proved
that dynamic covalent disulfide linkages are not only instrumental
in dynamic combinatorial discovery of self - assembling materials , but
also further stabilize the consequent self - assembly . they designed
a building block ( 315 ) equipped with a short peptide
sequence capped by a dithiol terminal and reasoned that self - assembly
would become feasible for a macrocycle ( 3156 ) that reached a critical size . they discovered that
photoirradiation of the solution containing 3156 ( 0.6 mm ) results in the formation of a hydrogel . miravet and escuder et al . prepared a ph - sensitive
complex molecular hydrogel from oppositely charged tetrapeptide components
( e.g. , 316 and 317 ) . they have shown that
small peptides bearing alternating phenylalanine and aspartic acid
residues such as 316 are able to form hydrogels at low
concentration , and 317 , designed as a charge complementary
analogue bearing alternating phenylalanine and lysine , is also able
to aggregate at low concentration . then they obtained a ph - sensitive
coassembled network from these two oppositely charged small self - assembling
peptides . by changing the ph of this system , they were able to switch
between two - component networks at neutral ph and one - component networks
at either basic or acidic ph . described the design and synthesis of molecular hydrogelators
composed of peptides and peptoids ( i.e. , one type of unnatural peptide
with the side chain attached to the amide nitrogen as well ) , such
as 318 . they tested the hydrogelation ability of 318 by the inverted - tube method and found that 318 and its analogues afford hydrogels with cgc values of 0.50.8
wt % . yang et al . developed a reversible
hydrogelation system using a redox system with selenium - containing
peptides ( 319 and its reduced version ) . they achieved
the reversible transformation between the solution and hydrogel of
the peptide derivative 319 at a concentration of 1 wt
% , which is accompanied by the reversible transformation between selenide
and selenoxide , by triggering with vitamin c and h2o2 ( 0.1 wt % ) . hamachi et al .
reported a series of dipeptide derivatives , for example , bhcmoc - ff
( 320 ) , in which ff is tethered with [ ( 6-bromo-7-hydroxycoumarin-4-yl)methoxy]carbonyl
( bhcmoc ) . they found that compound 320 forms a hydrogel
above 0.35 wt % and the resulting hydrogel collapses upon application
of stimuli such as uv irradiation . liang
et al . developed a salt - responsive peptide ( 321 ) as a
luminescent hydrogelator with a cgc of 0.3 wt % . they found that only
the presence of salt rather than the temperature , ph , or solvent caused
the dispersed hydrogelators to self - assemble to form a hydrogel network
to turn on bright emission . in addition to enzyme - responsive hydrogelation , supramolecular hydrogelators containing a photoresponsive group
are of great interest since light can act as an external stimulus
to modulate the properties of the hydrogels . for example , upon photoirradiation , a solution can transform into
a hydrogel , and vice versa . a variety of photoresponsive groups have
served as the photochemical module in peptides for the design of photoresponsive
hydrogelators . uv and visible light can regulate the geometry of spiropyran
( between the nonplanar spiropyran form and the planar merocyanine )
to control the hydrogelation process because the planar merocyanine
isomer favors the formation of aggregates ( due to stronger intermolecular
stacking ) while the nonplanar spiropyran form
disfavors interaction . as shown in scheme 40 , zhang et al . reported that the spiropyran - conjugated dipeptide 322 forms a hydrogel upon photoisomerization to a merocyanine form .
besides responding light , this hydrogel undergoes a gel sol
transition upon the addition of vancomycin because of the strong interaction
of vancomycin with the peptide unit d - ala - d - ala . the reversible photoregulated trans to cis isomerization of azobenzene
significantly influences the intermolecular interaction among the
hydrogelators , thus changing the morphologies of the aggregates or
controlling the gel sol transition . huang et al . reported a
dipeptide amphiphile incorporated with an azobenzene moiety ( 323 ) which self - assembles to form well - defined nanoribbons
that result in a macroscopic hydrogel . after uv irradiation , the authors
found a dramatic decrease of the viscosity of the sample , accompanied
by a transition from laminated ribbons to short fibers , as revealed
by em . studied the mechanism
by which azobenzene isomerization induces the breaking and reorganization
of the assemblies of n-(l - valyl - l - valyl - l - valyl)azobenzene-4-carboxamide ( azo(l - val)3 , 324 ) . as a hydrogelator , 324 forms
photoresponsive nanofibrils , and undergoes dispersion / reorganization
upon trans to cis photoisomerization that breaks and re - forms the
hydrogen bonds to induce reversible gel sol transitions . zhang et al . reported an azobenzene - linked
symmetrical gemini -helical peptide ( 325 ) that
undergoes light - switched self - assembly . with the reversible molecular
structure transition between trans and cis ( u - shape ) , the morphology
of the self - assembled gemini -helical peptide can reversibly
change between nanofibers and nanospheres in acidic conditions , and
between nanospheres and vesicles in basic conditions . many
different types of amphiphilic molecules are able to form organogels
or hydrogels , but they usually gel either water or an organic solvent .
in other words , few of them have the ability to form gels in both
water and an organic solvent , except certain peptide - based bolaamphiphiles . because of the versatile functional groups
on peptides , such as carboxyl , amine , thiol , hydroxyl , and other hydrophobic
groups , peptide - based bolaamphiphiles exhibit diverse self - assembly
behaviors in responding to different environments . as shown in scheme 41 , das et al . developed a library of bolaamphiphiles
by varying the amino acids ( e.g. , phe , tyr , leu , or gly ) as the head
groups , and reported that sequential ph changes trigger the hydrogelation
of these peptide bolaamphiphiles ( e.g. , 326 with a gelation
concentration of 0.003 wt % ) . one interesting observation reported
by them is that the addition of dimethyl sulfate , a reagent used for
the methylation of phenols and esterification of acids via an sn2 reaction , can dramatically change the morphologies of the
hydrogels . systematically
studied the gelation properties , self - assembly motifs , chirality effects ,
and morphological characteristics of the gels formed by retro - dipeptidic
bolaamphiphiles similar to 326 , and the dimethyl ester
and dicarboxamide derivatives of these bolaamphiphiles.327 , comprising a dibenzofuran
template and two peptide strands made up of alternating hydrophilic
and hydrophobic residues and a blocked carboxyl terminal , represents
another class of bolaamphiphiles that have a collapsed u - shaped structure .
in the bolaamphiphiles 327 , the dibenzofuran template
positions the strands about 10 apart . these molecules afford
wide nanofibers , having a cross--sheet structure , in water
via intermolecular hydrogen - bonding and hydrophobic interactions . self - assembly of -conjugated small molecules
has attracted a lot of attention for potential use in organic electronic
devices , such as photovoltaic cells . recently , several laboratories
have introduced -conjugated nanostructures into peptide - based
bolaamphiphiles to replace the lipid chain in the linker segment . for example , stupp et al . designed a peptide - based bolaamphiphile
( 328 ) that has three segments ( e.g. , polar amino acids
for solubility , -sheet - forming amino acids for self - assembly ,
and an oligothiophene core for conductivity ) . the self - assembly of
this molecule results in a self - supporting hydrogel at low concentrations
( 1 wt % ) . the authors envisioned that , in combination with biological
epitopes , the 1d nanostructure formed in the hydrogelation process
may be used to simultaneously signal cells with electrical currents
and epitope receptor interactions . in the early 1990s , newkome et al . pioneered the development of
dendritic bolaamphiphiles as effective hydrogelators . since then , there has been considerable interest
in exploring the dendritic hydrogelators for self - assembly to form
hydrogels due to their highly controllable sizes , topologies , and
surface properties . one particular appealing attribute of peptide - based
dendrons or dendrimers is their extremely broad structural diversities
by varying the -amino acids used in their construction . as
shown in scheme 42 , woolfson et al . described an approach that utilizes nonlinear or
dendritic peptides , such as 329 , to direct the self - assembly
of two complementary linear peptides . the two peptides , which combine
to form exclusively linear fibers , coassemble with 329 ( at 100 m ) to form specific structures , such as hyperbranched
networks , polygonal matrixes , and regularly segmented and terminated
fibers . dendritic peptide 330 , with repeating hydrophobic and hydrophilic residues as well as
lysine terminals , self - assembles to give a uniform toroid structure . lee et al . , who developed this small molecule , also found that removing
the electrostatic repulsions or increasing the hydrophobic interactions
of this peptide drives the -sheet peptides to form 1d nanostrucutres . liu et al . reported an amphiphilic dendron
containing three dendrite l - glutamic acid units and a long
alkyl chain ( 331 ) . the dendron can form hydrogels over
a wide ph range ( from 2 to 13 ) . the lowest cgc is 2.2 mm at ph 2 ,
and when the ph value is increased , the cgc also increases . at ph
13 , chau et al . developed a two - component self - assembling
system ( 332 and 333 ) using the interaction
of aromatic groups ( fmoc ) to construct nanoparticles . the triskelion
fmoc conjugate 333 can quickly self - assemble to form
spherical particles around 70 nm in diameter at physiological ph and
at a concentration of 100 m . some self - assembling peptides
have a ( tert - butyloxy)carbonyl ( boc ) group on their
n - terminal . describe the formation of complex nanostructures
by the coassembly of two simple peptides , boc - ff ( 334 ) and ff ( 208 ) . they found that 334 itself
self - assembles to form nanospheres and 208 self - assembles
to give tubular structures , but being combined together , the two peptides
coassemble into a construction of beaded strings at concentrations
of each higher than 0.3 wt % . feng et
al . developed cyclic dipeptide 335 , conjugated with a
carbohydrate , and found that the solution of 335 transforms
into a transparent hydrogel with the assistance of shear force at
a concentration of 5.0 wt % . according
to the authors , cyclic dipeptides are a group of special peptides
with unique properties , and most of them afford hydrogels after shearing . studied the incorporation of a single -aminoethane
sulfonyl amide moiety in highly amyloidogenic peptide sequences ( e.g. , 336 ) and observed that this incorporation results in a complete
loss of amyloid fibril formation . instead , they found 336 affords supramolecular nanofibers at a concentration of 0.1 wt % . similarly , maggini et al . inserted an oligo(p - phenylenevinylene ) into a peptide to afford 337 . they found that ph changes trigger a reversible self - assembly of 337 , which has a cgc of 13 mm ( 2.3 wt % ) . tian et al . recently reviewed the complexion between metal
ions and a series of interesting peptides , which can also lead to hydrogelation . since the discovery of the dna double helix structure
over 60 years
ago , the interactions between base pairs have been a subject of interest
in the fields of cell biology and supramolecular chemistry . because
of their exceptional abilities for forming intermolecular interactions
in water , nucleobases are able to serve as the building blocks of
hydrogelators and have received considerable research attention . because
araki et al . reviewed the development
of nucleobase - containing gelators in 2005 , in this section we mainly
focus on supramolecular hydrogelators made of nucleobases over the
past decade ( table s3 ) . considering that
the most attractive feature of nucleobases is the intermolecular interactions
between base pairs , we arrange these nucleobase hydrogelators according
to the classification of homotypic and multicomponent hydrogels . as shown in scheme 44 , barthelemy et
al . prepared a family
of new uridine phosphocholine hydrogelators ( 338 ) which
self - assemble in water to form dna - like helical nanofibers and result
in hydrogels at a concentration around 6 wt % . one interesting feature
of this hydrogel is that the hydrogelators , below the phase transition
temperature ( tm ) , self - assemble to form
helical fibers which are transformed to compact bilayers above the tm . in addition , they also designed and synthesized
a series of glycosyl nucleoside lipids ( gnls ; 339 , 340 ) by using a convenient double - click chemistry . particularly , 340 self - assembles to form
circular nanofibers ( figure 7 ) that afford a hydrogel with a cgc of 0.1 wt % . it would be interesting to determine the mechanism
of the formation of those circular nanostructures . in another study ,
barthelemy et al . prepared a hydrogel of the gnls for trapping nanoparticles
or nanomaterial from an aqueous suspension , and suggested the use
of hydrogelators as an additive for decontamination . tem images of gel 340 ( scale bar 50 nm ) .
adapted from
ref ( 819 ) . copyright
2009 american chemical society . to compare the effect of hydrophilic / hydrophobic balance
on hydrogelation ,
kim et al . designed and synthesized
four nucleosides ( 341 , 342 ) by modifying
the 5-position of the uracil base with an ( alkylbenzyl)triazole unit . unlike 341 , which forms metastable partial gels in water
when the concentrations are higher than 2.5 wt % , 342 affords a stable hydrogel with a cgc of 1.0 wt % , likely due to
the hydrophobicity of the butylbenzyl group . kim et al . reported an intriguing example in which a 2-deoxyladenosine
derivative ( 343 ) forms aggregates in the process of heating
to cooling , but only forms a hydrogel under ultrasonic radiation .
the authors suggested a very interesting explanation : that the production
of oxidized species of 2-deoxyladenosine during sonication
might contribute to tuning the hydrophilic / hydrophobic balance to
result in a hydrogel . developed
an aminonucleoside phospholipid ( 344 ) which self - assembles
to form superhelical strands and results in a hydrogel at a concentration
of 6 wt % . they found that 344 binds with double - stranded
dna on the basis of stacking and h - bonding ,
so they suggested that this work has potential application in gene
delivery . marlow et al . designed and
synthesized a new cytidine - derived gelator ( 345 ) that
forms a gel in a mixture of methanol and water ( meoh : h2o = 1:1 ) at a concentration of 0.5 wt % . prepared a series of nucleobase peptide amphiphiles
in which 346 self - assembles to form nanofibers and results
in a hydrogel in water ( 5% dmso ) with a cgc of 0.5 wt % . on the basis
of the base pair interaction , the authors suggested this work provides
new avenues for nucleobase - specific electrophoresis and oligonucleotide
delivery . as shown in scheme 45 , lehn et al . reported an interesting
example in which guanosine hydrazide ( 347 ) affords a
stable hydrogel ( 0.46 wt % ) on the basis of the formation of a guanine
quartet ( g - quartet ) in the presence of various metal cations ( e.g. ,
na , k ) . the authors used various spectroscopies
( electronic and vibrational circular dichroism ) to reveal that 347 forms long - range chiral aggregates consisting of g - quartets
which result in columns due to the binding of metal cations between
g - quartet species . further studies
by the authors prove that 347 in solution confers a pseudo - four - stranded
helix with guanine guanine hydrogen bonding to form a continuous
helical strand rather than the usually planar g - quartet . moreover , 347 is capable of forming
reversible acylhydrazone bonds with various aldehydes so that the
nature of the aldehyde can tailor the macroscopic properties of the
resulting materials . for example , 347 reacts with pyridoxal
monophosphate to afford 348 , which forms a hydrogel ( 0.8
wt % ) in the presence of k.this seminal work illustrates a dynamic combinatorial library , based
on the proper aldehyde , for the selection of the strongest hydrogel
within a pool of certain building blocks . using dft calculations ,
urbanova et al . predicted and elucidated
the molecular arrangement of 347 in the gel state , and
reported that the predictions are in good agreement with the experimental
data . using nucleotides 349 and 350 as counterions
to interact with cationic gemini surfactants , oda and co - workers designed and synthesized nucleobase gemini
hybrids which are able to form hydrogels with a proper hydrophobic
chain length ( e.g. , 349 forms a hydrogel at a cgc of
0.64 wt % ) . intriguingly , the addition of complementary nucleosides
to the solution of 349 affords a stable hydrogel at an
even lower cgc ( e.g. , the addition of adenosine to 349 reduces the cgc to 0.32 wt % ) . by simply mixing a nongelator , 2,3,5-tri - o - acetylguanosine ( 351 ) , with a guanosine gelator
( 352 ) , rowan et al . prepared
a hydrogel in the presence of potassium . the resulting hydrogel exhibits
an extended lifetime and enhanced thermal stability compared with
that of 352 alone , likely due to the incorporation of
the more hydrophobic 351 into g - quartets . besides demonstrating
the ratio of the two components as a tool to tune the mechanical and
thermal properties of the hydrogels , the authors used a combination
of light scattering , small - angle neutron and x - ray scattering , and
viscometric experiments to study the mechanism of hydrogelation and
found that an increase in the volume fraction of microgel domains
ultimately leads to macroscopic gelation . by mixing guanosine ( 352 ) with 0.5 equiv of kb(oh)4 , davis et al . developed a guanosine further studies found that the resulting hydrogel is able
to selectively absorb a cationic dye and nucleosides via electrostatic
interaction and hydrogen bonding . employing ag as the
metal ion to coordinate with 5-guanosine monophosphate ( 353 ) , mann et al . prepared a
hydrogel based on the ag this hydrogel binds a cationic dye and protein ( e.g. , cytochrome c ) without the loss of biological activity , suggesting possible
use in controlled drug release and molecular recognition . in another
experiment , kumar et al . the freeze - dried gel shows a high swelling ratio of 326% and loading
capacity for methylene blue , suggesting that this hydrogel has potential
applications in drug delivery and other biological applications . li
et al . reported a new two - component
hydrogel based on thymidine ( 354 ) and melamine at a cgc
of 0.1 wt % . using ft - ir and x - ray diffraction , the authors confirmed
that the thymidine and melamine , via intermolecular hydrogen bonding ,
form supramolecular complexes . utilizing click chemistry to connect
benzyl azide and 8-aza-7-deaza-2-deoxyadenosine , seela et
al . prepared a nucleoside hydrogel of 355 at a concentration
of 0.3 wt % in water . recently
reviewed guanosine and isoguanosine derivatives ( 356 and 357 ) that self - assemble in water . this review provides useful information on different self - assembled
architectures generated by guanosine and isoguanosine scaffolds , including
recent examples of their use in the preparation of functional devices .
in another work , nachtsheim et al . reported n-(uracil-5-ylmethyl)urea
( 358 ) as a minimalistic hydrogelator which undergoes
phosphate - induced self - assembly , as evidenced by ir , uv / vis , and nmr
spectroscopy , electron microscopy , and rheological experiments . according
to the authors , it is a rare example of an anion - triggered self - assembly
in aqueous solution without additional aromatic or lipophilic groups . the macroscopic appearance of the hydrogels implies the formation
of microcrystals as the gel matrixes . adhikari and kraatz et al . designed and synthesized a hydrogelator
of deoxylguanosine ( 359 ) that affords a hydrogel with
a cgc of 0.57 wt % in the presence of ag . one interesting
feature of this work is that the cogel of 352 and 359 , being injectable , exhibits enhanced stability , an extended
lifetime , and self - healing properties . xu et al . designed two nucleopeptides ( 360 and 361 ) that alone fail to form hydrogels , while the mixture
of the two nucleopeptides self - assembles to form nanofibers and result
in a hydrogel . one intriguing feature of this work is that the resulting
heterodimer dramatically enhances the proteolytic stability of these
nucleopeptides . bearing
multiple hydroxyl groups , saccharides provide hydrogen bond donors
and acceptors for intermolecular interactions that are critical for
molecular self - assembly in water to result in hydrogelation . moreover ,
the inherent hydrophilicity of saccharides allows the easy dissolution
of the hydrogelators prior to the triggering of the hydrogelation
by changing the ph , temperature , or ionic strength . in this section ,
we categorize the saccharide - based hydrogelators into monosaccharide - based
and oligosaccharide - based hydrogelators ( table s4 ) . as shown in scheme 46 , lu et al . designed and synthesized a hydrogelator ( 362 ) containing a phenyl -d - glucopyranoside which self - assembles
to form a tubular structure and results in a hydrogel at a concentration
of 0.2 wt % . they also prepared hydrogels
based on -d - glucopyranoside substituent thiosemicarbazide
derivatives 363 , and used the hydrogels
as templates for fabricating netlike cds nanofibers . reported a series of low molecular weight gelators ( 364 , 365 ) based on simple glycolipids which are capable
of forming gels in a water / alcohol mixture at a concentration of 0.15
wt % . hamachi et al . developed a supramolecular hydrogelator
( 366 ) composed of n - acetyl - d - glucosamine which self - assembles to form nanofibers and results
in a hydrogel with a cgc of 0.1 wt % . this work pioneered the use of a supramolecular hydrogel array to
monitor enzymatic reactions . according to the authors meanwhile ,
hamachi et al . also developed a photoresponsive gel droplet based
on the hydrogelator 367 and demonstrated the use of light
to trigger mass transport in gels . moreover , their further studies found that 366 , mixed with an appropriate amount of 368 ( e.g. , 366:368 = 1:1 ) , affords a ph - responsive shrinkage / swelling
supramolecular hydrogel at a concentration of 0.5 wt % . particularly , the authors envisioned that this
approach is useful for triggering the release of hydrophilic drugs . reported an intriguing example in
which a tetrameric d - xylofuranuronic acid derivative containing
an azobenzene core
( 369 ) , assisted by initial dissolution in a small amount
of dmso , exhibits pronounced hydrogelation at 0.1 wt % . since the
hydrophobic azobenzene group is packed inside the gel state , the resulting
hydrogel displays a remarkable photostability under uv irradiation . moreover , the addition of a salt ( e.g. , cacl2 ) to the hydrogel
changes the morphology of the networks in the hydrogel from globular
spongy to rodlike reported a d - glucopyranoside - based
hydrogelator ( 370 ) by incorporation of an unsaturated
diacetylene unit as the hydrophobic group . after its self - assembly
in water , 370 forms well - defined helical ribbons with
diameters of 20150 nm , which results in a hydrogel with a
cgc of 0.5 wt % . upon uv irradiation at 254 nm , 370 forms
typical nanofibers . on the basis of cd spectra and other measurements ,
the authors suggested that photopolymerization turns the well - ordered
bilayer structure into disordered molecular packing . also designed other hydrogelators ( e.g. , 371 ) which self - assemble to form fibrils with diameters of
1038 nm . meanwhile , the hydrogel based on 371 with crown ether can serve as a template to prepare various structures
of silica nanomaterials . by functionalization
of a commercially available glucose derivative ,
wang et al . designed and prepared a series
of d - glucose - based hydrogelators ( 372 ) ( scheme 47 ) . among them , 372 ( n = 5 , 12 ) exhibits an excellent gelation
ability and affords a hydrogel at a concentration of 0.4 wt % . further
studies found that the conjugates containing
a terminal acetylene and an aryl group exhibit an enhanced ability
of gelation . one interesting feature of this work is that these gelators
form birefringent fibers and tubules .
by connecting the d - glucamine derivatives and hydrophobic
unit via ureido or bis(ureido ) moieties , cintas et al . wan et al . reported a new saccharide - appended hydrogelator
of 4-(4-butoxyphenyl)phenyl -o - d - glucoside ( 374 ) which self - assembles to form
planar ribbons with widths ranging from tens to thousands of nanometers
and results in a hydrogel in water with a cgc of 0.5 wt % . yamanaka
et al . designed and synthesized a d - glucose - based hydrogelator ( 375 ) which self - assembles
to form a hydrogel with a cgc of 0.25 wt % in the presence of tris the intriguing feature of this work is that the hydrogel can
serve as an electrophoresis matrix to separate the native protein . this underexplored application appears to have much potential . as shown in scheme 48 , takaguchi et al . reported
a unique
example of an anthracene - based photoresponsive hydrogelator ( 376 ) which self - assembles in water to afford a hydrogel with
a cgc of 2.9 wt % and a tgel of 46 c . upon photoirradiation , the hydrogel is transformed to a solution due
to the dimerization of the anthryl moieties . reported an intriguing example in
which n-(-d - glucopyranosyl)-n-[2-(glycylglycylglycinamido)ethyl]octadecanediamide
( 377 ) self - assembles to form nanotubes with a 9 nm inner
diameter and results in a hydrogel at a concentration of 0.5 wt %
and ph 8.0 . notably , the fixed green fluorescent protein ( gfp ) and
myoglobin ( mb ) in the hollow cylinders of the nanotubes exhibit remarkable
resistance to denaturants such as guanidinium chloride and urea at
high concentration . furthermore , kameta et al . also found that the nanotubes of this hydrogelator ( 377 ) are able to act as artificial chaperones to assist the
transformation of encapsulated proteins into their refolded states
via simply changing the ph values . the authors suggested that the
modification of the diameter and inner surface of the nanotubes enhances
the efficiency of encapsulation and refolding of the proteins . tritt - goc et al . prepared
an -d - glucopyranoside - based hydrogel ( 378 ) at a concentration
of 1.5 wt % . the authors measured the thermal properties of the resulting
hydrogel and determined the gel sol transition enthalpy as
43 kj / mol . by conjugating pyrene and a glucose derivative , fang et
al . reported a superhydrogelator ( 379 ) which affords a hydrogel in water with a cgc of 0.07
wt % . zhang et al . designed
and synthesized an -d - glucopyranoside - based hydrogelator
( 380 ) containing an aldehyde group which forms a hydrogel
with a cgc of 0.8 wt % . due to the existence of aldehyde and acetal
groups , the resulting hydrogel not only responds to the ph , but also
reacts with cysteine , which may lead to a new approach to design smart
delivery systems . birchall and edward et al . reported a class of supramolecular hydrogels ( 381 and 382 ) derived from glucosamine and fmoc . notably , the authors
suggested that ch interaction , rather than
stacking and h - bonding , drives the self - assembly and subsequently
hydrogelation . pfannemuller and welte et al . reported that n - octyl - d - gluconamide ( 383 ) is able
to form a hydrogel which is converted to crystallites over a few hours . by adding a nonionic surfactant ( e.g. , polyethylene
glycol ( peg ) ) , rowan et al . elucidated
not only that the resulting hydrogel is stable for more than one year ,
but also that changing the ratios of the components in the gel allows
systematic tuning of the thermomechanical properties of the hydrogels . one notable result is that hydrogelator 383 exhibits
potent activity to inhibit ice crystal formation at a concentration
of 0.5 mm . ikeda and hamachi et
al . reported an intriguing
example in which a well - designed bolaamphiphile ( 384 )
forms a hydrogel via retro - diels alder reaction
induced by heat . the authors used tem to show the morphology transition
from a twisted ribbon to a helical ribbon , and suggested that this
simple and versatile molecular design should produce smart materials
for various applications . altenbach et al . designed and synthesized a d - glucose - based hydrogelator
( 385 ) via two simple steps . the resulting hydrogelator ,
acting as a surfactant and emulsifier , affords a hydrogel with a cgc
of 2.5 wt % . hamachi et al . prepared
a glycolipid - based supramolecular hydrogelator ( 386 )
that forms a hydrogel with a cgc of 0.1 wt % . the most interesting
feature of this work is that the resulting hydrogel exhibits a color
change when heated or upon the addition of relevant glycosidases to
induce a gel sol transition . employing click chemistry , mishra
and rao et al . reported a glucose - based
lipid ( 387 ) that forms a hydrogel with a cgc of 0.03
wt % in a mixture of water and methanol ( 50:50 ) . noto et al . designed and synthesized a molecule ( 388 ) which affords a hydrogel with a cgc of 1.0 wt % in the
presence of -cyclodextrin . meanwhile , simply changing the ratio
of cyclodextrin and 388 can easily tune the gelation
properties . as shown in scheme 49 , britt et al . prepared the conjugates 389 and 390 of lactose and fatty amine or fatty
acid , which act as
gelators and afford gels in a mixture of water and propanol ( 50:50 ) . thompson et al . designed and synthesized a series of -cyclodextrin aldonamide
conjugates . upon the addition of glucose , the solution of hexaaldonamide - substituted
-cyclodextrin turns into a hydrogel . it was suggested to be
useful in developing glucose sensors and glucose - sensitive drug delivery
systems . oriol et al . designed and synthesized a class
of maltose - based supramolecular hydrogelators ( 391393 ) via click chemistry . 391 and 393 self - assemble in water to form typical ribbons with a left - handed
twist and result in hydrogels with a cgc of 1.0 wt % , while 392 forms ribbons with a right - handed twist and affords a
hydrogel with a cgg of 0.5 wt % . by incorporating azobenzene into
the gelator 391 , oriol et al . also prepared another hydrogelator ( 394 ) that forms
a stable hydrogel with a cgc of 5.0 wt % . the authors suggested that the dense packing of azobenzene
in the gel state hinders the photoisomerization , which may serve as
a useful caution for designing photomechanical actuators based on
gels . the resulting hydrogelator is able to gel water at a concentration
of 3.0 wt % and 12 c . prepared a family of urea glycolipid - based
hydrogelators ( 396 and 397 ) and found that 396 forms a hydrogel at a concentration of 0.5 wt % , while
its stereoisomer 397 fails to form a hydrogel . this observation underscores that the orientation
of the hydroxyl group in the saccharide has a profound influence on
the self - assembly of this class of hydrogelators . in recent years , on the basis of the increased
understanding of
protein functions from cell biology and structural biology , considerable
efforts have focused on the incorporation of peptide epitopes as the
functional motifs on supramolecular hydrogelators for a wider range
of biological applications . these endevors also stimulated
the determination of the protein targets of supramolecular hydrogels . in this section , we mainly focus on the recent advances in the design
and development of supramolecular hydrogels for biological and biomedical
applications . since most of the biomedical applications
demand multiple functionalities of the hydrogel network and dynamic
interactions between the surrounding matrixes and cells , we first
discuss hydrogelators for cell - related applications , followed by fluorescent
hydrogelators used for imaging , and then hydrogels for tissue engineering ,
drug delivery , immunomodulation , and wound healing . we finally describe
the unique applications of supramolecular hydrogels and hydrogelators
in a cell environment . because
the most obvious features of hydrogels are soft and wet , which resemble
the cellular environment , it is not surprising that the most attempted
applications of the hydrogels are to mimic the ecm for cell culture
( or tissue engineering ) , and the necessary initial test for a hydrogelator
is its cell compatibility . since spps allows
oligomeric peptides to be made quickly , most of the hydrogelators
examined for cell - related applications are self - assembling peptides
or peptide derivatives . in the following subsections , we discuss various
hydrogelators that have been evaluated for the applications related
to cells , such as hydrogelators for cell culture , cell - compatible hydrogelators , cytotoxic
hydrogelators , and hydrogels for cell adhesion . ulijn et al . reported
the first case of cell culture using fmoc - dipeptides . as first reported by xu et al . , the hydrogel of fmoc - gly - gly - oh ( 398 ; scheme 50 ) consists of nanofibers with average diameters of 33 nm and exhibits
a cgc of 0.15 wt % at ph < 4 . later ,
ulijn used the hydrogel of a mixture of 398 and fmoc - phe - phe - oh
( 6 ) for the 2d or 3d cell culture . in addition , at ph 7 , 6 itself forms hydrogels
with a concentration between 0.22 and 2.14 wt % which contain polydispersed
nanofibers with average diameters of 56 nm . on the basi sof the fact
that the fmoc - dipeptide building blocks are approximately 2 nm in
length , the authors suggested that the nanofibers consist of bundles
of supramolecular stacks . although the results of cell viability indicate
that 6 shows a relatively high cytotoxicity to bovine
chondrocyte cells or caco-2 and hgf-1 cells after 7 days , ulijn et al . demonstrated that 6 can
still be applied to 2d and 3d cell culture . according to the authors , the hydrogel is stable under cell culture conditions and consists
of nanofibers that have dimensions similar to those of the fibrous
components of the ecm . . also studied two other diphenylalanine analogues ,
nap ( naphthalene)-phe - phe - oh ( 3 ) and cbz ( ( benzyloxy)carbonyl)-phe - phe - oh ( 399 ) , and
compared their self - assembly properties and cell culture applications
with those of 6 . after demonstrating that all three hydrogelators
form hydrogels consisting of nanofibers with -sheet arrangements
and varying fibril dimensions , the authors used ldh ( lactate dehydrogenase )
assays to prove that all three structures can support cell proliferation
and cell culture of chondrocytes in both two and three dimensions
for up to 10 days . on the basis of the
early work of ulijn et al . , liebmann et al . evaluated the hydrogel
of 6 as 3d cultures of cos-7 and mdck cells for 7 days . however , 6 still has limitations ,
especially in terms of long - term gel performance , stability , and cytotoxicity
when being used for culturing other cell types ( e.g. , skin cells such
as human dermal fibroblasts and mouse 3t3 cells ) . mixed 6 with positively charged fmoc - lys - oh ( 202 ) , uncharged / polar fmoc - ser - oh ( 400 ) , and negatively
charged fmoc - glu - oh ( 203 ) in the same hydrogel for examining
the proliferation of chondrocytes , 3t3 , and human dermal fibroblast
( hdf ) cells . besides confirming that
these heterotypic hydrogelators undergo self - assembly to form fibrous
scaffolds by mainly adopting an antiparallel -sheet arrangement ,
the authors used the live / dead staining assay to show that these three
types of mixed hydrogels maintain the viability of bovine chondrocytes . the hydrogel of 6 + 400 ( fmoc - ff / fmoc - s )
and the hydrogel of 6 + 203 ( fmoc - ff / fmoc - e )
are compatible with hdf cells , and only the hydrogel of 6 + 400 ( fmoc - ff / fmoc - s ) supports the proliferation of
3t3 fibroblast cells . besides the above
three fmoc - peptide mixtures , ulijn et al . designed
another hydrogel , a mixture of 6 and fmoc - arg - gly - asp - oh
( fmoc - rgd , 401 ) , as a 3d scaffold for hdf cells . they
found that this mixed hydrogel provides a highly hydrated , stiff nanofiber
network with -sheets interlocked by
stacking of the fmoc groups . the authors suggested that the rgd motif
plays a dual role : as a structural component that locates at the surface
of the unique , interwoven cylindrical nanofiber structure and as a
biological ligand that forms the specific rgd integrin binding
to promote adhesion , spreading , and proliferation of cells . using a similar design principle , hamley et
al . found that 401 itself forms a self - supporting hydrogel
consisting of well - defined amyloid fibrils with -sheet features
at a concentration of 2 wt % . in addition , the preliminary cell culture
experiments showed that 401 can be used to culture bovine
fibroblasts . on the basis of the
earlier report of a hydrogel made of a hexadecapeptide
( rada16,248a ) , hirose et
al . used the 248a self - assembling peptide solution ( puramatrix ) to evaluate the osteogenic differentiation of mesenchymal stem cells
( mscs ) that are derived from rat bone marrow . the authors reported
that over 80% of the mscs in the hydrogel are alive and have spread
within the hydrogel of 248a , and suggested that 248a acts as a scaffold for three - dimensional culture of mscs . the authors observed a significantly higher expression of alkaline
phosphatase ( alp ) activity and osteocalcin ( oc ) contents at both the
protein and mrna levels for 3 or 4 weeks , and thus concluded that
mscs in the 248a hydrogel differentiate into mature osteoblasts ,
followed by the growth of a mineralized extracellular matrix . although
it is suggested that the biodegradable / biocompatible hydrogel 248a may become an attractive option in bone tissue engineering , the complexity of the bone remolding and growth
process likely requires more than one component in the hydrogel . the commercial availability of 248a allows many research laboratories to evaluate the use of the hydrogel
of 248a for 3d cell culture . for example , zhao et al . studied the cellular behavior of human
lung cancer cells a549 within a 248a nanofiber scaffold . they found that the cells show morphologies in a 3d scaffold different
from those on a 2d petri dish , an observation that is consistent with
rada being a cell adhesion motif . mixed 248a and rgda16 ( ac - radargdaradargda - conh2 , 402 ) solutions at a concentration of 10 mg / ml
( 1% , w / v ) and found that the mixture scaffold can significantly promote
the cell attachment and proliferation of mc3t3-e1 cells compared with
the 248a scaffold . semino
et al . used the hydrogel of 248a functionalized with
biologically active motifs ( e.g. , grgdsp , 403 , or yigsr , 404 ) to replace the use of collagen i in the traditional culture
sandwich technique for maintaining functional hepatocytes in vitro . used the mixture of the
peptide solutions of rln ( 405 ) and 248a to
guide rabbit nucleus pulposus cells ( npcs ) , and demonstrated that
npcs migrate from the surface into the hydrogel in the 3d cell culture
experiments and exhibit stimulated synthesis of the ecm . they reported
enhanced angiogenesis in vitro and in vivo , and suggested that the
observation results from low - affinity integrin - dependent interactions
of microvascular endothelial cells ( mvecs ) with the rad motifs . these results imply that the development of
multicomponent hydrogels may address several limitations of single - component
hydrogels . established 3d hepatic cell cultures in the hydrogel to improve the
3d phenotype of hep g2 cells , a human liver carcinoma cell . the authors
reported that hep g2 cells formed multicellular spheroids which consist
of filamentous actin accumulation and large tubular bile canalicular
structures to indicate apicobasal cell polarity . realizing the merits of multicomponent hydrogels ,
collier et al . designed and examined multipeptide coassembling hydrogels based on
peptides rgds - q11 ( 408 ) and ikvav - q11 ( 409 ) consisting of two segments : a nanofiber - forming peptide , q11 ( 407 ) , which self - assembles to form a -sheet , at the
c - terminal and a ligand of integrins , rgds or ikvav , at the n - terminal . the authors suggested that such a design allows the ligands to be
presented on the surface of the nanofibers . in coassemblies of the
ligand - bearing peptides containing 407 , the amount of
the incorporated ligands is able to influence the attachment , spreading ,
morphology , and growth of human umbilical vein endothelial cells ( huvecs )
without significantly altering the materials properties , such
as fibrillization , -turn secondary structure , or stiffness . the authors reported that while 408 , being coassembled
into the gels of 407 , specifically increases huvec attachment ,
spreading , and growth , 409 exerts a more subtle influence
on the attachment and morphology of the cells . additionally , they
reported that 407 and 408 are minimally
immunogenic in mice , making the 407-based biomaterials
attractive candidates for applications in vivo . recently , on the basis of the sensitivity of 407 to the ionic strength , collier et al . developed a microgel
made of rgd - q11 ( 410 ) by triggering peptide self - assembly
within the aqueous phase of water - in - oil emulsions . according to the
authors , one of the advantages of microgels is that they can be embedded
within other self - assembled peptide matrixes for generating composites
of different peptide formulations . the authors , indeed , demonstrated
an example of microgels that are cytocompatible and encapsulate nih/3t3
fibroblasts ( figure 8) and c3h10t-1/2 mouse pluripotent stem cells with good viability . the viability of nih/3t3 cells encapsulated in 30 mm 407 microgels was quantified with calcein / ethidium homodimer
staining . the assay was conducted 2 h after the incubation of ( a ) 1 day , ( b )
2 days , and ( c ) 3 days . the scale bar in ( a ) represents 100 mm . copyright
2011 royal society of chemistry . on the basis of their seminal works of the applications of
peptide
amphiphiles for cell cultures , stupp et al . designed and synthesized a peptide amphiphile molecule , 411 ( scheme 51 ) , containing both the photocleavable 2-nitrobenzyl group as well
as the bioactive epitope arg - gly - asp - ser ( rgds ) . the 2-nitrobenzyl
group of 411 can be photocleaved to afford 412 , which self - assembles to form high - aspect - ratio nanofibers in the
presence of charge - screening salts . in vitro experiments with nih/3t3
mouse fibroblasts indicate that 412 and the byproducts
of the photoreaction at a concentration of 7.9 10 m are not toxic to the cells and that cell proliferation is normal
after the irradiation . in another case ,
stupp et al . reported a photoresponsive , synthetic ecm mimic through
linking peptide amphiphiles 413 to the ecm - derived cell
adhesion epitope rgds by a photocleavable nitrobenzyl ester group . this derivative self - assembles to form cylindrical nanofibers , and
light irradiation on the photolabile group in the peptide backbone
efficiently removes the rgds epitopes without affecting the nanofibers . the authors demonstrated that the adhesion of mouse nih/3t3 fibroblast
cells on the surface of peptide amphiphile ( pa ) hydrogels can be dynamically
controlled by rapid photolytic removal of the rgds peptide from the
supramolecular nanofibers . as shown in scheme 52 , schneider and
pochan et al . reported a hydrogel based on tss1 ( 414 ) ,
a de novo designed three - stranded -sheet . 414 ,
containing 29 amino acids with 12 lysine residues and
12 valine residues , undergoes thermally triggered folding and self - assembly
to afford a network of well - ordered -sheet - rich fibrils that
constitute a mechanically rigid hydrogel . a gelation test indicated
that 414 remains unfolded at lower temperatures but folds
and self - assembles into rigid hydrogels upon raising the temperature
of the aqueous solutions ( ph 9.0 or 7.4 ( 150 mm nacl ) ) of 414 . tem images and sans show that 414 self - assembles into
monodispersed fibrils with a width of around 3 nm , which corresponds
to the width of the peptide in its folded state . the authors demonstrated
the in vitro culture of c3h10t1/2 mesenchymal stem cells on the gel
surface for 24 h , and suggested that the surface of the hydrogel supports
cell adhesion and allows cell migration . schneider and pochan et al . developed a class of self - assembling
-hairpin peptides to create physical hydrogels as injectable therapeutic delivery
vehicles . on the basis of their works on peptide hydrogels , the authors
studied the behavior of -hairpin peptide - based hydrogels max1
( 251 ) and max8 ( 257 ) during and after flow . importantly , the authors verified that the observed shear - thinning
and rehealing , after flow , represent the authentic bulk gel properties .
in another experiment , the author utilized the hydrogel of 257 ( at a concentration of 0.5 wt % ) to encapsulate mg63 cells , a progenitor
osteoblast cell line from rat . however , 3 h after injection , some
cells were already dead in the 3d gel cell construct . a scaffold with biocompatibity and in vivo
stability needs to be designed in the future . conjugating peptide
epitope ikvav from laminin to a peptide amphiphile ,
c16h31o - a3g4d2 ( 415 ) , song et al . generated peptide amphiphile c16h31o - a3g4d2ikvav , 416 , which is similar to the
ikvav peptide amphiphiles reported by stupp et al . after observing that a 1 wt % concentration of the peptide
amphiphile self - assembles to form a hydrogel in cell media , the authors
investigated 2d and 3d culture of neural stem cells ( nscs ) using the hydrogel ,
and found that mice nscs proliferate and differentiate into neurofilament
( nf)-positive neurons and glial fibrillary acidic protein ( gfap)-positive
astrocytes on the surface of the hydrogel . synthesized
a peptide with the sequence of kld-12 ( 417 ) and found
that 417 , at 0.5 wt % , self - assembles to produce a hydrogel
consisting of nanofibers ( diameters of 3040 nm ) . the authors
reported that rabbit mscs , being encapsulated within the hydrogel
of 417 for 3d culture for 2 weeks , grow well and proliferate
with the culture time . kim et al . reported
that mscs encapsulated in the hydrogel of 417 decelerate
the progression of cartilage destruction in osteoarthritis in a rat
knee model . the authors suggested that the beneficial effect may result
from the prevention of chondrocyte apoptosis , the alteration of the
subchondral bone mineral density , a reduction of inflammation , and
a potential chondrogenic mechanism . gelain
et al . developed a co - assembly of peptides , which are analogs of 417 , for culturing neuronal cells . designed and synthesized a series of amphiphilic
multidomain peptides ( mdps ; 418 , 419 , and 420 ) with an innovative modular aba block motif in which the
amphiphilic b block drives self - assembly and the flanking a blocks
bear charges for controlling the conditions of self - assembly . in their
peptide design , the authors created four different variants with a
matrix metalloprotease 2 ( mmp-2)-specific cleavage motif , an rgds
adhesion sequence , and either one or two lysine residues in the flanking
regions . the lyophilized peptides self - assemble to form hydrogels
after being dissolved in a sucrose solution . with a final peptide
concentration of 1.0 wt % , the hydrogels consist of -sheet
fibrils formed by the cross - linking of lysine - containing peptides
due to the presence of negatively charged phosphate ions in the buffer . the authors illustrated that the structures of the peptides control
the lengths and diameters of self - assembled nanofibers , the gelation
conditions , and the viscoelastic properties of the formed hydrogels ,
which highlights the promises of this approach for materials and biological
applications . more interestingly , in an in vitro experiment with mesenchymal
stem cells from human exfoliated deciduous teeth ( shed ) , the authors
demonstrated that the incorporation of an mmp-2-specific cleavage
site and a cell adhesion motif increases the cell viability , cell
spreading , and cell migration into the hydrogel matrix . later , the authors designed another mdp , e2(sl)6e2grgds ( 421 ) , which self - assembles to form -sheet nanofibers approximately
8 nm wide , 2 nm high , and micrometers in length in the presence of
mg . the corresponding hydrogel undergoes shear thinning
and recovers nearly 100% of its elastic modulus after shearing , making
it ideal for being used as an injectable material . interestingly ,
in the in vitro experiments with human embryonic stem cells ( escs ) ,
the hydrogel acts like a sponge , soaking up most of the growth factors
and cytokines released by the escs . using in vivo experiments , the
authors demonstrated a promising application of the hydrogel as
a depot to release stem cell secretome gradually over time . by changing the serine residues in the amphiphilic
region to threonine , hartgerink et al . also designed another mdp ,
k(tl)2slrg(tl)3kgrgds ( 422 ) , which forms porous hydrogels with antiparallel -sheet
nanofibers . the authors also used this hydrogel to encapsulate the
shed cells ( figure 9 ) , and observed more fibroblast - shaped cells after 7 days in culture . confocal microscopy
of shed cells 1 , 3 , or 11 days after 3d encapsulation
in 422 hydrogels . aggeli et al . reported
a class of positively charged tape - forming
and gel - forming amphiphilic peptides in physiological solutions , all
of which bear 2 positive charges and 11 amino acid residues . by changing
the peptides to be amphiphilic or completely polar , they systematically
synthesized several derived peptides . each of them has a different
polar uncharged group : p11 - 8 ( 423 , based on
glutamine q , sequence ac - qqrfowofeqq - nh2 ; o represents
ornithine ) , p11 - 12 ( 424 , based on serine s ,
sequence ac - ssrfowofess- nh2 ) , p11 - 16
( 427 , based on asparagine n , sequence ac - nnrfowofenn-
nh2 ) , and p11 - 18 ( 428 , based on
threonine t , sequence ac - ttrfowofett- nh2 ) . they
found that all of these amphiphilic peptides carrying a + 2 charge
at neutral ph form self - supporting gels at concentrations above 25
mg / ml ( ca . 1.8% , w / v ) in physiological solutions . all these hydrogels
contain a network of semiflexible , micrometer long nanofibers . in
addition , all of these self - assembling peptide hydrogels show biocompatibility
with l929 murine fibroblast cells , on the basis of the contact cytotoxicity
test . however , only 423 supports l929 cell growth in
3d cell cultures inside 2% ( w / v ) gels for 14 days without observation
of macroscopic degradation of the peptide gel matrix during the experiment ,
while the other peptides are unable to support cell growth . later , aggeli and ingham et al . designed complementary
self - assembling peptides comprising the unimers p11 - 13 ( 425 ) and p11 - 14 ( 426 ) , which exhibit
negative and positive charges , respectively , under physiological conditions . being mixed in equal quantities , they instantaneously form a self - supporting
hydrogel that consists of long fibrils with widths ranging from 10
to 20 nm . although the hydrogels of 425 + 426 appear to be cytocompatible with primary human dermal fibroblasts ,
they fail to support the proliferation of this cell type , and the
cell numbers began to decline after 7 days . as shown in scheme 53 , yang et al . reported several supramolecular hydrogels based
on
adamantane - derivatized peptides that respond to the presence of -cyclodextrin
( -cd , 155 ) motifs . the authors used dithiothreitol
( dtt ) or gsh to reduce the disulfide bond in the precursors ( 429 ) to convert the solutions to hydrogels for encapsulations
of cells and drugs . sol transition upon the addition
of 155-containing molecules , the authors applied them
for cell culture and postculture cell recovery in an in vitro experiment
of mouse fibroblast 3t3 cells . later ,
yang et al . designed several other hydrogels formed via gsh reduction ,
and demonstrated the use of the concentrations and structures of the
hydrogelators to regulate the mechanical properties and potential
of the hydrogels . among these hydrogels , 430 , having
a storage modulus ( g ) of hundreds of pascals ,
is suitable for 3t3 cell spreading and proliferation . in addition , the authors also reported the
formation of 431 , a curcumin - based hydrogelator , after
disulfide bond reduction , to inhibit cancer cells and tumor growth
in vitro and in vivo . taking advantage
of the self - assembling ability of phe - phe , yang et al . have developed
useful hydrogels made of d - peptides as potential adjuvants for hiv
vaccine or for self - assembling on cell
surface . as shown in scheme 54 , zhang et al .
reported an intriguing example tin which a hydrogel
of dipeptide tet - ga ( 432 ) serves two functions : as the medium for 3d cell culture and as
the carrier for the delivery of mirna into live cells . the authors
found that 432 is able to form a transparent and stable
hydrogel at concentrations higher than 0.15 wt % in pbs buffer . after
confirming the cell compatibility of the hydrogelator , the authors
used repressed target gene expression in an in vitro experiment to
indicate the delivery of the mirna , encapsulated together with cells
in the hydrogel matrix , into the encapsulated cells . luo et al . reported
a hydrogel made of a d - form peptide , d - eak16 ( 433 ) , for 3d cell cultures . after confirming the proteolytic resistance
of the d - peptide , the authors used the hydrogel of 433 for 3d cell culture and reported the human hepatoma cell
smmc-7721 to show a high cell viability and low - level cell apoptosis
for weeks in the hydrogel . . reported an efk8-based small peptide , ac - fefkfefk - cs - eee
( 434 ) , that self - assembles to form a hydrogel via disulfide
bond reduction with a concentration of 0.5 wt % at physiological ph .
using the live / dead assay , the authors demonstrated that the hydrogel
of 434 is suitable for the 3d cell culture of nih/3t3
cells . furthermore , saiani et al . reported
the use of thermolysin , a protease , to trigger the gelation of fefefkfk
octapeptide ( 228 ) , which is able to encapsulate human
dermal fibroblast cells for 3d cell culture for 5 days . by replacing the alanine residue in eak16 ( 435 ) with a more hydrophobic residue , leucine , lu et al . designed
a new peptide , elk8 ( 436 ) , and directly attached three
kinds of functional motifs ( e.g. , an osteogenic growth peptide , an
osteopontin cell adhesion motif , and a two - unit rgd binding sequence )
to the c - terminal of 436 . using in vitro experiments ,
several labs illustrated that the mixtures of these peptides ( 228 , 435 , and 436 ) are suitable
for the 3d cell culture of mouse preosteoblast mc3t3-e1 cells and
promote the attachment , proliferation , and osteogenic differentiation
of those cells . hamachi et al . designed
a series of glycolipid mimics with muconic
amide as the spacer and found that 437 , consisting of n - acetylglucosamine as its hydrophilic head and methylcyclohexyl
groups as hydrophobic tails , forms a stable hydrogel with a cgc of
0.05 wt % . besides using tem to elucidate that hydrogelator 437 self - assembles into a helical , bilayer - type nanofiber
with a well - defined network of nanofibers of a high aspect ratio ,
the authors unexpectedly found that polystyrene nanobeads ( 100500
nm in diameter ) greatly facilitate the homogeneous 3d dispersion of
the supramolecular nanofiber networks . since 437 also forms a stable hydrogel in cell culture media , such as rpmi1641 or
dulbecco s modified eagle s medium ( dmem ) , the resultant
hybrid supramolecular matrix efficiently encapsulates and distributes
live jurkat cells ( a human t cell lymphoblast - like cell line ) in 3d
cell culture under physiological conditions . recently , hamachi et al . also reported photoresponsive hydrogels
for controlling cell motions and chemically
responsive hydrogels for enhancing analyte sensitivity . chou
et al . proposed a simple and economical methodology to synthesize
dimeric cholesterol derivatives ( dcds ) with high yields . the dynamic
light scattering analysis and tem images showed that in aqueous solution
most dcd dispersions are irregularly angular shaped with two peaks
in the size distribution centered at 204 and 837 nm . in addition ,
an mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based
cell viability assay indicated that 438 ( scheme 55 ) is innocuous to human keratinocyte
( hacat ) and squamous cell carcinoma ( scc25 ) cells at a concentration
of 0.05 mm after 24 h incubation with 438 . as a representative example of the de novo
-hairpin peptides , 251 folds as a -hairpin
peptide upon the addition of saline solution , and the -hairpin
peptide self - assembles to form a hydrogel at 2 wt % . thus , dmem cell
culture media initiate the folding and consequent self - assembly of 251 to afford a hydrogel which is cytocompatible with nih/3t3
cells . woolfson et al . reported supramolecular hydrogels formed
by rationally
designed standard linear peptides ( 439444 ) . consisting of two 28-residue peptides designed to coassemble ,
the pair of peptides results in an offset -helical dimer with
complementary sticky ends which promote longitudinal assembly into
-helical coiled - coil fibrils bundling to form nanofibers . on
the basis of the coiled - coil heptad repeat that is rich in alanine
( for hydrophobic interaction ) and glutamine ( for hydrogen bonding )
residues , the authors demonstrated that the glutamine - rich peptide
( 441 + 442 ) forms a gel at low temperature
which melts on warming , whereas the alanine - rich peptide ( 439 + 440 ) forms a weak gel at low temperature that strengthens
on warming . by replacing one of the surface - exposed alanine residues
with the more hydrophobic tryptophan , the authors obtained 443 and 444 , which form hydrogels that support the proliferation
and differentiation of rat adrenal pheochromocytoma ( pc12 ) cells . also extended the family of the aromatic fmoc - dipeptides
with a series of new fmoc - peptides which consist of natural and synthetic
amino acids with an aromatic nature for making supramolecular hydrogels .
with the assistance of dmso as the cosolvent , the authors produced
the hydrogels at a final peptide concentration of 0.5 wt % . tem and
sem analysis indicated that the self - assembly of these fmoc - peptides
results in various structures and distinctive molecular and physical
properties . a pair of notable peptides in their work are fmoc - frgd
( 445 ) and fmoc - rgdf ( 446 ) , which self - assemble
to form -sheet - based nanofibers . using the mtt assay , the authors
demonstrated that chinese hamster ovary ( cho ) cells on the hydrogels
of 445 and 446 show a high viability after
24 h. however , the cell viability decreases significantly at 72 h. palocci et al . used a lipase to trigger
the self - assembly of peptide
hydrogels of fmoc - fff ( 447 ) via reverse hydrolysis to
control or modulate the functions and responses of the hydrogels according
to their preparation conditions . under physiological conditions , the
authors obtained amphiphilic building blocks consisting of tripeptides
( phe - phe - phe ) linked to fmoc . sem and afm images indicated that the
hydrogels of 447 consist of a nanofiber network at 0.14
wt % . on the basis of the cell viability
of rat microglial cells incubated with 447 at concentrations
up to 300 g / ml later , the authors reported that the hydrogelator
also stimulates the production of neurotrophic factor ngf ( nerve growth
factor ) from the microglial cells . liu
et al . reported a hydrolase model based on the nanotubes formed by
the self - assembly of a synthetic fmoc amphiphilic short peptide ( fmoc - ffh , 448 ) . according to the authors , the imdazolyl groups on the
surface of the nanotubes act as the catalytic centers for the hydrolysis
of p - nitrophenyl acetate ( pnpa ) . replacing the histidine
of 448 with arginine , the authors produced a structurally
similar peptide , fmoc - ffr ( 449 ) , the guanidyl groups
of which reside in the nanotubes through the coassembly of these two
molecules to stabilize the transition state of the hydrolysis . the
authors also reported that this model of peptide hydrolase is compatible
with hela cells and suggested the applications of these peptides as
a substitute for natural hydrolases . liu et al . synthesized an fgl pepitide amphiphile ( 450 ) that self - assembles to form nanofibers ( 1020 nm ) as the
scaffold for nscs . besides self - assembling to form a hydrogel at a
cgc of 1 wt % , 450 at a concentration of 50 , 100 , or
200 mg / l promotes the proliferation of nscs , which agrees with the
ncam mimietic properties of the fgl peptide . meanwhile , the authors found that the nanofibers of 450 increase the rate of neuron differentiation from nscs and concluded
that the self - assembled nanofibers of 450 have good biocompatibility
with nscs . as shown in scheme 56 , xu et al . designed
a series of hydrogelators based on the conjugates
of a dipeptide and ( naphthalen-2-yloxy)acetic acid . among these hydrogelators , nap - gly - d - ala ( 451 ) and nap - gly - ala ( 452 ) form hydrogels efficiently with cgcs of 0.07 wt % . the hydrogen
bonding between dipeptides and aromatic aromatic interactions
of the naphthyl groups cooperatively result in the excellent hydrogelation
ability of these hydrogelators . besides demonstrating that the handedness
of the helical fibril structures in the hydrogels correlates with
the chirality of the hydrogelators , the authors also found that these
molecular hydrogelators are compatible with hela cells when the concentration
of the hydrogelators is 200 m and the incubation time is 24
h. recently , xu et al . reported a new
class of hydrogelator ( 453 and 454 ) based
on conjugates of nucleobases ( e.g. , thymine , adenine , cytosine , and
guanine ) and ultrashort peptides which self - assemble in water upon
application of a ph or enzymatic stimulus to afford a new class of
supramolecular hydrogels that are biocompatible ( figure 10 ) . the studies on the gelation
properties indicate that all these nucleopeptides self - assemble to
generate -sheet nanostructures at a concentration of 2 wt % . in addition , the hydrogelators also exhibit significant resistance
to proteinase k , which makes them attractive materials for applications
in vivo . besides illustrating the first example of nucleopeptides
as hydrogelators made by an enzymatic reaction , the approaches illustrated
by the authors provide a facile way to explore the potential applications
of nucleopeptides as soft biomaterials . one possible application may be kinase detection , as shown by yang
et al . meanwhile , xu et al . also developed
simple conjugates of a nucleobase , amino acids , and a glycoside as
a new class of supramolecular hydrogelators ( 455 and 456 ) . consisting of the three unified building blocks of life , these hydrogelators self - assemble in water
to yield ordered nanostructures and supramolecular hydrogels at a
concentration of 3 wt % . the conjugates not only exhibit exceptional
biocompatibility and biostability , but also facilitate the entry of
nucleic acids into the cytosol and nuclei of cells through interbase
interactions with nucleic acids . in addition , the integration of a
saccharide at the c - terminal into the hydrogelators significantly
enhances their resistance to proteinase k , which greatly expands the use of this kind of hydrogel in vivo .
as a facile way to generate a fundamentally new molecular architecture
from the unified building blocks of life , this approach promises the development of sophisticated soft biomaterials
from a rather simple pool of building blocks . ( a ) dephosphorylation process catalyzed by alp with 454a to result in nanofibers and a hydrogel . ( c ) optical images of hela cells on
the surface 0 and 20 h after creation of scratchs in the presence
of hydrogel 454 t ( by adding 27.7 mm 454 t to the media ) . adapted with permission from ref ( 965 ) . copyright 2011 wiley - vch
verlag gmbh & co. kgaa . encouraged by the self - assembly of the conjugates of a nucleobase ,
amino acids , and a glycoside , xu et al . explored the biological functions
of the self - assembly of this kind of conjugate . they engineered a
multifunctional small molecule that consists of adenine ( as an assembly
domain ) , an arg - gly - asp sequence ( rgd , as a binding domain ) , and glycosamine
( as a glycogen ) , and found that the assemblies of the conjugates ( 457 ) promote the proliferation of mes cells and the development
of zygotes into blastocysts of mouse . in addition , they found that each module ( i.e. , nucleobase , rgd ,
and glycosamine ) in the conjugate is indispensable for the observed
functions according to the cell proliferation test of the , the
authors suggested that the self - assembly of this kind of de novo glycoconjugate
( 457 ) promises a potential approach to use supramolecular
assemblies as multifunctional mimics of glycoconjugates , including glycoproteins . furthermore , xu et
al . replaced the adenine of 457 with thymine , generating an analogue of 457 which belongs
to an unprecedented type of small molecules that consist of unified
building blocks of life . this analogue
self - assembles in water to form nanofibrils and results in a hydrogel
at a concentration of 3.0 wt % . one important observation of this
work is that the glycoside at the c - terminal of the peptide greatly
enhances the proteolytic resistance of rgd in the hydrogelator ( 457 ) . later , xu et al . synthesized another glycoconjugate , 458 , and its analogues based on the three fundamental biological
building
blocks ( i.e. , saccharides , amino acids , and nucleobases ) by spps . they found that all these conjugates were compatible with hela cells
even at a concentration of 415 g / ml . xu et al . reported the first hydrogelator ( 459 ) consisting
of both proteinogenic amino acids ( e.g. , phenylalanine ) and a nonproteinogenic
amino acid ( e.g. , taurine ) by attaching taurine at the c - terminal
of a well - established self - assembly motif ( 3 ) . the authors
found that , besides the ph , the temperature and ultrasound affect
the gelation behavior of 459 to result in different morphologies
of the nanostructures . in addition , the mtt - based cell viability assay
indicated that 459 is biocompatible with hela cells for
3 days at a concentration of 500 m . saccharide
conjugates , xu et al . also designed another kind of hydrogelator which
is a nucleobase saccharide amino acid conjugate . among all the hydrogelators , 460 forms a typical hydrogel ( with a cgc of 0.8 wt % at ph 7.0 ) which
turns into a solution at 59 c or at ph above 9.0 . this study
illustrates the incorporation of l-3-(2-naphthyl)alanine
as an effective strategy to promote molecular self - assembly in water . furthermore , the addition of t10 appears to result in a
mechanically stronger hydrogel which consists of nanofibers with widths
increasing from 7 2 to 17 2 nm . the in vitro experiments
indicate that 460 is compatible with hela cells at concentrations
up to 500 m for 3 days . as shown in scheme 57 , stupp et al . reported a peptide amphiphile ( 461 ) with
its sequence ( krrasvagk[c12]-nh2 ) containing the specific consensus substrate ( rrxso ; x = any residue ;
o = hydrophobic ) for protein kinase a ( pka ) , a ubiquitous kinase in
intracellular signaling and metabolism that has also been demonstrated
to be an extracellular cancer biomarker . 461 is able
to form a hydrogel with a -sheet secondary structure in the
nanofibers , and its assembly and disassembly can be reversibly controlled
by pka . in addition , the authors suggested that the disassembly of
the nanofibers of 461 by using pka might contribute to
an enzyme - triggered release of an encapsulated cancer drug . the authors
also reported an in vitro experiment to show the peptides themselves
to be compatible with cells while the drug - loaded nanofibers of 461 induce preferential cytotoxicity in a cancer cell line
that is known to secrete high levels of pka , such as the mda - mb-231
human breast cancer cell line . ryadnov
et al . designed a self - assembling peptide ( 462 ) which
contains two domains that oligomerize by forming a parallel coiled - coil
heterodimer . in this arrangement , each domain pairs with its complementary
partner from another copy of the same peptide , connected through two
short linkers and cyclized antiparallel to each other such that interactions
occur between different peptides . one unusual feature of 462 is that it forms hyperbranched fibrillar networks spanning from
nano- to micrometer dimensions . although this elaborately designed
peptide is less effective than collagen for promoting the proliferation
of human dermal fibroblast cells , the decoration of a cell attachment
motif ( e.g , a mimic of yigsr ) results in a 20% increase of cell proliferation
compared with the bare scaffold . the two positive sequences
( l ( 463 ) and d ( 463 ) ) contain alternating neutral ( w and a ) and positively
charged ( k ) residues , while the two negative sequences ( l ( 464 ) and d ( 464 ) ) replace lysine by negatively charged glutamic
acid ( e ) . the oppositely charged oligopeptide modules can interact
with each other electrostatically , coassemble , and form a hydrogel .
according to the in vitro experiments reported by the authors , the l - homochiral hydrogels of 463 are the most cell
compatible , leading to the highest human mesenchymal stem cell ( hmsc )
viability and proliferation , but the peptides are susceptible to proteases .
however , the d - oligopeptide hydrogels of 463 , which resist proteases , are unable to support cell hmsc proliferation . the authors found that negative charges significantly improve hmsc
growth in the d - oligopeptide hydrogels of 464 but have little effect on their interactions with the l - oligopeptide hydrogels of 464 . this interesting observation
indicates that negative charges can compensate for the disadvantage
of the d - homochiral hydrogels . gu et al . reported a hydrogelator connecting 7-(carboxylmethoxy)coumarin
molecules and hydrazine linked by l - lysine ( dcou - lys - conh - nh2 , 465 ) to act as lipophilic and water - soluble
moieties . 465 self - assembles to form hydrogels in distilled
water with a cgc of 1 wt % . 465 starts to self - assemble
into short fibrils even at concentrations lower than the cgc . in addition ,
ultrasound accelerates the gelation and induces homogeneous self - assembly
to form nanofibers with average diameters between 30 and 40 nm ( figure 11 ) . on the basis
of a live / dead assay , the authors reported that the hydrogel is preferable
for the migration and proliferation of nih/3t3 fibroblast cells . formation mechanism of hydrogel 465 : ( a )
hydrogen - bond - driven
self - assembly , ( b ) self - assembled fibrils , ( c ) fibrils with a hydrogelator
concentration lower than the minimum gelation concentration ( mgc ) ,
( d ) entangled fibrils with a hydrogelator concentration higher than
the mgc , ( d ) well - organized 3d hierarchical nanoarchitectures with
ultrasound treatment , ( f ) cells seeded in hydrogels , ( g ) optical image
of the hydrogel ( the transition from solution to hydrogel was reversible ) .
adapted with permission from ref ( 85 ) . lin et al . have reported the detailed
study of a new series of
small molecular hydrogelators , among which the intramolecular alternative
packing of the phenyl / perfluorophenyl pair promotes the formation
of supramolecular nanofibers and hydrogels at ph 5 with a cgc of 1.0
wt % . the authors also reported that 466 is compatible
with the ctx tna2 cells in a concentration range of 10500
m for 48 h. the same lab recently
reported a co - assembled hydrogel based on naphthalene diimide for
treating mcf-7 cells . designed a series of uncapped hydrophobic heterochiral tripeptides
with all combinations of d- and l - amino acids to
minimize the disadvantages of l- or d - peptides . rheology and xrd results indicated that , among all the heterochiral
tripeptides , 467 forms a hydrogel with a -sheet
amyloid structure . according to the live / dead assay , the authors showed
that 467 maintains the viability and proliferation of
l929 mouse fibroblast cells in vitro for 3 days . taking advantage of click chemistry , barthlmy
et al . designed and synthesized two glycosyl - nucleoside fluorinated
amphiphiles ( gnfs , 468 ) which feature either -d - glucopyranosyl or -d - lactopyranosyl moieties
linked to a thymine nucleobase . on the basis of the air solution
surface tension ( ) measurements , the authors reported that
the critical aggregation concentrations ( cacs ) are 5.9 and 3.7 m ,
respectively . gelation tests indicate that both of the gnfs self - assemble
to form entangled nanofibers roughly 1020 nm in diameter and
the -d - glucopyranosyl - based gnf shows a cgc of 0.1%
( w / w ) . the authors also reported that -d - glucopyranosyl
is compatible with a human cell line ( huh-7 , human hepatocarcinoma
cell line ) . the same lab also reported
the hydrogels made of glycosyl - nucleoside bola - amphiphiles ( gnbas )
for culturing human mesenchymal stem cells isolated from adipose tissues . wang et al . designed and synthesized three
amino acid derivative saccharide conjugates , among which 469 self - assembles to form stable hydrogels containing nanofibers
with diameters of 80300 nm at a concentration of 0.2 wt % .
the authors found that the extensive hydrogen bonds between sugar
rings contributed to the formation of stacking
between aromatic naphthalene groups , which results in the formation
of stable hydrogels in aqueous solutions . using an mtt - based cell
viability assay , the authors verified that these kinds of saccharide - based
hydrogels are compatible with nih3t3 , hepg2 , ad293 , and hela cells . in addition , these cells show a good adhesion and proliferation rate
on the surface of hydrogels in a 2d environment . it would be interesting to know how the hydrogelators affect
the morphological properties of these cells . while most
of the research activities are centered on the use of supramolecular
hydrogels for promoting cell proliferation , xu et al . have been working
on the design of supramolecular hydrogelators to inhibit cell selectively . for example , as shown in scheme 58 , xu et al . designed and synthesized a new class of
supramolecular hydrogelators ( 304 , 470 ,
and 471 ) consisting of n - terminated diphenylalanine and
naphthalene motifs . they found that the hydrogelators self - assemble
to result in nanofibers and hydrogels at a concentration of less than
0.8 wt % , but within a relatively narrow ph range ( 5.06.0 ) . interestingly , the authors found that these hydrogelators exhibited
significantly higher cytotoxicity to hela cells than to ect1/e6e7
cells , which proves that hydrogelators selectively inhibit cancer
cells . thordarson et al . reported the
synthesis of a new hydrogelator ( 472 ) with an indole
capping group which forms exceptionally strong hydrogels in a variety
of environments with a cgc of 0.4 wt % . cell viability studies of
hela cells indicate that 472 exhibits compatibility with
cells at lower concentrations while being cytotoxic at concentrations
up to 0.1 wt % . liang et al . reported
heptapeptide hydrogelators 473 based on the devd peptide
sequence , which is a specific substrate for caspase-3 . the cryo - tem
photograph indicates that 473 self - assembles to form
a hydrogel containing flexible and long nanofibers with an average
width of 6.1 1.2 nm . the mtt cell viability assay shows that 473 is slightly more compatible with hep g2 cells than its
isomeric control hydrogelator ( 474 ) at 400 m for
3 days . western blot analysis indicated that the isomer 474 , which is not a substrate of caspase-3 , at 400 m , obviously
is able to activate caspase-3 to induce cell death via apoptosis . numata et al . reported the high - yield chemoenzymatic synthesis
of linear oligo(l - phenylalanine ) by proteinase k from tritirachium album . by connecting the synthesized
linear oligo(l - phenylalanine ) with tris(2-aminoethyl)amine ,
they obtained a star oligo(l - phenylalanine ) ( 475 ) that self - assembles into fluorescent fibers with various branching
ratios . the authors reported that the oligo(l - phenylalanine )
analogues exhibit slight cytotoxicity to human embryonic kidney 293
cells ( hek293 ) at a concentration above 25 mm after 8 h. zhang et al . designed and synthesized a glycopeptide
( 476 ) consisting of the fmoc - phe - phe - asp sequence and
a therapeutic glucosamine moiety . 476 dissolves well
to form a homogeneous solution at an elevated temperature and a concentration
of 1 wt % and forms a stable hydrogel at ph 7.4 upon being cooled
to room temperature . an mtt - based cell viability assay indicated that
the glycopeptide slightly inhibits the nih/3t3 fibroblast cells on
the surface of the hydrogel . most importantly , the authors reported
that the hydrogel of 476 is able to inhibit postoperative
fibrosis in eye surgery ( figure 12 ) , as evidenced by the fact that the filtration fistula
is constantly smooth and the mean intraocular pressure is significantly
lower within 21 days postsurgery compared with the results from conventional
antiproliferative drug injections . histological
section of rabbit eyes which underwent filtration
surgery ( a ) alone at 14 days postsurgery , received the fmoc - ff ( 6 ) hydrogel at ( b ) 7 and ( c ) 14 days postsurgery , and received
the glycopeptide hydrogel ( 476 ) at ( d ) 7 , ( e ) 14 , and
( f ) 21 days postsurgery . hematoxylin eosin ; magnification 100.
adapted with permission from ref ( 92 ) . copyright 2012 royal society of chemistry . as shown in scheme 59 , stupp et al .
linked a cyclic rgd motif at the side chain of a peptide amphiphile
to construct a branched architecture in the monomer 477 , which self - assembles to form cylindrical nanofibers having a very
high aspect ratio and therefore mimicks the soft fibrous environment
in the ecm . by changing the local dynamics either through the architecture
of the molecules or dilution of the epitopes , the authors were able
to tailor the density of rgd epitopes on the nanofibers to an extremely
high level . in addition , the authors found that branched architectures
of the monomers and additional space for epitope motion improve signaling
for cell adhesion , spreading , and migration of nih/3t3 fibroblast
cells in 2d and mda 231 cells in 3d cell migration . in a related study , zhou and zhang et al . reported cyclic
rgd exhibiting synergistic effect with a bmp-7 derived peptide in
the differentiation of mesenchymal stem cells . besides the branched architecture with the cyclic rgd epitope
reported by stupp et al . , hamley et al . investigated the fmoc - tetrapeptide
fmoc - rgds ( 478 ) consisting of the rgds cell adhesion
motif from fibronectin . circular dichroism and fiber x - ray diffraction
indicated that the self - supporting hydrogel formed by sonication and
heating / cooling at a concentration of 1 wt % is comprised of parallel
-sheet nanofibers with a diameter of approximately 10 nm . the
authors suggested that 478 may be used to produce collagen - based
gels for growing corneal fibroblasts . yang et al . reported
an intriguing example in which the peptide 479 self - assembles
to form nanofibers and results in a hydrogel
with a cgc of 0.3 wt % . the resulting hydrogel is able to selectively
form a thin layer of hydrogel at the surface of platelets , thus preventing
human platelet aggregation induced by various agonists such as collagen . yang et al . also designed and synthesized a
class of supramolecular hydrogelators consisting of the tripeptide
sequence glycine - xaa-4(r)-hydroxyproline ( gxo ; xaa
is any one of the natural amino acids ) from collagen . among all these
hydrogelators , 480 self - assembles in aqueous solution
to form nanofibers with a diameter of 2030 nm and with a cgc
of 0.06 wt % . furthermore , 480 promotes the cell adhesion
of nih/3t3 fibroblasts , a property similar to that of collagen , which
makes it suitable for 2d cell culture . they reported another collagen mimic hydrogelator , 481 , that self - assembles to form a thixotropic hydrogel , consisting
of flexible nanofibers of about 9 nm , at a concentration of 2 wt % . importantly , the authors found that the hydrogel of 481 selectively enhances flk1 expression in differentiated murine embryonic
stem ( mes ) cells . mihara et al . designed
a glutamic acid residue - conjugated -sheet peptide , e1y9 ( 482 ) , which , at a concentration of 2 wt % , undergoes hydrogelation
in the presence of ca . the hydrogel
maintains its shape well to allow it to be molded to a short string . the authors conjugated the rgds sequence to the c - terminals of 482 peptides and obtained a new peptide , e1y9- rgds ( 483 ) , which can be mixed with 482 to form hydrogel
strings . one impressive result is that pc12 adheres to the hydrogel
string and differentiates in 6 days ( figure 13 ) , suggesting that the surface of the hydrogels
resembles that of fibronectin surfaces . 3t3-l1 cells
were incubated on the flat hydrogels composed of 482 containing
0% , 10% , or 20% 483 , fibronectin ( fn ) , tissue - culture - treated
plates ( tctps ) , or nonadhesive plate surfaces in dulbecco s
modified eagle s medium containing 5 mm ca . ( b )
fluorescence microscopic images of cell - adhered peptide gel strings . pc12 cells were cultured in dulbecco s modified eagle s
medium containing 5 mm ca for 6 days . copyright 2012 the society
of polymer science , japan ( spsj ) . as shown in scheme 60 , feng et al . reported a series of peg - containing hydrogelators
by
coupling ethylene glycol ( eg ) monomers and the rgd motif onto c2-benzene cores to resist protein adsorption
and promote cell adhesion . tem images indicated that 484 self - assembles to form entangled fibrous gel networks with fiber
diameters of 68.9 4.3 nm at a cgc of 0.07 wt % . the incorporation
of the rgd sequence into 484 not only influenced the
supramolecular structure and viscoelasticity of the fibers , but also
contributed to overcoming nonspecific protein adsorption and promoting
adhesion of encapsulated cells , which makes 484 suitable
for 2d and 3d culture of human hepatoma cells and normal human skin
fibroblasts . furthermore , it is feasible
to vary the supramolecular self - assembly of 484 for controlling
the cell adhesion and proliferation in 2d and 3d microenvironments . meanwhile , feng et al . also reported a new kind of hydrogel derived
from the combination of a c2-phenyl - derived
gelator and a polysaccharide ( alginate ) . after addition of ca , the conjugate self - assembles to form flexible nanofibers
with branches and twists . the live / dead cell viability assay indicates
that the hydrogel exhibits no cytotoxicity to normal human skin flbroblasts
( nhsfs ) and promotes cell adhesion and spreading in vitro . the hydrogels formed by the coassembly of c2-phenyl - based hydrogelators and sodium hyaluronate
showed a high swelling property to ensure cell migration and proliferation
inside the bulk of the hydrogels . the
authors also investigated the influence of the chirality of the nanofibers
on cell adhesion and proliferation by using two enantiomers of c2-phenyl - derived hydrogelators . they found that
left - handed helical nanofibers ( containing an l - phenylalanine
derivative ) can increase cell adhesion and proliferation , whereas
right - handed nanofibers ( containing a d - phenylalanine derivative )
have the opposite effect . it would be
more informative if the authors had examined the biostability of the
hydrogelators . zhang et al . reported a class of photoresponsive small
molecular
hydrogels ( tet(i)-gff ( 485 ) and tet(ii)-gfrgd ( 487 ) ) formed by the self - assembly of short peptides linked
with a biaryl - substituted tetrazole - moiety - based phototrigger . at
ph 7 , 485 forms a clear and stable hydrogel with a cgc
of 0.08 wt % . upon mild light irradiation , 485 undergoes
fast intramolecular photo click ligation , and the complete transformation
from 485 to 486 takes < 2 min . this photoreaction
disturbs the self - assembled hydrogel matrixes and induces the photodegradation
of the hydrogel , which modulates the cellular microenvironments when
the hydrogel of 485 serves as the scaffolds for cell
cultures . the authors demonstrated that the irradiation of the hydrogel
causes the cell to express much higher levels of differentiation markers . the authors also demonstrated that the hydrogel of 487 at a concentration of 0.9 mg / ml can mimic the 3d microenvironment
for the hmscs . on the basis of the
fmoc - ff ( 6 ) hydrogelator , gazit et al . designed the dopa - containing
dopa - dopa and fmoc - dopa - dopa peptides 301 that self - assemble
to form a hydrogel at 0.25 wt % . the authors reported that the hydrogels
of these dopa - containing peptides reduce ionic silver into silver
nanoparticles . in addition , the conjugation of lysine ( lys ) with 301 generates 488 , which self - assembles to form
ordered nanostructures in the presence of dimethyl sulfoxide ( dmso )
and water . the authors envision that it may serve as a multifunctional
platform for various biotechnological applications . reported an fmoc - protected tetrapeptide amphiphile
for fabricating a bioadhesive hydrogel with dopa groups as affinity
fusion tags ( fmoc - lff - dopa , 489 ) . a 2000 u / ml concentration
of of metalloprotease can trigger 489 to form a transparent
yellow molecular hydrogel . on the basis of a live / dead assay , the
authors inferred that the hydrogel of 489 , containing
the catechol groups , could successfully promote the adhesion and proliferation
of adult human dermal fibroblast cells in vitro . it would be useful to know the mechanism behind this adhesion . as shown in scheme 61 , liang et al . designed a
radioactive probe ( 490 ) that intracellularly forms radioactive
nanoparticles under the action of furin in living tumor cells . they
found that , upon 160 min of cellular efflux , the radioactivity retained
in mda - mb-468 cells incubated with 490 remains at a high
level . intended
to design the peptide sequence fefkfefkgrgd ( 491 ) by adding a hydrophilic peptide ( rgd ) to the efk8 peptide to decrease
the strong aggregation properties of efk8 , therefore allowing hydrogels
to form in neutral ph conditions . interestingly , they found that the
hydrogel of 491 specfically supports and stimulates the
growth of delftia xd , a bacterium . chen and yang et al . reported another strategy to form
hydrogels of efk8 peptide derivatives at neutral conditions by the
replacement of f with f and the introduction of a hydrophilic
rgd tripeptide ( fefkfefkyrgd , 492 ) . they found that 492 self - assembles to form
hydrogels in pbs buffer with a cgc of 0.15 wt % . in addition , the
live / dead assay showed that the hydrogel of 492 , at a
concentration of 2 wt % , is suitable for cell proliferation and produces
a colony of hela cells in vitro . this
ingenious doping of d - amino acid residues for controlling
the self - assembly behaviors of the hydrogelator may be general and
applicable for other peptide sequences . chemo / biosensors
for visual detection are a class of increasingly attractive tools
for the analysis of many targets ( e.g. , biological markers , enzymes ,
ions , gases , etc . ) . they are extremely
useful for rapid and high - throughput diagnostics or detection in situations
where low cost , speed , and ease are required . supramolecular hydrogels , thus , attract tremendous
attention as a platform for chemosensors
because they have the following properties / advantages : ( i ) a variety
of biological , chemical , or physical triggers ( e.g. , temperature ,
ph , ionic strength , electric field , enzyme , etc . ) for example , hydrogel formation triggered by enzymes can serve as
an indicator of certain enzymes . ( ii ) supramolecular
hydrogels are able to incorporate / immobilize a variety of colorimetric
reagents , such as visible dyes , both covalently and noncovalently . a range of diverse yet selective molecular
interactions can lead to a color change of the hydrogel , for example ,
stimulus - induced release or absorbance of dye molecules and color
changes of the hydrogels initiated by target binding . ( iii ) supramolecular - hydrogel - based
chemosensors can work in aqueous conditions , which is of extreme importance
because most biological substances ( e.g. , enzymes , biomarkers , etc . )
remain active only in physiological conditions ( i.e. , in aqueous solution ) . in the following subsections , supramolecular
hydrogels show solidlike , yet soft , properties and contain three - dimensional
networks , formed by hydrogelators , to not only imbibe water , but also
immobilize other components , such as small molecules , enzymes , and
ions , especially when the hydrogels serve as chemo / biosensors . before
discussing supramolecular - hydrogel - based chemo / biosensors , we first
highlight some recent works on hydrogels used as efficient absorbents
of dyes , metal ions , and other molecules . due to the use of a wide range of dyes in several industries ( e.g. ,
paper , plastics , textiles , and cosmetics ) , it is necessary to remove
the dyes from industrial discharge to prevent pollution . among all
kinds of methods , absorption is more preferred due to its low cost ,
high efficiency , and easy handling . supramolecular hydrogels which
contain both hydrophilic and hydrophobic groups can absorb a variety
of dyes and may have superiority in the recycle
and adsorption rate compared with some traditional methods . as shown
in scheme 62 , banerjee
and co - workers report a phenylalanine - based bolaamphiphile , 493 , containing a centrally located oligomethylene group ,
which affords a hydrogel at ph 6.57.2 in the presence of divalent
metal salts ( e.g. , mncl2 , cocl2 , cuso4 , and nicl2 ) . by studying the hydrogelation behaviors
of these molecules , banerjee et al . concluded that these hydrogels
not only can entrap and release a biological substance , but also can
efficiently adsorb various toxic dyes , such as crystal violet and
naphthol blue black from water . the
same group reported several tripeptide - based hydrogels and their use
in removal of dyes from wastewater . sanchez et al . used a supramolecular hydrogel formed by triangular - shaped
dendronized oligo(phenyleneethynylene ) amiphiphiles 494 and 495 for dye encapsulation , such as disperse orange
3 , a hydrophobic dye ( figure 14 ) . temperature - dependent uv / vis spectra of aqueous solutions ( 10 m ) of ( a ) 494 and ( b ) 495 containing 1 equiv of disperse orange 3 . the insets depict the changes
in the absorbance at 400 nm as a function of temperature . adapted
with permission from ref ( 1021 ) . they easily obtained hydrogels by
changing the ph of
the solution of 496 , or by heating and then cooling the
solution of 497 . both of the hydrogels exhibit a unique
layered structure of activated carbon and are capable of the controllable
adsorption of 9799% of certain organic dyes , such as methylene
blue and methyl violet 2b , within 2 min . used nap - f ( 498 ) , which gels water
even at a concentration 0.025 wt % , as a network for dye entrapment . besides , they demonstrated that the addition of chaotropic reagents ,
as well as increasing the ph value , disassembles the gel and promotes
the release of the entrapped molecules . studied the hydrogenation behavior of lithocholate ( 87 ) by introducing alkali - metal ions and nh4 into the aqueous solution of 87 . this hydrogelator
shows a cgc varying from 75 to 130 mm ( from 2.8 to 4.9 wt % ) , depending
on the ions added . the authors demonstrated that these hydrogels show
high efficiency and the capability of absorption of cationic dyes ,
and thus may be a promising candidate for the removal of toxic substances . the networks of supramolecular
hydrogels can reversibly entrap a variety of probe molecules , which
allows the development of various readout systems , such as fluorescence
enhancement or quenching , color changes , or fluorescence resonance
transfer ( fret ) , based on the hydrogels . acting as a class of chemosensor ,
hydrogels help monitor the signal changes associated with molecular
recognition . as a pioneer in the applications
of hydrogels for chemosensing , hamachi et al . developed a supramolecular
hydrogel formed by 499 molecules ( scheme 63 ) as a platform for a semiwet sensor chip . they created a hydrogel - based array on a glass
plate by incorporating artificial receptors into the heated solution
of the hydrogelator 499 and spotting them on a glass
plate . they found that this semiwet sensor chip not only recognizes
a variety of cations by simply changing the incorporated artificial
receptors / probes , but also can work as a ph probe . they also demonstrated
that the integrated supramolecular sensor chip can accept mixtures
without tedious isolation steps . hamachi
et al . also used the same supramolecular hydrogel to construct a fluorescent
lectin array for detecting saccharides . by noncovalently fixing the
fluorescent lectins into the hydrogel matrix to act as a molecular
probe for various glycoconjugates , they demonstrated that one can
read a series of saccharides on the basis of the selectivity and affinity
of the immobilized lectins . using similar
molecules , 367 and 500 , hamachi et al . designed
a novel polyanion - selective fluorescence sensing system composed of
a hybrid material of supramolecular hydrogels , enzymes , and aminoethyl - modified
mcm41-type mesoporous silica particles with cationic nanopores encapsulating
anionic fluorescent dyes ( e.g. , 501 ) . this system efficiently
coordinates ( i ) the release of an anion - selective probe ( e.g. , 501 ) from mcm41 and ( ii ) the translocation of the probe facilitated
by enzymatic reaction ( e.g. , dephosphorylation catalyzed by phosphatases )
with ( iii ) fret sensing in the hydrogel form by 367 and 500 . on the basis of similar
strategies , hamachi et al . also developed several fluorescent sensors
for rapid and convenient detection of chemicals , such as phosphate
derivatives , polyamines , and polyols . kim et al . prepared a library of amphiphiles , each comprising a
pyrene group and a polar carbohydrate head group . they found that
all of the amphiphiles form robust hydrogels with cgc values ranging
from 0.07 to 0.30 wt % , but only amphiphile 502 ( a derivative
of d - gluconolactone ) affords a fluorescent hydrogel which
is sensitive to the presence of insulin in aqueous media . they suggested
that this supramolecular hydrogel can serve as an efficient probe
for insulin . demonstrated
that molecule 503 , based on a perylenediimide chromophore
decorated with polyethylene glycol , self - assembles in aqueous media
to form extended supramolecular fibers which afford a stable hydrogel . as reported by the authors , the hydrogel of 503 can respond
to multiple stimuli , such as temperature changes and chemical reductions . the authors suggested that the dual sensitivity toward chemical reduction
and temperature with a distinct and interrelated response to each
of these stimuli is especially useful to applications in the area
of adaptive functional materials , such as chemosensors . as shown in scheme 64 , kim et al . reported the design of an anisotropic
supramolecular
hydrogel of -cd ( 504 ) and an azo dye ( 505 ) in which the host guest interaction between the two molecules
leads to hydrogelation . they tested the obtained hydrogel for identifying
different classes of metal ions and demonstrated visual detection
of lead ions by the naked eye . in addition
to sensing ions in aqueous solution , hydrogel - based chemosensors can
serve as probes for gases . designed tetracarboxylic
acid - appended thiacalixarene ( 506 ) , which is able
to self - assemble in the presence of co. they unexpectedly
found that the red color of the filter paper coated with the resulting
hydrogel selectively changed to a blue color by exposure to a toxic
vgcl ( volatile gas containing a chlorine atom ) , such as hcl , socl2 , ( cocl)2 , and cocl2 , which hydrolyzed
to yield hcl . they concluded that the strategy may lead to useful
applications in sensing other chemical vapors . singh et al . developed a hg coordinate complex
of a 1,4-dioxa-7,13-dithia-10-azacyclopentadecane bodipy dyad
( 507 ) which selectively recognizes l - cysteine
over other amino acids via a reversible complexation / decomplexation . as reported by them , the detection relies on the switch of fluorescence
upon sequential addition of hg and a cysteine solution . the most attractive
feature of supramolecular hydrogels is that the 3d , semiwet nanofiber
network can entrap biological substances without a detrimental effect
on the activities or functions of the entrapped substances . for certain hydrogels formed by enzyme instruction , the state changing
from a solution to a hydrogel itself can serve as a signal for reporting
the existence of certain enzymes . for example , xu et
al . developed a simple assay based on the hydrogelation of small molecules
fmoc - yp ( 14 ) for the rapid detection of the
inhibitors of enzymes ( i.e. , acid phosphatase ) . on the basis of the
fact that phosphatase catalytically transforms the solution of 14 into a solid hydrogel within 30 min , xu et al . demonstrated
that the sol gel transition can serve as a visual assay for
screening the inhibitor of the enzymes . developed a strategy that utilized an aptamer - functionalized
hydrogel to detect human thrombin through a diffraction measurement
( 508 ; figure 15 ) . being a serine protease , thrombin acts as a model protein
to test its binding with an aptamer . wang et al . constructed the thrombin - responsive
hydrogel by functionalizing the hydrogelator with both the aptamer
and its complementary sequence as the physical cross - linking points . when exposed to human thrombin solution , the aptamer tends to bind
with thrombin rather than its complementary sequence , which causes
the hydrogel to swell due to the decrease of cross - linking and the
change in the diffraction efficiency . park et al . employed a self - assembled peptide hydrogel consisting
of fmoc - ff ( 6 ) as a biosensing platform . by encapsulating
enzymes ( e.g. , glucose oxidase or horseradish peroxidase ) and fluorescent
reporters ( e.g. , cdte , and cdse quantum dots ) physically within the
hydrogel matrix via simply mixing them in a peptide solution , they
successfully applied the system to detect analytes ( e.g. , glucose
or phenolic compounds ) on the basis of a photoluminescence quenching
of the hybridized quantum dots . shimizu
reported that an unsymmetrical bolaamphiphile , 509 ( scheme 65 ) , with glucose
and triglycine groups at both ends , exclusively self - assemblies into
nanotubes . they demonstrated that the self - assembled
nanotubes with an interior recognition probe on the inner surface
not only detect the encapsulation , transportation , and release behavior
of gfp in real time , but also report the stability of gfp in the hollow
cylinder . schematic illustration
of the sensing strategy of diffraction grating
for human thrombin detection . ( a ) the hydrogel 508 contains
an aptamer and its complementary sequence as the supermolecular cross - linking
points and swells when exposed to the human thrombin . ( b ) response
of the hydrogel grating to human thrombin in the solution . adapted
with permission from ref ( 1036 ) . reported a self - assembling vacomycin derivative
( 511 ) for bacterial detection and inhibition . they demonstrated
that the conjugation of vancomycin to the side chain of the peptide
derivatives increases its antimicrobial activity by 7-fold . by monitoring
the fluorescence response of the solution of 511 to the
bacteria , they found that the fluorescence of 511 increases
gradually with increasing concentration of the bacteria . similar to
the bacterial surface - induced self - assembly of 23 , this
specific peptide antibiotic interaction initiates the self - assembly
of an environment - sensitive conjugate ( 511 ) , which may
find applications for simultaneous detection and inhibition of bacteria . recently developed a self - assembled
nanofibrous hydrogel using a biologically inert amphiphile ( 512 ) which possesses unique physical / mechanical properties
and easily carries a diverse range of payloads . the authors found
that 512 exhibits excellent self - assembly ability in
multiple solvents , including aqueous and organic solvents , typically
at a concentration of 14 wt % . by noncovalently encapsulating
a ph dye ( phrodo ) into the self - assembled hydrogel / fibers of 512 , they obtained a ph sensor which can be internalized into
macrophages at both physiological and subphysiological temperatures
through an energy - dependent , passive process and report the ph in
both the cytoplasm and phagosomes as well as the nucleus . yang et al . synthesized compounds of dabcyl - gfng3ndevdgk(fitc / rhodamine )
( 513 ) ( n = 03 ) with and without
f - substitution on the 4-position of the benzyl ring of phenylalanine
as the self - assembling probes for caspase-3 . they demonstrated that
the incorporation of one or two amino acids of phenylalanine ( f ) ,
especially 4-fluorophenylalanine ( f ) , would greatly lower
the background fluorescence intensities of conventional quenched probes
with quenchers ( dabcyl ) by the synergistic effect of fret and aggregation - caused
quenching ( acq ) . by varying the amount of f , they optimized
the properties of the resulting probes , such as self - assembly ability ,
fluorescence recovery , and kinetics of enzyme cleavage . they found
that these probes can detect caspase-3 in complex environments such
as that in apoptotic cells , which offers a simple strategy to design
fluorescent molecular probes with better signal - to - noise ratios . due to the promising
applications of hydrogels in drug delivery , biosensors , tissue engineering ,
immunology , and other biomedicine , it is necessary to gain a comprehensive
understanding of the self - assembly behavior of small molecules in
the biological environment , aiming for optimal molecular design . such
being the case , imaging would be one of the most direct and revealing
methods to distinguish , depict , and record the supramolecular self - assembly
during the biological events or cellular processes . compared to other
imaging modalities ( e.g. , positron emission tomography ( pet ) , mri ,
etc . ) , the use of fluorescence has many advantages , such as easy access ,
low damage , and ready adaptability to specific molecular events despite
poor depth penetration . most importantly , fluorescent imaging provides
the highest spatial resolution for imaging the molecular process at
the cellular level . various successful examples have been established
to use specific fluorophores noncovalently staining supramolecular
self - assemblies to reveal their existence , formation , and degradation ,
such as using congo red to stain amyloids . in contrast , the covalent
incorporation of a suitable fluorophore into a self - assembling small
molecule ( e.g. , a hydrogelator ) not only allows the self - assembly
process to align the hydrogelators into nanofibers or other ordered
structures , but also forces the appended fluorophore to comply with
the ordered organization . in this subsection , we focus on the applications
of fluorescent hydrogelators for imaging in a cellular environment ,
and introduce it as a powerful and facile method to reveal the emergent
properties of supramolecular self - assemblies because it couples fluorescence
with the self - assembly process . before describing
imaging of molecular self - assembly in a cellular environment , we present
several typical examples of fluorescent hydrogels formed by small
molecules . there are two kinds of fluorescent hydrogels : one consists
of fluorescent hydrogelators , and the other forms by fluorescent dyes diffusing into the matrix
of the hydrogel . for example , shinkai et al . developed a supramolecular hydrogelator
( -d - glucopyranoside azonaphthol conjugate , 514 or 515 ; scheme 66 ) which affords a fluorescent hydrogel in
a mixture of water and ethanol ( 80:20 , v / v ) . the azonaphthol moiety
serves not only as an aggregative functional group but also as a probe
for microscopic solvent polarity . on the basis of a uv hydrazone tautomerism , it
is possible to estimate the microenvironmental polarity in the fibrous
aggregates of the hydrogelators . jung
et al . reported two fluorescent hydrogels formed by amide - linked tripyridine
derivatives 516 and 517 , with para or meta
substituents . dmso
and the hydrogelation ability depends mainly on ch
and stacking or strong intermolecular hydrogen
bonding between the amide groups . another
interesting example is the ruthenium(ii ) tris(bipyridine ) complex
( 10 ) , developed by xu et al . the integration of a tripeptide
derivative , a versatile self - assembly motif , with a ruthenium complex
affords the first supramolecular metallohydrogelator that not only
self - assembles in water to form a hydrogel but also exhibits a gel they found that this hydrogel formed
by 10 exhibits strong fluorescence upon the irradiation
of uv light . it is also noteworthy that
the long lifetime and photostability of [ ru(bipy)3 ] will likely find applications in molecular imaging in cells . ( a ) optical images of the oxidation - induced gel sol transition
and the tem images corresponding to the samples at different states
of transition . the hydrogel ( reduced state ) is formed by 0.8% ( w / v ) 10 in water at ph 1 . ( b ) fluorescent
images of a hela cell incubated with 3 ( 200 m ,
24 h ) . supramolecular hydrogelators
serve as an excellent system for exploring
the properties of molecular nanofibrils in a cellular environment . xu et al . developed a method to image enzyme - instructed self - assembly
of small molecules inside live cells ( see section 5.9.2 ) . in a different
study , tomasini et al . found that a physical hydrogel prepared with
small molecules of ch2(c3h6co - l - phe - d - oxd - oh)2 ( 518 ; scheme 67 ) is a
potential trojan horse carrier into cells . to check
the internalization process by confocal microscopy , they prepared
a fluorescent hydrogelator , introducing the fluorescent dansyl moiety
into the molecules ( 519 ) . yang and co - workers conjugated the environment - sensitive fluorophore
nbd to the peptide ffyeeggh at its n - terminal and found that
the resulting peptide derivatives 520 yield supramolecular
nanofibers with enhanced cellular uptake , brighter fluorescence , and
a significant fluorescence response to external stimuli ( figure 17 ) . ( a ) the nanofibers of 520 could specifically bind
to cu , leading to the formation of fluorescence - quenched
elongated nanofibers . confocal images ( bright field + fluorescence )
of ( b ) hela cells treated with 520 ( 0.05 wt % ) at a 2
h time point and ( c ) hela cells pretreated with 100 m cu and then treated with 520 ( 0.05 wt % ) at a
6 h time point . adapted from ref ( 1065 ) . infectious disease remains a major
threat to public health , and there
is an urgent need for novel antimicrobial agents with activities against
multi - drug - resistant bacteria . the discovery of antimicrobial peptides
has stimulated the use of self - assembly of peptide amphiphiles to
develop antibacterial hydrogels . particularly , the pioneering work
by schneider et al . on antibacterial hydrogels has provided useful
insights into the development of hydrogelators for antibacterial applications . we only give a brief discussion of the
works on antibacterial hydrogelators reported over the past decade . schneider and pochan et al . reported a series of -sheet
peptide - based hydrogels , among which
the surface of 251 is inherently antibacterial and exhibits
broad - spectrum activity against both gram - negative ( klebsiella pneumoniae and e. coli ) and gram - positive ( staphylococcus epidermidis , staphylococcus aureus , and streptococcus pyogenes ) bacteria without incorporating
exogenous antimicrobial agents . using the live / dead assays by laser
scanning confocal microscopy ( lscm ) , they found that the surface of
the hydrogel of 2 wt % 251 displays broad - spectrum antibacterial
activity when incubated with bacterial solutions ranging in concentration
from 2 10 to 2 10 colony - forming
units ( cfus)/dm ( figure 18 ) . on the basis of the -galactosidase leakage
experiments , they suggested that the surface of the 251 hydrogel likely causes inner and outer membrane disruption and controls
the release of -galactosidase from the cytoplasm of lactose
permease - deficient e. coli ml-35 , resulting
in cell death upon cellular contact with the surface of the hydrogel . furthermore , coculture experiments showed that , when nih3t3 fibroblasts
and a mixture of achromobacter xylosoxidans and stenotrophomonas maltophilia are
introduced onto the hydrogel , the surface of the hydrogel inhibits
bacterial proliferation yet allows mammalian cell adhesion and proliferation ,
indicating that the surfaces are selective against bacteria . later , they switched two lysine residues of 251 to two arginines , generating another -hairpin peptide ,
marg1 ( 521 ; scheme 68 ) . they found that the surface of the hydrogel of 2
wt % 521 imparts potent antibacterial activity against
methicillin - resistant s. aureus ( mrsa )
while it is noncytotoxic toward mammalian cells ( murine c3h10t1/2
mesenchymal stem cells ) . on the basis
of this result , schneider et al . designed another arginine - rich -hairpin
peptide , pep6r ( 522 ) , which
self - assembles to form hydrogels at 1.5 wt % or higher concentration
containing nacl . they found that the hydrogel surfaces of 522 exhibit potent activity for killing both gram - positive and gram - negative
bacteria , including multi - drug - resistant pseudomonas
aeruginosa , while they exhibit cytocompatibility toward
human erythrocytes as well as mammalian mesenchymal stem cells . coli incubated on ( a ) a borosilicate control surface
and ( b ) the hydrogel
of 2 wt % 251 after 24 h. the gel is viewed parallel
to the z - axis . green fluorescence denotes live cells ,
and red fluorescence denotes dead cells with compromised membranes . coli incubated on the surface of the hydrogel of 2 wt % 251 viewed perpendicular to the z - axis . arrows denote
the gel bacterial interface . adapted from ref ( 1068 ) . reported a series of cationic ,
naphthalene - derivatized
self - assembling ultrashort peptides , among which 523 self - assembles
to form hydrogels with a -sheet structure at a concentration
of 1 wt % and ph of 7.4 in water . the authors found that the hydrogel
of 2 wt % 523 significantly reduces the viable s. epidermidis biofilm by 94% while exhibiting little
hemolytic side effect toward human red blood cells ( hrbcs ) . on the
basis of the cytotoxicity assays against murine fibroblast ( nctc 929 )
cell lines and hemolysis assays using equine erythrocytes yang and wang et al . reported self - assembled
vancomycin derivatives 511 based on ff or ffy with aromatic
capping groups that showed great self - assembly ability in pbs buffer
with a critical micelle concentration ( cmc ) of 75 g / ml . using
the standard broth microdilution assay , the authors studied the bacterial
inhibition capacity of 511 and found that the minimum
inhibitory concentration ( mic ) of 511 is about 4.5 m ,
which was similar to that of the parent van molecule ( 1.3 m ) . meanwhile , wang and chen et al . reported a
selenium - containing vancomycin derivative with a redox - controllable
self - assembly property and antibacterial activity . das et al . designed and synthesized several dipeptide - based
cationic
amphiphiles with different head group structures by varying the combinations
of l - amino acid residues . among all the dipeptide derivatives ,
although 524 requires a relatively high concentration
( mgc ) , 22 wt % , to form a hydrogel , the authors reported that 524 inhibits the growth of several gram - positive ( mic = 0.10.5
g / ml ) and gram - negative ( mic = 510 g / ml ) bacteria
as well as fungi ( mic = 15 g / ml ) . moreover , the authors
reported that 524 is compatible with different mammalian
cell lines such as hep g2 , hela , and siha . later , the authors reported a new class of antibacterial hydrogelators
based on anti - inflammatory fmoc - amino acid / peptide - functionalized
cationic amphiphiles ( 525 ) . by the incorporation of a
pyridinium moiety at the c - terminal of fmoc - amino acid / peptides , the
positively charged hydrogelators self - assemble to form an antiparallel
-sheet arrangement of the peptide backbone and exhibit efficient
antibacterial activity against both gram - positive and gram - negative
bacteria . also designed
and synthesized several cholesterol - based amino acid - containing hydrogelators
( 526 ) that exhibit a high gelation efficiency ( mgc of
0.93.1 wt % ) and biocompatibility with human hepatic cancer - derived
hep g2 cells . after the incorporation of silver nanoparticles ( agnps ) ,
the soft nanocomposite of the amphiphile and agnps exhibits a notable
bactericidal property against both gram - positive and gram - negative
bacteria . sharma et al . designed and
synthesized two self - assembled amphiphilic ,-dehydrophenylalanine - containing
small glyco - dehydropeptides , 527 and 528 , with glucosamine attached at the c - terminal through a 6-aminocaproic
acid linker . the authors found that 527 and 528 self - assemble to form gels in a mixture of methanol and water at
a concentration of 0.1 wt % , with the sizes of the nanostructures
being 197 and 235 nm , respectively . in addition , the
authors used a disk diffusion assay to test the antimicrobial activity
of the peptides 527 and 528 , and they found
that the peptides display antimicrobial activity against micrococcus flavus , bacillus subtilis , and p. aeruginosa . das and ramesh et al . reported several
structurally diverse quinoline - based
amphiphiles containing a fluorescent head group and hydrophobic chain
of different lengths . among these amphiphiles , 529 ( scheme 69 ) is the most potent
antibacterial amphiphile to exhibit a dose - dependent bactericidal
activity on target pathogens and even inhibits the growth of a presumptive
mrsa strain . the authors found that this bactericidal activity may
result from the electrostatic binding of 529 to bacteria . most importantly , 529 has high antimicrobial selectivity ,
but hardly decreases the viability of human ht-29 cells . designed a peptide ( 530 ) by connecting two gram - positive antibacterial peptide sequences
( kigaki)3-nh2 with a central tetrapeptide
linker . they found that the electrostatic repulsion of the charged
lysine residues balances the hydrophobic collapse of the isoleucine
and alanine residues and backbone -sheet hydrogen bonding to
favor the self - assembly of 530 , which forms individually
dispersed nanofibers with a -hairpin conformation . furthermore ,
after 36 h of incubation , the hydrogel of 530 effectively
inhibits e. coli proliferation when
the concentrations of the initially introduced e. coli resuspensions are in the range of 1010 cfus / ml . however , when the bacteria reach a density of 10 cfus / ml , the hydrogel starts to lose its inhibitory capacity . yang and yi et al . designed and synthesized
a unique hydrogelator ( 531 ) based on ( )-menthol
and a lysine . 531 self - assembles to form an opaque hydrogel
at a concentration of 0.83 wt % . interestingly , the hydrogelators
form the 3d multiporous networks through acid base interactions
and strong double hydrogen bonding between amino acids for encapsulating
some known antibacterial agents such as zn and a series
of water - soluble organic antibiotic medicines such as lincomycin ,
amoxicillin , etc . using the oxford cup method , the authors found that
the antimicrobial susceptibility of the hydrogels loaded with zn or lincomycin was much more effective than that of the corresponding
aqueous solution when they were incubated with e. coli and s. epidermidis . in addition ,
the hydrogel of 531 is innocuous to mammalian cells such
as hela cells . reported the synthesis , self - assembly , and antimicrobial activity
of a series of oligomeric cationic compounds ( 532 ) . bisurea core flanked by
hydrophilic imidazolium groups with short alkyl ( cnh2n+1 , n <
6 ) or simple aryl tails , all the hydrogelators self - assemble to form
nanostructures in aqueous solutions . these cationic hydrogelators
exhibit potent , broad - spectrum antimicrobial activity and high selectivity
toward gram - positive bacteria ( including clinically isolated mrsa ) ,
killing the microbes via the membrane - lytic mechanism . notably , the
bacteria tested fail to develop resistance even after multiple exposures
to sublethal doses of the compounds , which is remarkably encouraging . chen and li et al . reported the preparation of biocompatible
hydrogels
with antimicrobial activity against gram - positive bacteria by taking
advantage of the intermolecular aromatic aromatic interactions
of fmoc and the phenyl group . they generated a hydrogel based on the
coassembly of fmoc - phe ( 200 ) and fmoc - leu ( 533 ) , and found that the coassembled ( 200 + 533 ) supramolecular hydrogel is bactericidal against gram - positive bacteria
via a mechanism involving cell wall and membrane disruption ( figure 19 ) . being biocompatible
with normal mammalian cells , this type of antibacterial hydrogel may
potentially serve as an antimicrobial coating in clinical devices
and wound dressings or a topical agent for the treatment of clinical
skin and wound infections mainly caused by gram - positive bacteria
such as s. aureus , as suggested by
li and chen . on the basis of the concept
of multidomain peptides ( mdps ) , dong et al . synthesized three hydrogelators , 534 , 535 , and 536 , that self - assemble
above critical assembly concentrations ( cacs ) of 0.87 , 1.24 , and 1.37
m , respectively . in addition , they found that the position
of tryptophan ( w ) determines the molecular secondary structure , supramolecular
nanostructure , stability , and antimicrobial activity . after incubation
with gram - negative bacteria ( e. coli and p. aeruginosa ) or gram - positive
bacteria ( s. epidermidis and s. aureus ) , a bacterial killing efficiency study
shows that 99% of gram - negative bacteria are killed by 535 and 536 , while less than 40% are killed by 534 . a surprising observation is the reverse dose - dependent relationships
between the concentration of the peptides and their cytotoxicity toward
primary mouse bone - marrow - derived monocytes ( bmdms ) , a result that is consistent with the formation
of aggregates . ( a ) representative sem images and ( b ) overlapping fluorescence
images for the live / dead bacterial staining assay of s. aureus before and after contact with the coassembled
( 200 + 533 ) hydrogel for 2 h. two fluorescent
dyes were used in live / dead staining in which syto 9 with green color
labeled both live and dead bacteria while propidium iodide with red
color stained only dead bacteria . adapted with permission from ref ( 1082 ) . if one looks into
a mirror , it is not difficult to realize that we are largely made
of soft tissues . because of the striking resemblance between hydrogels
and soft tissues , the most attractive and sought - after biomedical
application of supramolecular hydrogels is tissue engineering . however , most of the demonstrations , so far , center on the culture
of certain cells in vitro , which is still far away from the repair
processes needed for regenerating damaged tissues or diseased organs . first , the complexity
and dynamics of biological processes at the tissue level are just
beginning to be understood , and the
understanding is far from complete . thus , it remains difficult to
devise working engineering principles for tissue engineering without
adequate insights into the process . second , most of supramolecular
hydrogels consist of only one or two molecular species , which limits
their roles to be only complementary or supplementary to the inherent
or endogenous processes . third , currently , supramolecular hydrogels
still lack the sophistication or context - dependent features required
for the regeneration of tissues , which usually consist of a myriad
of transient biological processes . despite
these enormous challenges , it is still worthwhile to review the progress
made to date in tissue engineering by supramolecular
hydrogels so that further development can be made to meet the challenges
ahead . in the hope of developing an approach for repairing the
degenerated nucleus pulposus ( np ) of intervertebral disks , ulijn et
al . tested the growth of bovine nucleus pulposus cells on the hydrogel
made of [ ( fluorenylmethoxy)carbonyl]diphenylalanine ( fmoc - ff , 6)/fmoc - diglycine ( fmoc - gg , 398 ) in 1:0 and 1:1
ratios . using cryo - sem , the authors
verified that the hydrogels of 6 ( 1.0 wt % ) consist of
a dense network of nanofibers , whereas the hydrogels of 6/398 ( 1:1 , 0.7 wt % ) contain an overlapping mesh of
flat ribbons . in addition , the authors
found that the majority of the np cells remain in a rounded morphology
within both the hydrogels of 6 and the hydrogels of 6/398 after 5 days of culture . on the basis of
that observation , the authors suggested that the morphology of the
network has a limited effect on the np cells . glycosaminoglycan
by the np cells cultured within both the hydrogels of 6 and the hydrogels of 6/398 over 3 weeks ,
it is still too preliminary to establish the application of the hydrogel
of 6 for intervertebral disk tissue repair . recently ,
thordarson et al . examined the degradation of the hydrogels of 6 and observed that 6 or its degraded products
result in the necrosis of cells in vitro . this result , indeed , suggests that the fate of 6 in
vivo remains to be firmly established . as shown in scheme 70 , rowan et al .
reported the studies of the hydrogel of a guanosine - based
hydrogelator , 8-methoxy-2,3,5-tri - o - acetylguanosine ( 537 ) , for the cell culture
of a murine endothelial cell line ( c166 ) . 537 forms hydrogels
at as low as 0.5 wt % in 100 mm nacl . this hydrogelator forms helical
assemblies , rather than the macrocyclic quartet assemblies commonly
found in guanosine hydrogels . contrary to the claim of the authors
that there is little - to - no cytotoxicity of the hydrogels , the cell
viability of the c166 cells , in the presence of hydrogels containing
2 wt % 537 , is only about 50% of the control . it would be more interesting to elucidate
the cell mechanism of cell death caused by the self - assembly of this
hydrogelator . developed a short peptide derivative containing
halogenated phenylalanine and reported that the partially halogenated
peptide exhibits better gelation properties than fmoc - phe ( 200 ) in aqueous solutions . they found
that fmoc-4-fluorophenylalanine is the most efficient gelator ( among
the molecules derived by them ) that gels pbs buffer solution at a
minimum gelation concentration of 0.15 wt % . on the basis of this
observation , the authors designed and synthesized an fmoc - peptide
( fmoc - ffgrgd , 538 ) and
used the peptidic hydrogel to culture nih/3t3 cells . although 538 only formed a clear hydrogel in pbs buffer containing
20% dmso , the authors reported that the hydrogel could efficiently
promote the adhesion and proliferation of nih/3t3 cells . in a related study , parish and nisbet et al .
reported that fmoc - self - assembling peptides ( i.e. , fmoc - dikvav , fmoc - frgdf ,
fmoc - dyigsrf ) have been used as a vehicle for the delivery and support
of cell transplants in vivo . on the basis of self - assembling
p11-family of peptides reported
by boden , mcpherson et al . reported
the production of self - assembling peptides ( qqrfewefeqq , 233 ) in a relatively high yield using
an e. coli expression system . being
triggered by various physicochemical cues , 233 self - assembles
to generate self - supporting isotropic or liquid crystalline hydrogels
at peptide concentrations of 1030 mg / ml ( 13 wt % ) . using human dermal fibroblasts , the authors demonstrated that the
hydrogels formed by the recombinant peptides display excellent cytocompatibility . synthesized kld-12 peptide ( 417 ) and studied its biocompatibility with the host rabbit
and mscs , also , in the hope for repairing the degenerated nucleus
pulposus of intervertebral disks . on the basis of the histological
examination , the authors concluded that the 417 peptide
hydrogel has a good biocompatibility with the host rabbit and mscs
so that the 417 peptide hydrogel could serve as a good
scaffold material for tissue engineering of intervertebral disks . used phage display to
identify the peptide sequences
( e.g. , faqrvpp ( 539 ) , qhlprdh ( 540 ) ) preferably interacting with murine
nscs and connected those peptide sequences to ldlk12 peptides for generating functional self - assembling peptides . confirmed by rheology , these synthesized self - assembling peptide sequences
behave as classic hydrogelators to form hydrogels at a concentration
of 1 wt % which consist of nanofibers of 12 nm width and 1.6
nm height . the authors found that the new functional peptide sequences ,
being linked to the ldlk12 peptide , have the capacity to bind to nsc - derived
neural precursor cells ( npcs ) and promote the proliferation and differentiation
of the cells in vitro . on the basis of the high stem cell viability
and neural differentiation achieved by the 539 peptide
in vitro , the authors tested that peptide in acute contusive spinal
cord injury in rats , and reported that the peptides foster nervous
tissue regrowth and improve locomotor recovery . on the basis of these
results , the authors concluded that phage - display - derived functional
motifs need further investigation to elucidate their relevant molecular
targets and cellular pathways . according to the authors , in vitro
experiments display panning , the same laboratory
identified klpgwsg ( 541 ) as the nsc binding peptide and linked it to ldlk12 peptides for
the differentiation of nscs . the authors concluded that the enhancement
provided by the peptide conjugate still required the presence of the
growth factors ( e.g. , epidermal growth factor ( egf ) and basic fibroblast
growth factor ( bfgf ) ) and differentiative substrates . again , this
emphasizes the need to understand the molecular mechanisms linked
to the observed neuronal phenotype . on the basis of their works on
bmhp1 , gelain et al . derived a series of self - assembling peptides
by connecting biotin to various mutated bmhp1 peptides . after identifying
that one of the peptides ( biotin - gggafastkt - conh2 , 542 ) is particularly effective at fostering adhesion ,
differentiation , and proliferation of human nscs in vitro , the authors
conducted in vivo experiments and reported that 542 causes
a negligible immune response in the host nervous tissue in the short
term , though its performance on nerve fibers is similar to that of
saline . using a -sheet - forming
peptide , pdfdfdfdfdfdp
( denoted as pfd-5 , 543 ) , to form hydrogels
as the depot of tricalcium phosphate ( -tcp ) , rapaport et al . tested the proliferation of human fetal osteoblasts in vitro and evaluated
the performance of the hydrogels of 543 in rat bone defect
models . the authors found that the hydrogels of 543 ( at
a concentration of 5% , w / v ) are able to absorb calcium ions and to
induce osteoblast differentiation in vitro . although the tcp - loaded
hydrogels exhibit an efficacy of bone generation similar to that of
nonporous tcp , the in vivo results of bone defect healing in rat demonstrate
that the peptide hydrogel alone induces better bone regeneration in
comparison to the control ( nontreated defects ) . this result , indeed ,
agrees with the observation of calcium absorption by the hydrogels
due to the presence of a high density of aspartic acids in the peptides ,
and the hydrogels and the mineral act synergistically to enhance bone
regeneration . the authors concluded that the hydrogels of 543 might act as biocompatible and biodegradable matrixes to support
cellular osteogenic activity and to promote the turnover of calcium
minerals , through cellular processes , into bone tissue . banta et al . have evaluated a series
of peptides consisting of -roll
peptide derivatives as the calcium - responsive
motif and an -helical leucine zipper domain ( lz ) for intermolecular
interactions . one of the most valuable features of these peptides
is that the -roll domain of the peptides is intrinsically disordered
in the absence of calcium , while upon the addition of calcium , the
peptide forms a -roll secondary structure . the authors reported that these peptides form hydrogels
only in calcium - rich environments . recently , the same laboratory reported
another class of -roll peptides , but the application of these specific peptides in tissue engineering
has yet to be reported . recently , george et al . reported the use of
lz - based self - assembling peptides to form hydrogels for tissue engineering . the authors performed a quite comprehensive study of these hydrogels ,
from in vitro culture of human marrow stem cells ( hmscs ) to in vivo
evaluation of the hydrogels . besides demonstrating that the concentration
of the lz peptide is able to tune the pore size of lz hydrogels by
altering the peptide concentration from 7 to 12 wt % , the authors
functionalized the lz polypeptide by the incorporation of the rgd
domain for creating a suitable microenvironment for cell adhesion .
according to the results reported by the authors , the incorporation
of the canonical rgd domain has drastically improved the performance
of the lz hydrogels in many aspects . for example , an increase of the
percentage of rgd in the hydrogels not only improves the proliferation
of the hmscs , but also allows the hmscs to travel long distances within
the lz - rgds hydrogels . the in vivo implantation of the lz - rgds scaffolds
in a mouse model also significantly reduces the foreign body reaction
to the scaffold . in vivo experiments with hmscs also show that lz - rgds
hydrogels have a better ability to support neovascularization than
the lz hydrogels do . on the basis of these results , the authors concluded
that it should be possible to generate a functional and stable lz
scaffold for tissue engineering applications in vivo . galler and dsouza et al . reported
the use of the hydrogel
of a self - assembling , multidomain peptide for dental pulp tissue engineering . the authors used a peptide with a sequence
of k(sl)3rg(sl)3kgrgds ( 420 ; with a final peptide concentration of 1 wt % ) to interact with
heparin ( with a final concentration of 0.1 wt % ) to form a hydrogel
for incorporating growth factors ( e.g. , vascular endothelial growth
factor ( vegf ) , fibroblast growth factor ( fgf ) , and transforming growth
factor 1 ( tgf1 ) ) and then tested the use of the hydrogels
for encapsulating dental pulp stem cells in vitro and in vivo . the
authors observed that the proliferation of the cells increases in
the fgf - containing hydrogel , but decreases in the tgf1-containing
hydrogel and that the dental pulp stem cells spread and form a collagenous
matrix in the peptide hydrogel . one important observation is the formation
of a vascularized soft connective tissue similar to dental pulp subcutaneously
after transplantation of the hydrogel within dentin cylinders into
immunocompromised mice . although the authors concluded that the multidomain
peptide is a highly promising candidate for regenerative endodontics ,
the requirement of dental pulp stem cells , various growth factors ,
and dentin highlights the complexity of this regenerative process
and underscores the importance of mechanistic understanding . tirrell
et al . reported an innovative branched peptide amphiphile ( 544 ) that forms a hydrogel upon changing the ph from acidic to neutral . in addition , at 1% ( w / v ) 544 , the hydrogel is capable
of achieving a storage modulus of 10 kpa . by modulating the concentration
of 544 , the authors were able to regulate the viscoelastic
properties of the hydrogels , thus broadening their versatility for
complying with the mechanical requirement of a wide range of tissues . the authors tested the culture of nih/3t3 fibroblast cells on the
hydrogel of 544 for evaluating the biocompatibility of
the hydrogel ( figure 20 ) . after seeding the fibroblasts in the absence of serum to ensure
the cells attach to the hydrogels through nonspecific interactions ,
the authors observed that these previously attached cells spread when
serum was added . on the bais of live / dead staining , the authors determined
that the fibroblasts are predominately alive and suggested that the
hydrogel is a viable biocompatible nanofiber - based tissue scaffold
for supporting 3d cell growth . nanofibrous
hydrogels are reported to be compatible with nih/3t3
fibroblasts . in the presence of serum , fibroblasts spread by 28 h.
at 72 h , spreading appeared to be spindle - like , resembling the natural
morphology of the cell type . the fibroblasts proliferated for a minimum
of 96 h. these images are from a single hydrogel of 544 . have designed a
synthetic peptide hydrogel which
consists of a 16-amino acid peptide ( 281 ) and is called puramatrix . the authors used 281 to assess the feasibility of transplantation
of isolated mucosal cells to repair a damaged middle ear . the authors
collected middle - ear bullae with mucosa from rats , transfected the
cells with enhanced green fluorescent protein ( egfp ) , encapsulated
the cultured middle - ear mucosal epithelial cells into puramatrix hydrogels
( 1% , w / v ) , and then transplanted the cells into the immunosuppressed
rats ( figure 21 ) .
besides validating that primary cultured cells retain the character
of middle - ear epithelial cells , the authors found that a high proportion
of egfp - expressing cells reside in the recipient middle ear after
the transplantation using the hydrogel , but not without the hydrogel . these extensive studies demonstrated the feasibility of transplantation
of cultured middle - ear mucosal epithelial cells encapsulated within 281 for regeneration of surgically eliminated mucosa of the
middle ear in sprague dawley ( sd ) rats . however , the authors also observed that the proliferation rate depends
on the seeding density and suggested that it might be due to contact
inhibition or the limitation of the nutrient supply . in another related
study , puramatrix served as the carrier for recombinant human bone
morphogenetic protein-2 , which significantly enhances bone regeneration
in a bone augmentation rabbit model . ( a l ) analysis of transplanted mucosal epithelial cells
in recipient tissues at postoperative days 14 and 28 . serial frozen
sections of middle - ear bullae after transplantation ( 0.5 10 cells / ml ) at ( a f ) postoperative day 14 and ( g l )
postoperative day 28 . ( a , b , g , h ) fluorescence images at several
time points . enhanced green fluorescent protein ( egfp)-expressing
cells were detected on the internal surface of recipient middle - ear
bullae ( green , egfp ; blue , 4,6-diamidino-2-phenylindole ) ( a ,
g ) . results of immunostaining with ( c , i ) antipancytokeratin , ( d ,
j ) antivimentin , ( e , k ) anticollagen iii , and ( f , l ) anticollagen
iv antibody . egfp - expressing cells were positive for pancytokeratin
( c , i , arrows ) , but not for vimentin ( d , j ) . collagen iii - positive
regions were detected mainly in the subepithelium ( e , k ) . collagen
iv - positive regions were detected under the monolayer structure of
donor cells at 14 and 28 days after transplantation ( f , l , arrowheads ) . the scale bars represent 50 m . adapted with permission from
ref ( 1121 ) . copyright
2013 dove medical press ltd . wang et al . tested the hydrogels made of the self - assembling
peptide 281(1118 ) or 281 containing
the laminin epitope ikvav ( 545 ) at the c - terminal to
act as a functional peptide - based scaffold to repair injured brain
tissue . they found that 545 self - assembles to form nanofibers
with a bilayer -sheet structure and affords a hydrogel with
mechanical stiffness similar to that of brain tissue , which makes
the hydrogel suitable for encapsulating nscs in an animal model study . the authors reported that the in vitro results showed that 545 serves as a guiding cue to promote the adhesion of the encapsulated
nscs and to bias the neuronal differentiation of these cells . using
the injected peptide solution to form the 3d hydrogel immediately
in situ for filling up the cavity and bridging the gaps in the wound
created in the brain , the authors demonstrated that the hydrogel of 545 enhances the survival of the encapsulated nscs and reduces
the formation of astrocytes . although the authors reported enhanced
neuronal differentiation and an improvement in brain tissue regeneration
after 6 weeks post - transplantation , the functional recovery of the damaged brain remained to be evaluated . in addition , the authors also linked other functional groups derived
from fibronectin and laminin ( e.g. , grgds ( 546 ) or yigsr
( 547 ) ) to the 281 motif to evaluate the
capability of these functionalized self - assembling peptides for the
purpose of maintaining hemostasis and liver tissue regeneration . after developing responsive -helical
peptide hydrogels , woolfson et al . they
reported that the cell viability is high and the -helical gel
network is stable in tissue culture conditions over 14 days . besides macroscopic
properties , such as soft and wet , the majority of the volume of supramolecular
hydrogels is micropores filled with water . these interstices allow
the hydrogels to serve as a carrier or medium of other bioactive molecules for a relatively straightforward application , such as drug delivery . to describe the applications of supramolecular hydrogels for drug
delivery , we arrange the following section in two parts : first , we
mainly describe various hydrogelators used for encapsulating drugs ; second , we focus on hydrogelators covalently conjugated with therapeutics . as
shown in scheme 71 , van esch et al . reported the use of the classical small molecular
hydrogelator n , n-dibenzoyl - l - cystine ( dbc , 1 ) for the release of small molecules
8-aminoquinoline ( aq , 548 ) and 2-hydroxyquinoline ( hq , 549 ) as model molecules of drugs . using self - assembly of 1 to form stable , clear hydrogels in 150 mm nacl solution
and pbs buffer , the authors tested this kind of hydrogel for the release
of certain small molecules . the authors concluded that the release
profiles depend on the interactions of the hydrogelator with the entrapped
molecules because they observed that the release of 549 from the gels of 1 was 7 times faster than that of 548 due to acid base interactions between 548 and 1 . as suggested by the authors , the judiciary combinations
of the hydrogelator and the drug molecules should be able to control
the release of the drugs . xu et al . reported
the combination of two simple fmoc - amino acids ( 533 and 201 ) to form semitransparent hydrogels at ph 9.1 with a minimum
concentration of 533 or 201 of 10 mm . after
the addition of 2 molar equiv of na2co3 , the
mixture of 533 or 201 forms a clear hydrogel
consisting of entangled irregular fibers with widths of 120500
nm . besides the inclusion of 201 , an anti - inflammatory
drug candidate , the hydrogels act as
carriers for other bioactive agents , such as 5-fluoro-2a - deoxyuridine
( 5-fu ) , an antineoplastic agent , or pamidronate , an osteoporosis drug , by the simple mixing of the drugs in the solution
of 533 or 201 prior to hydrogelation . haldar et al . reported that the addition of
-aminobutyric acid ( 550 ) to the solution of fmoc - lysine
( 202 ) leads to the formation of a hydrogel at ph 6.9
without any heating cooling cycle at a concentration of 2 wt
% . the authors suggested that this hydrogel could be used for the
recognition and release of the anti - inflammatory agent 202 . ,
the sugar moiety facilitates intermolecular hydrogen bonding , the
phenyl ring enhances intermolecular aromatic aromatic interactions ,
and the hydrophobic hydrocarbon chain decreases the solubility in
water and increases the molecular association through the van der
waals interactions . the authors used the networks made of helical
ribbons and fibers ( 50 nm ) in the hydrogel of 551 for encapsulating curcumin , a chemopreventive hydrophobic drug ,
to demonstrate the potential application of the hydrogel of 551 in drug release . on the
basis of l - phenylalanine and l - tyrosine with subtle
variation in the structure of the head group , das et al . designed
and synthesized 10 structurally correlated amino acid - based amphiphiles
for screening hydrogelators , and found 3 of them to confer ph - responsive
hydrogels at room temperature . forming at a cgc of 4 wt % , the hydrogel
of 552 exhibits remarkable sensitivity to ph , which makes
the hydrogels suitable for the release of vitamin b12 and
cytochrome c. the authors found that , at ph 7.4 ,
all three hydrogelators form suitable hydrogels to release the entrapped
biomolecules via diffusion . at endosomal ph ( 5.5 ) or a further
lower ph , the release rate of biomolecules from the hydrogel of 552 increases by about 10-fold compared to that observed at
ph 7.4 , largely due to the dissociation of the gels . reported a guanosine derivative , guanosine-5-hydrazide
( 347 ) , that forms tetramers ( i.e. , g - quartet ( g4 ) ) in the presence of cations such as na , k , and nh4 . the authors used the hydrogel of 347 to entrap acyclovir , vitamin c , or vancomycin for controlled
release . besides the physical trap
and release of biological molecules , lehn et al . later reported that
the g - quartet structure of 347 acts as a delivery system
for the slow release of bioactive carbonyl derivatives since aldehydes
or ketones can reversibly react with the free hydrazide functions
at the periphery of the g - quartet to form acylhydrazones . barthelemy et al . reported the first example
of the use of a glycosyl - nucleoside lipid for the delivery of oligonucleotides
into cells ( human huh7 cells ) . they linked a simple monosaccaride ,
a lipidic chain , and a nucleoside together by 1,2,3-triazole bridges
to obtain 339 , which self - assembles to form nanofibers
roughly 2030 nm in diameter and results in hydrogels at a
concentration above 2.5 wt % . they reported that 339 is
compatible with huh7 cells after 5 days of incubation and the nucleic
acid339 complex enhances the cellular uptake
of the nucleic acids in the presence of serum . yi et al . designed and synthesized three amphiphilic 3,4,5-trihydroxybenzoic
derivatives with alkyl chains of different lengths , among which 553 and 554 gel aqueous ethanol in the presence
of the water - soluble drug tetracycline hydrochloride . they found that
a small amount of small molecules ( 10 mg / ml 554 or 3.3
mg / ml 553 ) are able to entrap a large amount of tetracycline ,
up to 91.5% . in addition , the release studies of tetracycline in various
solutions indicate that the release rate of tetracycline for a bovine
serum albumin ( bsa ) solution ( 10 mg / ml ) is faster than that with the
other solutions because of the strong interaction between tetracycline
and bsa . schneider and pochan
have designed a class of self - assembling peptides
that undergo triggered hydrogelation in response to physiological
ph and in salt conditions ( ph 7.4 , 150 mm nacl ) to form mechanically
rigid , viscoelastic hydrogels . among
the -hairpin peptides , 251 and 257 are two peptide sequences with different charge states for directly
encapsulating and controllably releasing model fluorescein isothiocyanate
( fitc)dextran macromolecules of varying size and hydrodynamic
diameters . using fluorescence recovery
after photobleaching ( frap ) , the authors studied the self - dissociation
of the hydrogels and bulk release of model fitc dextran macromolecules . the authors reported that the mobility of the macromolecules or the
probes within and release of these hydrogels depended on the sizes
of the probes , the peptide sequences , and the mesh size of the hydrogel . later , the authors also found that the self - assembling 257 peptide hydrogel is an effective vehicle for the local delivery
of curcumin . hamachi et al . reported
a single - component , multiple - stimulus responsive hydrogelator , 555 , containing a phosphate group that displays a macroscopic
gel sol response toward four distinct input stimuli ( temperature ,
ph , ca , and light ) . the authors suggested that the hydrogelator
confers gel - based supramolecular logic gates displaying and , or , nand ,
and nor functions . by using these logic - gate - like functions , they
found that the hydrogel is able to hold and release bioactive substances
( e.g. , vitamin b12 or the protein rh - con a ) in response
to various input triggers . several
labs evaluated the ac-(rada)4-conh2 ( 281 ) peptide hydrogel to act as an efficient slow
release carrier of a variety of proteins , such as lysozyme , trypsin inhibitor , bsa , and immunoglobulin g ( igg ) ,
which differ in physicochemical properties and morphologies . the results
of the fluorescence correlation spectroscopy ( fcs ) analysis indicated
that the peptide hydrogel of 281 , at a concentration
of 1 wt % , can encapsulate the proteins and release the proteins when
the hydrogel disintegrates due to the degradation of the peptides
by proteolytic enzymes in vivo . furthermore , zhang et al . found that
the protein diffusion through the hydrogel of 281 depends
primarily on the size of the proteins and the encapsulation and release
barely affect the protein conformations and functions . later , zhang et al . reported the use of the
peptide hydrogels for facilitating slow and sustained release of active
cytokines related to many areas of regenerative medicine . however ,
the release of negatively charged vegf from the hydrogel of 281 is slower compared to that of cytokines of somewhat similar
molecular weight but opposite charge , suggesting that the positive
guanidinium interacts with vegf and hinders the diffusion of vegf . the authors also found that the release of functional human fgf
and vegf occurs over 23 weeks within the hydrogel of 281 and two other hydrogels formed by peptides with net positive
or negative charges located at the c - terminal . shibata et al . have recently found that the aqueous solution
of 281 and insulin form a hydrogel , in vitro and in vivo ,
with an increase of the ionic strength by phosphate ion and an increase
of the ph . the in vitro experiments indicated that the release rate
of insulin depends on the concentration of 281 and the
controlled release of insulin occurs at final concentrations of 281 between 0.1 and 2.0 wt % . furthermore , the authors found
that 281 forms a hydrogel in vivo for a sustained - release
insulin , which also depends on the concentration of 281 . tan and kinoshita modified the 281 peptide scaffolds by positioning a phenylalanine residue
at the c - terminal to generate 556 and 557 and studied the entrapment and the release of certain enantiomers
( e.g. , d- , and d - phenylalanine ) of amino acids .
they found that the amount and chirality of the guests tailor the
network nanostructures , thus affecting the release of the enantiomers . in addition , the release rate of the enantiomers from the hydrogels
containing one phenyl group ( 556 and 557 ) is much slower than from the hydrogels without a phenyl group ( 281 ) , agreeing with the aromatic interactions between the
hosts and the guests . the concentration of the trapped enantiomers
after the diffusion matches with the
release kinetics controlled by fickian diffusion , which depends on
both the rational design of the peptides used for making the hydrogels
and the choice of the size and lipophilicity of the entrapped molecules . xu et al . reported the first in vivo
imaging for investigating
the drug release properties of the supramolecular hydrogel formed
by hydrogelators 558 and 559 consisting
of naphthalene ( nap ) and a d - peptide of diphenylalanine ( figure 22 ) . tem images show
that the hydrogels consist of nanofibers with a length of over tens
of micrometers , a width of about 50 nm , and an average mesh size of
about 200 nm . since the hydrogels resist hydrolysis catalyzed by proteinase
k and offer long - term biostability , the hydrogel of 559 is suitable for the controlled release of drugs in vivo . ( a ) profiles of the mean blood concentration
of i - nai
vs time after subcutaneous ( sc ) administration to rats ( 160 ci / kg )
( , control , i - nai solution , auc ( area under
the curve ) = 1213.3 cih / l ; , experimental , i - nai in gel ii , auc = 1453.5 cih / l ) . ( b ) dynamic
( upper two lines ) and static ( lower line ) single - photon emission computed
tomography ( spect ) images of rats with i - nai ( 500 ci / rat ;
left , in solution ; right , in gel ii ) administered sc . ( c ) profiles
of the mean blood concentration of i - epidepride vs time
after sc administration to rats ( 160 ci / kg ) ( , control , i - epidepride solution , auc = 645.5 cih / l ; ,
experimental , i - epidepride in gel ii , auc = 693.6 cih / l ) . ( d ) dynamic spect images of rats with i - epidepride ( 500
ci / rat ; left , in gel ii ; right , in solution ) administered sc .
adapted from ref ( 1176 ) . copyright 2009 american chemical society . as shown in scheme 72 , banerjee et al . reported two synthetic self - assembling tetrapeptides ,
gail ( 560 ) and gfil ( 561 ) , that form thermoreversible
and ph - sensitive hydrogels which consist of long , interconnected nanofibrillar
network structures with diameters of 1530 and 1025
nm , respectively . these hydrogels entrap doxorubicin to allow its
slow release at physiological ph , and achieve almost 85% ( for peptide
gel 560 ) and 90% ( for peptide gel 561 ) release
of the drug molecules after 45 h. adams
et al . reported the hydrogels of fmoc - phe ( 200 ) and fmoc - tyr
( 15 ) formed by careful adjustment of the ph of the solution
using gdl . they found that the hydrogels of 200 and 15 entrap and release certain dye molecules under the control
of fickian diffusion . on the basis of the similar diffusion coefficients
of the dyes of different radii from the hydrogel of 200 , the authors concluded that the networks in the hydrogel of 200 only restrict molecules larger than 5 nm . banerjee et al . reported two n - terminally
protected dipeptides ( 562 and 563 ) with
a -amino acid residue that form hydrogels at physiological
ph ( 7.46 ) and temperature ( 37 c ) . having different cgcs ( 0.85
wt % for 562 and 1.21 wt % for 563 ) , the
hydrogels consist of nanofibers of different widths ( 45130
nm for 562 and 3060 nm for 563 ) . in addition , these two hydrogels can encapsulate and sustainably release
two vitamins ( vitamin b2 and vitamin b12 ) over
3 days . banerjee et al . reported that
a designed tripeptide - based hydrogelator ( 564 ) having
both 11-aminoundecanoic acid and phe - phe residues forms hydrogels
that entrap vancomycin and vitamin b12 for sustained release
at physiological ph and temperature for about 2 days ( figure 23 ) . according to an mtt - based
cell viability assay at 24 h , the authors suggested that this peptide
gelator , 564 , is innocuous to cells . ( a ) illustration of the injectable nature
of the hydrogel and its
vitamin release phenomenon with vitamin b12 . ( b ) percentage
release plot of some important biomolecules from hydrogel 564 at physiological ph ( 7.46 ) and temperature ( 37 c ) , where the
concentration of the drugs loaded into the hydrogel was 1.14 mg / ml
for cyanocobalamin ( vitamin b12 ) and 0.24 mg / ml for vancomycin .
adapted from ref ( 1179 ) . zhang and jiang et al . reported a new peptide comprised of
a peptide
backbone containing an arg - gly - asp ( rgd ) sequence and a hydrophobic
fmoc tail . the peptide derivative 565 self - assembles
to form a transparent hydrogel and exhibits biocompatibility in rabbit
eyes . the authors found that this peptide hydrogel , acting as an implanted
carrier , delivers an antiproliferative model drug ( 5-fluorouracil ,
5-fu ) in rabbit eyes and inhibits postoperative scarring formation .
according to the in vivo experiments reported by the authors , the
5-fu - loaded peptide hydrogel releases 5-fu to inhibit scleral flap
fibrosis efficiently after the surgery . later , castelletto et al . reported another functionalized peptide , 401 , which also contains rgd . they found that 401 , at a concentration of 10 wt % , formed homogeneous hydrogel monoliths
that are stable in water for nearly 40 days . consisting of a rigid
porous structure made of the peptide fibers , the hydrogel monoliths
are able to encapsulate and release various molecules , including model
hydrophilic dyes and drug compounds ( e.g. , bioactive riboflavin and
hydrophilic pseudodrug salicylic acid ) . diaz et al . reported the supramolecular coassembly of complementary
structures followed by controlled thiol ene coupling as a new
strategy for fine - tuning the drug release kinetics of self - assembled
hydrogels made of small molecules 1 . tem and sem images
indicate that 1 self - assembles into fibers 30150
nm in diameter at a concentration of 0.2 wt % . using in vitro experiments ,
the authors showed that the hydrogel of 1 entraps and
releases small drugs , such as 2-hydroxyquinoline ( 549 ) , a model of water - soluble and uv - active drugs . jung and john et al . reported the formation of a coordination
polymeric hydrogel made of a simple pyridine derivative ( 516 ) and cu ions . consisting of a fibrillar network of
fibers several micrometers in length and 4565 nm in width ,
the hydrogel of 3 can encapsulate curcumin . being ph - triggered
at physiological temperature , the hydrogel dissociates and releases
the encapsulated curcumin . shimizu
et al . designed and synthesized a simple amphiphile , 566 , consisting of a photoresponsive azobenzene and a hydrogen - bonding
glycine to construct self - assembled nanotubes . tem showed that the
self - assembled morphologies strongly depend on the ph conditions ;
that is , the self - assembly of 566 at ph 6.1 gives fibers ,
but at ph 9.2 gives sheets . upon uv - light irradiation , the trans cis
photoisomerization of the azobenzene within the tubular wall results
in a morphological change from nanotubes to cylindrical nanofibers
to release the pre - encapsulated guests ( e.g. , carboxyfluorescein ( cf ) )
in the hollow cylinder of the nanotubes . miravet and escuder et al . reported a hydrogelator bearing a nucleophilic
reactive site that reacts with aldehydes to cause the disassembly
of the hydrogel network . the authors found that the hydrogels of 567 entrap and release dyes or drugs ( e.g. , methylene blue
or ketoprofen ) in response to the presence of specific aldehydes . in addition , 567 is highly biocompatible , which may present
a protective effect against toxic aldehydes . rapaport et al . have developed amphiphilic -sheet
peptides pfd-5 ( 543 ) decorated by acidic amino
acids . 543 self - assembles to form ordered monolayers
at the interfaces as well as hydrogels near physiological ph . the
authors found that the mildly amphiphilic doxorubicin can be entrapped
within the amphiphilic matrix of the peptide hydrogel , due to electrostatic
forces and hydrophobic interactions . the peptide doxorubicin
interactions may affect the release of doxorubicin from the peptide
hydrogels as less doxorubicin was released from the hydrogels with
a higher loading of doxorubicin . as shown in scheme 73 , zhao et al . designed a self - assembling peptide , p4 ( 568 ) , containing 16 amino acids that forms stable -sheet nanofibers
with a diameter of 25 nm and a length of micrometers . the authors
found that the hydrogel of 568 is capable of stabilizing
hydrophobic anticancer agents , such as ellipticine , a natural plant
alkaloid . sem images showed that the state of ellipticine in the complexes
relies on the concentration of 568 , which also affects
the size and morphology of the complex . it was found that the complexes
of ellipticine and the peptide significantly reduce the viability
of two cancer cell lines ( smmc7721 and ec9706 cells ) . miller et al . reported three octapeptides ,
vevevkve ( vek1 , 569 ) , vevkvevk ( vek2 , 570 ) , vkvkvevk ( vek3 , 230 ) , which carry
a net charge of 2 , 0 , and + 2 at neutral ph , respectively . the author found that all three peptides form transparent and self - supporting
hydrogels . the hydrogels of 570 and 230 encapsulate
two hydrophilic model drug molecules ( naphthol yellow and martius
yellow ) and release them following fickian diffusion and depending
on the fibrillar network and the overall charges of the complex molecules . reported
the simple and remarkable small molecular
hydrogelators of phenylalanine - containing cyclic dipeptides ( diketopiperazines ,
dkps , 571 and 572 ) , which only contain proteinogenic
amino acids ( serine , cysteine , glutamate , histidine , or lysine ) as
building blocks . 571 and 572 form stable
and self - healing hydrogels with a porous network or dense lamellar
sheets with bundled nanofiber connections between the sheets , respectively . furthermore , the authors found that the mixture of 571 and 572 forms heterotypic hydrogels and demonstrated
their use for the release of bsa and tetracycline . liu et al . designed a supramolecular hydrogel based on
a peptide dendron ( 573 ) and found that metal ions can
trigger a continuous shrinkage after the gels have been annealed for
several hours . it is reported that the metal ions ( e.g. , mg , cu ) significantly promote the gelation capacity , and
decrease the cgc from 0.3 to 0.08 wt % or below . in addition , the
reversible shrinkage property of the hydrogels allows the controlled
release of small molecules such as vitamin b1 after addition
of divalent metal ions ( such as mg ) into the gel . focused on an octapeptide , fefefkfk ( 228 ) , which self - assembles into antiparallel -sheet - rich fibers
and forms hydrogels at concentrations above 2 wt % in water . the authors
used the hydrogel of 228 for the delivery of two commercial
drugs , lidocaine and flurbiprofen . they found that the addition of
lidocaine to the hydrogel stiffens the samples without affecting the
overall peptide release , while the hydrogel encapsulating flurbiprofen
exhibits improved resistance erosion and enhances drug retention . reported the use of a class
of phenylalanine ( phe)-containing
self - assembling peptide nanofibrous material ( ratea - f8 , 574 ) for the delivery of 5-fluorouracil ( 5-fu ) and leucovorin ( lv ) ,
which shows synergistic action against colon cancer cells . the study
of the gelation properties indicated that 574 self - assembles
to form nanofibers in water and tris hcl buffer at a concentration
of 0.78 wt % with a diameter of 520 nm and a length of 6080
nm . the in vitro experiments indicated that the hydrogel of 574 slowly releases 5-fu , lv , and phe . koutsopoulos et al . designed a class of lipid - like peptides
with an aspartic acid or lysine hydrophilic head and a hydrophobic
tail composed of six alanines ( ac - a6k - conh2 ( 575 ) or ac - a6d - cooh ( 576 ) ) . the authors
found that the addition of this kind of lipid - like peptide into water
or an electrolyte solution results in formation of a turbid suspension
due to self - assembly of the peptide monomers to minimize the interaction
between the hydrophobic domains and polar environment . in addition ,
it was found that 576 is more suitable for the encapsulation
and release of carboxyfluorescein and nile red . in a related study , qiu et al . reported the use of 575 to transfer pyrene into living hepg2 cells . as shown in scheme 74 , van esch et al . reported the gelation
properties of a class of
cyclohexanetrisamide - based hydrogelators with an l - phenylalanylamidoquinoline
( l - phe - aq , 577 ) moiety as well as two ethylene
glycol chains . according to this design ,
-chymotrypsin ( -chy ) can enzymatically cleave the 577 moiety to release the fluorogenic model drug
6-aminoquinoline ( 6-aq , 578 ) . moreover , the cleavage
of the two ethylene glycol chains increases the hydrophobicity of
the overall structure to improve the gelation properties . the hydrogelator
forms fibers with identical diameters of 4.2 nm and a thermoreversible
hydrogel with a cgc of 0.03 wt % at room temperature . the authors
envisioned that the hydrogel might act as a two - stage enzyme - mediated
drug release system . that is , aq ( 548 ) , in the gel fibers ,
is initially protected from enzymatic cleavage ; after the temperature
is increased , more hydrogelators become available for enzymatic cleavage
to result in a dramatic increase in the rate of release of 548 . kim et al . synthesized a class of
( s)-(+)-ibuprofen - based hydrogelators , among which 579 forms hydrogels consisting of entangled irregular fibers
with widths of 60100 nm and with a cgc of 0.9% ( w / w ) . two
hours after the addition of an enzyme ( carboxypeptidase y ) , the hydrogel
is able to release the drug ( s)-(+)-ibuprofen . wang et al . reported a simple
drug candidate , 4-oxo-4-(2-pyridinylamino)butanoic
acid ( 580 ) , which self - assembles to form hydrogels at
a 4 wt % concentration under various conditions . they found that this
hydrogel with different backbone structures releases drug molecules
at different speeds . reported a synthetic decapeptide kp ( 581 ) derived from
the variable region of the light chain of a recombinant antibody . they found that , in nonreducing conditions , the solubilized 581 molecules easily dimerize due to the formation of disulfide
bridges and spontaneously and reversibly self - assemble to generate
an organized network of fibril - like structures . furthermore , this
self - assembled network , likely being resistant to proteolysis , slowly
releases the active dimeric form of 581 , which acts as
a microbicide in vitro against a number of pathogenic microorganisms ,
such as candida albicans . john et al . reported the synthesis of small
hydrogelators 582 and 583 containing amphiphilic
prodrugs , such as acetaminophen . the prodrugs self - assemble to form
branched or entangled fibrous / sheetlike gel networks with a fiber
thickness of 50400 nm and fiber lengths of several micrometers . the hydrogels are able to encapsulate a second drug such as curcumin .
using the in vitro experiments with mscs , the authors showed that
the hydrogels of 582 and 583 release single
or multiple drugs under physiological conditions and upon enzyme catalysis ,
and retain certain features of the mscs . reported the use of coordination for converting n - acetyl - l - cysteine ( 584 ) , a mucolitic
agent or an antidote in paracetamol intoxication , into a hydrogelator . 585 is a metal thiolate that self - assembles to form
metallophilic hydrogels in the presence of au(iii ) , ag(i ) , and cu(ii )
salts with a microporous structure in the form of flakes . although
these specific hydrogels only form at ph < 4 to be able to act
as drug delivery systems , the concept
demonstrated by odriozola et al . should be applicable to other systems
that form metallophilic hydrogels at physiological ph . on the
basis of the serendipitous discovery of a supramolecular
hydrogelator made of vancomycin , xu et
al . reported the first example of a hydrogelator ( 586 ) derived from an aminoglycoside antibiotic ( e.g. , kanamycin ) and
the resultant hydrogel for sequestering 16s rrna selectively via divalent
interaction ( figure 24 , scheme 75 ) . 586 self - assembles to form a transparent hydrogel containing
small fibrils with diameters from 50 to 60 nm at neutral ph with a
cgc of 0.3 wt % . kanamycin a in the hydrogel of 586 likely
binds to the a - site of 16s rrna . the association constant between 586 and 16s rrna is much higher than the binding constant
of kanamycin a with 16s rrna , suggesting cooperative binding . xu et al . designed and synthesized another
hydrogelator ( 587 ) by using a tripeptide derivative that
consists of a naphthyl group , two phenylalanines , and one modified
lysine residue carrying an olsalazine moiety ( a clinically used anti - inflammatory
prodrug ) in the side chain of the lysine residue . they found that 587 self - assembles to form supramolecular hydrogels under
mildly acidic conditions with a cgc of 0.8 wt % . in addition , the
reduction of olsalazine not only leads to a gel - to - sol phase transition
but also controls the release of 5-aminosalicylic acid as the anti - inflammatory
agent , which potentially provides a way to encapsulate the prodrug
and release the active ingredients upon biological cues . supramolecular nanofibers sequester the potential
targets ( e.g. ,
16s rrna ( in red ) ) in the gel phase . xu et al . designed another
hydrogel precursor ( 588 ) based on paclitaxel , a well - established
antineoplastic agent that
binds specifically to the -tubulin subunit of microtubules
to arrest mitosis and result in apoptosis . upon the action of alkaline
phosphatase , 588 turns into a hydrogelator ( 589 ) that self - assembles to form nanofibers with a uniform width of
29 nm and affords a supramolecular hydrogel of the paclitaxel derivative . the mtt cell viability assay indicated that , after 48 h of incubation
with hela cells , 588 exhibited an ic50 value
of 25 nm , which is comparable to that of paclitaxel ( 13.5 2.2
nm ) . in addition , 589 itself also exhibited an ic50 of 25 nm , which is also comparable to that of paclitaxel
and 588 . reported a class of innovative hydrogelators ( 590 ) by
connecting a gel - forming lysine moiety with model drugs ( e.g. , benzylamine
and phenethylamine ) through a self - immolating spacer ( p - aminobenzyloxycarbonyl ) . tem images showed that 590 self - assembles to form hydrogels containing fibers with dimensions
ranging from hundreds of nanometers in width and several micrometers
in length . the authors found that trypsin catalyzes the hydrolysis
of the amide linkage between the gelator moiety and the spacer of 590 , thus releasing the model drug . as shown in scheme 76 , yang et al . designed and synthesized the first example of
a folic
acid ( fa)paclitaxel conjugate , fa - gpyk - paclitaxel ( 591 ) . this innovative
precursor contains paclitaxel , an effective , clinically used anticancer
drug , folic acid , a ligand targeting cancer cells , and tyrosine phosphate ,
an enzymatic trigger for self - assembly . upon dephosphorylation catalyzed by phosphatases , 591 forms a transparent hydrogel in pbs buffer at a concentration of
0.2 wt % with a nanosphere morphology . in addition , 591 also acts as a prodrug for releasing paclitaxel upon ester cleavage . later , yang et al . designed and synthesized
another precursor ( 592 ) that contains two complementary
anticancer drugs , dexamethasone ( dex ) , an anti - inflammatory and immunosuppressant ,
and paclitaxel or hydroxycamptothecin ( hcpt ) . upon the reduction by
gsh and dtt , 592 turns to 593 , which self - assembles
to form hydrogels with cgcs of about 0.25 and 0.75 wt % , respectively ,
in pbs buffer . four hours after the formation of the hydrogels , the
authors added an equal volume of fresh pbs buffer solution and observed
the original drug molecules of dex , paclitaxel , and hcpt releasing
from the gels due to the hydrolysis of the ester bond . they also developed a series of hydrogelators
based on paclitaxel and short peptides / amino acids with simple synthetic
strategies and high yields that could be used for the release of paclitaxel
without any burst releases . as shown in scheme 77 , yang et al . also designed
a precursor ( 594 ) by using
a releasable disulfide carbonate linker to form stable molecular hydrogels . although 594 is unable to be completely converted from
the gelator terminated by a free thiol group to the gelator terminated
by a hydroxyl group , the authors succeeded in generating a stable
molecular hydrogel mainly formed by the hydrogelator terminated with
a hydroxyl group . in addition , upon the endosomal reduction of the
disulfide bond , a self - cyclization process results in the unmodified
drug . yang and zhao et al . combined
a galactose - caged nitric oxide ( no ) donor and a short peptide of nap - ffggg
to generate a hydrogelator ( 595 ) that forms hydrogels
in pbs buffer at a concentration of 0.5 wt % . using the -galactosidase
to remove the protective galactose from the no donor , the authors
demonstrated enzyme - triggered release of no from the hydrogel of 595 . furthermore , the in vivo experiments showed that the
two - component hydrogels of 595 improve wound healing
of mice ( figure 25).scheme 77representative molecular
structures of hydrogelators conjugated with drugs ( a ) percentage of wound area left in different groups
at day 7
compared to the original wound area ( mean sem ) at day 0 . ( b )
photographs of wounds in animals treated with pbs buffer , 3 ( hydrogel containing 1.0 wt % 3 ) , free no + gal ( solution
containing 0.2 wt % no donor with daily addition of 1.5 10 u of -galactosidase ) , no gel ( hydrogel containing
1.0 wt % 3 and 0.6 wt % 595 without the
addition of -galactosidase ) , and no gel + gal ( hydrogel containing
1.0 wt % 3 and 0.6 wt % 595 with the addition
of 1.5 10 u of -galactosidase each
day ) . adapted with permission from ref ( 1211 ) . kim et al . designed and synthesized
an amphiphile ( 596 ) containing riboflavin , an essential
biomolecule ( vitamin b2 ) that is involved in various biochemical
processes . 596 forms hydrogels at a concentration of
1.6 wt % in acidic
( ph 5 ) and neutral ( ph 7.4 ) buffer solution with mild heating . the
authors found that the hydrogel of 596 , being cell compatible ,
helps deliver vegf - sirna efficiently into human cells . conjugated anti - hiv reverse transcriptase inhibitor 2,3-dideoxy-3-thiacytidine
( 3tc ) or azidothymidine ( azt ) to a versatile self - assembly motif of
hydrogelators 597 to form supramolecular nanofibers as
the matrixes of hydrogels in weak acidic conditions . in the presence
of prostatic acid phosphatase ( pap ) , the hydrogels exhibit drastically
enhanced elasticity , which should help to match the change of the
physiological environment . in addition , the hydrogelators are biocompatible ,
and are able to release the hiv inhibitors under physiological conditions . reported the synthesis and assembly
of a type of innovative amphiphile ( 598 ) containing a
very bulky anticancer drug , paclitaxel , and a short peptide . with
a relatively high loading ( 41% ) of paclitaxel , 598 self - assembles
to form nanofibers typically a few micrometers in length and with
a diameter of 11.8 1.3 nm in pbs buffer . the addition of gsh
induces the release of paclitaxel , and the release depends on the
concentration of 598 . in addition , the in vitro cytotoxicity
of 598 showed that the amphiphile inhibited the growth
of cancer cells ( e.g. , mcf-7 , a549 , and pc3-flu ) . as adjuvants
are crucial components of vaccines , safer and more potent adjuvants
are gaining increasing interest and attention . as injectable biomaterials
for drug delivery and tissue engineering , supramolecular hydrogels
made of peptides or peptide derivatives are excellent candidates as
adjuvants because of their low cost of production , ease of being produced
in large quantities , and relatively high activity and stability . these
merits of peptides or peptide derivatives promise their applications
in cancer immunotherapies and vaccination against infectious diseases ,
particularly for enhancing the potency of vaccines or for delivery
of vaccines . for example , sun et al . reported a self - assembling peptide ,
flivigsiigpggdgpggd ( 599 ) ( scheme 78 ) , consisting of
two native sequences from an elastic segment of spider silk and a
transmembrane segment of the human muscle l - type calcium channel . they prepared supramolecular hydrogels with 599 either
by changing the ph of its solution or by adding the cation ca at a concentration of 0.5 wt % and found that the two resulting
hydrogels formed by 599 through different approaches
have distinct physical properties . they also found that the shear - thinning ,
rapid - strength - recovering hydrogel made of 599 and ca can be used as an h1n1 influenza vaccine adjuvant which
is biologically safe and improves the immune response by 70% compared
with an oil - based commercial adjuvant . they also evaluated the potential of 599 to act as
an adjuvant for the porcine reproductive and respiratory syndrome
virus ( prrsv ) attenuated live virus ( mlv ) vaccine . their studies suggest
that the supramolecular hydrogel of 599 , when combined
with the prrsv mlv vaccine , can enhance the vaccine efficacy against
two different prrsv strains by modulating both the host humoral and
the host cellular immune responses . jiang and yang et al . also reported a nanovector composed of a peptide - based
nanofibrous hydrogel formed by napgffy - nme ( 600 ) with
a cgc value of 0.01% . according to their report , 600 can
condense dna to lead to a strong immune response against hiv by activating
both humoral and cellular immune responses in mice . their results
indicate that the peptide - based nanovector promises biocompatibility
and may provide a safe , straightforward , and effective approach for
hiv dna vaccines if hiv dna vaccines
are effective in humans ( figure 26 ) . moreover , the impressive
potency of 600 indicates its applications in other immunotherapies
such as its use as an adjuvant in cancer immunotherapy . process of a peptide - based
nanofibrous hydrogel enhancing the immune
responses of hiv dna vaccines . besides being immunomodulating
adjuvants , synthetic peptides or
peptide derivatives are excellent candidates as antigens due to their
precise chemical definitions , which allow one to specify the exact
epitopes against an immune response . however , most peptides , despite
being antigenic , are poorly immunogenic by themselves , thus requiring
the assistance of strong adjuvants . thus , the unique properties of
self - assembling peptides motivate the pursuit of self - adjuvanting
or adjuvant - free systems derived from peptides . cao et al . developed
a vaccine with mutant a peptides 601606 that avoid the use of an adjuvant . they demonstrated that
these adjuvant - free vaccines with different a peptides are
able to induce a good antibody response without stimulating an unwanted
inflammation reaction , thus acting as a safe vaccination approach
against alzhheimer s disease . another example of a self - adjuvanting vaccine is derived from the
understanding of muc1 ( mucin 1 , cell surface associated ) . muc1 proteins
are key targets of the vaccines for epithelial tumors , which have
a variable number of tandem repeats bearing tumor tumor - associated
carbohydrate antigens . however , short muc1 peptides usually exhibit
low immunogenicity , which remains a major obstacle in cancer vaccine
development . to overcome this problem , li et al . synthesized and evaluated
a class of synthetic self - adjuvanting vaccine candidates ( 607610 ) comprising a b - cell epitope with different
glycosylation patterns and a nonimmunogenic self - assembling domain . they found that all of the peptide derivatives self - assemble into
fibers over 200 nm long in aqueous solution at a concentration of
400 m and display b - cell epitopes on the fiber surfaces . they
demonstrated that the vaccine with tn glycosylation in the pdtrp domain
( 610 ) after intraperitoneal injection elicits a significant
immune response in mice . collier
and rudra et al . investigated the molecular determinants
and immunological mechanisms leading to the significant immunogenicity
of the self - assembling peptide ova - q11 ( 611 ) , which elicits
strong antibody responses in mice . their results showed that the
deletion of amino acid regions in the peptide recognized by t cells
or the mutation of the key residues in the self - assembling domain
to prevent fibrillization could diminish or attenuate the immunogenicity
of the peptides . using a different self - assembling sequence to make
ova - kfe8 ( 612 ) , which also self - assembles to form nanofibers
and elicits a strong immune response , they demonstrated that 407 and kfe8 themselves are unrelated to immumogenicity while
it is the peptide assembly that matters . collier et al . concluded
that a key strategy for modulating the immunogenicity appears to center
on the effective t cell epitope , and this appears to be broadly applicable
to fibrillar peptide assemblies . also utilized the same self - assembling domain 407 to design and develop another self - adjuvanting supramolecular vaccine
which carries a folded protein antigen . they first synthesized pnp - q11
( 613 ) , having a pnp ( p - nitrophenyl phosphonate )
ligand on the n - terminal of the 407 domain . in parallel they designed and expressed a fusion
protein ( cut - gfp ) containing cutinase and gfp domains separated by
a flexible linker of glycine and serine residues . they found that 613 self - assembles to form nanofibers , with a morphology similar
to that of the nanofibers of 407 , and the resulting nanostructure
remains unchanged after reaction with cutinase fusion proteins . their results demonstrate that
the nanofibers bearing gfp elicit robust anti - gfp antibodies , which
indicates that the supramolecular assemblies can act as self - adjuvanting
vaccines for whole - protein antigens . in addition to boosting the immune response , supramolecular
hydrogels
formed by peptide or peptide derivatives also suppress immunity . xu
et al . designed and synthesized a conjugate , 614 , of
a self - assembling motif and l - rhamnose to examine its immunomodulatory
properties ( scheme 79 ) . they found that 614 self - assembles in water to form
a weak hydrogel ( 0.4 wt % ) which allows the encapsulation of a fluorescent
model antigen , ( r)-phycoerythrin ( pe ) . surprisingly ,
they found that the resulting hydrogel , in contrast to the properties
of monomeric l - rhamnose , suppresses the antibody response
of mice to pe . xu et al . demonstrated that the covalent conjugation of d - amino acids to naproxen ( i.e. , a nonsteroidal anti - inflammatory
drug ( nsaid ) ) not only affords supramolecular hydrogelators ( e.g. , 615 , 616 , 617 , and 618 ) for potential topical anti - inflammatory gels but also significantly
raises the selectivity toward cox-2 about 20-fold at little expense
of the activity of naproxen . conjugated naproxen with -amino acid to generate
a variety of supramolecular conjugates such as 619 , 620 , 621 , and 622 , all of which
are able to gel pure water , nacl solution , or pbs buffer with cgc
values of 0.802.0 wt % . they found that all of the hydrogelators
display an anti - inflammatory response comparable to that of the parent
drug . although many wound
dressings have entered clinical use , these wound dressings still are
unable to fully satisfy the requirement of wound healing . an ideal
wound - healing therapeutic should offer an optimal microenvironment
to achieve a rapid wound closure , a functionally satisfactory recovery ,
and minimal scar formation . hydrogels are of
great interest as wound dressing or its component because hydrogels
preserve the gaseous permeability , provide a hydration environment ,
absorb wound exudate , and serve as the matrixes for drug delivery . however , current wound - healing hydrogels have
only limited functions and are still unable to adequately match the
complexity of wound - healing processes . despite the enormous challenges
in the development of hydrogels for wound healing , the exploratory
works described in the following have provided useful insights for
further development of supramolecular hydrogels for wound healing . on the basis of the biological functions of glucosamine in the
wound - healing process , xu et al . the preliminary
animal model study found that the application of the hydrogel of 623 to the mice with a skin wound promotes wound healing and
reduces the formation of scars , compared to the results for the control
mice without the treatment ( figure 27 ) . one intriguing observation
is that the use of l - phe in 623 fails to afford
a wound - healing hydrogel , which indicates that the subtle structure
change in the glycopeptide may have profound impacts on both hydrogelation
and their biological functions . it would valuable to understand the
underlying mechanisms of this kind of observation . in another experiment
on wound healing , xu et al . used disodium pamidronate ( 624 ) ( scheme 80 ) , a
clinically used drug that binds with uo2 ,
to generate a supramolecular hydrogel with fmoc - leu ( 533 ) and fmoc - lys ( 201 ) . four equivalents of 624 plus 533 and 201 yields a transparent
hydrogel at ph 10.4 . the resulting hydrogel is able to reduce the
uranyl ion poisoning in the wound on mice because the pamidronate
remains active in the form of a hydrogel . later , the authors conjugated pamidronate with the motif of naphthelene - l - phe - l - phe to generate hydrogelator 625 , which self - assembles to form nanofibrils and induces hydrogelation . the hydrogel of 625 significantly reduces the amount
of uranyl nitrate in the kidney and enhances the survival rate of
the wounded mice . ( a ) molecule 623 self - assembles to form a hydrogel .
gross appearance of the wound site treated without ( b ) or with ( c )
the gel on day 6 . copyright 2007 royal
society of chemisty . the synthetic self - assembling peptide 248a ,
initially
discovered by zhang et al . , is able
to form nanofibers under physiological conditions and results in a
hydrogel with a cgc of 0.1 wt % . serving as a wound dressing , the
hydrogel of peptide 248a can reduce the edema of burn
wound , advance the beginning and disappearance of eschar , and speed
wound contraction . suggested that the hydrogel of 248a provides an optimal hydration microenvironment and simulates
various cytokines and growth factors in the extracellular matrix to
confer a beneficial effect . in another experiment , ellis - behnke and so et al . found that 248a is able to immediately stop bleeding of a wound in the brain , spinal
cord , femoral artery , liver , or skin of mammals . on the basis
of a similar principle , hauser et al . developed two supramolecular
hydrogels based on the short peptides ac - ilvagk - nh2 ( 626 ) and ac - livagk - nh2 ( 627 ) . 626 and 627 form rigid and transparent hydrogels
in pbs buffer at the concentrations of 0.5 and 0.75 wt % , respectively .
compared to the standard - of - care wound dressing mepitel , the authors reported that the hydrogels of
these two peptides , in a rat model , result in earlier onset and completion
of autolytic debridement , and promote epithelial and dermal regeneration
without the exogenous growth factor . it would be important to correlate the in vivo stability of these
peptides with this exciting result . after mixing a dendron , 628 , and the polymer 629 in pbs buffer , grinstaff
et al . prepared a thioester hydrogel
within several seconds at a concentration of 30 wt % . this resulting
hydrogel is transparent , adhesive , and cell compatible , and exhibits
strong mechanical properties even after swelling 4-fold in pbs buffer .
as a hydrogel sealant for wound closure , this hydrogel could be washed
away from the skin by simply using a thiolate solution during surgical
care . compared to the conventional polymeric hydrogels , self - assembly is a ubiquitous feature of supramolecular hydrogels .
considering self - assembly of proteins to generate assemblies that
are crucial for cellular functions ( e.g. , actins and tubulins to form
cytoskeletons ) , the biological functions
of supramolecular assemblies of small molecules are scientifically
intriguing and increasingly significant in biology and medicine . the
development of supramolecular hydrogelators , thus , provides a new
frontier for scientists to explore molecular self - assemblies at the
intersection of supramolecular chemistry and cell biology . in the following section , we discuss how the
development of supramolecular hydrogelators and hydrogels leads to
the interplay between supramolecular assemblies of small molecules
and proteins ( figure 28 ) as a new paradigm in chemistry and in biology . we first introduce
enzyme - instructed self - assembly ( eisa ) , a process that allows the control of the formation and the location
( extra- or intracellular ) of the supramolecular assemblies and hydrogels . second , we highlight several examples of enzyme - instructed self - assembly
in a cellular environment . third , we illustrate the use of the hydrogels
formed by the assemblies of small molecules to bind proteins , including
the molecular hydrogel protein binding ( mhpb ) assay and the assemblies of small
molecules promiscuously interacting with proteins inside cells to
control the cell fate . ( i ) enzyme - instructed
self - assembly ( eisa ) : the enzyme transforms a precursor to the self - assembling
small molecules ( i.e. , hydrogelator ) to form the supramolecular assemblies
( in the form of nanofibers / hydrogel ) . ( ii ) molecular hydrogel protein
binding ( mhpb ) assay : the hydrogels formed by the supramolecular assemblies
bind proteins for proteomic analysis and identification of the protein
targets of the assemblies . to investigate the biological functions
of the supramolecular assemblies of small molecules enzyme - instructed
self - assembly ( eisa)the integration of enzymatic transformation
and self - assembly of small molecules , which usually results
in the formation of supramolecular hydrogels , has provided a facile
approach to examine and to create supramolecular assemblies in a cellular
environment . in general , there are two strategies of enzyme - instructed
self - assembly for generating supramolecular nanofibers making
or breaking bonds . both routes allow the enzyme to convert a precursor
to a hydrogelator which self - assembles in the aqueous phase to form
nanofibers and results in hydrogelation ( figure 29 ) . such a relatively simple design permits
enzymatic formation of supramolecular nanofibers to be applicable
on ( almost ) any gelators because the attachment of a hydrophilic segment to a hydrogelator
easily generates a precursor that is soluble in the aqueous phase .
the removal of the hydrophilic segment by the enzyme - catalyzed bond
cleavage converts the precursor back to the hydrogelator , which self - assembles
into nanoscale assemblies ( e.g. , nanofibers ) and affords the hydrogel . similarly , an enzyme can catalyze bond formation to link two precursors
together to create a hydrogelator that self - assembles into nanofibers . one obvious advantage is the self - assembly
of the hydrogelator to exhibit a selective response to the biological
environments because the expression of enzymes in a living organism
usually is highly specific in a spatiotemporal manner . illustration
of eisa to form supramolecular nanofibers via bond
formation or bond cleavage and the macroscopic outcomes ( i.e. , viscosity
change or hydrogelation ) . as discussed earlier ( scheme 6 ) , xu et al . demonstrated
enzyme - instructed self - assembly of small molecules based on an alkaline
phosphatase and an amino acid derivative . catalyzing the removal of phosphate groups from a variety of substrates
containing the phosphate group , phosphatase can control the balance
between hydrophilicity and hydrophobicity , thus converting a precursor
to a hydrogelator which self - assembles in water to form supramolecular
nanofibers . as shown in figure 30 , the commercially available fmoc - tyrosine phosphate
( 14 ) dissolves in a weak alkaline aqueous solution . the
addition of alkaline phosphatase to the solution converts 14 to a hydrogelator , 15 , which self - assembles into a
three - dimensional network of nanofibers and affords a hydrogel . because phosphatases , prevailingly existing
in the cellular environment and constituting a large family of enzymes ,
act as the integral component of the canonical phosphatase / kinase
enzyme switch that dictates cellular signaling , this seemingly simple
process promises many possibilities and has opened a new paradigm
of molecular biomaterials , as evidenced by the subsequent research . ( a ) molecular structures of the precursor 14 and its
corresponding hydrogelator 15 and the enzymatic transformation . ( b ) transmission electron microscopy ( tem ) image of the nanofibers
made by the self - assembly of 15 . optical images of ( c )
the solution of 14 in alkali buffer ( ph 9.8 ) and ( d )
the hydrogel formed by adding the phosphatase to the solution of 14 to produce the nanofibers of 15 . adapted with
permission from ref ( 153 ) . copyright 2004 wiley - vch verlag gmbh & co. kgaa . unlike phosphatases that catalyze hydrolysis to
trigger molecular
self - assembly , reported the use of thermolysin to couple two peptide derivatives
( 200 and 208 ) to make a hydrogelator ( 447 ) which self - assembles to form a three - dimensional network
of nanofibers and affords a hydrogel ( figure 31 ) . since thermolysin catalyzes the reverse
hydrolysis of many substrates , especially hydrophobic amino acids
and peptides , it greatly expands the scope of enzyme - instructed self - assembly
and the formation of supramolecular nanofibers / hydrogels . although
bond formation may serve as a useful route to produce hydrogels as
scaffolds for tissue engineering , the
low solubility of the hydrophobic precursors in the aqueous phase
likely limits their application ( especially in vivo ) . ( a ) structures of the
precursors 200 and 208 and the hydrogelator 447 and the enzymatic transformation . ( b ) sem image of the corresponding nanofibers ( scale bar 500 nm ) . many advances have
taken place in the development of enzyme - instructed
self - assembly . on the basis of the enzyme switch in the cellular process , xu et al . designed a substrate that undergoes
phosphorylation and dephosphorylation catalyzed by an alkaline phosphatase
and a tyrosine kinase , and examined the use of the kinase / phosphatase
switch to regulate the formation of nanofibers / hydrogels . as shown in figure 32a , a pentapeptide derivative , nap - ffgey
( 630 ; nap = 2-(naphthalen-2-yl)acetic acid ; f = phenylalanine
( phe ) ; g = glycine ( gly ) ; e = glutamic acid ( glu ) ; y = tyrosine ( tyr ) ) ,
self - assembles into nanofibers and results in a hydrogel at 0.6 wt
% . the addition of a kinase to the hydrogel in the presence of adenosine
triphosphates ( atps ) phosphorylates 630 to give the corresponding
precursor 631 , thus disrupting the self - assembly to induce
a gel sol phase transition and produce a solution ; treating
the resulting solution with a phosphatase converts the precursor 631 to the hydrogelator 630 , again , thus repeating
the self - assembly of the hydrogelator to form the network of nanofibers
and afford the hydrogel . besides illustrating a general way of using
enzymes to instruct the formation or disassembly of supramolecular
nanofibers , this work demonstrates that enzyme - instructed self - assembly
generates more ordered self - assembling nanostructures than a simple
adjustment of the ph does ( figure 32b ) . ( a ) structures of the
precursor 630 and the hydrogelator 631 and
the corresponding transformations catalyzed by phosphatase
and kinase . ( b ) tem images showing ( i , left ) the nanofibers of 630 formed by adjusting the ph , ( ii , middle ) the absence of
nanofibers due to enzymatic phosphorylation of 630 , and
( iii , right ) the restored nanofibers of 630 by enzymatic
dephosphorylation of 631 . ( c ) optical images of ( i ) the
hydrogel of 630 formed by changing the ph , ( ii ) the solution
obtained by treating the hydrogel with a kinase and atp ( at 50% conversion ) ,
and ( iii ) the hydrogel of 630 restored by adding phosphatase .
adapted from ref ( 1254 ) . copyright 2006 american chemical society . to serve as therapeutic agents , the supramolecular nanofibers
have
to be innocuous to normal tissues . therefore , it is essential to evaluate
the biochemical properties ( e.g. , biocompatibility , biodurability ,
and toxicity ) of enzyme - instructed supramolecular nanofibers in vivo .
after confirming that 630 is biocompatible , xu et al . injected the solution of 631 into mice to evaluate enzymatic
formation of the nanofibers and the hydrogel of 630 in
vivo . they observed that the hydrogel forms at the location of subcutaneous
injection ( figure 33a ) . hplc analysis of the hydrogel reveals that 80% of precursor 631 turns into hydrogelator 630 . on the basis
of the weight change of the mice after being injected with 631 ( figure 33b ) , subcutaneous
administration of 631 at the experimental dosage results
in little acute toxicity to the mice . moreover , because the enzyme - catalyzed
reaction quickly converts 631 to the biocompatible molecule 630 , there is hardly long - term in vivo toxicity of 631 . ( a ) optical image of the hydrogel formed
1 h after subcutaneously
injecting the solution of the precursor 631 into the
mice . ( b ) weight gain of the mice ( n = 6 , initial
body weight 20 2 g ) after subcutaneously injecting 0.5 ml of 631 at 0.8 wt % concentration . copyright 2006 american chemical society . to generate supramolecular nanofibers
that resist hydrolytic enzymes
( e.g. , proteases ) in challenging biological conditions ( e.g. , biological
fluids ) , xu et al . designed a precursor based on a -amino acid . as shown in figure 34 , tyrosine phosphate attaches at the c - terminal
of a -amino acid derivative to afford a precursor ( 632 ) which serves as a substrate of phosphatase . after being treated
with a phosphatase , 632 hydrolyzes to give a hydrogelator
( 633 ) which self - assembles to afford nanofibers . moreover ,
this enzymatic formation of nanofibers proceeds in complex and challenging
biofluids ( e.g. , blood and cytoplasm ) that contain a variety of proteases
and results in the hydrogelation of these fluids ( figure 34c , d ) . the -peptide - based nanofibers exhibit a longer half - life
than the -peptide nanofibers do . the excellent biostability renders -amino acids and other
non - natural amino acids as potential candidates for creating supramolecular
hydrogels for long - term biomedical applications . ( a ) structures of the
precursor 632 and the hydrogelator 633 and
the enzyme - catalyzed transformation . ( c ) hydrogel formed by mixing
blood , pbs buffer , and alkaline phosphatase . ( d ) gel formed by mixing
the solution of 632 ( 1.0 wt % in pbs buffer , ph 7.4 ) ,
alkaline phosphatase , and the cytoplasm collected from 1.0
10 broken hela cells . -lactamases are an important family of bacterial enzymes
that catalyze the hydrolysis of -lactam antibiotics and have
caused widely spread antimicrobial drug resistance . xu et al . explored enzyme - instructed self - assembly by -lactamases . as shown in figure 35 , the precursor 634 , consisting
of a cephem nucleus as the linker coupling a hydrophilic group and
a proper hydrogelator , is too soluble to self - assemble in water and
bacteria cell lysates . upon the action of a -lactamase , the
lactam ring opens to release the hydrogelator 635 , which
self - assembles to form nanofibers and to afford a hydrogel . this facile
process allows the detection of -lactamase in the lysates of
bacteria . specifically , -lactamase in a bacterial lysate could
convert the precursor 634 to its corresponding hydrogelator 635 , resulting in the formation of supramolecular nanofibers
( figure 35c f ) .
without -lactamase , this result
not only confirms the stability of the nanofibers of 635 in bacteria lysates consisting of a wide range of enzymes , but also
suggests that one may use -lactamase to control the self - assembly
of small molecules as a general platform to target antimicrobial - drug - resistant
gram - negative bacteria since only bacteria express -lactamases . ( a )
structures of the precursor 634 and the hydrogelator 635 and the -lactamase - catalyzed transformation . ( b )
tem images showing the enzymatic formation of nanofibers of 635 : top , the solution , bottom , the gel . ( c f ) images
showing formation of nanofibers of 635 in the lysates
of e. coli that express different -lactamases
( c , ctx - m13 ; d , ctx - m14 ; e , shv-1 ; f , tem-1 ) . adapted from ref ( 159 ) . xu et al . also reported
the use of -galactosidase to remove
galactose from precursor 637 in water to trigger the
self - assembly of the corresponding hydrogelator and to result in a
hydrogel at a concentration of 0.1 wt % at ph 7.5 . the synthetic difficulty of saccharides is apparently the
limiting factor for the exploration of enzymatic hydrogelation using
glycolytic hydrolases . in another experiment , xu et al . designed and
synthesized a short peptide with a sequence of ffffcgldd ( 638 ) as a substrate of matrix metalloprotease-9 ( mmp-9 ) . after
successfully cleaving the hydrophilic residue of ldd off 638 , the resulting hydrogelator ( ffffcg ) self - assembles to form nanofibrils
and affords a hydrogel at a concentration of 0.4 wt % . since mmp-9
is an important enzyme related to the invasiveness and metastatic
potency of human malignant tumors , this work may lead to a new strategy
for making biomaterials or therapeutics for cancer therapy or other
diseases . later , xu et al . reported an esterase - based approach to generate
supramolecular hydrogels . by using esterase to hydrolyze the precursor 7 , the resulting hydrogelator 8 self - assembles
to form a hydrogel at a concentration of 0.8 wt % and ph 7.4 . one
intriguing feature of this work is that the hydrogel of 8 is stable over a wide ph range , likely due to the presence of the
alcohol group instead of a carboxylic group at the c - terminal . moreover ,
breaking the ester - bond apparently is the only path to make hydrogel
of 8 . relying on both aromatic aromatic interactions
and enzyme catalysis , xu et al . reported that enzymatic dephosphorylation
of the precursor 639 generates a hydrogel consisting
of spontaneously aligned supramolecular nanofibers as the matrix of
the gel . as shown in scheme 81 , xu et al . also designed
a new class of conjugates ( 640 ) containing a nucleobase ,
amino acids , and a saccharide which afford a supramolecular hydrogel
( 0.5 wt % ) upon addition of phosphatase . furthermore , 640 is able to inhibit the proliferation of hela cells . in another experiment , xu et al . reported
that 641 affords a supramolecular hydrogel at a concentration
of 0.5 wt % in the presence of acid phosphatase ( ap ) . the immobilized
ap in the hydrogel shows higher activity and stability compared with
free ap in the same solvent . integrating
enzymatic catalysis and self - assembly , xu et al . reported a feasible way to prepare the supramolecular hydrogel
of an adenosine derivative ( 642 ) . this exploration may
lead to a new type of biomaterial because of the ubiquitous importance
of adenosine 5-monophosphate ( amp ) in bioenergetics , metabolism ,
and transfer of genetic information . meanwhile , xu et al . also prepared
a series of nucleopeptides by connecting a nucleobase with phe - phe
which self - assemble to form nanofibers and trigger hydrogelation at
a concentration of 2.0 wt % and ph 5.0 . it is worth noting that the nucleopeptides exhibit better proteolytic
resistance than their corresponding peptides . further
explored the use of thermolysin to catalyze
the bond formation of a series of peptides for self - assembly and hydrogelation . in one case , they reported that thermolysin catalyzes the condensation
of fmoc - ser and phenylalanine to form 643 ( scheme 82 ) . the favorable
product 643 of the condensation self - assembles to form
nanosheets that result in an opaque hydrogel . later , they used subtilisin to hydrolyze fmoc - peptide methyl
esters ( e.g. , 644 ) to obtain the corresponding hydrogelator 645 , which self - assembles to
form nanotubes and affords a hydrogel . they also reported a case in which enzyme - catalyzed dephosphorylation
of precursor 646 affords a hydrogel at a concentration
of 0.55 wt % . this work provides useful insights into the mechanism
of 646 transforming from micelles to nanofibers . by incorporating a metalloproteinase substrate
of pvglig into self - assembling peptides , langer et al . prepared an
enzyme - sensitive hydrogel of 647 at a concentration of
1.0 wt % for eliciting cell and tissue remodeling activities . further
studies found that enzyme - mediated degradation occurred on the gel
surface . while schneider et al . reported
the use of mmp-13 to degrade -hairpin self - assembled hydrogels
by proteolysis , collier et al . designed
a series of depsipeptides containing ester bonds within the peptide
backbone which are able to self - assemble into -sheet fibrillar
materials and degrade via ester hydrolysis with rates controllable
by the amino acid proximal to the ester bonds . among them , 648 forms a very stiff hydrogel
( g > 10 pa ) which becomes
soft
and dissociates over time . unexpectedly , c3h10t1/2 cells were encapsulated
in the hydrogel of 648 and exhibit better spreading and
proliferation than the peptide without an ester bond . reported a new approach in which thermolysin catalyzes the reverse
hydrolysis to produce fmoc - leu - leu - leu ( 649 ) efficiently
in the presence of laminin . hydrogelator 649 self - assembles
to form nanofibrils that interact with laminin to result in a hydrogel . the authors reported that the immobilized laminin is more stable after
being microinjected into a disease site of zebrafish . mcneil and soellner et al . reported
a generalizable method to detect protease activity via hydrogelation . they designed a recognition sequence for targeting the protease of
interest ( e.g. , mmp-9 and prostate - specific antigen ( psa ) ) ; meanwhile
an aminopeptidase removes the residues to release the hydrogelator 650 at physiological conditions . assuming that the mmp-9 and
psa are potential biomarkers for cancer , the authors suggested that
this simple visual assay might be useful for early cancer detection . this innovative two - enzyme approach may find broader applications
than the detection of a specific hydrolase . in addition , ulijn et
al . examined the effect of the concentration of alkaline phosphatase
on the gelation time , mechanical properties , and molecular arrangement
of the enzymatic hydrogelation of 15 , and reported that
an increase of the enzyme concentration enhances the elastic modulus
and the apparent order of the resulting hydrogel . recently , employing enzymatic dephosphorylation of fmoc - tyrosine - phosphate
( 14 ) , mann et al . prepared
a supramolecular hydrogel to act as a matrix for calcium phosphate
mineralization . the authors suggested that this approach may produce biomaterials
for tissue engineering , wound treatment , and drug release . meanwhile ,
yang et al . reported the methylation of 14 to afford
a slightly more hydrophobic precursor , 651 . although 651 is more hydrophobic than 14 , phosphatase
still dephosphorylates 651 to result in a supramolecular
hydrogel . yang and co - workers reported
a short peptide ( nap - gffyp , 652 ) that results
in a hydrogel of nap - gffy at a minimum
gelation concentration of 0.08 wt % after dephosphorylation , and its
methylation form nap - gffyp - ome has a minimum gelation concentration
of 0.01 wt % after enzymatic conversion , which is one of the most
efficient small molecular hydrogelators . as a group pioneering the study of the nap - gffy motif , yang et al . also extensively explored the properties and applications of the supramolecular
hydrogels based on nap - gffy derivatives . enzyme - instructed self - assembly
allows the exploration of molecular self - assembly in a wide range
of biological processes involving enzymes , thus providing abundant
opportunities to evaluate intracellular molecular self - assembly of
small molecules , a previously unexplored subject . the ability to form
supramolecular assemblies inside cells offers a new way to examine
the emergent properties of small molecules at a new level of complexity supramolecular
and intracellular , thus providing a multiple - step process to control
the fate of cells . as shown in the following sections , these studies
have confirmed ( at least ) two aspects of enzyme - instructed intracellular
supramolecular nanofibers : ( i ) the supramolecular nanofibers change
the viscosity of the cytosol and result in selective cell death and
( ii ) intracellular enzyme catalysis plays the key role . to form
supramolecular nanofibers within a cell , an intracellular enzyme should
convert a soluble precursor , which does not self - assemble outside
cells , into a hydrogelator that self - assembles to generate the nanofibers
inside the cells . to meet this requirement , xu et al . mammalian cells uptake 653 ; the
endogenous esterases in the cells convert 653 to a hydrogelator , 654 . the molecules of 654 self - assemble to form
nanofibers , resulting in hydrogelation when a threshold concentration
is reached , and thus changing the viscosity of the cytoplasm to cause
cell death . as shown in figure 36 , most hela cells died at day 3 after the addition
of 653 to the culture medium , while most of the nih/3t3
cells remained alive and dividing . according to the esterase activity
assays , the levels of expression of
esterase between these two cell lines are different . with higher esterase
activities , hela cells likely convert more 653 to 654 than nih/3t3 cells do . more nanofibers form in hela cells
than in nih/3t3 cells , and the resulting nanofibers / hydrogel in hela
cells cause the cell death . the kinetics of the formation of intercellular
nanofibers of 654 is specific to different types of cells ,
which opens a new paradigm for controlling the fate of cells by enzyme - instructed
self - assembly of small molecules . ( a ) an esterase to convert the precursor 653 to the
hydrogelator 654 . ( b ) tem image of the nanofiber formed
by 654 ( inset : optical image of the hydrogel ) . mtt assays
of ( c ) nih/3t3 cells and ( d ) hela cells treated with 653 at concentrations of 0.08 , 0.04 , and 0.02 wt % . adapted with permission
from ref ( 1289 ) . to further demonstrate intracellular enzyme - instructed self - assembly
to control the cell fate , xu et al . used two types of e. coli strains : the wild - type bl21 ( as the control )
and a bl21 strain ( bl21(p+ ) ) that overexpresses human tyrosine phosphatase
( hptp ) . since the only difference between the two strains is the expression
of the phosphatase , any discrepancy in the uptake of the precursor
is minimized . after the precursor 656 diffuses into the e. coli , the phosphatase converts the precursor 656 into the hydrogelator 657 . the bl21(p+ ) bacteria stopped growing upon the addition of 656 ( ic50 = 20 g / ml ) , but the wild - type bl21 bacteria
grew normally ( ic50 > 2000 g / ml ) under the same
conditions ( figure 37 ) . on the basis of on hplc analysis ,
the authors found significant accumulation of 657 inside
bl21(p+ ) cells . tem analysis on the broken bl21(p+ ) cells suggests
the formation of nanofibers of 657 inside bl21(p+ ) cells . these results confirm that enzymatic formation
of the nanofibers and the subsequent hydrogelation inside the bacteria
inhibit their growth . this work illustrates that intracellular enzyme - instructed
self - assembly allows enzymatic transformation rather than tight ligand ulijn
et al . reported the self - assembly of several fmoc - protected dipeptide
( e.g. , fmoc - phe - tyr , and fmoc - tyr - asn ) amphiphiles and the design
of their corresponding phosphorylated precursors . all the precursors
could be dephosphorylated by alkaline phosphatases , generating hydrogelators
that self - assemble to form nanofibers . in addition , the peptide amphiphiles
showed a similar antimicrobial response when incubated with the phosphatase - overexpressed e. coli . ( a ) a schematic
representation of intracellular nanofiber formation
and the inhibition of bacterial growth . ( b , c ) structures and graphic
representations of the precursor 656 and the corresponding
hydrogelator 657 . ( d ) tem image of the nanofibers of 657 ( indicated by arrows ) formed inside the bacteria after
culturing with 656 . ( e ) concentration of 656 needed to inhibit bl21(p+ ) and bl21 by forming nanofibers of 657 inside the bacteria . adapted with permission from ref ( 1259 ) . xu et al . reported a straightforward method for studying
the enzyme - instructed
self - assembly of small molecules inside cells . as shown in scheme 83 , they designed
a new precursor , 658a , containing a fluorophore which
exhibits low fluorescence . after dephosphorylation catalyzed by phosphatases ,
the resulting hydrogelator 658b self - assembles to form
nanofibers that display bright fluorescence . on the basis of this
principle , they found that 658a is easily accumulated
inside cells to form nanofibers / hydrogel , and thus exhibits a bright
spot near the nucleus ( figure 38 ) . further experiments show that most of the self - assembly
of 658b occurs in the endoplasmic reticulum ( er ) due
to the dephosphorylation catalyzed by a tyrosine phosphatase ( ptp1b ) . this work illustrates a facile approach for
studying enzyme - instructed self - assembly of small molecules by other
enzymes inside live cells . ( a ) principle of imaging enzyme - instructed self - assembly inside
cells . ( d ) fluorescent confocal
microscopy images showing the time course of fluorescence emission
inside the hela cells incubated with 500 or 50 m 658a in pbs buffer . adapted with permission from ref ( 156 ) . copyright 2012 nature
publish group . to address the undesired
issues in the fluorescence labeling technique
( e.g. , toxicity and alternation of macromolecular interaction ) , xu
et al . reported a facile method to image enzyme - instructed self - assembly
of small molecules without a high concentration of fluorescent labels
inside mammalian cells via a doping method . specifically , after incorporating
a dansyl ( dns)-labeled molecule of 660a into the self - assembly
of the native molecule 659a as the fluorescent dopant ,
they determined the formation , localization , and progression of molecular
assemblies generated from the nonfluorescent small molecular hydrogelator
by enzyme - instructed self - assembly . after using the cell fraction
experiment to confirm that self - assembly occurs in the endoplasmic
reticulum ( er ) , they used correlative light and electron microscopy
( clem ) to further prove that molecular assemblies localized near or
inside the er and are likely processed via the cellular secretory
pathway ( e.g. , er golgi lysosomes / secretion ) by the
cells . as shown in figure 39 , clem directly correlates the fluorescence signal of 659b/660b molecular assemblies imaged in live
cells ( figure 39b e )
with ultrastructural changes of the treated cell in em ( figure 39f , the authors observed a high accumulation
of vesicles with low - electron - density material in the cytoplasmic
area . therefore , this work establishes
a general strategy that may reveal the spatiotemporal profile of the
assemblies of small molecules inside cells . ( a ) precursor 659a self - assembles to form nanofibers / hydrogels
upon the addition of alp . ( b i ) correlative light and electron
microscopy ( clem ) images of hela cells incubated for 48 h with 500
m 659a and 200 nm 660a . ( b e )
differential interference contrast ( dic ) and fluorescence light microscopy
images of treated hela cells growing on an aclar plastic film . ( f i )
tem images of the cell of interest shown in ( b)(e ) copyright
2013 american chemical society . unlike intracellular
enzymes and the cytosolic catalytic domains of membrane enzymes , ectoenzymes
( i.e. , an enzyme that locates on the cell surface with catalytic domains
outside the plasma membrane ) are less explored . however , emerging
evidence indicates the important role of ectoenzymes in cellular processes . coincidentally , several laboratories are exploring ectoenzyme - instructed
self - assembly to form nanofibers on and near the cell surface ( i.e. ,
pericellular space ) . these studies suggest that one of the promising applications of
the pericellular nanofibers is selective inhibition of targeted cells
( e.g. , cancer cells ) without harming normal cells . xu et al .
reported the first example of enzyme - instructed self - assembly to form
hydrogel / nanonets in pericellular space , which selectively inhibits cancer cells , including certain drug - resistant
cell lines . specifically , the ectophosphatases
( e.g. , placental alkaline phosphatases ( alpp ) ) dephosphorylate the precursor 661a made of
a small d - peptide to a hydrogelator , 661b , which
self - assembles to form nanofibrils at a concentration of 280 m
and results in pericellular nanofibers / hydrogel ( scheme 84 ) . the resulting hydrogel
can prevent the diffusion of a nucleus dye ( 4,6-diamidino-2-phenylindole ,
dapi ) into the cells . a further experiment proves that the pericellular
hydrogel is able to block secretary protein / enzyme in the culture
medium . therefore , such blocking of cellular mass exchange has a profound
negative effect on the critical cellular activities . an enzyme - linked
immunosorbent assay ( elisa ) shows that active caspase-3 and active
parp increase significantly with an increase of the incubation time ,
suggesting that the cells undergo caspase - dependent apoptosis ( figure 40 ) . the authors
also found that the enantiomer of 656 , an l - peptide
derivative , is unable to inhibit hela cells due to proteolysis . on
the basis of this observation , they investigated how a d - amino
acid affects the cellular response to the enzyme - instructed nanofibers
by systematically using d - amino acid residue(s ) to replace
the l - amino acid residue(s ) in tripeptidic precursor 656 or its hydrogelator 657 . further studies
found that the enantiomeric precursors exhibit dramatically different
cellular responses , while enantiomeric hydrogelators show similar
cellular responses ( figure 41 ) . the use of the uncompetitive
inhibitor of alpp abrogates the inhibitory effects of 661a , suggesting that the overexpressed alpp on hela cells is the major
enzyme responsible for enzyme - instructed self - assembly and cell inhibition . ( a ) overlaid images and ( b ) 3d stacked z - scan
image of congo red- and dapi - stained hela cells after the incubation
of the hela cell with 661a for 12 h. ( c ) tem image of
the pericellular hydrogels on the hela cells treated by 661a ( 280 m ) . ( d ) change of the relative amount of apoptosis signal
molecules over time in hela cells treated by 661a ( 280
m ) . ( e ) cell viability of hela cells incubated with 661a ( 280 m ) , 661b ( 280 m ) , and 661a ( 280 m ) plus l - phe ( 1.0 mm ) . ( f ) illustration of
enzyme - instructed self - assembly to form pericellular nanofibers / hydrogel
and selectively induce cell death . adapted with permission from ref ( 288 ) . ic50 values of precursors
and their hydrogelators on
hela cells . f and y the most revealing result is that the hydrogelator itself , 661b , at 280 m , is innocuous to the hela cells . this
result indicates that alpp , as the enzyme on the cell membrane , catalytically
generates hydrogelators to achieve a high local concentration on the
cell surface to form pericellular nanofibers / hydrogel to inhibit the
cancer cells . these results further establish enzyme - instructed self - assembly
as a multiple - step process to control cell fates . in another experiment ,
xu et al . developed a nucleopeptide ( 662a ) as the substrate
of cd73 , an ectoenzyme . the resulting hydrogelator 662b self - assembles to form nanofibrils and induces a hydrogel at a concentration
of 2.0 wt % . one interesting feature of this work is that 662a inhibits hepg2 cells , likely resulting from cd73-instructed self - assembly
to form nanofibers of 662b . in a related study , ulijn and pires reported a novel
carbohydrate
amphiphile ( 663 ) that is able to self - assemble into nanofibers
upon enzymatic dephosphorylation . more importantly , the authors also
confirmed that the membrane - bound alkaline phosphatase expressed by
the osteosarcoma cell line ( saos-2 ) is responsible for triggering
the hydrogelation of 382 in the pericellular environment
( figure 42 ) . the resulting
pericellular hydrogel reduced the metabolic activity of the cells ,
which induced cancer cell death . an
important observation by the authors is that the membrane - bound alkaline
phosphatase ( i.e. , an ectoenzyme ) is responsible for the selective
inhibition of the saos-2 cells over the atdc5 cells . , further validates the use of
enzyme - instructed self - assembly , rather than an enzyme inhibitor ,
for selectively inhibiting cancer cells without harming normal cells . these results , together with the earlier studies , are remarkable because they firmly establish the use of a process ( i.e. , eisa ) , not simply a molecule ( e.g. , 661b ) , for targeting cancer cells . ( a ) chemical
structures of precursors and hydrogelators made of
a carbohydrate amphiphile . ( b ) enzyme - instructed self - assembly for
pericellular nanofiber formation / hydrogelation on saos-2 cells copyright
2015 american chemical society . since it is a challenge to know how the assemblies of small
molecules
behave in cellular environments to affect the cells , xu et al . developed
a facile and reliable method for evaluating the spatiotemporal profiles
of the assemblies of small molecules . in this work , they incorporated
a series of fluorophores ( e.g. , nbd ) into a precursor ( e.g. , 658a ) which is transformed to a hydrogelator ( 658b ) upon the addition of phosphatase . except 666b , all
other dephosphorylated molecules form hydrogels at a concentration
of 0.6 wt % and physiological conditions . cell imaging experiments show that the molecules with different
self - assembly properties exhibit a distinct spatial distribution and
result in different cellular responses . as shown in figure 43 , 658a enters
the cells to form intracellular nanofibers and is able to curtail
the effect of an f - actin toxin . self - assemblies of 664b mainly were localized in the cell membrane , resulting in considerable
cytotoxicity . the resulting 665b hardly accumulated in
the cell , but self - assembles to form nanofibers outside the cells
and exhibits little effect on the cell adhesion . this work not only
illustrates a useful approach to visualize and modulate the spatiotemporal
profiles of small molecules in a cellular environment , but also serves
as a caution to the indiscriminate use of fluorescent aggregates as
imaging probes because the interactions between the aggregates and
endogenous proteins may interfere with the goal of molecular imaging . ( a )
molecular structures of the precursors and the hydrogelators
containing different fluorophores . ( b ) illustration of the distinct
spatial distribution of the small molecules in a cellular environment . fluorescent confocal images of the hela cells incubated with 500 m
( c ) 658a , ( d ) 664a , ( e ) 665a , and ( f ) 666a for 30 min . adapted from ref ( 1063 ) . recently , maruyama
et al . reported that mmp-7 generates hydrogelators
made of peptide lipids to result in intracellular self - assembly of
the hydrogelator , which leads to the selective inhibition of cancer
cells . as shown in figure 44 , they designed a precursor ( 667 , n - palmitoyl - gghgplglaak - conh2 ) which turns into a
hydrogelator ( 668 , n - palmitoyl - gghgplgl )
after enzymatic hydrolysis by mmp-7 . the resulting hydrogelators enter
the cells and are accumulated to form nanofibers which result in cancer
cell death . the likely key design is the incorporation of a lipid
into the peptide because the palmitoyl chain favors the localization
of the precursors on the cell surface for the cleavage catalyzed by
mmp-7 . interestingly , the inhibition
concentration is around 250 g / ml , which also falls into the
average cytotoxicity of the molecular aggregates . this work illustrates a promising approach that combines extracellular
and intracellular self - assembly to control the cell fate . ( a ) illustration
of how enzyme - instructed molecular self - assembly
induces cancer cell death . ( b ) chemical structures of precursor er - c16
( 667 ) and hydrogelators g - c16 ( 668 ) . adapted
from ref ( 894 ) . the above results are remarkable because they
firmly establish the use of a process ( i.e. , eisa ) ,
not simply a molecule ( e.g. , 661b ) ,
for targeting cancer cells . to further understand the details and
improve the efficiency of this fundamentally new process , one also
needs to elucidate how the supramolecular assemblies of the small
molecules promiscuously interact with the endogenous proteins of cells .
in the following , we discuss the approaches for helping address the
fundamental questions about the mechanisms or the consequences of
the self - assembly of small molecules . xu et al . reported the use of a supramolecular hydrogel
to discover the interaction between proteins and supramolecular assemblies
of small molecules . in this study , xu et al . 669 is able to form a transparent hydrogel at a concentration
of 0.6 wt % and ph 7.4 . upon uv irradiation , the resulting hydrogel
can retain the proteins that bind to the nanofibers in the hydrogels . tandem ms analysis suggested that
the supramolecular nanofibers interact with tubulins , actins , and
several other proteins . on the basis of this observation , the authors
eliminated the step of photofixation , and directly used the supramolecular
hydrogel for discovering the interaction of proteins and assemblies
of small molecules . using this hydrogel
protein binding assay to evaluate the interaction of cytosol protein
with different morphological molecular aggregates formed by the same
molecule ( nap - ff , 3 ) , xu et al . found that the nanofibers
of 3 in the hydrogel are able to bind proteins , but the
precipitates of 3 bind with few proteins . moreover , the
authors found that two types of nanofibers formed by wff show similar
morphologies and bind with a similar set of proteins . these results
indicate that mhpb offers a simple and reproducible method for identifying
the protein targets of the supramolecular assemblies of the small
molecules . xu et al . studied the gelation
properties and bioactivities of
hydrogelator nap - ff ( 3 ) , which contains a naphthyl group
and two phenylalanine residues . they
found that 3 is able to self - assemble in pbs buffer to
form -strand - like nanofibers with a uniform width of 24 nm
below a cgc of 0.4 wt % . furthermore , the mtt cell viability assay
indicates that the nanofibers of 3 significantly inhibit
the proliferation of hela and t98 g cells at a concentration of 400
m while showing little toxicity toward pc12 cells . besides
confirming that the nanofibers of 3 disrupt the dynamics
of microtubules and consequently induce apoptosis of glioblastoma
cells , the authors also demonstrated
that the nanofibers of 3 inhibit tumor growth in the
xenograft mice model . these results
support the approach that uses the supramolecular nanofibrils as de
novo molecular amyloids for inhibiting the growth of cancer cells . encouraged by the emerging results that show that the assemblies
of small molecules play an important role in biology , xu et al . examined the mechanism of how
the assemblies of 3 inhibit the proliferation of cells .
using the mhpb assay ( figure 45 ) to investigate the interaction
between nanofibers of 3 and cytosol proteins ( upper panel
of figure 46a ) , they
found that nanofibers of 3 promiscuously interact with
different proteins , particularly with tubulins , vimentin , and actins ,
as confirmed by western blot analysis ( lane b , lower panel of figure 46a ) . specifically ,
the absence of glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) in
lane b ( lower panel of figure 46a ) indicates that the nanofibers of 3 ,
indeed , bind to proteins in a rather specific manner , despite the
promiscuity . furthermore , the tubulin polymerization assay ( figure 46b ) shows that assemblies
of 3 significantly reduce the polymerization rate . this
result , together with tem and confocal images ( figure 46c ) , confirmed that nanofibers of 3 impede the polymerization of microtubules . in addition , confocal fluorescent imaging ( figure 46d , e ) , together with the mhbp
assay , implies that assemblies of 3 also disrupt the
dynamics of actin filaments and intermediate filaments of vimentins
in cells . by selectively inhibiting the endocytosis processes and
measuring the intracellular concentration of 3 ( figure 46f ) , they determined
that both the assemblies of 3 and the monomers of 3 enter the cell via micropinocytosis . using pathscan apoptosis
multitarget sandwich elisa to monitor
the change of several key signaling molecules in the intrinsic pathway
of apoptosis , they found that the assemblies of 3 initiate
the activation of bad and p53 , which later activate the caspase cascade
and downstream parp to induce apoptosis in hela cells ( figure 46 g ) . on the basis of these
results , the authors proposed a partial mechanism for the biological
functions of the nanoscale assemblies of 3 . as summarized
in figure 46h , the
assemblies of 3 enter the cell via macropinocytosis ,
promiscuously interact with cytoskeleton proteins , and eventually
induce apoptosis via the intrinsic pathway of apoptosis . as the first
case of nanoscale , supramolecular assemblies of small molecules to
impede the dynamics of multiple cytoskeletal proteins , this work not
only provides a mechanism for inherent cytotoxicity of hydrophobic
molecular assemblies , but also illustrates a facile approach for developing
nanoscale assemblies of small molecules to perform a diverse range
of biological functions , including serving as a new type of anticancer
agent via enzyme - instructed self - assembly . illustration of the
mhpb assay and hydrogel protein pull - down coupled
with electrophoresis and tandem mass spectrometry for identifying
cytosolic proteins that bind to supramolecular nanofibers . ( a ) photoreaction
of the hydrogelator and supramolecular nanofibers that bind with proteins . ( b ) silver staining of the sds page gel shows that different
conditions alter the protein binding on the supramolecular hydrogel .
adapted with permission from ref ( 883 ) . ( a ) molecular hydrogel protein binding
( mhpb ) assay : upper panel ,
silver staining of sds page reveals a major protein band at
55 kda in lane b ; lower panel , western blot confirms the cytoskeletal
proteins as the primary protein targets . ( c e ) confocal images showing the
assemblies of 3 impede the dynamics of cytoskeletal proteins . ( f ) cellular uptake of 3 in hela cells treated by endocytosis
inhibitors . ( g ) time - dependent activation of the apoptotic proteins
of hela cells treated with 3 . ( h ) mechanism of the selective
cytotoxicity of 3 toward cancer cells . adapted with permission
from ref ( 1248 ) . copyright
2014 american society for biochemistry and molecular biology . adapted
with permission from ref ( 1290 ) . the active research and development of supramolecular
hydrogels
over the past 20 years have revealed a simple fact ; that is , one can
( almost ) make any small molecule a supramolecular hydrogelator , providing
proper derivatization . this is remarkable
because it implies that self - assembly of small molecules in water
is beyond the scope of lipids . such a basic phenomenon , like the formation
of liposomes , also raises many fundamental questions . we arbitrarily
and briefly discuss three of them for the purpose of stimulating possible
discussion rather than providing answers . like many other fields in science one persistent
and still unmet challenge is how to obtain the atomistic details of
intermolecular interactions in the hydrogels or in the assemblies
of the hydrogelators . since the hydrogelators self - assemble to form
nanofibers , this problem is analogous to the structural elucidation
of the molecular arrangement of a peptide ( 142 ) in
the amyloid fibrils . recent studies reveal the polymorphism of -amyloid
fibrils , which implies the polymorphic
nature of the nanofibers of supramolecular hydrogelators . although
the current methodologies still fall short of addressing this challenge ,
the rapid advancements in cryo - tem and x - ray
lasers may lead to the solution of
this problem in the near future . in other words , the lack of structural
details of the aggregates of the hydrogelators should not be the deterrent
to the exploration of the functions and applications of the assemblies
of the hydrogelators or supramolecular hydrogels , as long as the functions
are important and reproducible . in fact , the successful demonstration
of important applications of supramolecular hydrogels is probably
the prerequisite for the initiation of the endeavor of structural
elucidation . the majority of the research activities on
supramolecular hydrogels have largely focused on molecular self - assembly
under thermodynamic equilibrium conditions . realizing a living system is at far from equilibrium and taking
advantage of the self - assembly of hydrogelators , an increased number
of researchers are beginning to explore the self - assembly of hydrogelators
in the context of a dynamic library or with energy input . the reaction diffusion features
of these cases , however , remain to be examined rigorously . arguably ,
one can consider enzymatic hydrogelation accompanied by energy input ,
but the unanswered question in enzyme - instructed self - assembly is
the role of the energy generated during bond breaking or bond formation . does the dissipation of the energy promote or disfavor the self - assembly
or simply raise the temperature ? does the energy input in enzyme - instructed
self - assembly actually result in highly ordered nanostructures ( e.g. , figure 32b ) ? although this question remains to be answered ,
nature already provides an insightful hint by evolving the energy - dissipating
self - organization that is ubiquitous in cellular processes , such as
the self - organization of microtubules or actin filaments . however ,
none of the currently reported hydrogelators are able to mimic the
unique feature of actin or tubulin proteins , such as unidirectional
elongation of the filaments , but the continuing exploration of the
sophisticated supramolecular hydrogelators offers an opportunity to examine the fundamental differences between
self - assembly and self - organization , which ultimately may lead to
a man - made molecular system that self - organizes . the origin of life
remains one of the most perplexing and challenging mysteries in all
of science . currently , there are three
main theories on the origin of life : rna world first metabolism first argues that life began from primordial
metabolism networks created by existing energy sources and nonequilibrium
environments , such as found in hydrothermal vents , and lipid first while each theory has its own validity and captures certain
features of modern life , they all have a crucial missing link . that
is , how do molecules evolve from simple ones to greater complexity ?
for example , what are the molecular processes that result in rna from
simple prebiotic building blocks , produce protoenzymes for the metabolic
cycles , and generate the sophisticated contents to be encapsulated
by the liposomes ? since one of the undeniable facts of life is that
cells are largely made of molecules noncovalently packed in a highly
viscous setting , it is tempting to suggest supramolecular hydrogels
may provide clues for the origin of life , as hypothesized by pollack . interestingly , luo et al . recently reported
that a clay hydrogel enhances transcription and translation . while it remains unknown whether the primordial
soup contains supramolecular hydrogels ,
we speculate that a series of serendipitous events in the exploration
of more sophisticated supramolecular hydrogels or hydrogelators may offer more revealing clues about hydrogels
in the context of the origin of life . over the past decade , the research on supramolecular
hydrogels
and hydrogelators has experienced rapid growth , as evidenced by the
fact that the numbers of published works on the supramolecular hydrogels
in 2014 was about 10 times that in 2004 , according to the web of science . as illustrated by the supramolecular hydrogels and hydrogelators in
this review , the research focuses of hydrogelators are expanding from
the curiosity for an intriguing type of soft matter to the rational
development of molecular biomaterials . this trend coincides with the
tremendous successes and explosive generation of data in the biological
sciences . particularly , the successful completion of the human genome
project and maturation of a variety of omics projects have laid the
knowledge foundations to support the molecular engineering of supramolecular
hydrogels and hydrogelators for potential biomedical applications . for example , the knowledge on protein protein interactions
may provide a useful guide for developing heterotypic supramolecular
hydrogels . on the other hand , the rapid
increase of the exploration of supramolecular hydrogels for developing
biomaterials itself attests to the fact that self - assembly of small
molecules in water offers a facile , promising , and powerful approach
for scientists and engineers to develop supramolecular hydrogelators
or hydrogels that aim to improve human health . however , the current
supramolecular hydrogelators or hydrogels , due to insufficient molecular
engineering , are still too primitive to serve as sophisticated functional
molecular biomaterials . after billions of years of evolution ,
a fundamental fact is that
living organisms are largely made of molecules . these molecules usually
self - assemble or self - organize to perform necessary cellular functions . for example , the most common form of protein assemblies is dimers
( e.g. , 38% of proteins in e. coli exist
as dimers ) , and the most abundant proteins
in cells self - organize ( i.e. , actins for formation of the cytoskeleton )
for many functions . therefore , it is reasonable to take nature as
the inspiration to develop supramolecular hydrogelators for generating
sophisticated molecular self - assembly or self - organization in water
for functions . by preserving the essence of the functions , not just
the appearance of the structures , of biology systems in supramolecular
hydrogelators or hydrogels , one may ultimately discover or create
molecular biomaterials as a new kind of biomedicine that has prescribed
functions . to achieve this goal , it is necessary to identify the problems
from diseases , to define the objectives from functions , and to engineer
the materials from molecules . needless to say , this endeavor requires
interdisciplinary collaborations among scientists and engineers from
different disciplines of the biological , physical , and medical sciences ,
but it needs more than just assembling an interdisciplinary team .
since the building blocks of supramolecular hydrogels are molecules ,
the successful development of supramolecular hydrogelators or hydrogels
as molecular biomaterials demands bioengineers or medical doctors
to have a deep understanding of molecular interactions , and chemists
to acquire knowledge of molecular and cell biology . for example , the
completion of the synthesis of molecules and the characterization
of molecular structures or supramolecular structures becomes the starting
point of the research for chemists , not the ending point . if the goal
of the research on the supramolecular hydrogelators is to develop
molecular biomaterials , it would be beneficial to have a chemist who
has the knowledge of molecular biology and cell biology and clinical
medicine , is able to communicate with the language of biochemistry ,
and possesses the skills of bioinformatics . while these capabilities
seemed to be quite demanding two decades ago , the impressive development
of information technology , digitalized knowledge , and the new generation
of young scientists make these prerequisites increasingly easy to
meet . on the other hand , having more insights into the molecular structures
and intermolecular interactions , bioengineers and medical doctors
will likely more accurately and effectively identify the problems ,
define the objective , and devise the plan . it is our conviction that
the creative exploration of supramolecular hydrogelators and hydrogels
not only will bring innovative molecular biomaterials , but also may
lead to new frontiers of science . on the basis of the above
rationale and optimism , we propose several
prejudiced possible directions of supramolecular hydrogels and hydrogelators ,
including controlled drug release ( e.g. , autogel - like systems ) , tissue engineering
( e.g. , organoids of human immune systems ) , intracellular
delivery , regenerative medicine ( e.g. ,
control of stem cell differentiation ) , immunomoduation ( e.g. ,
molecular adjuvants of vaccines or even pan - flu vaccines ) , wound healing ( e.g. , treatment of diabetic ulcers ) , and cell signaling ( e.g. , biomimetics of autocrines , paracrines ,
or juxtacrines ) . considering the complexity of biological systems ,
it is unlikely that one type of hydrogelator would meet all needs
in biomedical applications . having said that , our biased view is that
hydrogelators , which consist of basic biological building blocks or
are able to mimic a particular biological process , are excellent starting
points for exploring the biomedical applications aforementioned . the
applications of supramolecular hydrogels and hydrogelators certainly
go beyond biomedicine ; they already have found applications in the
catalysis , food , cosmetic , and art industries . we have witnessed the exciting development of supramolecular hydrogelators
and hydrogels in the past decade . this astonishing versatility of
supramolecular hydrogelators and hydrogels has offered scientists
a fruitful playground to reinvent chemistry in the context of molecular biomaterials . by shifting the research
focus from molecules to processes , from thermodynamics
to kinetics , and from molecules to cells , the research
on supramolecular hydrogels and hydrogelators will lead to the integration
of molecular science and bioinformatics , and contribute to the use
of molecules for better human life . | in this review we intend to provide
a relatively comprehensive
summary of the work of supramolecular hydrogelators after 2004 and
to put emphasis particularly on the applications of supramolecular
hydrogels / hydrogelators as molecular biomaterials . after a brief introduction
of methods for generating supramolecular hydrogels , we discuss supramolecular
hydrogelators on the basis of their categories , such as small organic
molecules , coordination complexes , peptides , nucleobases , and saccharides .
following molecular design ,
we focus on various potential applications
of supramolecular hydrogels as molecular biomaterials , classified
by their applications in cell cultures , tissue engineering , cell behavior ,
imaging , and unique applications of hydrogelators .
particularly , we
discuss the applications of supramolecular hydrogelators after they
form supramolecular assemblies but prior to reaching the critical
gelation concentration because this subject is less explored but may
hold equally great promise for helping address fundamental questions
about the mechanisms or the consequences of the self - assembly of molecules ,
including low molecular weight ones .
finally , we provide a perspective
on supramolecular hydrogelators .
we hope that this review will serve
as an updated introduction and reference for researchers who are interested
in exploring supramolecular hydrogelators as molecular biomaterials
for addressing the societal needs at various frontiers . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
ductal carcinoma in situ ( dcis ) of the breast is a preinvasive lesion with theoretically no potential for lymph node or distant metastases by definition . so far axillary dissection rates have been decreased since 1990s because it has been shown that omission of axillary dissection in patients with pure in situ disease had no adverse effect on survival or recurrence [ 1 , 2 ] . in the era of sentinel lymph node biopsy ( slnb ) which has been well established in early invasive breast cancer with low axillary recurrence , the center of argument in dcis has become to do or not to do slnb in patients with dcis ? today , some authors still believe that there is a subset of patients at high risk for microinvasive disease and subsequent axillary metastasis who may benefit from the sln biopsy ( slnb ) [ 5 , 1013 ] . however , there have been reports which discourage slnb in patients with pure dcis due to very low rate of axillary metastasis [ 3 , 4 , 1417 ] . the flaw is that definitive diagnosis of dcis can not be made unless the final histopathological examination is done . however , the surgeon mostly has to decide on whether slnb is to be performed based on a preoperative stereotactic core - needle biopsy or vacuum aspiration biopsy or tumor characteristics or surgery type . many surgeons advocate slnb for patients with dcis in whom mastectomy was planned to avoid an unnecessary axillary clearance in case an invasive focus has been identified in the final pathology . presence of high grade dcis or presence of comedonecrosis , and diagnosis of invasive component by core - needle biopsy , and mammographic dcis size of at least 4 cm were identified as independent risk factors for invasion [ 12 , 18 ] . however , no risk factors could be found that were predictive for sentinel lymph node metastasis . furthermore , presence of comedonecrosis and larger mammographic dcis size were independent predictors of patients ' undergoing slnb with a presurgical diagnosis of dcis in core biopsy . in the present study , we reviewed the patients with a definitive diagnosis of pure dcis in a single institution series to identify factors to select patients who may benefit from slnb , and to define our surgical management in these patients . of 699 patients operated for breast cancer at our institution between july 2000 and march 2011 , 68 patients ( 9.7% ) were diagnosed with pure dcis or dcis associated with microinvasion . patients with dcis associated with microinvasion ( n = 5 ) were excluded from the study , and 63 patients with pure dcis were reviewed . the data regarding patient and tumor characteristics were collected form medical records : age at diagnosis , menopausal status , tumor size , nuclear grade , presence of comedo necrosis , type of biopsy ( core biopsy , vacuum aspirated biopsy , excisional biopsy by wire - needle localisation or radioguided occult lesion localisation ( roll ) ) , type of breast surgery ( mastectomy versus breast conservation ) , margin status , estrogen receptor ( er ) staining , progesterone receptor ( pr ) staining , her2/neu staining , presence of slnb , and pathological examination of sentinel lymph nodes . the excisional biopsy technique to remove the nonpalpable lesion by roll has been described before in detail . the decision on whether a slnb was to be performed lymphatic mapping was done with a combined technique of lymphoscintigraphy after radiocolloid injection at the day of surgery and subareolar methylene blue injection followed by breast massage . all the lymph nodes colored with blue dye and showed radioactivity with gamma probe were removed and were sent for intraoperative evaluation by the pathologist . briefly , the sentinel lymph node(s ) was bisected fresh along its long axis through the hilus or the entering point of afferent lymphatic if it was colored with blue . scrape preparations especially from the hilus and from 2 to 4 faces pairs were made and were stained with hematoxylin and eosin ( h&e ) . in the presence of suspicious cells , frozen section from the related slice of the lymph node the entire sentinel lymph nodes were serially sectioned with 50 m intervals , and two sequential slices with 3 m thickness were prepared . one of these sequential sections was stained with h&e , and the other one was spared for immunohistochemistry in cases with suspicious atypical cells by h&e to be stained by using a pan - cytokeratin antibody ( novocastra , rtu - pan - ck , newcastle , uk ) . sln metastases were classified according to the 7th edition of the american joint committee on cancer ( ajcc ) staging system as follows : isolated tumor cells ( itc ) were defined as isolated tumor cells or clusters 0.2 mm in maximum diameter ; micrometastasis were defined as metastases > 0.2 mm but < 2 mm ; macrometastasis as > 2 mm . if the breast lesion consisted of microcalcifications with or without a mass , the entire specimen was sampled after inking and embedded in paraffin after orientation . in cases with a palpable mass , complete sampling of the mass and surgical margins were performed and samples from the surrounding tissue were evaluated for margin evaluation after the specimen was inked . in cases of mastectomy , in addition to complete sampling of microcalcifications or mass , samples of the surrounding tissue and nipple areola complex were also examined . the distance of the tumor to the specimen 's inked edge was reported for every marking margin and the margin width was considered as the narrowest distance between the tumor and any inked margin . microinvasion was defined according to the 7th edition of ajcc staging system , which was considered as a microscopic focus of invasion of cancer cells extending beyond the basement membrane into the adjacent tissue , with no focus greater than 0.1 cm in dimension . the histopathologic diagnosis and classification of dcis were done according to criteria as defined by rosen and oberman . well- , intermediately , and poorly differentiated dcis according to the classification of holland et al . . immunostains for er and pr were performed by using er ( novocastra ( 6f11 ) , newcastle , uk ) and pr ( novocastra ( pgr-312 ) , newcastle , uk ) antibodies on full sections , and cases with 10% or more positive staining were considered as positive . positivity was determined based on immunohistochemistry staining by using her2/neu antibody ( ventana ( her2/neu 4b5 ) , tucson , arizona , usa ) . the statistical analyses were performed by using the statistical package for the social sciences ( spss ) program , version 15.0 ( spss inc . , predictive factors of patients undergoing slnb were investigated by univariate analyses using fisher 's exact test . the statistically significant variables were further analyzed by logistic regression analyses to identify the independent factors . a p value equal or less than 0.05 was considered significant . thirty - six patients ( 57.1% ) presented with pleomorphic microcalcifications in mammogram without clinical symptom whereas 6 patients ( 9.5% ) presented with pathological nipple discharge and 18 ( 28.6% ) had a palpable mass in physical exam . two patients ( 3.2% ) had a mass in ultrasound or mri , whereas one patient had an asymmetric density in the affected breast mammogram . preoperative diagnosis was made by core biopsy in 12 patients ( 19% ) , and by vacuum aspirated biopsy in 4 patients ( 6.3% ) , respectively . however , the majority of patients ( n = 47 , 74.6% ) underwent excisional biopsy by wire - needle localisation ( n = 42 ) , or roll ( n = 5 ) for pathologic diagnosis . mastectomy was performed in 32 patients ( 50.8% ) due to multifocal or multicentric disease , whereas 31 patients ( 49.2% ) underwent breast conserving surgery . forty patients ( 63.5% ) with pure dcis underwent slnb , and a median number of 2 slns ( range , 18 ) were harvested during the procedure . two patients were found to have a positive slnb ( 5% ) . of patients with sln positivity , one patient ( 2.5% ) was demonstrated to have isolated tumor cells ( itcs ) , whereas one patient had macrometastasis ( 2.5% ) . axillary lymph node dissection was performed in one patient with macrometastasis . in all 2 cases with sln metastases , only one sentinel lymph node was involved with tumor cells , whereas all the other sentinel and nonsentinel lymph nodes were found to be reactive . both of the patients with sln positivity had high grade tumors , either a palpable mass or a lesion more than 3 cm . patients , who underwent slnb , were more likely to have a palpable mass , or a tumor size > 30 mm or dcis with intermediate or high nuclear grade or comedonecrosis or mastectomy due to extensive disease ( table 1 ) . tumor size , nuclear grade , mastectomy , presence of palpable mass were analyzed in logistic regression model . performing mastectomy , tumor size > 30 mm , and presence of intermediate or high nuclear grade were significant independent predictive factors to do a sentinel lymph node biopsy in logistic regression analysis ( table 2 ) . other factors including age > 50 , estrogen or progesteron receptor status , or her-2/neu positivity did not significantly influence the surgeon 's decision to perform slnb . although ductal carcinoma in situ ( dcis ) is a lesion which has a theoretical risk of 0% for metastases , axillary metastases have been found in 1 - 2% of the patients treated with axillary dissection . furthermore , sln positivity in dcis is higher than reported axillary positivity ranging between 1% and 13% in published reports as shown in table 3 [ 35 , 9 , 2427 ] . in a metaanalysis of 3166 patients , the incidence of sln metastases was 7.4% in patients with a preoperative diagnosis of dcis compared with 3.7% in patients with a definitive postoperative diagnosis of dcis alone . klauber - de more et al . reported 12% sln positivity in dcis but when patients with microinvasive focus and patients with stromal and vascular invasion were excluded , the incidence decreased to 6.5% . reported 4.6% positivity with 4 positive slns ( by h&e and ihc ) in 86 patients with pure dcis . in a series of 854 patients with pure dcis , the overall risk of sln metastases was found to be 1.9% by intra et al . . this ratio dropped to 1.4% when presence of itc in slns was considered negative according to the last tnm classification [ 3 , 28 ] . in concordance with some studies , we found the sln positivity as 5% in pure dcis which is a slightly higher rate of sln positivity in published large series [ 3 , 13 ] . the variation in sln positivity may be attributed to evolution of sentinel node biopsy techniques , different preoperative diagnostic methods , variations in pathological examination including extent of tissue sampling and evaluation of the slns with h&e or ihc or both , and small patient numbers in some series [ 13 , 29 ] . some reports doubled their node positivity frequencies by using ihc to detect sln involvement [ 5 , 8 , 30 ] . in a study by lata et al . , in 13% of the patients , slns were shown to be involved by tumor cells by ihc methods but no significant association with local , regional or distant recurrence was shown . reported metastatic lymph nodes in 5% of 559 patients with a final diagnosis of dcis after surgical resection and 70% of them were detected only by ihc . in our series , slns were meticulously examined with h&e staining after serially sectioned with 50 m intervals , but ihc was only performed in the presence of suspicious cells . most of the sln metastases of dcis consist of micrometastases and itc , and the sln is the only affected node usually even in the presence of macrometastasis [ 9 , 25 , 31 ] . similarly , in our series , only one sln was involved in both cases whereas all the other sentinel and nonsentinel nodes were reactive . this has been mostly attributed to an unrecognized invasive focus in the breast or metastases subsequent to an invasive local recurrence or due to the different examination methods such as h&e or immunohistochemistry [ 23 , 31 ] . the presence of itc or micrometastases in the sln in patients with dcis is an intriguing issue with unclear clinical implications . reported an increase in incidence of lymph node metastases after revision with ihc from 1.4% to 10.6% in pure dcis patients . they mentioned that the cells might have represented a false positive finding associated with microembolism of breast epithelial / tumor tissue that had been dislodged to the lymphatic system by a sampling procedure , but they could not find any evidence of mechanical displacement . however , bleiweiss et al . demonstrated that ck positive cells in the positive slns had different histologic and immunohistochemical characteristics from the primary intraductal carcinoma involving an intraductal papilloma . they suggested that slnb should not have been a routine procedure until the patient has a histologically proven invasive tumor . because of low incidence of the sln involvement , the routine use of slnb in pure dcis is discouraged [ 3 , 14 , 16 ] . however , diagnosis of pure dcis can only be made after final pathological examination , and in case of an invasive focus in paraffin sections , reoperation for slnb is needed . it has been reported that an underestimation of an invasive focus is present in 1042% of patients when preoperative diagnosis is made by core needle biopsy or vacuum aspiration biopsy as in clinical practice [ 7 , 3338 ] . when literature that attempted to define a subgroup of patients in whom a second operation for slnb could be avoided , most of the authors have reported that a palpable mass , mammographic mass , a high grade lesion , and a large size were associated with a significant risk of invasive disease in the final resection specimen despite some inconsistencies between the studies [ 12 , 13 , 3941 ] . yen et al . mentioned about 4 independent predictors of invasive cancer on final pathology which were age < 55 , diagnosis by core needle biopsy , mammographic dcis size > 4 cm , and high grade dcis . furthermore , presence of comedonecrosis and larger mammographic dcis size were found as independent predictors of patients ' undergoing slnb in multivariate analysis . suggested that dcis with microinvasion or diffuse dcis requiring mastectomy ( including dcis more than 30 mm ) was the main risk factor for sln metastasis in a 110-patient series . reported that the risk of sln metastases did not seem to be correlated with the comedocarcinoma subtype , presence of necrosis , tumor grade , hormone receptor status , ki67 , her2/neu status , multifocality , or type of surgery . the most common architectural patterns were solid and cribriform patterns , but not comedo among those patients with pure dcis and positive slns . the authors found age younger than 50 and mass as clinical presentation to be important factors predicting the likelihood of the sln metastasis . on the other hand , there have also been some other reports that did not identify any significant predictive risk factors [ 5 , 18 , 42 , 43 ] . our surgical approach to perform slnb in the same session with definitive operation is in concordance with the published literature [ 3 , 5 , 12 , 44 ] . factors significantly affecting our approach were performing mastectomy , the size of the tumor ( > 30 mm ) , and presence of intermediate and high nuclear grade . patients with large comedo dcis , large solid tumors , diffuse or multicentric microcalcifications , recurrent lesions , and high grade dcis were also scheduled for slnb . in all cases , meticulous examination of the tumor specimen was done to exclude microinvasive foci and to decrease the prevalence of unexpected sln metastases . in our series , we found a relatively higher slnb positivity in patients with pure dcis than the large series reported elsewhere . this may either be due to the meticulous examination of slns by serial sectioning technique or due to our patient selection criteria or both . although the importance of presence of itc in slns has not been clarified yet , it may be reasonable to perform sln in selected patients with pure dcis . slnb should be considered in cases of dcis where there is a strong doubt of invasion at the definitive pathology as in patients with large tumors or diffuse pluricentric microcalcifications undergoing mastectomy or high grade dcis with comedonecrosis or solid cribriform pattern . | introduction .
sentinel lymph node biopsy ( slnb ) in patients with pure ductal carcinoma in situ ( dcis ) has been a matter of debate due to very low rate of axillary metastases .
we therefore aimed to identify factors in a single institutional series to select patients who may benefit from slnb .
material and methods .
patients , diagnosed with pure dcis ( n = 63 ) between july 2000 and march 2011 , were reviewed .
all the sentinel lymph nodes were examined by serial sectioning ( 50 m ) of the entire lymph node and h&e staining , and by cytokeratin immunostaining in suspicious cases .
results .
median age was 51 ( range , 3079 ) .
of 63 patients , 40 cases ( 63.5% ) with pure dcis underwent sln , and 2 of them had a positive sln ( 5% ) .
in both 2 cases with sln metastases , only one sentinel lymph node was involved with tumor cells .
patients who underwent slnb were more likely to have a tumor size > 30 mm or dcis with intermediate and high nuclear grade or a mastectomy in univariate and multivariate analyses .
conclusion . in our series
, we found a slightly higher rate of slnb positivity in patients with pure dcis than the large series reported elsewhere .
this may either be due to the meticulous examination of slns by serial sectioning technique or due to our patient selection criteria or both . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
acute kidney injury ( aki ) remains a vexing clinical problem resulting in unacceptably high patient mortality , development of chronic kidney disease , and enhanced progression to end - stage kidney disease . although clinical risk factors for developing aki have been identified , there is no reasonable surveillance technique ( ' biomarker ' ) to either definitively and rapidly diagnose the injury or determine the extent of its severity . since patient outcomes correlate with the extent of injury and effective therapy requires early intervention , the ability to rapidly diagnose and stratify patients by their level of kidney injury is of paramount importance for therapeutic progress . therefore , a search for a biomarker of kidney injury has intensified and is now considered by many experts to be the highest priority in the field of aki . it is likely that a combination of structural and functional markers of aki will provide the highest clinical utility . glomerular filtration rate ( gfr ) , which measures the amount of plasma filtered through glomeruli within a given period of time , is clinically the most widely used indicator of kidney function . yet reduction in the gfr , secondary to kidney injury , is the hallmark of aki and results in increased levels of blood urea nitrogen ( bun ) and serum creatinine . unfortunately , the rates of increase in bun and serum creatinine do not parallel the fall in gfr in a time frame that is clinically useful . in addition , since both creatinine production from muscle and gfr determine the serum creatinine level , using serum creatinine as an indicator of gfr is highly patient - specific and often problematic or even misleading . achieving the ability to rapidly and accurately measure gfr in an early stage of aki would be beneficial for many reasons . it would rapidly identify and determine the extent of injury , allowing early pharmacologic or dialytic treatment ( or both ) , enrollment and stratification for clinical studies , and prognostic information . it could also be used to determine the effect of a clinical maneuver on gfr , such as volume resuscitation and the use of pressors to support blood pressure , and enable appropriate drug dosing for agents cleared by the kidney or nephrotoxins . on this well - known background , pickering and colleagues set out to determine the clinical utility of a 4-hour creatinine clearance ( crcl ) , compared with plasma creatinine , for diagnosing aki . the authors found that crcl increased the likelihood of diagnosing aki ; a decreasing crcl correlated with increased kidney injury severity , death , or dialysis ; and the crcl was most helpful when patients began with a serum creatinine in the normal range . although the receiver operating characteristics were not impressive , the study was a small pilot study . this is not the first time a shortened crcl has been used to estimate gfr in clinical settings . herget - rosenthal and colleagues used a 2-hour crcl in stable patients and found an acceptable and repeatable correlation with the 24-hour crcl . however , the accuracy and utility of a shortened collection in unstable patients were questioned by two studies conducted in patients with aki . this limitation may relate to reduced production of creatinine in sepsis , increased production of creatinine with trauma , increased metabolism including the use of glucocorticoids , or the changing of gfrs during the collection periods . pickering and colleagues did not attempt to validate their 4-hour crcl rates with 6-hour iohexol or iothalamate infustion studies , currently the gold standard for clinical studies . this validation could have been done nearly simultaneously with the 4-hour crcl urine collection . until such studies are conducted , confidence for using a crcl may be limited . a clinically useful technique to measure gfr in aki has been a long - sought - after goal . the cumbersome , time - consuming , and expensive techniques currently available have not met this important clinical need . the development and use of estimating formulas based on serum creatinine or cystatin c have been disappointing for several well - explained reasons . therefore , the development of a rapid , accurate , safe , easy , and in - expensive technique has high clinical importance both in and out of the hospital . an approach being developed commercially , used primarily in preclinical studies to date , is the use of bedside techniques to measure gfr . the use of inexpensive , nontoxic , easily sized fluorescent molecules has emerged as a likely candidate , and several groups are now working with this approach in preclinical models . it is likely that several of these approaches will enter clinical phase studies , and improvements in design and clinical utility are continually being developed . test rapidity , convenience , cost , safety , accuracy , and repeatability are all critical characteristics . hopefully , one or more of these approaches will emerge and alleviate the need for multiple - hour urine collection and analysis . aki : acute kidney injury ; bun : blood urea nitrogen ; crcl : creatinine clearance ; gfr : glomerular filtration rate . bam is a co - founder and part owner and the medical director of fast biomedical ( indianapolis , in , usa ) . | acute kidney injury has become a major focus for nephrologists and critical care physicians .
the development of structural biomarkers is proceeding , but the results to date have been disappointing . the use of a shortened creatinine clearance as a functional acute kidney injury biomarker is not new but has not been compared with that of other diagnostic approaches
. a rapid , repeatable , and accurate measured glomerular filtration rate would be the gold standard for a functional biomarker and is not far off . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
kocuria rhizophila is a gram - positive , coccoid , and spherical bacterium belonging to the family micrococcaceae in the phylum actinobacteria , and was designated as a new species by kovcs and coworkers 1 . members of the genus kocuria have been isolated from a wide variety of natural sources , and k. rhizophila in particular , was found to have important industrial applications . k. rhizophila has been utilised as the standard quality control strain for antimicrobial susceptibility testing 2 and also as a decolorizing agent of industrial dyes 3 . our strain was isolated from sungai gabai , selangor , malaysia and analysis of the genome revealed the presence of an aromatic compound degradation pathway . initially , the genomic dna of k. rhizophila tpw45 was extracted using masterpure complete dna and rna purification kit ( epicentre , inc . , madison , wi , usa ) according to the manufacturer 's recommended protocol . subsequently , the quality of dna was examined using the nanodrop 2000 spectrophotometer ( thermo scientific ) and quantified using qubit 2.0 fluorometer ( life technologies ) . for whole - genome sequencing of k. rhizophila tpw45 , the library was prepared using illumina nextera dna sample preparation kit as per manufacturer 's guidelines . the sequencing of this strain was then performed on the illumina hiseq 2500 ( illumina , inc . , ca ) using a paired - end library with an average insert size of 702 bp , obtaining 3,407,419 raw reads of 100 bp in length . after trimming , de novo assembly of the genome was performed using clc genomics workbench 7.0 4 while the genome annotation was performed using rapid annotation using subsystem technology ( rast ) 5 . the assembled genome consists of 2,701,701 bases across 46 contigs with an n50 contig length of 114,097 bases . the average genome coverage was found to be 202.76. the assembled genome was found to have a g + c content of 70.6% , falling within the range of known k. rhizophila genomes ( table 1 ) . there were 2,349 predicted coding dna sequences ( cds ) , whereas a total of 50 trnas were predicted using trnascan - se 6 , and 4 rrnas was identified by using rnammer 7 . this draft genome revealed a putative gene cluster involved in aromatic compound degradation that encodes a phenylacetatic acid ( paa ) pathway . this paa gene cluster was found in contig 17 of our strain and the gene arrangement was found to be similar to that of k. rhizophila strain dc2201 , which is a closely related strain ( figure 1 ) . phenylacetate or phenylacetyl - coenzyme a ( coa ) can be found as a derivative product from many substrates namely phenylalanine , 2-phenylethylamine , some phenylalkanoic acids , and also pollutants such as styrene and ethylbenzene . 8 the paa pathway proceeds via phenylacetyl - coa and ring fission is hydrolysed via a multicomponent oxygenase encoded by paaghijk . 9 in summary , phenylacetate degradation has been more widely studied in proteobacteria such as escherichia spp . and pseudomonas spp . 8 we hope that this genome sequence of strain tpw45 will provide more insight into the phenylacetate degradation pathway by k. rhizophila . further analysis of this genome and supporting experimental evidence will be needed to support this prediction and further understand the process of aromatic compound degradation by bacteria via paa catabolism . the whole genome shotgun project has been deposited in ddbj / embl / genbank under accession number jwtc00000000.1 . | kocuria rhizophila is a ubiquitous bacterium which is well known for its industrial value . here , we present the draft genome of kocuria rhizophila strain tpw45 which was isolated from sungai gabai , selangor , malaysia . the assembled genome comprised of 46 contigs and
the estimated genome size is 2.7 mb .
based on the rast annotation , a gene cluster responsible for aromatic compound degradation was identified in this strain . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
an estimated 569,490 americans are expected to die of cancer in 2010 , accounting for approximately 25% of the overall mortality 1 . bone metastases are a common manifestation of advanced disease with autopsy studies showing an incidence of 33 - 36% in patients with lung cancer 2 , 3 , 68% in prostate cancer 3 , and 73% in breast cancer 2 , 3 . while many patients receive therapy at major cancer centers , numerous other patients choose local or regional hospitals , and most imaging studies include the skeleton secondarily if not primarily ( e.g. chest radiography , body computed tomography [ ct ] ) . thus , the appearance and behavior of bone metastases can be detected on a wide variety of imaging studies that are performed for many different indications . response criteria represent the standard by which the efficacy of new therapeutic agents is determined in cancer treatment trials . the most commonly used set of criteria is the response evaluation criteria in solid tumors ( recist ) . these and similar anatomic criteria focus predominantly on the physical measurement of solid tumors . disease that is not easily measurable with a ruler or calipers , such as most bone metastases , is designated as unmeasurable . cancer patients with no measurable disease ( e.g. individuals with bone - only metastases following the resection of a primary tumor ) are often ineligible for clinical trials , which may be the only available source of therapy . this article reviews anatomic ( recist 1.1 ) , bone ( md anderson [ mda ] ) , and metabolic ( positron emission tomography response criteria in solid tumors [ percist ] ) cancer response criteria , with a focus on the developing role of bone metastases and the interpretation of the treatment response of bone metastases seen on imaging studies . change in tumor size following therapy , also known as objective response 4 , 5 , is a robust indicator of outcome in the treatment of numerous solid tumors 6 - 9 and forms the basis for anatomic response criteria . recist 10 , updated to recist 1.1 in 2009 11 , was designed to standardize the assessment of therapeutic response to allow meaningful comparison of drug efficacy among individuals in the same study and across different studies 12 , 13 . recist 1.1 specifies that up to 5 target lesions , representing all affected organ systems but with no more than 2 target lesions per organ , be selected for measurement throughout the course of a therapeutic trial . to be considered as target lesions , at baseline nodules must measure 10 mm on ct ( or twice the slice thickness if the interval is > 5 mm ) , the short axes of lymph nodes must measure 15 mm on ct ( recommended slice thickness is 5 mm ) , palpable masses must be 10 mm as measured with calipers ; and lung lesions must be 20 mm , clearly delineated , and surrounded by lung parenchyma on chest radiographs . lesions may be measured using ct or magnetic resonance imaging ( mri ) , but ct is preferred in most situations because of the variability of mri scan parameters . measurements made using ultrasonography are not acceptable because of operator dependency and lack of objective reproducibility . according to recist 1.1 , drug efficacy is primarily determined by the sum of the measurements of the greatest longitudinal dimension of each target lesion . one of the differences between recist and recist 1.1 is that bone metastases with soft tissue masses measuring 10 mm are now accepted as target lesions . the soft tissue component is to be measured in an identical manner to that used for other target lesions ( fig . 1 ) . measurements are to be made in the plane of acquisition ( typically axial for ct unless isotropic reconstructions are performed ) . the largest lesions are preferred if they are clearly and reproducibly measurable ( e.g. the largest well - defined lesion is preferred over larger , ill - defined lesions ) , and no previously irradiated lesion is eligible as a target lesion unless it demonstrates progression after irradiation . therefore , a careful search of the medical record for previous therapeutic radiation exposure is indicated prior to the selection of a bone metastasis as a target lesion . recist 1.1 states that ct is the best currently available and reproducible method to measure lesions selected for response assessment 11 . however , mri has been shown superior to ct in delineating the extent of primary bone tumors ( which are similar to target bone lesions because they typically produce large soft tissue masses ) and their relationship to adjacent structures 14 , 15 . the value of the high soft tissue contrast resolution of mri was shown in a prospective study comparing mri and ct for the detection of locally recurrent tumors in 49 patients following the resection of musculoskeletal malignancies 16 . in the 33 biopsy - proven locally recurrent tumor nodules , mri demonstrated sensitivity , specificity , and accuracy of 82.5% , 96.3% , and 92.6% , respectively ; ct values for sensitivity , specificity , and accuracy were 57.5% , 96.3% , and 85.0% , respectively . mri scans with and without the use of intravenous gadolinium contrast can be considered for the follow - up of measurable bone lesions . the 4 response categories included in recist 1.1 are complete response ( cr ) , partial response ( pr ) , progressive disease ( pd ) , and stable disease ( sd ) ( table 1 ) . cr is defined as the disappearance of all target lesions and reduction of the short axes of target lymph nodes to < 10 mm . fludeoxyglucose f ( fdg ) positron emission tomography ( pet ) can be used in place of biopsy to verify cr when a residual mass is thought to represent scarring or fibrosis . the pr category requires a decrease in the sum of the diameters of all target lesions by 30% ; the patient 's baseline sum of these diameters is the reference standard . pd requires an increase of 20% ( with at least a 5-mm increase ) in the sum of target lesion diameters ; again , the patient 's smallest recorded sum of these diameters is the reference standard . additionally , the interval development of a malignant fdg uptake pattern is considered an indicator of pd unless it corresponds to an anatomically stable lesion . the metastasis is to be confirmed on contemporaneous or follow - up ct ( fig . the sd category includes all patients whose disease activity does not meet the requirements of the other 3 categories using the smallest previous sum of lesion diameters as the reference standard . these include small tumors ( nodules with a short - axis dimension < 10 mm ) , leptomeningeal disease , lymphangitic spread , inflammatory breast disease , pericardial / pleural effusions , palpable abdominal masses / organomegaly not reproducible on imaging studies , lesions surrounded by postradiation scar tissue , and bone metastases without soft tissue masses measuring 10 mm ( the large majority of bone metastases ) . while no focus of unmeasurable disease can be used as a target lesion , the progression of such tumors can have an effect on the recist response assessment under the designation of unequivocal progression of nontarget lesions . in general , progression of nontarget lesions is to be estimated as a 20% increase in the sum of the greatest tumor diameters , which is calculated to be a 73% increase in volume , by the authors of the recist criteria 11 . therefore , frank progression of bone metastases on any imaging modality can contribute to the classification of overall patient response through the designation of unequivocal progression ( fig . the field of oncology has seen several generations of cancer response criteria . the international union against cancer ( uicc ) 17 and the world health organization ( who ) 4 published criteria in 1977 and 1979 , respectively . while at the time representing the most sophisticated attempts to standardize the evaluation of tumor response , these criteria were published before the widespread availability of ct . both sets of criteria , which have been largely supplanted by recist and recist 1.1 , consider bone metastases to be measurable disease . additionally , the who criteria include radiograph - based guidelines for the interpretation of bone metastases ; however , these guidelines were not adopted by recist or recist 1.1 . the resultant void regarding the evaluation of bone metastases led to the development of bone - specific response criteria at the university of texas md anderson cancer center in 2004 18 . the mda criteria updated the uicc and who bone response criteria by expanding radiographic assessment and incorporating both ct and mri . the mda criteria divide response into 4 standard categories ( cr , pr , pd , and sd ) and include quantitative and qualitative assessments of the behavior of bone metastases ( table 2 ) . pr is defined as a decrease of 50% in the sum of the perpendicular measurements of any lesion and pd as an increase of 25% in this sum . according to the mda criteria , cr is defined as complete sclerotic fill - in of lytic lesions on radiographs or ct , the restoration of normal bone density on radiography or ct , the disappearance of abnormal tracer uptake on skeletal scintigraphy ( ss ) , and the normalization of signal intensity on mri ( fig . 5 ) or partial ( rather than complete ) sclerotic fill - in of lytic metastases on radiography or ct ; 50% decrease in the sum of the perpendicular measurements of measurable lesions on radiography , ct , or mri ( fig . 6 ) ; 50% subjective decrease in the sum of the perpendicular measurements of unmeasurable ( ill - defined ) lytic or blastic lesions on radiography , ct , or mri that can not be accounted for by changes in obliquity or slice placement ; and 50% subjective decrease in tracer uptake on ss ( fig . 7 ) . a caveat to interval visualization of sclerotic lesions or lytic lesions with sclerotic rims , in the setting of other signs of pr , does not indicate disease progression but the healing of previously inconspicuous lesions 19 . osteoblastic flare can not be diagnosed if any preexisting lesions show signs of progression ( e.g. enlargement of lytic lesions , development of new lytic lesions ) ( fig . pd is defined as 25% increase in the sum of the perpendicular measurements of any measurable lesion on radiography , ct , or mri ; 25% subjective increase in the size of unmeasurable ( ill - defined ) lytic or blastic lesions on xr , ct or mri that can not be accounted for by obliquity or slice placement ; 25% subjective increase in tracer uptake on ss ; or the development of new metastases . an increase in tracer uptake on ss may need correlation with other imaging studies to exclude the scintigraphic flare phenomenon , which is typically seen within the first 3 months after therapy . scintigraphic flare occurs when healing sclerosis results in more tracer uptake than was caused by the untreated lesion ( fig . sd is defined as < 25% increase or < 50% decrease in size or no change in measurable lesions and no new lesions . in a study comparing the mda , uicc , and who criteria in 41 breast cancer patients with bone - only metastases , the mda criteria were shown to better differentiate responders to chemotherapy from nonresponders and were the only set of criteria to correspond to progression - free survival 25 . according to the mda criteria , time to disease progression was 5.5 months for nonresponders and 23.3 months for responders ( p = 0.025 ) , compared with 10.4 months and 12.4 months , respectively , according to the who criteria ( p = 0.55 ) . the mda criteria identified nonresponders earlier and better correlated with clinical response in the first 2 - 6 months of therapy than did the who criteria . early signs of disease progression are valuable , allowing the halting of ineffective therapy in a timely fashion and the possible substitution of effective therapy . in addition to their utility for guiding treatment decisions , the mda bone response criteria closely reflect the behavior of bone metastases on radiography and ct and can be used as guidelines for the interpretation of these studies whether or not a patient is enrolled in a therapeutic trial . the mda criteria can be considered for use in conjunction with other cancer response criteria or in patients with bone - only metastases and no measurable disease . pet has the potential to revolutionize the definition of measurable tumors because it introduces imaging criteria based on function . the regular , well - defined tumor margins that are necessary for reproducible anatomic measurements are of lesser importance in functional imaging . fdg is a radiolabeled form of glucose that can not be metabolized and therefore accumulates in cells , which take up the molecules as if they were normal glucose . through this accumulation , fdg activity acts as a surrogate for glucose metabolism 26 . since many malignancies are highly metabolic and accumulate fdg the following review of the percist criteria includes many of the concepts discussed in the source article 27 . some chemotherapeutic agents are cytostatic rather than cytocidal and therefore do not result in a profound change in tumor size despite their effectiveness 28 - 30 , and some malignancies , such as gastrointestinal stromal tumors , do not demonstrate pr through a large decrease in size 31 . by reflecting change in tumor metabolism , fdg pet scanning can provide a method by which tumor response can be measured in the absence of marked anatomic change 32 . a decrease in fdg uptake has been shown to indicate treatment response and/or improved survival times in patients with solid tumors such as breast cancer 33 , 34 , esophageal cancer 35 - 37 , lung cancer 38 , 39 , osteosarcoma 40 , 41 , and others 42 . fdg pet has also been shown to provide more rapid response data than anatomic measurements 43 - 45 . fdg pet / ct has also been used to successfully modify disease management 46 by preventing futile thoracotomies in patients with lung cancer 47 and stratifying patients with colorectal cancer into surgical versus palliative groups 48 . uniformity of measurement and reproducibility of results are of paramount importance in cancer response criteria so that data from one study can be meaningfully compared to data from other studies . many acceptable scan acquisition parameters are in clinical use , and several previous attempts have been made to standardize pet for cancer trials through guidelines such as those published by the european organization for research and treatment of cancer ( eortc ) 49 , the netherlands society of nuclear medicine 50 , and the national cancer institute 51 . percist , published in the journal of nuclear medicine 27 , represents the most recent effort to create standardized criteria that accurately reflect response in the largest number of malignancies . because tumor response is inherently continuous , discrete categorization ( e.g. cr , pr , pd , and sd ) may result in the loss of important information 27 , 28 , 52 . therefore , percist specifies that the percentage of change in metabolic activity from baseline and the number of weeks from the initiation of therapy be recorded to provide a continuous plot of tumor activity . the primary determinant of response using percist is the standardized uptake value ( suv ) , a semiquantitative measure of activity that is most commonly calculated by dividing the measured tumor activity by injected dose / body weight 53 . among the many variants of suv ( e.g. maximum suv , mean suv ) , suv corrected for lean body mass ( sul ) was selected for use with percist because sul has been shown to be less susceptible to variations in patient body weight than the other suv metrics 54 , 55 . percist specifies that the sul peak is to be obtained on the single most active lesion on each scan . sul peak is the average of the activity within a spherical region of interest measuring 1.2 cm in diameter ( for a volume of 1 cm ) centered at the most active portion of the tumor . the sul peak may be located in a different lesion on a follow - up scan because the current most avid lesion is to be measured . using a concept similar to recist , it is also recommended that a sum of the activity of up to 5 target lesions ( no more than 2 per organ ) be measured as a secondary determinant of response . future studies will show which of the 2 methods of response determination most accurately reflects treatment outcome . an alternative metric that can be used to determine fdg avidity according to the percist criteria is total lesion glycolysis ( tlg ) . this is a measure of the fdg uptake of the entire tumor above a pre - set threshold and is calculated by multiplying the mean suv by total tumor volume ( ml ) 27 , 56 tlg has been tested in several malignancies and has produced mixed results in comparison to suv metrics , showing a weaker correlation with response in bone metastases in breast cancer patients 57 and in sarcomas41 , 58 but equal or better in esophageal , lung , gastric and rectal cancer 59 60 , 61 . percist suggests that sul peak and tlg can be measured simultaneously in order to further evaluate the efficacy of tlg . for further specifics regarding pet scanning , such as information regarding patient preparation and scan acquisition , percist defines 4 response categories ( table 3 ) in addition to plotting tumor response in weeks from the initiation of therapy . complete metabolic response is defined as the disappearance of metabolic tumor activity in target and nontarget lesions . residual fdg uptake can be seen despite effective therapy , possibly due to macrophage activity 62 , and therefore percist define complete metabolic response as a decrease in tumor sul to the level of surrounding normal tissue . partial metabolic response is defined by a decline of > 30% in sul peak with at least a 0.8-unit decline ( fig . progressive metabolic disease includes an increase of > 30% in sul peak with at least a 0.8-unit increase , a visible increase in the extent of fdg uptake ( increase in the color field representing fdg uptake ) , or the development of new lesions . in the absence of clear evidence of disease progression on the fused ct image , new fdg - avid foci are to be verified on a follow - up scan 1 month after discovery . stable metabolic disease is the absence of change or mild changes that do not meet the minimum qualifications of the other categories . anatomic change in tumor size remains an important factor under percist and is to be measured according to recist 1.1 . if lesions increase or decrease in size without a corresponding change in metabolic activity , disease progression or response is to be verified on a follow - up scan . when evaluating the potential role of functional imaging modalities such as pet , the recist working group decided that there was not sufficient standardization or evidence to abandon anatomical assessment of tumor burden 11 . considering the numerous areas of potential variability that must be overcome in the acquisition and interpretation of pet / ct scans , this hesitation is understandable . nevertheless , if the attempt at standardization represented by percist is successful , fdg pet / ct may be considered as an alternative source of disease measurement in future revisions of the recist criteria . functional imaging criteria can also be considered for use in conjunction with anatomic criteria such as recist or mda ( table 4 ) . the mda criteria can allow more bone lesions to be considered measurable disease than does the recist 1.1 system by allowing physical measurement of well - defined bone lesions regardless of soft tissue extension , by allowing regimented subjective assessment of ill - defined lesions , and by taking into account characteristic behaviors such as the development of healing sclerosis . metabolic imaging criteria can allow bone metastases to be measured in the absence of anatomic change by assessing tumor metabolism . response criteria are of crucial importance to the care of many cancer patients , and the tumor response assessment of bone metastases is assuming a greater role in therapeutic management . knowledge of the fundamental concepts of tumor response criteria ( anatomic , bone , and functional ) and the appearance of bone metastases as they respond to treatment or progress can aid in the interpretation of studies in a manner that will render them of optimal value to the patient and clinician . | response criteria represent the standard by which the efficacy of therapeutic agents is determined in cancer trials .
the most widely used criteria are based on the anatomic measurement of solid tumors .
because bone metastases are typically located in irregularly shaped bones and are difficult to measure with rulers , they have been previously considered unmeasurable disease .
new developments in cancer response criteria have increased awareness of the importance of the response of bone metastases to therapy .
the recently updated response evaluation criteria in solid tumors ( recist 1.1 ) now consider bone metastases with soft tissue masses > 10 mm to be measurable disease .
response criteria specific to bone metastases have been developed at the university of texas md anderson cancer center ( mda criteria ) and can be used to assess therapeutic response in numerous types of bone metastases .
functional imaging criteria , such as the recently developed positron emission tomography response criteria in solid tumors ( percist ) allow response to be measured in the absence of anatomic change through assessment of metabolic activity . as monitoring tumor response of bone metastases becomes more important in the management of cancer , so does the demand on radiologists and nuclear medicine physicians for accurate interpretation of the behavior of these lesions .
this article reviews anatomic , bone , and metabolic response criteria , providing illustrations for the interpretation of therapy - induced change in bone metastases . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
laromustine ( also called 101 m , vnp40101 m ,
cloretazine , and onrigin ) ,
1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine ) ,
ks119 ( 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine ) ,
and ks119w ( 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-phospho-4-nitrophenyl)ethoxy]carbonyl]hydrazine )
are relatively new anticancer alkylating agents . laromustine , which has shown significant
clinical activity in phase i and ii clinical trials in aml , generates 90ce upon base catalyzed decomposition ;
whereas , ks119 and ks119w generate 90ce when reductively activated ,
with the aim of adding selectivity toward hypoxic solid tumor regions . the 90ce moiety has considerable potential in tumor targeted prodrugs
because it is an excellent leaving group , and the rapid spontaneous
decomposition of 90ce ( t1/2 30
s ) , which can be prevented by the substitution of the n-2 hydrogen
with a cleavable chemical trigger , results in the delivery of alkylation
stress close to the site of activation . the cytotoxic
action of 90ce appears to result from the generation of chloroethylating
intermediates that alkylate biomolecules , particularly the o-6 position
of dna guanine . this latter alkylation
accounts for the vast majority of its anticancer activity and leads
to the eventual formation of a 1-(n - cytosinyl),-2-(n - guaninyl)ethane dna dna interstrand
cross - link ( g - c ethane cross - link ) via an n , o - ethanoguanine intermediate
( figure 1 ) . selectivity for tumor
cells arises from differentials between normal and tumor cells in
their effective levels of mgmt , the resistance protein responsible
for the repair of dna o-6 guanine lesions , and in their ability to
repair the resultant cross - links generated from lesions that evade
restoration by mgmt . each mgmt molecule can only repair a single o-6 guanine
lesion ; therefore , the mgmt content correlates with sensitivity to
agents of this type . few tumors are completely devoid
of mgmt activity , but those that are exhibit high sensitivities to
agents that generate guanine o-6 lesions among their repertoire of
dna damage . summarizing scheme illustrating
the generation of therapeutic guanine
o-6 lesions and g - c ethane cross - links exploiting tumor cell dna repair
deficiencies by 90ce and prodrugs thereof and bcnu . ( panel a ) the
generation from 90ce and prodrugs thereof of primary and potential
secondary chloroethylating species . ( panel b ) the generation from
bcnu of aminoethylating , carbamoylating , hydroxyethylating , and vinylating
species not strongly associated with therapeutic activity , and the
formation of therapeutic anticancer chloroethylating species which
are potentially in common with the secondary chloroethylating species
generated by 90ce . ( panel c ) the overall stoichiometry for the generation
of chloroethanol via the chloroethylation of water by 90ce via the
primary or the secondary chloroethylating species . ( panel d ) scheme
showing the major therapeutically relevant dna lesions generated by
oxophilic chloroethylating electrophiles and the cellular processes
involved in their repair . this lesion can be restored by mgmt or cyclize
to form 1-(n - cytosinyl)-2-(n - guaninyl)ethane . this latter lesion can either react
with the opposing cytosine to form a high cytotoxic g - c ethane cross - link
or with mgmt preventing cross - link formation , but additional processes
are required to complete the repair . cells have a limited capacity
to repair the resultant g - c ethane cross - links via hdr . once formed , the g - c ethane cross - link , which does
not contain
an o-6 linkage , can not be repaired by mgmt . however , cells can repair a limited number of g - c ethane cross - links ,
probably using homology directed repair ( hdr ) . as expected , cells possessing defective hdr show additional
sensitivity to laromustine , and cells lacking both hdr and mgmt are
hypersensitive to this agent . a very
small proportion of tumors are likely to possess both of these defects ,
but it is possible to screen for subsets of cancers that would likely
exhibit exceptional sensitivity to 90ce prodrugs . compared to bcnu
( 1,3-bis(2-chloroethyl)-1-nitrosourea ) , which generates a wide array
of electrophiles , including carbamoylating species ( figure 1 ) , a greater proportion of the overall cytotoxicity
of 90ce appears to depend upon the o-6 chloroethylation of guanine .
in support of this assertion , a 22-fold differential in clonogenic
lc90 values was found between l1210 cell lines expressing
and not expressing mgmt for 90ce , compared to only a 2-fold
differential for bcnu in the same matched cell line pair . furthermore , laromustine produces 100% cures
in many mgmt - deficient in vivo tumor models and has
a therapeutic index ( ld50/ed50 ) against the
l1210 leukemia of > 8 , more than double that of over 300 nitrosoureas
tested . electrophilic and nucleophilic species
can be described as hard
or soft , and this concept can be used to predict their reaction site
preference . hard electrophiles have a
high positive charge density and tend to react via sn1
reaction mechanisms with hard nucleophiles which have a high negative
charge density . in contrast , soft electrophiles have a low charge
density or are easily polarized and tend to react via sn2 reaction mechanisms with soft nucleophiles that have a low negative
charge density or are easily polarized . the relative hardness and
softness of electrophiles determines the spectrum of nucleophilic
positions available in dna for preferred reactions . the dna - backbone phosphate groups are the hardest nucleophilic
sites in dna , and the o-6 position of guanine is the hardest base
centered site , while the n-7 position of guanine is the softest . a single therapeutic alkylating agent can generate
a wide array of lesions on sites of diverse hardness / softness in dna
because complex decomposition pathways can result in the production
of many alkylating species with a wide range of hardness / softness
and hence different and/or overlapping alkylation site preferences . this is the case with bcnu , which generates several potential chloroethylating
species together with species that hydroxyethylate , vinylate , aminoethylate ,
and carbamoylate . while o-6 chloroethylation of guanine still dominates in terms of
the anticancer activity of bcnu , it is a relatively minor product
compared to alkylations on the n-7 or n-1 positions of dna guanine . 90ce was designed to generate a unique primary
chloroethylating species ( clch2ch2n = nso2ch3 ) , but this intermediate could potentially give
rise to three other secondary chloroethylating species ( 2-chloroethyldiazohydroxide ,
2-chloroethyldiazonium , and chloronium ions ) that are in common with
those believed to be generated by bcnu ( figure 1 ) . nevertheless , the pattern of dna alkylation by 90ce
appears to have a greater o-6 guanine to n-7 guanine alkylation bias
under biologically relevant conditions and in in vitro and in vivo models than that of bcnu . we have previously studied
the decomposition mechanism of 90ce
in non - pi and low pi buffers ; under these
conditions , 90ce appears to decompose in a relatively simplistic manner ,
as initially intended by design , to generate approximately two moles
of methanesulfinate , one mole of nitrogen gas , and one mole of 2-chloroethanol ,
produced as a consequence of the chloroethylation of water ( figure 1 ) . it was found that the rate determining step during
the decomposition of 90ce and 1,2-bis(sulfonyl)-1-alkylhydrazines
was the elimination of the n-1 sulfonyl moiety from the 1,2-bis(sulfonyl)-1-alkylhydrazine
anion , which in the case of 90ce results
in the formation of the primary chloroethylating species clch2ch2n = nso2ch3 ( figure 1 ) . subsequently , we have found that in the presence
of high concentrations of pi , the alkylation of 4-(4-nitrobenzyl)pyridine
( 4nbp ) by 90ce was greatly increased . 4nbp is the most commonly used
colorimetric reagent for determining alkylating activity , and the
pyridine nitrogen which serves as the nucleophilic target in this
molecule is a moderately soft site . thus ,
4nbp would normally be expected to be poorly alkylated by 90ce , as
is found in the absence of pi . this pi dependent change in character
was not observed with 1,2-bis(methylsulfonyl)-1-methylhydrazine ( ks90 ) ,
the methylating analogue of 90ce . in view of the ubiquitous cellular
presence of pi , this interaction was further investigated , and a second
decomposition pathway for 90ce has been elucidated . the implications
of these findings in terms of future drug designs , tumor cell selectivity ,
agent toxicity , and possible novel resistance mechanisms are discussed . both 90ce and ks90 are
potentially carcinogenic and mutagenic and should be handled carefully
using personal protective equipment . all other chemicals were purchased
from the sigma - aldrich chemical co. , st . eugene , or ;
hpcd , ( 2-hydroxypropyl)--cyclodextrin was obtained from
american maize products company ( 1100 indianapolis boulevard , hammond ,
in ) . murine l1210 leukemia
cells were used as a source of dna for dna cross - linking assays . l1210
leukemia cell lines were grown in suspension culture in rpmi 1640
medium supplemented with 10% fbs in air/5% co2 at 37 c . dna
was isolated using a puregene dna isolation kit ( gentra systems , minneapolis ,
mn ) using procedures recommended by the manufacturer . isolated l1210
dna was diluted to 400 g / ml with 5 mm tris , 1 mm edta ,
and 1 mm nan3 at ph 7.4 buffer , and stored at 4 c
until required . the alkylation of 4nbp by 90ce in the presence of
various concentrations of pi was studied using a method that is dependent
upon the high ph aqueous stabilization of alkylated 4nbp using ( 2-hydroxypropyl)--cyclodextrin
( hpcd ) . all buffers contained 20
mm tris - hcl ( ph 7.4 ) and various concentrations of potassium phosphate
0200 mm ( ph 7.4 ) . 4nbp was added to give a final concentration
of 1 mg / ml ( close to its solubility at 37 c ) from a 50
stock solution in dmso , and -thioglycerol ( tg ) was added as
a competing nucleophile in some experiments to give final concentrations
of either 1 or 20 mm from 100 aqueous stock solutions . the reactions
were initiated by the addition of 200 m 90ce ( 100 mm stock
solutions in dmso ) and incubated at 37 c for 15 min . some analogous
experiments were performed using bcnu in place of 90ce with 20 h overnight
incubations being utilized due to its more than 60-fold longer t1/2 time . the samples were assayed for 4nbp
alkylation products by rapidly mixing equal volumes of the above reaction
mixtures with 38% hpcd in 0.5 m koh at room temperature and
recording the absorbance at 635 nm within 1 min . aging experiments
to follow the loss of the ability of 90ce to alkylate 4nbp under high
pi concentrations were performed in a very similar manner . the reaction
was initiated by the addition of 200 m 90ce to 200 mm potassium
phosphate buffer ( ph 7.4 ) at 37 c with very rapid mixing . one
milliliter samples were then taken from this mixture and rapidly mixed
with 20 l of 50 mg / ml of 4nbp in dmso , 0 , 30 , 60 , 90 , 120 ,
and 300 s after reaction initiation , and incubated for a further 15
min . mnp
is a highly chromophoric thiol / thiolate at physiological ph values ,
absorbing with a max at 389 nm . in these experiments , observations were made at 440 nm
because at this wavelength , the extinction coefficient is still substantial ,
and derivatization of the thiol / thiolate causes a complete rather
than a partial bleaching of the absorbance . mnp was generated in situ from 2,2-dithiobis(5-nitropyridine ) by reduction
with thioglycerol ( tg ) . to a 60 m dtbnp solution ( 10 mm stock
in dmso ) in 200 mm potassium phosphate buffer ( ph 7.4 , 37 c ) ,
a limiting quantity of 100 m tg ( 10 mm stock in h2o ) was added ; this should convert 50 m of the dtbnp
to 100 m of mnp , exhausting the tg and leaving mnp
as essentially the only thiol present . this assumption was confirmed
by observing an equivalent increase in absorption at 440 nm upon adding
a 100 molar - fold excess of tg to 50 m dtbnp . the absorbance
of 100 m mnp at 440 nm was 0.775 au and was attained
within a few minutes of the addition of 100 m tg at 37 c
and remained stable thereafter at this level . addition of excess 90ce
( 500 m ) fully bleached the mnp absorbance in < 1 min . this
method for producing mnp was used to follow the extent and kinetics
of alkylation / bleaching of mnp upon the addition of limiting aliquots
of 90ce in 200 mm potassium phosphate buffer . to examine the effects
of anions / solutes other than phosphate on
the generation of soft electrophilic species from 90ce , identical
experiments were performed in 20 mm tris - hcl buffer ( ph 7.4 , 37 c )
containing 100 m mnp ( generated in situ as
described ) in which the buffer was supplemented with various solutes
( ph adjusted to 7.4 ) at 50 mm . the bleaching of mnp was followed after
the addition of 100 m 90ce over 5 min ( 50 mm pi results in
a submaximal effect ) . in the case of atp , some experiments were also
performed in the presence of a 1.05-fold molar excess of mgcl2 . reaction mixtures had a final total volume of 40 l and were
made up in 5 mm tris - hcl , 1 mm edta , and 1 mm nan3 buffer
( ph 7.4 ) and contained l1210 leukemia dna ( 120 g / ml ) , various
final concentrations of pi ( 0100 mm ) , and 20 mm tg . the reactions were initiated by the addition of 90ce to give a final
concentration of 100 m ( 4 l of 1.0 mm 90ce in 1.0 mm
hcl ) . these samples were then incubated for 15 min at 37 c ,
then diluted by the addition of 560 l of 5 mm tris - hcl , 1 mm
edta , and 1 mm nan3 ( ph 8.0 ) buffer to give a total volume
of 600 l . at this point , these dnas then contained a monoadduct
cross - link precursor ( o-(2-chloroethyl)guanine and n , o - ethanoguanine ) but insignificant
cross - links . these samples were then incubated at 50 c for a
further 3 h to allow the monoadducts sufficient time to fully react
with the complementary strand and generate dna interstrand cross - links .
from these samples , the level of dna cross - linking was then determined
using a modification of a previously described assay . this assay is based upon the fact that upon rapid cooling thermally
denatured dna containing one or more covalent interstrand cross - links
rapidly renatures , yielding a highly fluorescent complex with h33258
dye . the 200 l aliquots of l1210 dna containing various levels
of interstrand cross - links were added to 1.5 ml of 5 mm tris - hcl ,
1.0 mm edta , and 1.0 mm nan3 buffer ( ph 8.0 ) containing
0.1 g / ml of h33258 , heated to 100 c for 3 min , then plunged
into a water bath at room temperature for 3 min . fluorescence measurements
were taken before the heating phase and after the 3 min chill using
a hoefer scientific instruments tko 100 fluorometer , and the fraction
of the dna molecules that were cross - linked ( i.e. , that contained
at least one cross - link per dna molecule ) and the average number of
cross - link moieties per dna molecule calculated assuming a poisson
distribution as previously described . the decomposition / reaction of
90ce involves the formation of a strong acid ( methanesulfinic acid ,
pka 2 ) and thus can be followed
using protonometric assays . we utilized a simple colorimetric assay
that relies upon the measurement of the relatively linear change in
absorption at 560 nm of phenol red that occurs in proportion to the
generation / addition of small quantities of hydrogen ions when a weakly
buffered solution of this ph indicator is subjected to incremental
acidification over a narrow ph range ( ph < 0.1 unit ) close
to the pka values of the buffer / indicator
components . using a 20 g / ml solution
of phenol red in 2 mm tris - hcl buffer , the absorbance at 560 nm was
followed at an initial ph value of 7.4 at 20 and 37 c upon the
addition of 50 m 90ce ( 5 l / ml of 10 mm 90ce in dmso ) . the assay mixtures were sealed with parafilm in 1 ml cuvettes to minimize
changes in ph due to co2 exchange and brought to the appropriate
temperature prior to the addition of agent via injection through the
parafilm and rapid mixing . decomposition / reaction
kinetics of 90ce ( 100 m ) were followed at both 20 and 37 c
in 200 mm potassium phosphate ( ph 7.4 ) and 20 mm tris - hcl ( ph 7.4 )
by recording the appearance of uv absorbing material at 240 nm after
the addition of 10 l / ml of a 10 mm 90ce solution in dmso . because
of the temperature dependence of the dissociation constants of the
buffer constituents , in particular that of tris - hcl , the buffer ph
values must be set at the appropriate assay temperature . the
loss of the ability of 90ce to chloroethylate the o-6 position of
dna guanine was examined by following the exhaustion of dna cross - linking
capability versus time at ph 7.4 and 37 c in 20 mm tris buffer .
to 500 l of 20 mm tris buffer ( ph 7.4 at 37 c ) , 100 m
90ce was added ( 10 l of 10 mm 90ce in dmso ) to initiate electrophile
generation ; at various times after initiation and mixing ( 0 , 30 , 60 ,
90 , 120 , and 300 s ) , aliquots ( 12 l ) were withdrawn and mixed
with an equal volume of l1210 dna 400 g / ml in 5 mm tris , 1
mm edta , and 1 mm nan3 ( ph 7.4 ) buffer and rapidly mixed . these mixtures were then incubated for 15 min at 37 c , diluted
to a volume of 600 l with 5 mm tris , 1 mm edta , and 1 mm nan3 ( ph 8.0 ) buffer then incubated at 50 c for a further
3 h. the level of dna cross - linking and the number of cross - link moieties
per dna molecule were then measured and calculated as described above . water is
a particularly good trapping agent since it is present at a concentration
of 55 m , which results in it sequestering the vast majority
of the harder electrophiles generated . since 2-chloroethanol lacks any strong spectroscopic features and
is relatively unreactive , it is necessary to transform it into a chromophoric
derivative . therefore , the 2-chloroethanol was oxidized to its corresponding
aldehyde using pichia pastoris alcohol oxidase ( ao ) ,
a relatively nonspecific enzyme which oxidizes short - chain , linear
aliphatic alcohols to their respective aldehydes . the resultant aldehyde was then reacted with 2,4-dinitrophenylhydrazine
( 2,4-dnph ) to generate a hydrazone product which was quantified by
hplc . to 0.5 ml samples of ph 7.4 buffers containing various concentrations
of pi ( 0200 mm ) , 1.0 mm 90ce ( 5 l of 100 mm 90ce in
dmso ) was added . the 0 mm pi buffer experiments contained 20 mm tris
( ph 7.4 ) to allow the decomposition to fully proceed , which would
normally slow substantially as the ph fell , due to the liberation
of methanesulfinic acid . these mixtures were incubated for 5 min at
40 c , then diluted 10-fold with distilled h2o . to
0.5 ml of this diluted mixture , 10 unit / ml of ao was added and the
mixture incubated for 30 min at 40 c in sealed tubes with occasional
shaking to maintain aeration . aliquots ( 0.5 ml ) were then mixed with
an equal volume of a 0.4% solution of 2,4-dnph in ch3cn
and 50 l of 1 m hclo4 , and the mixture incubated
at 40 c for 5 min and then assayed directly by hplc . the hplc
protocol utilized a 250 mm 4.6 mm varian microsorb 100 - 5 c-18
reverse phase column ( varian inc . , lake forest , california , usa ) and
a constant composition buffer ( 52.5% ch3cn and 47.5% 30
mm potassium phosphate , ph 5.4 ) at a flow rate of 0.8 ml / min . all hplc measurements were performed using a beckman 127p solvent
module and a beckman 168 uv / vis detector ( beckman , fullerton , ca ,
usa ) . this assay gave good linearity in the test range using authentic
2-chloroethanol samples and was not significantly affected by the
initial buffer composition . the limit of 2-chloroethanol detection
using this method was 1 m . assays were compared with
identical reagent blanks containing all components except 90ce . acetaldehyde yields were
determined using a protocol identical to that used for measuring 2-chloroethanol ,
except that the addition of and incubation with ao were not required . the assay was calibrated using authentic acetaldehyde standards , and
the resultant hydrazone eluting at 14.9 min gave a linear auc ( area
under the curve ) for the resultant hydrazone peak versus the initial
concentration of acetaldehyde . the auc of the hydrazone product peak
over that of dmso controls was used to calculate the acetaldehyde
yields by comparison with authentic standards . ethylene glycol was
measured in a manner similar to that used with 2-chloroethanol , except
that a longer 60 min incubation with ao was utilized to oxidize the
ethylene glycol to glyoxal since ethylene glycol is a poorer substrate
for this enzyme . even with this longer incubation time , the oxidation
was not complete , and the yield of glyoxal was approximately 50% at
all concentrations ( based upon comparisons with authentic glyoxal
samples ) . in addition , the hydrazone generated from glyoxal required
the use of an increased ch3cn concentration ( 75% ch3cn and 25% 30 mm potassium phosphate , ph 5.4 ) in the hplc
protocol compared to the previous method to avoid excessively long
elution times . under these revised hplc conditions , the hydrazone
product eluted at 9 min . ethylene glycol standards gave a
linear auc for the resultant hydrazone peak versus the initial concentration
of ethylene glycol . the auc of the glyoxal hydrazone product peak
over that of dmso controls was used to calculate the ethylene glycol
yield by comparison with authentic standards . the generation of free chloride
during the decomposition of 90ce in buffers containing various concentrations
of pi was assayed using a modification of the method of jrg
and bertau . this is a highly sensitive
colorimetric assay based on the formation and strong absorbance of
[ fecl ] at max 340 nm . a 200 mm tris - acetate
buffer ( ph 7.4 ) ( produced using tris free base and glacial acetic
acid ) and a 200 mm potassium phosphate buffer ( ph 7.4 ) ( produced using
kh2po4 and koh ) were combined in various proportions
to generate chloride free buffers with pi concentrations between 0
and 200 mm . this was circumvented by formulating 0.5 m
90ce stock solutions in hydroxyacetone ( acetol ) for these experiments . two additional problems were encountered , one associated with the
determination of chloride in the presence of high pi levels in an
fe based assay and a second due to an additional background
generated from nonchloride 90ce decomposition products ( discovered
by utilizing ks90 as a control agent , with ks90 being a 90ce analog
in which the chloroethyl moiety is replaced by a methyl group and
therefore unable to liberate chloride ) . the first problem was resolved
by precipitation of the pi using ca(no3)2 prior
to the assay . the second problem was negated by splitting each sample
into two , removing the chloride from one portion by utilizing silver
acetate precipitation and using this chloride free counterpart as
the reagent blank against which the second portion was measured . to 2 ml samples of buffers
containing various levels of pi , formulated as described above , 40
l of 0.5 m 90ce dissolved in acetol was added to give a final
90ce concentration of 10 mm . this mixture was then incubated for 20
min at 37 c to allow for complete decomposition . these samples
were then diluted 10-fold with distilled water and 0.1 ml of saturated
1.6 m ca(no3)2 solution added per ml and the
precipitate removed by centrifugation . the supernatants were then
split into two portions and one - half treated with 40 l / ml of
50 mm silver acetate and the other with 40 l / ml of distilled
water . these samples were then centrifuged to remove any precipitate
and 0.5 ml samples of supernatant mixed with an equal volume of 40
mm fe(no3)3 in 50% perchloric acid , and the
absorbance of the experimental sample measured at 340 nm versus its
chloride depleted counterpart . this assay was linear using chloride
standards and chloride spiked experimental controls independent of
the buffer pi concentration . methanesulfinic acid
was assayed by a modification of the method
of babbs and gale , which is based upon
the reaction of an aromatic diazonium salt ( ar n = n ) , in this case , fast blue bb salt with methanesulfinic acid
to produce a chromophoric diazosulfone derivative ( ar n = n - sooch3 ) , which is highly hydrophobic and thus can be selectively
extracted into an organic solvent and determined spectrophotometrically . ten microliter aliquots of 100 mm 90ce in dmso were added to 1.0 ml
samples of ph 7.4 buffers containing various pi contents ( 0200
mm ) to give a final 90ce concentration of 1 mm . the 0 mm pi buffer
experiments contained 20 mm tris - hcl buffer ( ph 7.4 ) to allow the
decomposition to fully proceed , which would normally slow substantially
as the ph fell due to the liberation of methanesulfinic acid . these
mixtures were incubated for 5 min at 37 c to allow for a complete
decomposition / reaction , and then aliquots were diluted 40-fold with
500 mm nah2po4/h3po4 ( ph
2.2 ) containing 1 part in 80 of saturated fast blue bb salt solution
in 500 mm nah2po4/h3po4 ( ph 2.2 ) . these mixtures were allowed to react at 40 c for
10 min in
the dark . an equal volume ( 0.8 ml ) of n - octanol pre - equilibrated
with 500 mm nah2po4/h3po4 was then added and the tubes vigorously shaken for 2 min en masse ;
the samples were then centrifuged at 10,000 g for
120 s , and the upper octanol layer was separated and the absorbance
measured at 460 nm versus a reagent blank within 5 min . calibration
curves were generated using solutions of authentic sodium methanesulfinate
at concentrations of 0.0 , 0.5 , 1.0 , 1.5 , 2 , and 3 mm in buffers with
various pi contents ( 0200 mm ) , and a linear response ( absorbance
460 nm versus methanesulfinate concentration ) was observed that was
independent of the pi concentration in the initial samples . the strongly absorbing thiol mnp produced by reducing dtbnp was
used as a convenient electrophile trapping agent suitable for the
separation by hplc . the hplc protocol utilized a 250 mm 4.6
mm varian microsorb 100 - 5 c-18 reverse phase column ( varian inc . , )
and elution with an unbuffered ch3cn / h2o solvent
system , starting with 5% acetonitrile for 5 min followed by a 550%
ch3cn linear gradient over the subsequent 30 min . after
this point , the concentration of ch3cn was maintained at
this level for 5 min , then returned to the starting concentration
over an additional 5 min . absorbance was monitored at 340 nm using
a beckman 168 uv / vis detector ; 340 nm was chosen because the strong
absorption of the 5-nitropyridine moiety is largely independent of
the derivatization or oxidation state of the attached thiol group
at this wavelength . the reduced thiol ( mnp ) and oxidized disulfide
dimer ( dtbnp ) elute at 5 and 38 min , respectively . the reaction protocol was as follows : 1 mm dtbnp in 200 mm k2hpo4 was reduced to mnp by the addition of 2 mm thioglycerol
to generate 2 mm mnp in a high pi buffer environment . after
a few minutes of gentle shaking at room temperature to ensure that
the reduction was complete , an aliquot of 100 mm 90ce in dmso was
added to give a final concentration of 2 mm 90ce . this mixture was
incubated for 4 min at 37 c , centrifuged at 10,000 g for 1 min , then diluted 10-fold with distilled water and immediately
analyzed by hplc . this reaction sequence is easily followed by the
eye , as reduction to mnp caused the sample to develop a strong yellow
color , which is then largely discharged over a few minutes upon alkylation
by 90ce . under these reaction conditions , only one new mnp/90ce derived
major product , eluting as a broad tailing peak ( 1013 min ,
peak 11.6 min ) , was observed . a similar experiment was performed at
four times these concentrations of mnp and 90ce and the alkylated
product peak collected for analysis by lcms . the alkylated product prepared ,
separated , and collected as described above was analyzed by mass spectroscopy
using a chromatographic system consisting of an agilent 1200 series
hplc system , including a binary pump ( model g1312b ) , a vacuum degasser
( model g1379b ) , an autosampler ( model g1367c ) , and a column oven ( model
g1316b ) . the mass spectrometer was an applied biosystems sciex 4000
q - trap mass spectrometer ( applied biosystems sciex ; foster , ca ) . data
acquisition was carried out by analyst 1.4.2 software on a dell computer .
a declustering potential of 50 v , excitation energy of 100 v , and
a collision energy of 10 v were utilized . previous studies , in the absence of high pi concentrations , demonstrated
that the initial elimination of the n-1 sulfinate moiety was the rate
determining step in the decomposition of 1,2-bis(sulfonyl)-1-alkylhydrazines
( figure 1 panel a ) as measured by hydrogen
ion production . this time course also
corresponded to the loss of biological activity upon agent aging . the instantaneous release of the first mole of
protons represents the ionization of the acidic n-2 hydrogen and can not
reflect the elimination of the n-1 sulfinic acid moiety from the un - ionized
parental 1,2-bis(sulfonyl)-1-alkylhydrazine because the t1/2 for the first order release of the second mole of
protons decreases with the increasing leaving group ability of the
n-1 sulfinate moiety , and this could not occur if the n-1 sulfinate
moiety had already left the molecule . the
release of the second mole of protons occurs upon nucleophile alkylation
by the primary oxophilic alkylating species rn = nso2ch3 ( e.g. , figure 1 panel d ) or
secondary alkylating species derived from these . this biphasic release
of two moles of hydrogen ions is observed during the decomposition
of both methylating and chloroethylating analogues in a pi free or
low pi buffers . in tris - hcl buffer , both
ks90 and 90ce poorly alkylated 4nbp as would be expected for generators
of relatively hard electrophiles . however , in 200 mm pi buffer , the
behavior of 90ce changed markedly , and a > 10-fold increase in
the
alkylation of 4nbp was observed . we therefore investigated the pi
concentration dependence of the alkylation of 4nbp ( 1 mg / ml ,
4.7 mm ) by 200 m 90ce over a 0200 mm pi concentration
range ( figure 2 ) . the presence of pi resulted
in a marked , but saturatable , concentration dependent increase in
4nbp alkylation , with approximately 30 mm pi eliciting a half - maximal
effect under these conditions . the inclusion of 20 mm tg , as a competing
soft nucleophile , decreased the alkylation of 4nbp at the highest
pi concentration by > 96% . the addition of 1 mm tg was almost as
effective ,
resulting in an 90% reduction in 4nbp alkylation at 200 mm
pi ( data not shown ) . thus , the generated electrophile must be extremely
soft in nature since only 1 mm tg could out compete the alkylation
of 5 mm 4nbp by 90% . this very strong thiol preference
is more reminiscent of a michael addition reaction than the reaction
of a nucleophile with a carbonium ion or highly polarized alkyl group .
in comparison , bcnu at an equivalent concentration poorly alkylated
4nbp over a 0200 mm pi concentration range , and this nbp alkylation
was marginally decreased rather than greatly increased at higher pi
concentrations ( figure 2 ) , probably due to
some alkylation of pi . in addition , no pi dependent increase in thiol
preference was observed with bcnu ( data not shown ) . since 90ce can
potentially generate three secondary chloroethylating species which
are in common with the hard oxophilic chloroethylating species proposed
for bcnu ( figure 1 , panel b ) , these findings imply that the pi dependent increase in soft nucleophile
preference seen with 90ce does not involve these secondary chloroethylating
species ( figure 1 , panel a ) but a component
unique to 90ce . effect of pi concentration on the preference of 90ce and
bcnu for
4nbp . reactions containing either 200 m 90ce ( 20 mm tg )
or 200 m bcnu and 1 mg / ml of 4nbp ( 4.7 mm ) in 20 mm tris - hcl
buffer ( ph 7.4 ) containing various concentrations of pi ( ph 7.4 ) samples
were reacted for either 15 min at 37 c for 90ce or overnight
at 37 c for bcnu due to its > 60-fold longer t1/2 . the resultant level of 4nbp alkylation was then determined
by measuring the absorbance at 635 nm versus a reagent blank after
mixing samples with an equal volume of 38% hpcd dissolved in
0.5 m koh . ( ) 200 m 90ce in the absence of tg ; ( )
200 m 90ce in the presence of 20 mm tg ; and ( ) 200
m bcnu in the absence of tg . all values are the result of at
least 3 determinations se . in view of the pi concentration dependent increase in the
generation
of soft thiophilic alkylating species with increasing pi concentration
from 90ce , we decided to examine the effects of pi concentration on
the formation of dna interstrand cross - links which are dependent upon
the generation of hard oxophilic chloroethylating electrophiles . dna
interstrand cross - link formation is of particular importance since
it is crucial to the mode of action of 90ce prodrugs . it can be seen
( figure 3 ) that increasing the pi concentration
results in a progressive decrease in the yield of dna cross - linking
moieties per dna molecule and that the electrophiles responsible for
generating these lesions are hard oxophilic electrophiles that are
resistant to thiol interception since 20 mm tg only decreased the
yields 15% . only a very small proportion of the oxophilic
chloroethylating electrophiles generated react with the o-6 position
of dna guanine since the bulk chloroethylate water , which is present at 55 m , to generate 2-chloroethanol
( figure 1 , panel c ) . this is especially true
for hard oxophilic chloroethylating species , which have a strong preference
for water - like nucleophiles . however ,
it is expected that water in the absence of competing favored nucleophiles ,
due to its high concentration , would also trap the majority of any
generated soft chloroethylating species as 2-chloroethanol . therefore ,
we looked at the production of 2-chloroethanol versus pi concentration
to ascertain the changes occurring in the overall yields of chloroethylating
electrophiles ( figure 4 , panel a ) . a large
decrease in chloroethanol production from 83% this
change approximately parallels the decrease seen in dna cross - link
formation and is the inverse of the increase observed in soft thiophilic
alkylators in the 4nbp alkylation experiments . the corresponding decrease
in chloroethanol production suggested that the soft thiophilic alkylator
was not a chloroethylating species . the reduction in 2-chloroethanol
yields could mean that the chlorine was lost as chloride or remained
as part of a different species . we therefore measured chloride release
and found that 80% of the total chlorine was liberated as
chloride under high pi conditions ( figure 4 , panel a ) . this finding confirmed that the chlorine was largely
lost in the pi catalyzed pathway and was no longer part of the donated
electrophile moiety . it was thought that the loss of chloride by elimination
or by a hydrolytic mechanism could possibly lead to either vinylating
or hydroxyethylating species , respectively . acetaldehyde and ethylene
glycol are formed by the vinylation and hydroxyethylation of water ,
respectively , and can be generated in quite high yields by bcnu via
decomposition pathways involving chloride loss ( figure 1 , panel b ) . for this reason ,
we assayed for the generation of these two chlorine - free two - carbon
atom species during the decomposition of 90ce . at 200 mm pi , the highest
level tested , the yields of acetaldehyde and ethylene glycol were
approximately 5% and 2% , respectively ( figure 4 , panel a ) . these yields combined were insufficient in quantity to
account for the large decrease in the yields of 2-chloroethanol (
83% to 17% ) between 0 mm pi and 200 mm pi , respectively ( figure 4 panel a ) . in previous studies following the decomposition
of gram quantities of ks90 , both the volume
and composition of the gas liberated were determined ; and very close
to a mole equivalent of nitrogen was found to be released during ks90
decomposition . while it is difficult to quantify and analyze microliter
volumes of gas liberated during the decomposition of small quantities
of dilute aqueous solutions of 90ce , at decomposition concentrations
of 1 mm or greater gas bubbles can be observed nucleating
on the vessel s walls . we therefore quantified the bubble formation
number during the decomposition of 2 mm solutions of 90ce in both
tris - hcl and 200 mm potassium phosphate and noted a > 80% reduction
in bubble numbers under high pi conditions ( figure 4 , panel b ) . this observation implied that the volume of gas
liberated under high pi conditions was also significantly reduced . the apparent failure of the bulk of the 90ce , under high pi conditions ,
to liberate either the two carbon alkylating moiety as chloroethanol ,
acetaldehyde , or ethylene glycol and the hydrazine derived nitrogen ,
suggested that the molecule failed to fully fragment under these conditions . consequently , we determined the effects of pi concentration on the
liberation of methanesulfinate during the decomposition of 90ce ( figure 5 ) . the quantity of methanesulfinate was found to
be reduced from 2 to 1.2 mols per mol of 90ce under
high pi conditions . this finding is consistent with 80% of
the 90ce failing to fully fragment after the initial elimination of
the n-1 methanesulfinate moiety , thus liberating a single mole of
methanesulfinate instead of two at the highest concentrations of pi . a small proportion ( 17% ) of the 90ce appeared to decompose ,
liberating chloride even in the absence of pi in the 200 mm tris - acetate
buffer used in the chloride assay . if this proceeded by the 1 mol
of methanesulfinate route , one would expect 1 mol of 90ce to liberate
a maximum of 1.83 mols of methanesulfinate rather than 2 mols .
this discrepancy could be due to a minor decomposition pathway(s )
where both the chloride and second methanesulfinate are lost in the
absence of pi ( potential minor pathway a , see later ) , a small pi - like
effect of the 200 mm tris - acetate buffer used in the chloride determinations
( see later ) , or an inaccuracy in the methanesulfinate standards due
to hygroscopicity of the sodium salt used , or a combination of these
possibilities . effects of pi concentration on the average number of cross - link
moieties generated per dna molecule ( reaction concentration 120 g / ml
of l1210 dna ) by 100 m 90ce at ph 7.4 in the presence and absence
of 20 mm tg as a competing nucleophile . ( ) average number
of cross - link moieties generated per dna molecule in the absence of
tg ; and ( ) average number of cross - link moieties generated
per dna molecule in the presence of 20 mm tg . effects of pi concentration on the yields of 2-chloroethanol , acetaldehyde ,
ethylene glycol , and chloride anions ( panel a ) , and nitrogen gas evolution
from 90ce ( panel b ) at ph 7.4 and 37 c . panel a , ( )
2-chloroethanol , ( ) acetaldehyde , ( ) ethylene glycol ,
and ( ) chloride anions . the experimental data indicate
that the formation of ethylene glycol and acetaldehyde are insufficient
to account for the decreases in chloroethanol and increases in chloride
formation observed under high pi concentration conditions and that
some other processes must account for these differences . ( panel b )
photographs taken just after the addition of 2 mm 90ce ( upper and
lower left - hand images ) to microcentrifuge tubes containing either
20 mm tris - hcl ( upper row ) or 200 mm potassium phosphate ( lower row )
buffers at ph 7.4 , and after 5 min of incubation at 37 c ( upper
and lower right - hand images ) , note the large decrease in nitrogen
evolution under high phosphate conditions . effects of pi concentration on the yields of methanesulfinate formed
during the decomposition of 90ce . one millimolar solutions of 90ce
were decomposed at 37 c and ph 7.4 for 10 min in buffers containing
various concentrations of pi . these samples were derivatized with
the aromatic diazonium dye fast blue to give the octanol soluble highly
chromophoric diazosulfone derivative . the diazosulfone was separated
into octanol and the absorbance measured at 460 nm versus a reagent
blank ( identical except for the absence of 90ce ) . ( insert ) decomposition
scheme illustrating the generation of 2 mols of methanesulfinate per
mol of 90ce in the absence of pi , with the downward arrow representing
a potential decomposition pathway branch point in the presence of
pi retaining a methanesulfinate moiety and both nitrogen atoms . differences in the overall kinetics
of alkylation by 90ce reacting
via hard oxophilic or soft thiophilic alkylating species could arise
from the change in the decomposition pathway since this could be accompanied
by a change in the rate determining step . furthermore , soft alkylating
species could be expected to persist for longer time periods in aqueous
solution and potentially extend the time course of alkylation in the
absence of preferred nucleophiles or in the presence of rate limiting
nucleophile concentrations . therefore , the kinetics of the following
processes were measured and compared : the kinetics of the release
of the second mole of hydrogen ions ( 0 mm pi ) , bleaching of mnp ( 200
mm pi ) , and the generation of uv absorbing species ( 200 mm pi ) , together
with the loss of the ability of 90ce to produce dna cross - link moieties
( 0 mm pi ) and to alkylate 4nbp ( 200 mm pi ) upon aging ( figure 6 , panels a essentially identical half - reaction
times of approximately 35 , 31 33 , 36 , and 30 s , respectively , were
determined for all of these processes . the generation of uv absorbing
product(s ) ( 16-fold greater under high pi conditions ) occurs with
the inverse kinetics of the other processes ( exponential association
rather than decay ) . the kinetics of all of these exponential decay
processes and the exponential association process are compared by
normalization in figure 6 , panel f ( 1 minus
the normalized value has been plotted in the case of the exponential
association ) . thus , the same rate determining step , i.e. , the previously
identified elimination of the methylsufinate from n-1 , appears to
control all of these processes . two of these processes , the kinetics
of hydrogen ion generation at 0 mm pi and the kinetics of the production
of uv absorbing material at 200 mm pi , were also examined at 20 c
where the kinetics of both processes were equivalently slowed by a
factor of 7-fold ( data not shown ) . these findings indicate
that the rate determining steps for these two processes ( one at 0
mm pi and the other at 200 mm pi ) have both the same values and temperature
dependencies , further implying the equivalency of their rate determining
steps . because of inherent assay physical limitations , measurements
of the kinetics of hydrogen ion generation are restricted to weakly
buffered solutions . therefore , equivalent experiments could not be
performed under high pi concentrations ; however , hydrogen ion liberation
assays would be blind to a switch from methanesulfinate to chloride
liberation since both would be accompanied by the liberation of a
single hydrogen ion . the increase in the production of uv absorbing
species under conditions of high pi concentration could imply the
acquisition of double bonds , and this finding would be consistent
with the elimination of hcl from the chloroethyl moiety . this would
be favored after the elimination of the n-1 methanesulfinate moiety
because it would then result in extended conjugation from the alkyl
moiety all the way to the oxygen atoms on the remaining sulfonyl moiety . this would generate ch3so2n = nch = ch2 , a very thiophilic michael type acceptor , which would retain
one of the methylsulfonyl moieties and the nitrogen atoms upon reaction
with a thiol . kinetics of various 90ce dependent processes at 37 c
and
ph 7.4 under high and low pi conditions . ( panel a ) hydrogen ion generation
versus time ( 0 mm pi ) ; ( panel b ) loss of dna cross - linking activity
versus time ( 0 mm pi ) ; ( panel c ) loss of 4nbp alkylating activity
versus time ( 200 mm pi ) ; ( panel d ) mnp bleaching versus time ( 200
mm pi ) ; ( panel e ) generation of uv absorbing material ( 240 nm ) versus
time in the presence ( upper trace ) and absence ( lower trace ) of 200
mm pi ; and ( panel f ) comparison of the kinetics of the processes in
panels a the calculated t1/2 and first order rate constant , determined by a nonlinear
regression best curve fit analysis , for each reaction is given in
the corresponding panel . the loss of activity upon aging
of 90ce for dna cross - link formation
( 0 mm pi ) ( figure 6 , panel b ) and 4nbp alkylation
( 200 mm pi ) ( figure 6 , panel c ) indicate that
neither the oxophilic nor thiophilic alkylating species persists in
these reaction mixtures since the residual activity matches the kinetics
for the rate determining elimination of the n-1 methanesulfinate moiety
and thus is only equal to the remaining 90ce in both cases . therefore ,
the half - lives of both the oxophilic and thiophilic alkylating species
must be considerably less than the 30 s rate determining elimination
step at 37 c and ph 7.4 . the bleaching upon alkylation
of highly chromophoric mnp at 440
nm is particularly useful since this can be used to follow real time
alkylation by the thiophilic alkylating species as no workup is required . therefore , some additional studies were performed in which mnp ( 100
nmol / ml ) was progressively titrated by the repeated addition of initially
limiting quantities of 90ce ( 20 nmol / ml ) until the mnp was completely
exhausted ( figure 7 ) . during these successive
additions , the same half - reaction time ( 30 s ) and extent of
the reaction ( 20 nmol 90ce bleached 12.6 nmol of mnp ) was
observed until the mnp was nearly completely consumed , despite the
fact that initially there was a 5-fold molar ratio of mnp to 90ce ,
and when the sixth aliquot was added , this was reduced to only 1.2-fold .
in the presence of 200 mm pi , 83% of the 90ce reacts via the
chloride liberating pathways , which would largely comprise the pi
catalyzed thiophilic electrophile pathway and the potential minor
pathway a , while the remaining 17% results in chloroethanol
formation . thus , 1416 nmol of the 20 nmol of 90ce
would be expected to decompose via the phosphate catalyzed chloride
liberating pathway , and 12.6 nmol ( 8090% ) of this was trapped
by reacting with mnp . since this trapped fraction appeared to be independent
of the mnp concentration until exhaustion , implying extremely fast
and efficient scavenging ( figure 7 , panel a ) ,
it is possible that this small discrepancy reflects the presence of
another minor reaction pathway that occurs in the presence of pi ( potential
minor pathway b ) and not merely a failure of the mnp to fully scavenge
the short - lived thiophilic electrophile generated . stoichiometry of mnp
( 100 m ) absorbance bleaching
at 440 nm by 90ce at 37 c and ph 7.4 in 200 mm pi and 20 mm
tris - hcl buffers . blue trace , bleaching of mnp ( 100 m )
by 100 m 90ce in 20 mm tris - hcl buffer ; red trace , bleaching
of mnp ( 100 m ) by 100 m 90ce in 200 mm pi buffer ;
and black trace , bleaching of mnp ( 100 m ) by successive
20 m additions of 90ce until mnp exhaustion in 200 mm pi buffer . ( insert a ) plot of the absorbance of a mnp ( 100 m )
solution at 440 nm , 300 s after the addition of various quantities
( 0200 m ) of 90ce at 37 c and ph 7.4 in 200 mm
pi , indicating the stoichiometry of mnp titration . all values are
the result of at least 3 determinations se . in situ generated mnp was chosen
as a reagent
to trap and identify the soft thiophilic electrophile because it efficiently
scavenged the thiophilic species but would be expected to feebly trap
any hard oxophilic electrophiles , resulting in few trapped products .
furthermore , the strong uv absorbance at 340 nm of the nitropyridine
moiety is largely independent of the state of reduction , oxidation ,
or alkylation of the thiol group , aiding the detection of any mnp
derivatives formed . moreover , since we suspected that the thiophilic
electrophile was a michael type acceptor , produced by the loss of
hcl from the primary chloroethylating species , the resultant mnp adduct
would be expected to be a neutral species suitable for lcms . schemes
illustrating the in situ reduction of dtbnp by tg
to yield 2 mols of mnp and its subsequent reaction with 90ce derived
electrophiles in the presence of 200 mm pi are shown in figure 8 , panels a and b. hplc analysis of dtbnp gave a
large 38 min peak for the oxidized material ( figure 8 , panel c and trace a ) , and reduction by a stoichiometric
quantity of tg largely converted the dtbnp to mnp eluting at 5
min and a trace of the mixed disulfide eluting at 31.5 min
( figure 8 , panel c and trace b ) . treatment
of this reduced material with 90ce in the presence of 200 mm pi resulted
in an 80% decrease in the area of the 5 min mnp peak and the appearance
of a single new broad tailing peak ( 1013 min , peak 11.6 min )
for the trapped product ( figure 9 , panel c
and trace c ) . lcms analysis of collected material eluting between
11 and 12 min indicated a mass of 291 for the m+1 peak of
the trapped mnp derivative ( figure 8 , insert
d ) , which corresponded to a mass increase due to the electrophile
moiety of 134 ( figure 8 , insert e ) . for a michael
type acceptor , this would equal the mass of the attacking electrophile . this mass corresponds to the mass of our proposed thiophilic electrophile
ch3so2n = nch = ch2 . reaction
of this electrophile with a thiol adds the following moiety ch3so2nhn = chch2 to the sulfur
atom . such adducts to both glutathione and n - acetylcysteine
were previously postulated based on ms / nmr studies of the electrophilic
metabolites of isotopically labeled laromustine and 90ce conducted
at a pi concentration of 100 mm . it should
be noted that the use of thiol traps would not be useful in trapping
and identifying oxophilic or even thiophilic chloroethylating species
because any adducts that were formed would rapidly eliminate the chloride
to form a reactive cyclic sulfonium ion via an intramolecular nucleophilic
substitution reaction and then react further with surrounding nucleophiles . trapping
of ( panel a ) scheme illustrating the
two stage reduction by tg of dtbnp to yield 2 mols of mnp . ( panel
b ) scheme illustrating the trapping of the thiophilic electrophile
derived from 90ce . ( panel c ) hplc traces recorded at 340 nm of 10-fold
dilutions of reactions conducted in 200 mm pi buffer . trace ( a ) , 100
m dtbnp ( prereduction ) ; trace ( b ) , dtbnp postreduction by two
molar equivalents of tg to yield mnp 200 m ; trace
( c ) , postreaction of in situ generated mnp with a
molar equivalent of 90ce indicating the production of a trapped product
eluting at 11 min . ( insert d ) lcms analysis of the trapped
product ( eluting at 11 min ) , indicating a molecular plus hydrogen
cation combined mass of 291 ( [ m + h ] = 291 ) . ( insert e ) calculation of the molecular weight of the donated alkyl
moiety and postulated entity . effects of various solutes on the generation of thiophilic species
from 90ce as measured by the bleaching of mnp . ( panel a ) bleaching
of the absorbance of a 100 m mnp solution in various concentrations
of tris - hcl buffer at ph 7.4 and 37 c at 440 nm , 300 s after
the addition of 100 m 90ce . ( panel b ) the bleaching of the
absorbance of a 100 m mnp solution in 20 mm tris - hcl at ph
7.4 and 37 c at 440 nm , 300 s after the addition of 100 m
90ce supplemented with various solutes at 50 mm ( adjusted to ph 7.4 ) . the horizontal line above the x - axis represents the
magnitude of mnp bleaching due to the presence of 20 mm tris - hcl at
ph 7.4 in these solute solutions ( the addition of 20 mm tris - hcl was
required as many of these solutes lack buffering capacity at ph 7.4 ) . the mg atp sample
contained 52.5 mm mgcl2 in addition to 50 mm atp . we examined 28 different anions / solutes including several
phosphate
esters to determine if other solutes behaved like pi and increased
the yields of soft electrophilic species from 90ce ( figure 9 ) . nonphosphate containing anions of strong acids
exhibited very little activity over the small effect due to the copresence
of 20 mm tris - hcl , the addition of which was required to add buffering
capacity . all reactions containing nonphosphate agents with pka values in the vicinity of the experimental
ph ( edta , carbonate , citrate , imidazole , mops , triethanolamine - hcl ,
and tris - hcl with pka values of 6.20 ,
6.35 , 6.40 , 6.95 , 7.20 , 7.76 , and 8.06 , respectively ) contain relatively high concentrations of potentially catalytic
brnsted - lowry bases , and these all exhibited at least some to
moderate activity . therefore , the mechanism by which these components
catalyze the elimination of hydrogen chloride from the primary chloroethylating
species probably involves a general acid / base catalyzed elimination
reaction . however , factors other than the pka value appear to influence the catalytic efficiency since
phosphoesters exhibited greater activity than other agents ( excluding
arsenate ) with comparable pka values . we examined the activity of the three methyl esters of phosphate ( monomethylphosphate ,
dimethylphosphate , and trimethylphosphate ) since these successively
replace the acidic protons of triprotic phosphoric acid and have relatively
little steric bulk compared to other possible esters . compared to
pi ( pka 7.21 ) , monomethylphosphate ( pka 6.31 ) was found to be approximately equiactive , while
dimethylphosphate ( pka 1.29 ) surprisingly retained half the activity of pi
despite being a relatively strong acid , and trimethylphosphate was
inactive . this finding implies that at least one of the protic sites
on pi must be unblocked for catalytic activity . glucose 6-phosphate
( pka 6.11 ) , a slightly more sterically hindered and acidic phosphomonoester ,
was only a little less active than monomethylphosphate . a number of
polyphosphates were also examined ( atp , adp , and pyrophosphate ) , and
these all proved to be approximately equiactive with pi ; thus , the
addition of successive phosphate groups in a linear conformation does
not significantly increase activity ( figure 9 ) . most ( 80% ) of the intracellular atp pool exists as a mg chelate , owing to the strong binding affinity of atp for
mg . since this chelation
involves the two terminal phosphates , we suspected that it may impact atp s ability to facilitate
a switch in the decomposition pathway . therefore , the activity of
atp - mg was assessed using a slight molar excess of mgcl2 ; this resulted in a 60% reduction in activity over that of
atp alone . phosphocreatine ( creatine phosphate ) ( pka 4.5 ) , a guanidino phosphate
in which a relatively acidic phosphate is involved in a n - phosphoguanidine linkage and a hydrogen bond with the guanidine
moiety at physiological ph values , had 1/4th the activity of pi ( figure 9 ) . the only tested anion found to be superior to
pi was arsenate , which can be viewed as a pi analogue . arsenic , like
phosphorus , is a group 15 element and has very similar chemical properties ;
moreover , arsenic acid ( h3aso4 ) and phosphoric
acid ( h3po4 ) have analogous structures and near
identical pka values of ( pka1 = 2.19 , pka2 = 6.94 , and
pka3 = 11.5 ) and ( pka1 = 2.12 , pka2 = 7.21 , and pka3 = 12.67 ) for arsenic and phosphoric acids ,
respectively . two major decomposition pathways dominate , one
resulting in the generation of the therapeutically important hard
oxophilic chloroethylating species , which accounts for > 80% of
the
flux in tris - hcl buffers . the second pathway is facilitated by the
presence of pi and some phosphoesters and generates a novel soft thiophilic
electrophile ( ch3so2n = nch = ch2 ) , which is currently thought to have little therapeutic relevance . this pathway contributes 80% of the flux at pi concentrations
of 200 mm or greater . a number of minor pathways undoubtedly occur
( potential minor pathways a and b , figure , 10 ) as evidenced by the generation of small yields of acetaldehyde / ethylene
glycol and minor discrepancies in the anticipated yields of some of
the major products . some of these discrepancies could be explained
if the pathways that generated acetaldehyde / ethylene glycol resulted
in the liberation of both sulfinate moieties in addition to chloride . scheme
illustrating the proposed decomposition pathways for 90ce
in the presence and absence of pi or catalytic brnsted - lowry
base . the aqueous decomposition of 90ce ( pka 6.5 ) begins with the rate determining elimination of methanesulfinate
( t1/2 30 s ) from the 90ce anion
to generate the primary oxophilic chloroethylating species . this intermediate
can chloroethylate oxygen based nucleophiles ( roh ) directly
or via the potential generation of secondary oxophilic chloroethylating
species resulting in the liberation of a further mole of protons and
methanesulfinate and a mole of nitrogen . in the presence of pi or
suitable brnsted - lowry base , hcl is eliminated , generating a
short - lived highly thiophilic conjungated soft electrophile which
retains the remaining methylsufonyl moiety and both nitrogens on reaction
with a thiol based nucleophile ( rsh ) . the positions of two
potential minor pathways ( a and b ) are also indicated that could account
for the generation of small yields of acetaldehyde / ethylene glycol
and minor discrepancies in the anticipated yields of methanesulfinate
and trapped mnp product . the generation of highly cytotoxic g - c ethane dna interstrand
cross - links
arising from dna guanine o-6 chloroethylation , caused by the oxophilic
chloroethylating species , is critical to the antineoplastic activity
of 90ce . these guanine o-6 lesions are specifically repaired by mgmt ,
and a 22-fold greater clonogenic lc90 value was observed
in l1210 cell lines expressing mgmt . this
result implies that the total cytotoxicity due to the alkylation of
all other biomolecules by both the oxophilic and thiophilic alkylating
species , in these mgmt deficient cells , is probably 5% of
that due to guanine o-6 chloroethylations . moreover ,
it is expected that the vast majority of the thiophilic electrophiles
would be scavenged by glutathione . in the presence of normal intracellular
pi and phosphate ester concentrations , it is likely that 1025%
of the 90ce reacts via the thiophilic pathway , and this can be regarded
as a loss of potentially active agent . therefore , increases in the
concentration of intracellular pi and active phosphoesters could contribute
to the total tumor resistance by decreasing the yields of the therapeutically
relevant oxophilic electrophiles . such a resistance mechanism would
be expected to be limited to low level resistance ( 2-fold )
to 90ce prodrugs . the overall sensitivity of a tumor cell would be
the product of several major and minor factors , including the dna
repair activities of mgmt and hdr , protective glutathione - s - transferases able to intercept a portion of the oxophilic
electrophiles prior to reaction with dna , and the levels of pi and phosphoesters able to divert the decomposition
pathway away from oxophilic chloroethylating electrophile generation . aberrant microvascular systems in solid tumors result in hypoxic
regions where the o2 concentrations can approach anoxia . under these conditions , the levels of phosphocreatine
and atp / atp - mg fall markedly , while the levels of adp ,
amp , pi , and phosphomonoesters exhibit a commensurate rise . since atp / mg and phosphocreatine
have modest activity , while the total activities of their hydrolysis
products are much greater , this change would be expected to result
in a decrease in the percentage yield of oxophilic chloroethylating
electrophiles . this action could contribute toward a modest resistance
to 90ce prodrugs in hypoxic regions . despite this , ks119 , a hypoxically
targeted prodrug of 90ce synthesized in our laboratory , exhibits a
remarkable 5 logs of differential cell kill in in
vitro experiments between oxic and hypoxic environments ,
and selective targeting in in vivo models . this high degree of hypoxic selectivity is likely
the result of the efficiency of the targeting system utilized by this
agent . the development of 90ce analogues which eliminate or diminish
the phosphate catalyzed decomposition route would be expected to remove
this weakness , increase potency , and remove potential toxicities from
the thiophilic electrophiles which likely do not significantly contribute
to the anticancer activity . the rate determining
step in the generation of reactive electrophiles
from 90ce at ph 7.4 and 37 c involves the initial elimination
of the n-1 methylsulfinate moiety ( t1/2 30 s ) from the 90ce anion . after this point , two major
routes of further decomposition exist : ( i ) generating therapeutically
relevant hard oxophilic chloroethylating electrophiles and ( ii ) a
pathway producing a soft thiophilic electrophile ( ch3so2n = nch = ch2 ) currently of no known
therapeutic importance ( figure 10 ) . at this
branch point , the proportion taking pathway ( ii ) is likely stimulated
by the presence of brnsted - lowry bases , with pi and its mono-
and diesters appearing to be the most potent influencing molecules
of biological significance . it is expected that 1025% of 90ce
decomposition proceeds via this thiophilic route under normal cellular
pi / active pi ester concentration conditions , and this percentage is
likely to increase further under hypoxic conditions . this decomposition
pathway could potentially lead to novel resistance mechanisms whereby
intracellular levels of pi / active pi esters or other catalytically
active brnsted - lowry bases are elevated in concentration , imparting
cells with modest resistance to 90ce and its prodrugs . this
study highlights the largely overlooked influence that seemingly
inert reaction mixtures and/or buffer components can potentially have
on reaction pathways . while the generation of electrophiles from 90ce
probably represent an extreme case , it may be prudent to conduct initial
studies in a range of buffers or in buffers resembling the cellular
milieu to detect such influences , or to ensure that the data obtained
are physiologically relevant . we are currently designing 90ce analogues
which are expected to diminish this pi catalyzed route with the aim
of increasing the yields of oxophilic chloroethylating species . these
analogues are likely to be superior to 90ce when incorporated into
hypoxia targeted prodrugs and would lack potential toxicities arising
from the generation of thiophilic electophiles . | prodrugs of the short - lived chloroethylating
agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine
( 90ce ) and its methylating analogue 1,2-bis(methylsulfonyl)-1-(methyl)hydrazine
( ks90 ) are potentially useful anticancer agents .
this class of agents
frequently yields higher ratios of therapeutically active oxophilic
electrophiles responsible for dna o6-guanine
alkylations to other electrophiles with lower therapeutic relevance
than the nitrosoureas .
this results in improved selectivity toward
tumors with diminished levels of o6-alkylguanine - dna
alkyltransferase ( mgmt ) , the resistance protein responsible for o6-alkylguanine repair .
the formation of o6-(2-chloroethyl)guanine , which leads to the
formation of a dna dna interstrand cross - link , accounts for
the bulk of the anticancer activity of 90ce prodrugs .
herein , we describe
a new decomposition pathway that is available to 90ce but not to its
methylating counterpart .
this pathway appears to be subject to general / acid
base catalysis with phosphate ( pi ) , phosphomonoesters , and phosphodiesters ,
being particularly effective .
this pathway does not yield a chloroethylating
species and results in a major change in nucleophile preference since
thiophilic rather than oxophilic electrophiles are produced .
thus ,
a pi concentration dependent decrease in dna
dna interstand
cross - link formation was observed .
changes in 90ce decomposition products
but not alkylation kinetics occurred in the presence of pi since the
prebranch point elimination of the n-1 methanesulfinate moiety remained
the rate - limiting step .
the pi catalyzed route is expected to dominate
at pi and phosphoester concentrations totaling > 2535 mm .
in
view of the abundance of pi and phosphoesters in cells ,
this pathway
may have important effects on agent toxicity , tumor selectivity , and
resistance to prodrugs of 90ce .
furthermore , it may be possible to
design analogues that diminish this thiophile - generating pathway ,
which is likely superfluous at best and potentially detrimental to
the targeting of hypoxic regions where pi concentrations can be significantly
elevated . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
intubation and mechanical ventilation are safe and effective treatments for critically ill patients with respiratory failure . however , while unnecessary prolongation of mechanical ventilation increases the risk of complications , including bronchopulmonary infections , barotrauma , and oxygen toxicity , its premature discontinuation may also increase morbidity and mortality . intensive care can indeed be prolonged if reintubation is needed . ' weaning ' is the withdrawal of mechanical ventilatory support , although the term ' liberation ' may be more appropriate . the effect of weaning on the stress response in critically ill patients is uncertain , as are the effects of different weaning modes . the aim of the present study was to compare changes in plasma concentrations of insulin , cortisol and glucose , and in urine vanilmandelic acid ( vma ) during weaning and after extubation , using the three different modes of pressure support ( ps ) ventilation , continuous positive airway pressure ( cpap ) and t - piece , in intensive care patients . the study was approved by the human studies review board of the cerrahpasa medical faculty of istanbul university , and informed consent was obtained from the patients ' families . patients with endocrine or immune system disease , patients with malignant disorders , patients who were also receiving insulin , and patients who were on sympathomimetic agents or steroids were excluded from the study . weaning was considered unsuccessful if reintubation was required within 48 hours , and these patients were excluded from the study . we calculated the acute physiology and chronic health evaluation ii scores from clinical data available after the first 24 hours of intensive care . all patients received a nasojejunal tube , and feeding was commenced according to the harris entry criteria for the weaning trial were as follows : positive end expiratory pressure 5 cmh2o , partial arterial oxygen tension ( pao2)/fractional inspired oxygen concentration ( fio2 ) 200 , the resolution of ( or significant improvement in ) the underlying condition requiring mechanical ventilation , hemodynamic stability , an adequate neurological status , an absence of fever , a presence of cough reflex when on a passing suction catheter , an intact gag reflex , and an absence of vasopressor or sedative infusions drips . dopamine < 5 g / kg per min was allowed , as were intermittent doses of sedatives . the criteria for tolerating weaning trials were pao2 60 mmhg or arterial oxygen saturation 90% , or fio2 0.40.5 , paco2 increase 10 mmhg or ph decrease < 0.1 , systolic blood pressure 90 mmhg or 180 mmhg with any increase or decrease 20% , heart rate 50 beats / min or 140 beats / min with any increase or decrease 20% , respiratory rate 35 breaths / min , and no sign of excessive respiratory work ( absence of thoracoab - dominal paradox , respiratory alternans or accessory respiratory muscle action ) . no diaphoresis , agitation , depressed mental status , or distress were present . patients were ventilated by either the siemens - elema servo 900 c or servo 300 a ventilators ( siemens - elema , solna , sweden ) . two modes of partial ventilatory support during the weaning period were employed ; group ps received fio2 0.4 , ps 10 cmh2o , and positive end expiratory pressure 5 cmh2o for 2 hours . the cpap group received fio2 0.4 and cpap 5 cmh2o for 2 hours . the t - piece group ( group t ) received 4 l / min oxygen via a t - piece for 2 hours . in group t , the mean respiratory rate was 20 breaths / min , the tidal volume was 550600 ml and the t - piece reservoir volume was 60 ml , giving a mean fio2 of 0.350.4 . eight patients in group ps thus received the servo 900 c ventilator and 12 received the servo 300 a ventilator . in the cpap group , seven received the servo 900 c ventilator and 13 received the servo 300 a ventilator . an 8.0 mm inner diameter endotracheal tube was used for all patients . at the beginning of the weaning trial , blood and urine samples were taken for insulin , cortisol , glucose , vma and arterial blood gas measurements ( pao2 , paco2 , ph ) . the measurements were repeated at the end of the weaning trial , after 2 hours , when the patients were extubated . the blood samples for measuring the endocrine stress response were taken between 07:00 and 09:00 am because of the circadian rhythm . no patient received any medication that could affect vma measurement , such as insulin , reserpine , norepineprine , epineprine , morphine , or pentobarbital . plasma insulin levels were determined using a commercially available radioimmunoassay ( novo ; nordisk , copenhagen , denmark ) . plasma cortisol measurements were measured by a competitive immunoassay with the use of an electrochemiluminescence immunoassay ( eclia ; roche , mannheim , germany ) . vma concentrations of spot urine samples ( 2-hour collection ) were determined by chomatographic - spectrohotometric assay ( far , verona , italy ) . blood glucose levels were determined by medisense glucotrend ( behring , mannheim , germany ) . wallis test and , when appropriate , by multiple comparison tests ( dunn test ) . statistical analysis was performed by the istanbul university cerrahpasa medical faculty , department of biostatistics . demographic and biochemical data were compared between groups using the kruskall wallis test and , when appropriate , by multiple comparison tests ( dunn test ) . statistical analysis was performed by the istanbul university cerrahpasa medical faculty , department of biostatistics . there were no differences between the groups in age , weight , gender and acute physiology and chronic health evaluation ii scores ( table 1 ) , and there were no differences over time in the heart rate or noninvasive blood pressure , arterial pao2 , paco2 , or ph ( data not shown ) . four patients required reintubation within 48 hours in group t. reintubation was not required within 48 hours in the cpap and ps groups . there was no significant difference between groups according to patients receiving sedatives and according to sedative doses ( data not shown ) . plasma insulin concentrations increased in group ps and in group t ( both p < 0.05 ) during weaning , and in group t after extubation ( p < 0.01 ) ( table 2 ) . plasma insulin was greater in group t than in the other groups after extubation ( p < 0.01 ) ( table 2 ) . plasma cortisol was increased in group cpap ( p < 0.01 ) and in group t ( p < 0.001 ) during weaning , and in group t after extubation ( p < 0.01 ) ( table 3 ) . concentrations were greater in group t than in group ps during weaning and after extubation ( both p < 0.05 ) , and were greater in group t than in group cpap ( p < 0.01 ) post extubation ( table 3 ) . blood glucose increased during weaning in group ps ( p < 0.05 ) and in group t ( p < 0.01 ) ( table 4 ) , increasing more in group t than in group ps and group cpap ( both p < 0.01 ) . the blood glucose concentration was greatest in group t post extubation ( group ps and group cpap both p < 0.05 ) ( table 4 ) . urinary vma increased in group ps ( p < 0.05 ) and in group t ( p < 0.001 ) during weaning , and increased in group cpap ( p < 0.01 ) and in group t ( p < 0.001 ) post extubation ( table 5 ) . urinary vma was greatest in group t during both weaning and post extubation ( p < 0.001 ) ( table 5 ) . plasma insulin concentrations increased in group ps and in group t ( both p < 0.05 ) during weaning , and in group t after extubation ( p < 0.01 ) ( table 2 ) . plasma insulin was greater in group t than in the other groups after extubation ( p < 0.01 ) ( table 2 ) . plasma cortisol was increased in group cpap ( p < 0.01 ) and in group t ( p < 0.001 ) during weaning , and in group t after extubation ( p < 0.01 ) ( table 3 ) . concentrations were greater in group t than in group ps during weaning and after extubation ( both p < 0.05 ) , and were greater in group t than in group cpap ( p < 0.01 ) post extubation ( table 3 ) . blood glucose increased during weaning in group ps ( p < 0.05 ) and in group t ( p < 0.01 ) ( table 4 ) , increasing more in group t than in group ps and group cpap ( both p < 0.01 ) . the blood glucose concentration was greatest in group t post extubation ( group ps and group cpap both p < 0.05 ) ( table 4 ) . urinary vma increased in group ps ( p < 0.05 ) and in group t ( p < 0.001 ) during weaning , and increased in group cpap ( p < 0.01 ) and in group t ( p < 0.001 ) post extubation ( table 5 ) . urinary vma was greatest in group t during both weaning and post extubation ( p < 0.001 ) ( table 5 ) . we therefore measured blood insulin , blood cortisol and blood glucose , and urinary vma during and after weaning in three modes : ps , cpap and t - piece . brinkmann and colleagues found that catecholamine and stress hormone blood concentrations did not increase following withdrawal of sedation and cessation of mechanical ventilation after abdominal surgery . rathgeber and colleagues used biphasic positive airway pressure , controlled mandatory ventilation , or intermittent mandatory ventilation for weaning after cardiac surgery . calzia and colleagues and quinn and colleagues compared the effects of synchronized intermittent mandatory ventilation and biphasic positive airway pressure on the stress response , also during weaning after cardiac surgery . neither mode affected postoperative plasma cortisol , epinephrine , or norepinephrine concentrations . in patients who underwent cardiac surgery , brinkmann and colleagues and calzia and colleagues found no significant differences in plasma ephineprine and norephineprine levels during weaning . insulin and blood glucose concentrations were increased 1218 hours postoperatively ( i.e. there was insulin resistance ) . in a study of trauma patients , ljungqvist and colleagues found increased insulin and glucose levels as a response to the stress of trauma . we have not found any study in the literature investigating stress responses during weaning in intensive care patients ventilated for more than 48 hours . in our study , plasma insulin and glucose increased in group ps and in group t during weaning . at 48 hours post extubation , the glucose and insulin levels were returning to their initial levels in group ps . the reason for the significant increase of glucose and insulin levels in the ps group is not clear . the greatest changes in measured variables during the 2 hours of weaning occurred in group t. the increase in blood glucose in group t during weaning was accompanied by an increase in blood insulin , which remained elevated after extubation , while the blood glucose decreased . there were signs of insulin resistance , and patients in group t showed the greatest increase in glucose . the plasma cortisol level was greatest in group t during both weaning and after extubation . cortisol increased significantly during weaning in the cpap group but not in the ps group . cortisol , glucose and vma were greater in the t group than in the cpap group . the increased stress response in group t has not been reported by other authors , for which there are two possible reasons . second , different weaning modes were used . in our study , because it was not possible to measure plasma catecholamine levels , we measured urinary vma , the end product of catecholamine metabolism . vma was greatest in group t , both during weaning and post extubation , but in spite of this there were no hemodynamic differences between groups , possibly because our patients were well hydrated and not in cardiac failure . the reasons for the higher endocrine stress response in group t may be explained by the t - piece not having ventila - tory support . in our study of patients weaned after more than 48 hours of ventilation , using the t - piece was associated with a greater stress response than using the ps or cpap modes . weaning process increases endocrine stress response weaning via t - piece caused a greater stress response that the ps and cpap modes the use of ps and cpap modes may be more appropriate during weaning process cpap = continuous positive airway pressure ; fio2 = fractional inspired oxygen concentration ; pao2 = partial arterial oxygen tension ; ps = pressure support ; vma = vanilmandelic acid . demographic data in all groups ps , pressure support ; cpap , continuous positive airway pressure ; t , t - piece . plasma insulin levels ( u / ml ) * p < 0.05 , * * p < 0.01 when compared with the initial level within the group . p < 0.01 , continuous positive airway pressure ( cpap ) group compared with t - piece group . plasma cortisol levels ( g / dl ) * p < 0.01 , * * p < 0.001 when compared with the initial level within the group . p < 0.01 , continuous positive airway pressure ( cpap ) group compared with t - piece group . blood glucose levels ( mg / dl ) * p < 0.05 , * * p < 0.01 when compared with the initial level within the group . p < 0.05 , p < 0.01 , pressure support group compared with t - piece group . p < 0.05 , p < 0.01 , continuous positive airway pressure ( cpap ) group compared with t - piece group . urinary vanilmandelic acid concentrations ( g / mg ) * p < 0.05 , * * p < 0.01 , * * * p < 0.001 , when compared with the initial level within the group . p < 0.001 , continuous positive airway pressure ( cpap ) group compared with t - piece group . | introductionthe aim of the present study was to investigate the effects of the stress response on plasma insulin , cortisol , glucose , and urinary vanilmandelic acid during weaning and after extubation , using pressure support ( ps ) , continuous positive airway pressure ( cpap ) and t - piece modes.methodssixty patients were randomly divided into three groups ( n = 20 ) .
the ps group received fio2 0.4 , ps 10 cmh2o , and positive end expiratory pressure 5 cmh2o for 2 hours .
the cpap group was given fio2 0.4 and cpap 5 cmh2o for 2 hours .
the t - piece group ( group t ) received 4 l / min oxygen via a t - piece for 2 hours .
after 1 hour and 2 hours in their respective weaning modes , blood and urine samples were taken for insulin , cortisol , glucose and vanilmandelic acid measurements .
forty - eight hours after extubation , blood and urine samples were again taken.resultsplasma insulin was greater in group t than in the ps and cpap groups ( p < 0.01 and p < 0.01 ) .
the plasma cortisol concentration was greater in group t than in group ps during weaning ( p < 0.05 ) and after extubation ( p < 0.05 ) .
blood glucose concentrations were greater in group t than in the other groups ( both p < 0.01 ) both during weaning and post extubation .
urine vanilmandelic acid was greater in group t than in the other groups during weaning and after extubation ( both p < 0.001).conclusionweaning via the t - piece caused a greater stress response than the ps and cpap modes . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the classic account , made by dandy & blackfan in 1914 * , where autopsies showed severe hypertentorial hydrocephalus , cystic dilatation of the fourth ventricle , small vermis , removal of the cerebelar hemispheres and absence of the roof of the fourth ventricle , thickening and opacity of the pia - arachnoid cisternae of the skull base and dilatation of the aqueduct1 . the dandy - walker syndrome ( sdw ) is a non - familial syndrome characterized by cystic dilatation of the fourth ventricle and aplasia or partial or total atrophy of the cerebelar vermis . occur other brain malformations such as agenesis of the corpus callosum , heteropsias , lissencephaly , stenosis of the aqueduct of sylvius
2
3 . gardner et al * * proposed that the sdw , along with other syndromes ( arnold - chiari malformation , cerebelar arachnid cyst and syringomyelia ) were manifestations of the same disease1 . some studies show an incidence of approximately 70% of relationship between the sdw and systemic abnormalities1 . little is known about congenital malformations of the posterior pit structures , their genetic alterations have been mapped to chromosome 3q4
5 , but the gene is not located exactly , but it is known that the basis of the development process of the structures of the pit later is nature for human cerebelar malformations5 . it is also known that structures cerebelar growth early embryo in the period until the early post - natal , this event would cerebellum susceptible to a wide spectrum of disorders of development6 . the pathogenesis of this syndrome is controversial , but the most accepted theory is that the leaf development of the foramina of magendie and lushka during the fourth month of fetal life , leading to bulging cystic fourth ventricle . new theories have proposed that the sdw result from a failure to develop the roof of the hindbrain , taking this as a cause teratogenic effect1 . some studies suggest that the use of warfarin in the long run would be responsible for the development of sdw in 1 - 2% of exposed fetuses7 . , showed no relationship between degree of hydrocephalus and the size of the cyst in the posterior pit , vermis or attenuation of the permeability of the fourth ventricle and hydrocephalus in some cases was absent1 . the sdw is an entity of heterogenic hyperplasia of the cerebelar vermis and is a newly identified gene associated with the connection with x - hprt8 also relates to the basal ganglia disease . clinically there may be moderate delayed psychomotor development , microcephaly , hypotonia , but the predominant symptoms refers to hydrocephalus , usually in the first two years of life , this however can be ignored , appearing late ( first or second decade of life)2
3
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9 . some ocular abnormalities are described in the sdw , as coloboma corioretinian10 , nystagmus11
12
13 . there may be mental retardation ( 50% ) , spasticity ( instead of hypotonia ) , seizures , vomiting , all depending on the degree of cerebelar malformation3 . in patients with vermis fissures with two conformations and almost normal brain functions are also almost normal without association with other malformations . in patients with severe malformations of the cerebellum , vermis with only one or no crack , it is common to severe mental retardation and other malformations of the central nervous system , such as agenesis of the corpus callosum . it is divided , so the sdw into two large groups according to the previous malformations for determining the prognosis intellectual12
13 . it is reported in the literature of the coexistence of large facial coetaneous hemangioma with sdw14 . other syndromes where coexist brain and ocular malformations are reported as neuhauser syndrome ( mmmm - megalocornea , macrocephaly , mental and motor retardation ) , which are found cortical atrophy , enlargement of the fourth ventricle , hyperplasia of the corpus callosum . , there is need for magnetic resonance images of good quality axial view of the cerebelar vermis and t2 images12 . the neuroradiological findings are characteristic , such as cystic dilatation of the fourth ventricle and changes in the cerebelar vermis2 , besides others already mentioned . the bilateral sensorineural deafness can be part of the clinical picture of the syndrome and we describe a case whose patient underwent implantation cochlear by presenting profound bilateral deafness . the aim of this paper is to describe a case of female patient , 13 years with a diagnosis of this syndrome and bilateral hearing loss underwent cochlear implant surgery under local anesthesia and sedation . cgs , 13 years old female was referred to the ent department of otolaryngology institute of parana with a diagnosis of dandy - walker syndrome for ent evaluation for bilateral hearing loss with no response to the use of hearing aids . the patient performs follow up with neurology since birth , with a history of gestational rubella , cataracts in both eye and neurological surgery prior hydrocephalus , absence of parental consanguinity . child with imaging exam compatible with dandy - walker syndrome with posterior pit collection and communication with the fourth ventricle , associated with signs of hyperplasia of the cerebelar hemispheres and vermis . was not possible to detect brainstem evoked potentials bilaterally . in the absence of otoacoustic emission response bilaterally mri of the brain showing cerebelar vermis hyperplasia associated with mega cistern magna , communicating with the fourth ventricle ( figures 1 and 2 ) . the fluid spaces of the cochlea are normal thickness , ranging from 1.3 to 1.7 mm the right and 1.4 to 1.7 mm left . the cochlear nerves have been identified and normal thickness of 0.6 mm ( figure 3 ) . surgery was performed under local anesthesia and sedation by retroauricular access , creation of niche outdoor unit , closed mastoidectomy , posterior tympanotomy , chocleostomy , inserting the internal components and telemetry . all steps performed uneventfully . the implant was activated 1 month after surgery with excellent sound perception at the time of activation . the patient has excellent levels of discrimination with less need for lip reading and excellent response to therapy with spectacular mend the quality of speech . no doubt the results bring impact on quality of life of patients while providing better social integration . the field of cochlear implants is growing rapidly due to improved quality of the implants , surgery less invasive , and wider dissemination of this type of treatment of deafness . the surgery is now much faster and less invasive than some years ago , with smaller incisions and less morbidity for the patient . the patient even though the syndrome dandy - walker does not present significant deficit in neuropsychomotor development , fulfilling the criteria established in the literature for the performance of cochlear implant surgery . there are already cases of patients with the same syndrome who underwent cochlear implant surgery , complications described in the literature21 and with good results despite co morbidities . in our facility this type of surgical anesthesia brings morbidity / anesthetic besides a minor post - operative recovery and faster lower hospital costs when compared with general anesthesia and were perfect forward from the patient . we believe that the presence of dandy - walker syndrome can not be considered a contraindication to the performance of cochlear implant surgery , and there were no surgical complications due to neurological disorders with very favorable results for the patient who exhibits excellent discrimination . | summary
introduction : dandy walker syndrome is a congenital abnormality in the central nervous system , characterized by a deficiency in the development of middle cerebelar structures , cystic dilatation of the posterior pit communicating with the fourth ventricle and upward shift of the transverse sinuses , tentorium and dyes . among the clinical signs are occipital protuberances , a progressive increase of the skull , bowing before the fontanels , papilledema , ataxia , gait disturbances , nystagmus , and intellectual impairment .
objectives : to describe a case of female patient , 13 years old with a diagnosis of this syndrome and bilateral hearing loss underwent cochlear implant surgery under local anesthesia and sedation .
case report : cgs , 13 years old female was referred to the otolaryngological department of otolaryngology institute of parana with a diagnosis of dandy - walker syndrome for otolaryngological evaluation for bilateral hearing loss with no response to the use of hearing aids .
final comments : the field of cochlear implants is growing rapidly .
we believe that the presence of dandy - walker syndrome can not be considered a contraindication to the performance of cochlear implant surgery , and there were no surgical complications due to neurological disorders with very favorable results for the patient who exhibits excellent discrimination .
it has less need for lip reading with improvement in speech quality . |
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in 2002 , a global study reported that the us had among the highest reported age standardized incidence rate of bladder cancer ( 24.1/100,000 ) . the national cancer institute ( nci ) estimated that in 2009 , there would be approximately 70,980 new cases and 14,330 deaths from bladder cancer in the usa . despite reduced exposure to established risk factors such as smoking , aromatic arylamines ( occupation ) , and schistosomal infection , the incidence of bladder cancer in the usa remains high . this is consistent with other studies suggesting that these risk factors only partly explain bladder cancer etiology [ 4 , 5 ] . one etiologic study investigating regional variation in bladder cancer rates across the usa also considered diet as a possible risk factor . several studies [ 714 ] have reported that dietary factors such as fruit and vegetables may be protective against bladder cancer . however , there is a lack of consistent evidence between the intake of related micronutrient components of fruits and vegetables and reduced risk of the disease . the second expert report on nutrition and cancer by the world cancer research fund / american institute of cancer research states that the evidence was too limited to conclude that any food or nutrient directly influences the risk of bladder cancer . conversely , a recent systematic review as part of a who consultancy report found that nutrient components of specific fruits and vegetables have a possible inverse association with bladder cancer . it is these dietary micronutrients or their metabolites that actually come into direct contact with the bladder epithelium and thus in theory could be protective . vitamin a is obtained from the diet as preformed vitamin a from animal products and as carotenoids including both provitamin a and non - provitamin a carotenoids from plant foods . an inverse association between vitamin a intake and bladder cancer has been reported in some studies [ 17 , 18 ] , but not others [ 3 , 1921 ] . some recent studies suggest that it is the carotenoids [ 8 , 2224 ] ( -carotene , -carotene [ 8 , 25 ] , -cryptoxanthin , lycopene , lutein / zeaxanthin , and not retinol [ 10 , 23 , 26 ] that influences the risk of bladder cancer . although , other studies have reported the opposite effects , with some finding that retinol may actually be protective [ 18 , 2729 ] and others that the carotenoids have no effect [ 3 , 10 , 19 , 30 , 31 ] . the relationship between bladder cancer and vitamin c and e also appears to be unclear with some studies reporting potential inverse associations ( vitamin c : [ 29 , 32 , 33 ] ; vitamin e : [ 20 , 27 ] ) and others no associations at all ( vitamin c : [ 3 , 19 , 21 , 23 , 25 ] ; vitamin e : [ 3 , 19 , 21 , 23 , 25 , 31 ] ) . deficiencies in folic acid , vitamins b3 ( niacin ) , b6 , b12 , have been reported to mimic radiation damage to dna and possibly lead to the initiation of cancer . however , there is only limited literature available on the association between water - soluble b - group vitamins and risk of bladder cancer , and the findings have been inconsistent [ 20 , 21 , 23 , 25 , 28 , 35 ] . he concluded that the potential for certain dietary minerals to influence urine composition and hence bladder carcinogenesis observed in animal studies needed further evaluation before extrapolation could be made to human populations . to date , few epidemiological studies have investigated the effect of dietary minerals such as sodium [ 20 , 23 ] , potassium , calcium [ 20 , 23 , 26 ] , magnesium , and iron [ 20 , 26 ] on the risk of developing bladder cancer . micronutrient deficiencies could provide a possible explanation for why an estimated 25% of the us population who consume the least fruit and vegetables have double the cancer rate . the aim of our study was to investigate the association between major dietary minerals and vitamins and the risk of bladder cancer in a us population from a region with a high incidence rate . a population - based case control study was conducted in new hampshire , usa . briefly , bladder cancer cases were identified from the new hampshire state department of health and human services cancer registry as histologically confirmed , primary bladder cancer diagnosed between 1 july 1998 and 31 december 2001 . to be eligible for inclusion in the study , all cases had to be new hampshire residents aged between 25 and 74 , have a listed phone number and speak english . physician consent was obtained before contacting potential participants . of the 472 potentially eligible cases we contacted , , controls were shared with another study on non - melanoma cancer covering a reference period from 1 july 1997 to 30 march 2000 . controls less than 65 years of age were selected from lists obtained from the new hampshire department of transportation . controls 65 years of age and older were chosen from data files provided by the centers for medicare & medicaid services ( cms ) of new hampshire . a total of 526 controls ( 76% ) were interviewed from a potential 694 confirmed eligible participants . extensive in - person interviews were conducted with consenting participants , usually in the homes of the participants . data were collected on participants socio - demographic information such as education level , residence , occupation ( history ) , medical history , lifestyle factors ( including tobacco smoking ) , household water supply , and family history of cancer . interviewers were blind to case / control status and interviews were tape recorded with subjects consent ( less than 5% refused to have the interview taped ) to ensure consistent quality of the interview and clarification of details . cases were also asked if they held a driver s license or a medicare enrollment card for comparability with controls . interviews which included dietary assessment took place between 2000 and 2003 for both cases and controls ( for 95% of subjects ) . as data were unavailable for 150 cases and 455 controls ; a total of 322 cases and 239 controls were included in the dietary analyses of this study . subjects from whom dietary data were collected were comparable to those who did not provide dietary data with respect to age , sex , and smoking history ( data not shown ) . dietary information was collected from a 121-item semi - quantitative food frequency questionnaire ( ffq ) that was developed by willett et al . for the nurses health study . these included dairy , fruit , vegetables , eggs and meat , breads , beverages , and baked goods . there was a list of nine common mineral and vitamin supplements to choose from which included the following : multiple vitamins ( participants were asked to report the individual vitamins within this category ) , vitamins a , c , b6 and e , selenium , iron , zinc , and calcium . additional questions covered folic acid , vitamin d , vitamin b complex , cod liver oil , omega 3 fatty acids , iodine , copper , brewer s yeast , -carotene , magnesium , and an open ended option , other . participants were provided with options for duration of supplement intake ranging from 0 to 10 + years . nutrient intake was calculated by multiplying frequency of each food item on the ffq by the nutrient content for the specific portion size . food composition data were based primarily on data from the united states department of agriculture ( usda ) . we investigated vitamins that had a biologically plausible association with bladder cancer and macro - minerals , which are required in quantities greater than 100 mg per day e.g. , sodium , potassium , calcium , phosphorus , and magnesium . an exception was iron , which was included due to its physiological function and potential to influence the risk of bladder cancer . we investigated the association between total mineral and vitamin intake , which included micronutrients from both dietary and supplemental sources and bladder cancer risk . the intake of carotenoids , folate , and vitamin b12 were measured in micrograms ( g ) per day . all other vitamins including , b1 ( thiamin ) , b2 ( riboflavin ) , b3 ( niacin ) , vitamin c and vitamin e , and minerals ( sodium , potassium , calcium , phosphorus , magnesium , and iron ) were calculated in milligrams ( mg ) per day except for vitamin d intake , which was calculated in international units ( iu ) . adjustment was made for a number of possible confounding variables including sex , age , cigarette smoking status ( current / non - current smoker ) , pack years of cigarette smoking ( categories : 0 < 10 , 10 < 20 , 20 < 30 , 30 < 40 , 40 < 50 , 50 ; calculated by dividing the number of cigarettes smoked per day by 20 and multiplying by the number of years smoked ) and total energy intake ( quartiles for kcal / day ) . additional adjustment for other potential confounders , total fat intake ( fat - soluble vitamins ) , alcohol and coffee consumption ( water - soluble vitamins ) did not alter the associations between micronutrients and bladder cancer . odds ratios and 95% cis were calculated to investigate potential differences between invasive and non - invasive bladder cancer . we also performed additional analyses to assess the effects of minerals and vitamins from both dietary and supplemental sources separately . intake of nutrients from dietary sources were analyzed in quartiles using the same statistical model for total intake of minerals and vitamins . we estimated the intake of minerals and vitamins from supplements by subtracting dietary intake from total nutrient intake . due to the relatively small contribution to mineral and vitamin intake by supplements in this study population likelihood ratio tests were conducted to test for trend by assigning an integer to each quartile of the nutrient e.g. , 14 then fit the term as a continuous variable in the model . the joint effects of specific nutrients and smoking status / history and age were also investigated . nutrients and age were dichotomized into high and low categories based on median values of the controls . smoking status was dichotomized into never / ever smoker , cigarettes per day into high and low categories ( < 20/20 cigarettes per day ) and duration of smoking ( < 27 years/27 years ) . we used the likelihood ratio test to evaluate interaction , i.e. , comparing the log likelihood of regression models with and without the interaction term included . the false - positive report probability ( fprp ) was employed to assess the probability of no true association between nutrients and the risk of bladder cancer given a statistically significant finding . this test takes into account three factors that determine the probability that a statistically significant result may actually be a false - positive finding . these are ( 1 ) prior probability of an association ; ( 2 ) the alpha - level , and ( 3 ) statistical power to detect an odds ratio for the alternative hypothesis at a given alpha - level or p value . we used previously reported arbitrary values for these factors : 0.05 ( alpha - level ) , 0.25 ( prior probability ) , and 0.5 ( false - positive report probability ) [ 46 , 47 ] . all reported p values were two - sided and values less than or equal to 0.05 were considered to be statistically significant . the statistical software package stata / se 10.0 ( stata corporation , college station , tx , usa ) was used for all statistical analyses . a population - based case control study was conducted in new hampshire , usa . briefly , bladder cancer cases were identified from the new hampshire state department of health and human services cancer registry as histologically confirmed , primary bladder cancer diagnosed between 1 july 1998 and 31 december 2001 . to be eligible for inclusion in the study , all cases had to be new hampshire residents aged between 25 and 74 , have a listed phone number and speak english . physician consent was obtained before contacting potential participants . of the 472 potentially eligible cases we contacted , , controls were shared with another study on non - melanoma cancer covering a reference period from 1 july 1997 to 30 march 2000 . controls less than 65 years of age were selected from lists obtained from the new hampshire department of transportation . controls 65 years of age and older were chosen from data files provided by the centers for medicare & medicaid services ( cms ) of new hampshire . a total of 526 controls ( 76% ) were interviewed from a potential 694 confirmed eligible participants . extensive in - person interviews were conducted with consenting participants , usually in the homes of the participants . data were collected on participants socio - demographic information such as education level , residence , occupation ( history ) , medical history , lifestyle factors ( including tobacco smoking ) , household water supply , and family history of cancer . interviewers were blind to case / control status and interviews were tape recorded with subjects consent ( less than 5% refused to have the interview taped ) to ensure consistent quality of the interview and clarification of details . cases were also asked if they held a driver s license or a medicare enrollment card for comparability with controls . interviews which included dietary assessment took place between 2000 and 2003 for both cases and controls ( for 95% of subjects ) . as data were unavailable for 150 cases and 455 controls ; a total of 322 cases and 239 controls were included in the dietary analyses of this study . subjects from whom dietary data were collected were comparable to those who did not provide dietary data with respect to age , sex , and smoking history ( data not shown ) . dietary information was collected from a 121-item semi - quantitative food frequency questionnaire ( ffq ) that was developed by willett et al . for the nurses health study . these included dairy , fruit , vegetables , eggs and meat , breads , beverages , and baked goods . there was a list of nine common mineral and vitamin supplements to choose from which included the following : multiple vitamins ( participants were asked to report the individual vitamins within this category ) , vitamins a , c , b6 and e , selenium , iron , zinc , and calcium . additional questions covered folic acid , vitamin d , vitamin b complex , cod liver oil , omega 3 fatty acids , iodine , copper , brewer s yeast , -carotene , magnesium , and an open ended option , other . participants were provided with options for duration of supplement intake ranging from 0 to 10 + years . nutrient intake was calculated by multiplying frequency of each food item on the ffq by the nutrient content for the specific portion size . food composition data were based primarily on data from the united states department of agriculture ( usda ) . we investigated vitamins that had a biologically plausible association with bladder cancer and macro - minerals , which are required in quantities greater than 100 mg per day e.g. , sodium , potassium , calcium , phosphorus , and magnesium . an exception was iron , which was included due to its physiological function and potential to influence the risk of bladder cancer . we investigated the association between total mineral and vitamin intake , which included micronutrients from both dietary and supplemental sources and bladder cancer risk . the intake of carotenoids , folate , and vitamin b12 were measured in micrograms ( g ) per day . all other vitamins including , b1 ( thiamin ) , b2 ( riboflavin ) , b3 ( niacin ) , vitamin c and vitamin e , and minerals ( sodium , potassium , calcium , phosphorus , magnesium , and iron ) were calculated in milligrams ( mg ) per day except for vitamin d intake , which was calculated in international units ( iu ) . adjustment was made for a number of possible confounding variables including sex , age , cigarette smoking status ( current / non - current smoker ) , pack years of cigarette smoking ( categories : 0 < 10 , 10 < 20 , 20 < 30 , 30 < 40 , 40 < 50 , 50 ; calculated by dividing the number of cigarettes smoked per day by 20 and multiplying by the number of years smoked ) and total energy intake ( quartiles for kcal / day ) . additional adjustment for other potential confounders , total fat intake ( fat - soluble vitamins ) , alcohol and coffee consumption ( water - soluble vitamins ) did not alter the associations between micronutrients and bladder cancer . odds ratios and 95% cis were calculated to investigate potential differences between invasive and non - invasive bladder cancer . we also performed additional analyses to assess the effects of minerals and vitamins from both dietary and supplemental sources separately . intake of nutrients from dietary sources were analyzed in quartiles using the same statistical model for total intake of minerals and vitamins . we estimated the intake of minerals and vitamins from supplements by subtracting dietary intake from total nutrient intake . due to the relatively small contribution to mineral and vitamin intake by supplements in this study population , these nutrients were dichotomized according to average intake of controls . likelihood ratio tests were conducted to test for trend by assigning an integer to each quartile of the nutrient e.g. , 14 then fit the term as a continuous variable in the model . the joint effects of specific nutrients and smoking status / history and age were also investigated . nutrients and age were dichotomized into high and low categories based on median values of the controls . smoking status was dichotomized into never / ever smoker , cigarettes per day into high and low categories ( < 20/20 cigarettes per day ) and duration of smoking ( < 27 years/27 years ) . we used the likelihood ratio test to evaluate interaction , i.e. , comparing the log likelihood of regression models with and without the interaction term included . the false - positive report probability ( fprp ) was employed to assess the probability of no true association between nutrients and the risk of bladder cancer given a statistically significant finding . this test takes into account three factors that determine the probability that a statistically significant result may actually be a false - positive finding . these are ( 1 ) prior probability of an association ; ( 2 ) the alpha - level , and ( 3 ) statistical power to detect an odds ratio for the alternative hypothesis at a given alpha - level or p value . we used previously reported arbitrary values for these factors : 0.05 ( alpha - level ) , 0.25 ( prior probability ) , and 0.5 ( false - positive report probability ) [ 46 , 47 ] . all reported p values were two - sided and values less than or equal to 0.05 were considered to be statistically significant . the statistical software package stata / se 10.0 ( stata corporation , college station , tx , usa ) was used for all statistical analyses . table 1 presents the frequency distributions of selected study characteristics for the 322 cases and 239 controls for whom dietary data were available for analyses in this study . bladder cancer cases consisted of approximately three times more men ( 74% ) than women . a majority of both cases and controls were in the older age group ( 60 years and over ) . there was a higher prevalence of current smokers and greater number of both cigarettes smoked per day and years smoked among cases compared with the controls . we detected no statistically significant differences between cases and controls for calorie , total fat , alcohol , or coffee intake.table 1distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factorscases number ( % ) controls number ( % ) total322239sex * men237 ( 74)138 ( 58 ) women85 ( 26)101 ( 42)age ( years ) [ mean ( sd)]62 ( 9.2)60.7 ( 10.6)smoking status * never smokers56 ( 17.4)95 ( 39.7 ) ex - smokers162 ( 50.3)103 ( 43.1 ) current smokers104 ( 32.3)41 ( 17.2 ) years smoking [ mean ( sd)]*32.2 ( 14)27.4 ( 15 ) cigarettes / day [ mean ( sd)]*26.7 ( 14.8)22.3 ( 15.6)total calories kcal / day [ mean ( sd)]1962.56 ( 790.72)1887.74 ( 706.64)total fat g / day [ mean ( sd)]71.74 ( 32.96)69.77 ( 31.76)total alcohol g / day [ mean ( sd)]7.91 ( 15.84)8.29 ( 14.45)total coffee g / day [ mean ( sd)]227.41 ( 210.13)216.57 ( 192.88)tumor stage at diagnosis carcinoma in situ ( % ) 16 ( 5 ) non - invasive / low grade ( % ) 190 ( 59 ) non - invasive / high grade ( % ) 23 ( 7 ) invasive ( % ) 93 ( 29)sd standard deviation * statistically significant differences between cases and controls ; p < 0.05 distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factors sd standard deviation * statistically significant differences between cases and controls ; p < 0.05 table 2 shows the ors and 95% cis for bladder cancer and total intake ( including dietary and supplemental sources ) of fat - soluble and water - soluble vitamins . we observed a borderline statistically significant inverse association between total intake of vitamin e and bladder cancer ( highest vs. lowest quartile , or : 0.66 ; 95% ci : 0.361.20 ; p trend = 0.09 ) . no statistically significant ors were observed when comparing the highest quartile of intake with the lowest quartile for carotenoids as a group or for any of the individual carotenoids , -carotene , -carotene , -cryptoxanthin , lycopene , or lutein / zeaxanthin . although the point estimate for vitamin d was well below one , this also did not reach statistical significance ( or : 0.58 ; 95% ci : 0.311.06 ; p trend = 0.22).table 2calculated odds ratios ( 95% ) for total intake of vitamins and bladder cancerdaily intakeq1q2q3q4p trendalpha - carotene ( g)3.4404404.1647.13647.141104.701104.710.26 cases / controls76/5989/6077/6177/59 ors ( 95% ci)1.001.33 ( 0.772.30)1.19 ( 0.682.11)1.48 ( 0.832.64)beta - carotene ( g)292.292294.212294.223773.803773.815517.205517.210.90 cases / controls94/5978/6165/6079/59 ors ( 95% ci)1.000.87 ( 0.501.50)0.80 ( 0.461.39)0.99 ( 0.561.72)beta - cryptoxanthin ( g)052.6052.61125.77125.78215.71215.720.65 cases / controls109/5976/6159/5878/61 ors ( 95% ci)1.000.69 ( 0.401.20)0.66 ( 0.371.16)0.87 ( 0.501.53)lycopene ( g)03092.683092.69482748287746.437746.440.74 cases / controls89/5973/6072/6088/60 ors ( 95% ci)1.000.96 ( 0.551.66)0.70 ( 0.391.24)0.98 ( 0.551.73)lutein ( g)0.931261.481261.491942.441942.453134.853134.860.66 cases / controls116/5861/6174/6070/59 ors ( 95% ci)1.000.52 ( 0.300.90)0.76 ( 0.451.31)0.80 ( 0.461.40)total carotenoids ( g)398.268879.228879.2312900.6012900.6117932.7217932.730.47 cases / controls107/5973/6052/6087/60 ors ( 95% ci)1.000.70 ( 0.411.21)0.49 ( 0.270.86)0.90 ( 0.511.59)vitamin d ( iu)15.53171.74171.75388.90388.91641.12641.130.22 cases / controls89/5972/60100/6061/60 ors ( 95% ci)1.000.78 ( 0.441.38)1.03 ( 0.601.76)0.58 ( 0.311.06)vitamin e ( mg)1.137.267.2720.9420.95193.35193.360.09 cases / controls77/5795/5881/5754/57 ors ( 95% ci)1.001.19 ( 0.682.08)0.88 ( 0.491.56)0.66 ( 0.361.20)thiamin ( mg)0.211.321.332.112.123.343.350.50 cases / controls91/5863/5999/6367/58 ors ( 95% ci)1.000.53 ( 0.290.96)0.97 ( 0.561.68)0.63 ( 0.351.14)riboflavin ( mg)0.251.631.642.752.764.044.050.61 cases / controls85/6074/5991/6171/59 ors ( 95% ci)1.000.73 ( 0.411.29)1.02 ( 0.581.77)0.75 ( 0.421.34)niacin ( b3 ) ( mg)5.7721.9121.9233.0733.0846.5046.510.23 cases / controls90/5868/6093/6067/60 ors ( 95% ci)1.000.59 ( 0.331.06)0.94 ( 0.541.64)0.56 ( 0.311.02)vitamin b6 ( mg)0.321.941.953.283.294.884.890.41 cases / controls93/5975/5977/6175/59 ors ( 95% ci)1.000.67 ( 0.381.18)0.82 ( 0.471.44)0.72 ( 0.411.27)vitamin b12 ( g)0.755.515.5210.8210.8318.018.10.75 cases / controls79/6089/6065/6087/59 ors ( 95% ci)1.001.30 ( 0.752.26)0.84 ( 0.471.51)1.05 ( 0.601.84)folate ( g)48.41339.03339.04579.10579.11819.50819.510.43 cases / controls82/5982/6185/5971/60 ors ( 95% ci)1.000.83 ( 0.471.46)0.91 ( 0.531.56)0.74 ( 0.401.35)vitamin c ( mg)14.61113.03113.04190.25190.26366.10366.110.42 cases / controls98/5878/6080/6162/59 ors ( 95% ci)1.000.83 ( 0.481.43)0.84 ( 0.481.45)0.78 ( 0.451.38)or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) calculated odds ratios ( 95% ) for total intake of vitamins and bladder cancer or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) among the water - soluble vitamins , the or for niacin and bladder cancer was of borderline statistical significance ( highest vs. lowest quartile or : 0.56 ; 95% ci : 0.311.02 ) . no other associations were observed between bladder cancer and any other b - group or water - soluble vitamins , which included thiamin , riboflavin , b6 , b12 , and folate or vitamin c. similarly , no associations were observed between bladder cancer and total intake for any of the minerals reported in table 3 , including sodium , potassium , magnesium , calcium , phosphorus , and iron . nor were there any detectable differences in the effects of any of the minerals and vitamins between invasive and non - invasive bladder cancer.table 3calculated odds ratios ( 95% ) for total intake of minerals and bladder cancerdaily intakeq1q2q3q4p trendsodium ( mg)344.611484.831484.841898.641898.652372.652372.660.32 cases / controls78/5864/6059/60119/60 ors ( 95% ci)1.001.17 ( 0.612.23)1.00 ( 0.482.12)1.66 ( 0.703.92)potassium ( mg)535.632382.52382.62874.242874.253781.333781.340.58 cases / controls101/5945/6095/6180/59 ors ( 95% ci)1.000.50 ( 0.270.91)1.03 ( 0.532.0)0.65 ( 0.291.45)calcium ( mg)86.63600.36600.37918.23918.241346.41346.50.27 cases / controls99/5973/6090/5960/60 ors ( 95% ci)1.000.60 ( 0.351.05)0.88 ( 0.501.53)0.60 ( 0.321.13)magnesium ( mg)58.76259.03259.04328.08328.09418.58418.590.20 cases / controls95/5962/6183/6080/59 ors ( 95% ci)1.000.65 ( 0.361.15)0.79 ( 0.431.44)0.56 ( 0.271.16)phosphorus ( mg)177.85965.2965.31195.671195.681556.51556.60.46 cases / controls93/6065/5981/6183/59 ors ( 95% ci)1.001.02 ( 0.551.92)0.93 ( 0.441.98)0.70 ( 0.281.75)iron ( mg)2.1211.2511.2615.5615.5723.2023.210.93 cases / controls82/5975/6077/6086/60 ors ( 95% ci)1.001.04 ( 0.571.90)0.92 ( 0.491.73)1.06 ( 0.571.96)or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) calculated odds ratios ( 95% ) for total intake of minerals and bladder cancer or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) analyses were repeated for all minerals and vitamins using measures taken from both dietary sources ( excluding supplements ) and supplements alone ( data not shown ) . the only nutrient of interest to emerge from dietary sources alone when comparing highest quartiles of intake with lowest quartiles of intake , although not statistically significant was phosphorus ( or : 0.49 ; 95% ci : 0.211.17 ; p trend = 0.13 ) . no other associations or trends could be observed for any of the other dietary or supplemental sources of minerals and vitamins . we continued analyses for minerals and vitamins where an association had been indicated either from borderline associations and biological plausibility or from previous studies . minerals and vitamins that met the criteria were investigated for interactions with smoking characteristics and age , which were both adjusted for each other . the joint effects of nutrient intake and established risk factors e.g. , smoking status ( ever / never ) , cigarettes per day ( < 20/20/day ) , duration of smoking ( < 27 years/27 years ) , and age ( < 63 years/63 years ) are presented in table 4 . the or for heavy cigarette smokers ( 20 cigarettes per day ) in the highest group for total intake of vitamin e , was 0.58 ( 95% : 0.340.99 ; p interaction 0.03 ) . inverse associations of borderline statistical significance were also observed among heavy smokers for the highest intake of total carotenoids ( or : 0.62 ; 95% ci : 0.361.09 ; p interaction 0.08 ) and niacin ( or : 0.66 ; 95% ci : 0.391.14 ; p interaction 0.08 ) . interaction between thiamin intake and the number of cigarettes smoked per day was also of borderline statistical significance . possible reductions in risk of bladder cancer for older participants were also associated with higher intakes of total carotenoids ( or : 0.59 ; 95% ci : 0.350.99 ) , vitamin d , thiamin , and niacin ( p interaction : 0.04 , 0.02 , and 0.03 , respectively ) . no other statistically significant associations were observed , although there were reduced odds of bladder cancer among the older age group for the highest intake of vitamin e ( or : 0.61 ; 95% ci : 0.371.02 ) and interactions between vitamin c and ever - smoking ( p = 0.08 ) and vitamin c and age ( p = 0.08 ) were borderline ( data not shown).table 4odds ratios ( 95% confidence intervals ) for the joint effects of total intake of selected nutrients ( highest vs. lowest ) and smoking characteristics and agecarotenoidsvitamin dvitamin ethiaminniacin<12900.6 g12900.6 gors ( 95% ci)<388.91 iu388.91 iuors ( 95% ci)<20.7 ( mg)20.7 ( mg)ors ( 95% ci)<2.12 ( mg)2.12 ( mg)ors ( 95% ci)<33.08 ( mg)33.08 ( mg)ors ( 95% ci)cases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlssmoking status never*28/4328/520.66 ( 0.321.35)29/5427/411.04 ( 0.502.15)33/5120/420.64 ( 0.311.33)23/5033/441.35 ( 0.662.77)27/5329/411.10 ( 0.542.26 ) ever*155/76111/680.75 ( 0.481.17)132/65134/790.82 ( 0.541.24)140/64117/730.73 ( 0.481.13)134/70132/740.91 ( 0.591.39)135/66131/770.80 ( 0.521.23 ) p interaction0.980.410.870.270.26cigarettes / day < 2025/3026/231.65 ( 0.683.99)19/2632/271.58 ( 0.663.79)20/2731/231.57 ( 0.633.88)19/2832/251.85 ( 0.784.41)20/2731/251.64 ( 0.683.95 ) 20127/4484/440.62 ( 0.361.09)111/38100/500.73 ( 0.431.23)118/3685/480.58 ( 0.340.99)114/4197/470.78 ( 0.461.34)114/3897/500.66 ( 0.391.14 ) p interaction0.080.140.030.100.08years smoking < 2751/3845/341.19 ( 0.582.47)49/3247/400.78 ( 0.401.52)49/3645/321.08 ( 0.542.16)46/3050/420.71 ( 0.351.43)47/3049/410.70 ( 0.351.41 ) 27101/3764/330.62 ( 0.331.16)81/3284/381.00 ( 0.551.81)89/2770/400.61 ( 0.331.12)87/3978/311.41 ( 0.762.59)87/3578/351.02 ( 0.561.85 ) p interaction0.200.580.240.190.51age < 6373/5969/540.91 ( 0.491.68)76/6966/441.55 ( 0.882.74)79/6156/471.12 ( 0.622.01)69/6573/472.02 ( 1.113.68)69/6373/481.59 ( 0.882.87 ) 63110/6070/660.59 ( 0.350.99)85/5095/760.65 ( 0.401.08)94/5481/680.61 ( 0.371.02)88/5592/710.75 ( 0.451.24)93/5687/700.64 ( 0.381.07 ) p interaction0.180.040.180.020.03iu international units , mg milligrams , low intakes were categorized below median and high intakes above mediansadjusted for age , sex , smoking status ( current / non - current ) , number of cigarettes per day , and years of smoking * adjusted for age , sex , and kcal only odds ratios ( 95% confidence intervals ) for the joint effects of total intake of selected nutrients ( highest vs. lowest ) and smoking characteristics and age iu international units , mg milligrams , low intakes were categorized below median and high intakes above medians adjusted for age , sex , smoking status ( current / non - current ) , number of cigarettes per day , and years of smoking * adjusted for age , sex , and kcal only the statistically significant inverse associations observed between total intake of carotenoids , vitamin d , thiamin , and niacin and the risk of bladder cancer for older individuals remained robust following the fprp test . each inverse association had less than a 50% likelihood of being false positive for an odds ratio of 1.5 and based on a prior probability of 25% , table 1 presents the frequency distributions of selected study characteristics for the 322 cases and 239 controls for whom dietary data were available for analyses in this study . bladder cancer cases consisted of approximately three times more men ( 74% ) than women . a majority of both cases and controls were in the older age group ( 60 years and over ) . there was a higher prevalence of current smokers and greater number of both cigarettes smoked per day and years smoked among cases compared with the controls . we detected no statistically significant differences between cases and controls for calorie , total fat , alcohol , or coffee intake.table 1distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factorscases number ( % ) controls number ( % ) total322239sex * men237 ( 74)138 ( 58 ) women85 ( 26)101 ( 42)age ( years ) [ mean ( sd)]62 ( 9.2)60.7 ( 10.6)smoking status * never smokers56 ( 17.4)95 ( 39.7 ) ex - smokers162 ( 50.3)103 ( 43.1 ) current smokers104 ( 32.3)41 ( 17.2 ) years smoking [ mean ( sd)]*32.2 ( 14)27.4 ( 15 ) cigarettes / day [ mean ( sd)]*26.7 ( 14.8)22.3 ( 15.6)total calories kcal / day [ mean ( sd)]1962.56 ( 790.72)1887.74 ( 706.64)total fat g / day [ mean ( sd)]71.74 ( 32.96)69.77 ( 31.76)total alcohol g / day [ mean ( sd)]7.91 ( 15.84)8.29 ( 14.45)total coffee g / day [ mean ( sd)]227.41 ( 210.13)216.57 ( 192.88)tumor stage at diagnosis carcinoma in situ ( % ) 16 ( 5 ) non - invasive / low grade ( % ) 190 ( 59 ) non - invasive / high grade ( % ) 23 ( 7 ) invasive ( % ) 93 ( 29)sd standard deviation * statistically significant differences between cases and controls ; p < 0.05 distribution of bladder cancer cases and controls by demographic characteristics and bladder cancer risk factors sd standard deviation * statistically significant differences between cases and controls ; p < 0.05 table 2 shows the ors and 95% cis for bladder cancer and total intake ( including dietary and supplemental sources ) of fat - soluble and water - soluble vitamins . we observed a borderline statistically significant inverse association between total intake of vitamin e and bladder cancer ( highest vs. lowest quartile , or : 0.66 ; 95% ci : 0.361.20 ; p trend = 0.09 ) . no statistically significant ors were observed when comparing the highest quartile of intake with the lowest quartile for carotenoids as a group or for any of the individual carotenoids , -carotene , -carotene , -cryptoxanthin , lycopene , or lutein / zeaxanthin . although the point estimate for vitamin d was well below one , this also did not reach statistical significance ( or : 0.58 ; 95% ci : 0.311.06 ; p trend = 0.22).table 2calculated odds ratios ( 95% ) for total intake of vitamins and bladder cancerdaily intakeq1q2q3q4p trendalpha - carotene ( g)3.4404404.1647.13647.141104.701104.710.26 cases / controls76/5989/6077/6177/59 ors ( 95% ci)1.001.33 ( 0.772.30)1.19 ( 0.682.11)1.48 ( 0.832.64)beta - carotene ( g)292.292294.212294.223773.803773.815517.205517.210.90 cases / controls94/5978/6165/6079/59 ors ( 95% ci)1.000.87 ( 0.501.50)0.80 ( 0.461.39)0.99 ( 0.561.72)beta - cryptoxanthin ( g)052.6052.61125.77125.78215.71215.720.65 cases / controls109/5976/6159/5878/61 ors ( 95% ci)1.000.69 ( 0.401.20)0.66 ( 0.371.16)0.87 ( 0.501.53)lycopene ( g)03092.683092.69482748287746.437746.440.74 cases / controls89/5973/6072/6088/60 ors ( 95% ci)1.000.96 ( 0.551.66)0.70 ( 0.391.24)0.98 ( 0.551.73)lutein ( g)0.931261.481261.491942.441942.453134.853134.860.66 cases / controls116/5861/6174/6070/59 ors ( 95% ci)1.000.52 ( 0.300.90)0.76 ( 0.451.31)0.80 ( 0.461.40)total carotenoids ( g)398.268879.228879.2312900.6012900.6117932.7217932.730.47 cases / controls107/5973/6052/6087/60 ors ( 95% ci)1.000.70 ( 0.411.21)0.49 ( 0.270.86)0.90 ( 0.511.59)vitamin d ( iu)15.53171.74171.75388.90388.91641.12641.130.22 cases / controls89/5972/60100/6061/60 ors ( 95% ci)1.000.78 ( 0.441.38)1.03 ( 0.601.76)0.58 ( 0.311.06)vitamin e ( mg)1.137.267.2720.9420.95193.35193.360.09 cases / controls77/5795/5881/5754/57 ors ( 95% ci)1.001.19 ( 0.682.08)0.88 ( 0.491.56)0.66 ( 0.361.20)thiamin ( mg)0.211.321.332.112.123.343.350.50 cases / controls91/5863/5999/6367/58 ors ( 95% ci)1.000.53 ( 0.290.96)0.97 ( 0.561.68)0.63 ( 0.351.14)riboflavin ( mg)0.251.631.642.752.764.044.050.61 cases / controls85/6074/5991/6171/59 ors ( 95% ci)1.000.73 ( 0.411.29)1.02 ( 0.581.77)0.75 ( 0.421.34)niacin ( b3 ) ( mg)5.7721.9121.9233.0733.0846.5046.510.23 cases / controls90/5868/6093/6067/60 ors ( 95% ci)1.000.59 ( 0.331.06)0.94 ( 0.541.64)0.56 ( 0.311.02)vitamin b6 ( mg)0.321.941.953.283.294.884.890.41 cases / controls93/5975/5977/6175/59 ors ( 95% ci)1.000.67 ( 0.381.18)0.82 ( 0.471.44)0.72 ( 0.411.27)vitamin b12 ( g)0.755.515.5210.8210.8318.018.10.75 cases / controls79/6089/6065/6087/59 ors ( 95% ci)1.001.30 ( 0.752.26)0.84 ( 0.471.51)1.05 ( 0.601.84)folate ( g)48.41339.03339.04579.10579.11819.50819.510.43 cases / controls82/5982/6185/5971/60 ors ( 95% ci)1.000.83 ( 0.471.46)0.91 ( 0.531.56)0.74 ( 0.401.35)vitamin c ( mg)14.61113.03113.04190.25190.26366.10366.110.42 cases / controls98/5878/6080/6162/59 ors ( 95% ci)1.000.83 ( 0.481.43)0.84 ( 0.481.45)0.78 ( 0.451.38)or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) calculated odds ratios ( 95% ) for total intake of vitamins and bladder cancer or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) among the water - soluble vitamins , the or for niacin and bladder cancer was of borderline statistical significance ( highest vs. lowest quartile or : 0.56 ; 95% ci : 0.311.02 ) . no other associations were observed between bladder cancer and any other b - group or water - soluble vitamins , which included thiamin , riboflavin , b6 , b12 , and folate or vitamin c. similarly , no associations were observed between bladder cancer and total intake for any of the minerals reported in table 3 , including sodium , potassium , magnesium , calcium , phosphorus , and iron . nor were there any detectable differences in the effects of any of the minerals and vitamins between invasive and non - invasive bladder cancer.table 3calculated odds ratios ( 95% ) for total intake of minerals and bladder cancerdaily intakeq1q2q3q4p trendsodium ( mg)344.611484.831484.841898.641898.652372.652372.660.32 cases / controls78/5864/6059/60119/60 ors ( 95% ci)1.001.17 ( 0.612.23)1.00 ( 0.482.12)1.66 ( 0.703.92)potassium ( mg)535.632382.52382.62874.242874.253781.333781.340.58 cases / controls101/5945/6095/6180/59 ors ( 95% ci)1.000.50 ( 0.270.91)1.03 ( 0.532.0)0.65 ( 0.291.45)calcium ( mg)86.63600.36600.37918.23918.241346.41346.50.27 cases / controls99/5973/6090/5960/60 ors ( 95% ci)1.000.60 ( 0.351.05)0.88 ( 0.501.53)0.60 ( 0.321.13)magnesium ( mg)58.76259.03259.04328.08328.09418.58418.590.20 cases / controls95/5962/6183/6080/59 ors ( 95% ci)1.000.65 ( 0.361.15)0.79 ( 0.431.44)0.56 ( 0.271.16)phosphorus ( mg)177.85965.2965.31195.671195.681556.51556.60.46 cases / controls93/6065/5981/6183/59 ors ( 95% ci)1.001.02 ( 0.551.92)0.93 ( 0.441.98)0.70 ( 0.281.75)iron ( mg)2.1211.2511.2615.5615.5723.2023.210.93 cases / controls82/5975/6077/6086/60 ors ( 95% ci)1.001.04 ( 0.571.90)0.92 ( 0.491.73)1.06 ( 0.571.96)or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) calculated odds ratios ( 95% ) for total intake of minerals and bladder cancer or odds ratios , ci confidence intervals , adjusted for age , sex , smoking status : current versus non - current smoker , pack years smoked ( categories : 010 , 1020 , 2030 , 3040 , 4050 , 50 + ) , and total energy intake ( quartiles kcal / day ) analyses were repeated for all minerals and vitamins using measures taken from both dietary sources ( excluding supplements ) and supplements alone ( data not shown ) . the only nutrient of interest to emerge from dietary sources alone when comparing highest quartiles of intake with lowest quartiles of intake , although not statistically significant was phosphorus ( or : 0.49 ; 95% ci : 0.211.17 ; p trend = 0.13 ) . no other associations or trends could be observed for any of the other dietary or supplemental sources of minerals and vitamins . we continued analyses for minerals and vitamins where an association had been indicated either from borderline associations and biological plausibility or from previous studies . minerals and vitamins that met the criteria were investigated for interactions with smoking characteristics and age , which were both adjusted for each other . the joint effects of nutrient intake and established risk factors e.g. , smoking status ( ever / never ) , cigarettes per day ( < 20/20/day ) , duration of smoking ( < 27 years/27 years ) , and age ( < 63 years/63 years ) are presented in table 4 . the or for heavy cigarette smokers ( 20 cigarettes per day ) in the highest group for total intake of vitamin e , was 0.58 ( 95% : 0.340.99 ; p interaction 0.03 ) . inverse associations of borderline statistical significance were also observed among heavy smokers for the highest intake of total carotenoids ( or : 0.62 ; 95% ci : 0.361.09 ; p interaction 0.08 ) and niacin ( or : 0.66 ; 95% ci : 0.391.14 ; p interaction 0.08 ) . interaction between thiamin intake and the number of cigarettes smoked per day was also of borderline statistical significance . possible reductions in risk of bladder cancer for older participants were also associated with higher intakes of total carotenoids ( or : 0.59 ; 95% ci : 0.350.99 ) , vitamin d , thiamin , and niacin ( p interaction : 0.04 , 0.02 , and 0.03 , respectively ) . no other statistically significant associations were observed , although there were reduced odds of bladder cancer among the older age group for the highest intake of vitamin e ( or : 0.61 ; 95% ci : 0.371.02 ) and interactions between vitamin c and ever - smoking ( p = 0.08 ) and vitamin c and age ( p = 0.08 ) were borderline ( data not shown).table 4odds ratios ( 95% confidence intervals ) for the joint effects of total intake of selected nutrients ( highest vs. lowest ) and smoking characteristics and agecarotenoidsvitamin dvitamin ethiaminniacin<12900.6 g12900.6 gors ( 95% ci)<388.91 iu388.91 iuors ( 95% ci)<20.7 ( mg)20.7 ( mg)ors ( 95% ci)<2.12 ( mg)2.12 ( mg)ors ( 95% ci)<33.08 ( mg)33.08 ( mg)ors ( 95% ci)cases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlscases / controlssmoking status never*28/4328/520.66 ( 0.321.35)29/5427/411.04 ( 0.502.15)33/5120/420.64 ( 0.311.33)23/5033/441.35 ( 0.662.77)27/5329/411.10 ( 0.542.26 ) ever*155/76111/680.75 ( 0.481.17)132/65134/790.82 ( 0.541.24)140/64117/730.73 ( 0.481.13)134/70132/740.91 ( 0.591.39)135/66131/770.80 ( 0.521.23 ) p interaction0.980.410.870.270.26cigarettes / day < 2025/3026/231.65 ( 0.683.99)19/2632/271.58 ( 0.663.79)20/2731/231.57 ( 0.633.88)19/2832/251.85 ( 0.784.41)20/2731/251.64 ( 0.683.95 ) 20127/4484/440.62 ( 0.361.09)111/38100/500.73 ( 0.431.23)118/3685/480.58 ( 0.340.99)114/4197/470.78 ( 0.461.34)114/3897/500.66 ( 0.391.14 ) p interaction0.080.140.030.100.08years smoking < 2751/3845/341.19 ( 0.582.47)49/3247/400.78 ( 0.401.52)49/3645/321.08 ( 0.542.16)46/3050/420.71 ( 0.351.43)47/3049/410.70 ( 0.351.41 ) 27101/3764/330.62 ( 0.331.16)81/3284/381.00 ( 0.551.81)89/2770/400.61 ( 0.331.12)87/3978/311.41 ( 0.762.59)87/3578/351.02 ( 0.561.85 ) p interaction0.200.580.240.190.51age < 6373/5969/540.91 ( 0.491.68)76/6966/441.55 ( 0.882.74)79/6156/471.12 ( 0.622.01)69/6573/472.02 ( 1.113.68)69/6373/481.59 ( 0.882.87 ) 63110/6070/660.59 ( 0.350.99)85/5095/760.65 ( 0.401.08)94/5481/680.61 ( 0.371.02)88/5592/710.75 ( 0.451.24)93/5687/700.64 ( 0.381.07 ) p interaction0.180.040.180.020.03iu international units , mg milligrams , low intakes were categorized below median and high intakes above mediansadjusted for age , sex , smoking status ( current / non - current ) , number of cigarettes per day , and years of smoking * adjusted for age , sex , and kcal only odds ratios ( 95% confidence intervals ) for the joint effects of total intake of selected nutrients ( highest vs. lowest ) and smoking characteristics and age iu international units , mg milligrams , low intakes were categorized below median and high intakes above medians adjusted for age , sex , smoking status ( current / non - current ) , number of cigarettes per day , and years of smoking * adjusted for age , sex , and kcal only the statistically significant inverse associations observed between total intake of carotenoids , vitamin d , thiamin , and niacin and the risk of bladder cancer for older individuals remained robust following the fprp test . each inverse association had less than a 50% likelihood of being false positive for an odds ratio of 1.5 and based on a prior probability of 25% , we investigated an extensive range of micronutrients in relation to the risk of bladder cancer . possible inverse associations of borderline statistical significance were observed between bladder cancer and total intake of vitamin e and dietary phosphorus . we found some evidence that total intake of vitamin e , total carotenoids , niacin and possibly thiamin might be modified by cigarette smoking in that inverse associations were observed largely among heaviest smokers . higher intakes of carotenoids , vitamin d , thiamin , niacin , and vitamin e were also associated with reduced odds of bladder cancer in the older age group . the potentially protective effect of vitamin e that we observed against developing bladder cancer was consistent with findings from two other us studies [ 20 , 27 ] . however , we detected no association between risk of bladder cancer overall and the intake of carotenoids either as individual carotenoids ( -carotene , -carotene , -cryptoxanthin , lycopene , and lutein / zeaxanthin ) or collectively as total carotenoid intake . our findings were in agreement with an intervention study , a meta - analysis , two prospective studies [ 3 , 20 ] , and one case control study . conversely , four case control studies [ 8 , 22 , 23 , 48 ] observed inverse associations with carotenoid intake and two other prospective studies [ 21 , 25 ] found specific carotenoids , dietary beta - carotene and beta - cryptoxanthin to be possibly protective . like previous studies , we investigated the most common carotenoids from an estimated 40 carotenoids present in the human diet . while some carotenoids have potential to form vitamin a ( provitamin a carotenoids include , -carotene , -carotene , and -cryptoxanthin ) others do not have this capability ( non - provitamin a carotenoids are lycopene , lutein , and zeaxanthin ) . contrary to an earlier study , we did not find either of these two categories of carotenoids to be associated with the risk of bladder cancer ( data not shown ) . any chemo - preventive effect may be difficult to detect for individual carotenoids either due to the relatively small sample size , small size of the effect or because carotenoids possibly only work in synergy with each other or other dietary factors . where a relatively large prospective study of us men and a case control study both reported no association between vitamin d and bladder cancer , we observed reduced odds of bladder cancer with higher intake of vitamin d ; although this was not statistically significant . the anti - proliferative effect of vitamin d has been shown in various in vitro and in vivo investigations . however , as vitamin d is available from both dietary sources and uv - b radiation , it is difficult to estimate the full effect of vitamin d without taking into account sunlight exposure . although b - group vitamins are thought to possibly offer protection via their role in genomic stability , dna repair , and regulation of cell division , we did not detect any associations between any b - group vitamin and bladder cancer overall . this was consistent with three previous prospective studies [ 20 , 21 , 25 ] and one retrospective study but disagreed with two other recent case control studies [ 28 , 35 ] . we also failed to detect any association between bladder cancer and another water - soluble vitamin with antioxidant potential , vitamin c. once again , there were conflicting reports relating to the effect of vitamin c on bladder cancer risk among the various studies [ 3 , 8 , 1921 , 23 , 29 , 32 , 33 ] . when reviewing the existing literature on vitamin c and many of the other vitamins , any evidence for an association generally appeared to be weaker in prospective studies compared with case control studies . lack of association between some micronutrients and bladder cancer might also be due to collinearity among nutrients as a result of shared food sources . for instance , many fruits and vegetables rich in carotenoids are also good sources of vitamin c , potassium , and folate . to our knowledge , phosphorus has been investigated in relation to bladder cancer in only one other epidemiological study that reported no association following multivariate analyses . although we observed an approximate 50% reduction in the odds of bladder cancer associated with higher dietary intake of phosphorus , it was not statistically significant . given this ubiquitous micronutrient is an important physiological component of dna , rna , atp , and cell membranes , it may be worthy of further consideration . in our study , a higher intake of vitamin e related to a reduced risk of bladder cancer among heaviest smokers , a finding consistent with those reported in another american case control study . the reduced odds of bladder cancer we observed for the highest quartile of total carotenoids among the heaviest smokers also agrees with results presented in another case control studies [ 22 , 48 ] also reported increased risk of bladder cancer among smokers with the lower intakes of total and specific carotenoids , lutein , and zeaxanthin . . suggested that carotenoids provide protection against bladder cancer for smokers by detoxifying arylamines . borderline statistically significant interactions between the number of cigarettes smoked per day and intakes of thiamin and niacin may warrant further investigation . our findings indicate that higher intakes of vitamin d , thiamin , niacin , carotenoids , and possibly vitamin e may be beneficial to participants in the older age group . older individuals may have limited access to regular sunshine ( e.g. , living in institutions ) and therefore have to rely strongly on dietary sources of vitamin d and supplementation . bladder cancer is a disease that typically affects older people , and bioavailability of b - group vitamins may be compromised in this demographic by certain drugs ( e.g. , acid lowering agents ) . additionally , vitamin e , like carotenoids acts as an antioxidant and , as suggested by our results , could be more beneficial under conditions of the greatest oxidative stress such as smoking and aging . like most case control studies , potential for recall bias is a consideration in dietary estimation . however , as dietary factors are not commonly associated with bladder cancer etiology recall of dietary intake is likely to be non - differential between cases and controls . other potential limitations relating to measurement of dietary exposures include only one dietary collection period and the possibility of preclinical disease altering dietary intake . although there was only one collection period , dietary data were collected using a detailed and validated ffq . as for dietary change , a majority of the cases in our study were diagnosed with superficial bladder cancer , and it is considered unlikely that preclinical disease would alter dietary behavior . although we can not totally exclude the possibility of recall bias , dietary intake for major dietary components ; calories , total fat , alcohol , and coffee were comparable between cases and controls suggesting that it should be minimal . finally , controls were drawn from two sources department of transportation ( for those under 65 years ) and medicare ( for those 65 years and older ) . these are considered nearly complete sources of records in our population . among cases , over 95% report having a driver s license ( for those under 65 years ) as do over 95% ( of those 65 years and older ) report being enrolled in medicare ( data not shown ) . we examined the characteristics of controls younger than 65 years and over 65 years and found them to be comparable with respect to sex , smoking , and medical access variables ( i.e. , visit to a doctor in the past 2 years ) ; however , older subjects had less than a college education ( 40% of those under 65 years and 50% of those 65 years and older ) . however , level of education is not a strong risk factor for bladder cancer ( including in our own data ) and thus was not considered a potential confounder in our analysis . while multiple comparisons were made in this study , we attempted to address this issue by utilizing the fprp . statistically significant associations between higher intakes of carotenoids , vitamin d , thiamin , and niacin and risk of bladder cancer among the older individuals remained robust following this test . although it is worth noting that statistical power was low and determining appropriate prior probabilities is difficult . biologically plausible mechanisms of action and similar results reported from other studies provide additional support for some of our findings [ 8 , 20 , 27 ] . we investigated an extensive range of micronutrients in relation to the risk of bladder cancer . possible inverse associations of borderline statistical significance were observed between bladder cancer and total intake of vitamin e and dietary phosphorus . we found some evidence that total intake of vitamin e , total carotenoids , niacin and possibly thiamin might be modified by cigarette smoking in that inverse associations were observed largely among heaviest smokers . higher intakes of carotenoids , vitamin d , thiamin , niacin , and vitamin e were also associated with reduced odds of bladder cancer in the older age group . the potentially protective effect of vitamin e that we observed against developing bladder cancer was consistent with findings from two other us studies [ 20 , 27 ] . however , we detected no association between risk of bladder cancer overall and the intake of carotenoids either as individual carotenoids ( -carotene , -carotene , -cryptoxanthin , lycopene , and lutein / zeaxanthin ) or collectively as total carotenoid intake . our findings were in agreement with an intervention study , a meta - analysis , two prospective studies [ 3 , 20 ] , and one case control study . conversely , four case control studies [ 8 , 22 , 23 , 48 ] observed inverse associations with carotenoid intake and two other prospective studies [ 21 , 25 ] found specific carotenoids , dietary beta - carotene and beta - cryptoxanthin to be possibly protective . like previous studies , we investigated the most common carotenoids from an estimated 40 carotenoids present in the human diet . while some carotenoids have potential to form vitamin a ( provitamin a carotenoids include , -carotene , -carotene , and -cryptoxanthin ) others do not have this capability ( non - provitamin a carotenoids are lycopene , lutein , and zeaxanthin ) . contrary to an earlier study , we did not find either of these two categories of carotenoids to be associated with the risk of bladder cancer ( data not shown ) . any chemo - preventive effect may be difficult to detect for individual carotenoids either due to the relatively small sample size , small size of the effect or because carotenoids possibly only work in synergy with each other or other dietary factors . where a relatively large prospective study of us men and a case control study both reported no association between vitamin d and bladder cancer , we observed reduced odds of bladder cancer with higher intake of vitamin d ; although this was not statistically significant . the anti - proliferative effect of vitamin d has been shown in various in vitro and in vivo investigations . however , as vitamin d is available from both dietary sources and uv - b radiation , it is difficult to estimate the full effect of vitamin d without taking into account sunlight exposure . although b - group vitamins are thought to possibly offer protection via their role in genomic stability , dna repair , and regulation of cell division , we did not detect any associations between any b - group vitamin and bladder cancer overall . this was consistent with three previous prospective studies [ 20 , 21 , 25 ] and one retrospective study but disagreed with two other recent case control studies [ 28 , 35 ] . we also failed to detect any association between bladder cancer and another water - soluble vitamin with antioxidant potential , vitamin c. once again , there were conflicting reports relating to the effect of vitamin c on bladder cancer risk among the various studies [ 3 , 8 , 1921 , 23 , 29 , 32 , 33 ] . when reviewing the existing literature on vitamin c and many of the other vitamins , any evidence for an association generally appeared to be weaker in prospective studies compared with case control studies . lack of association between some micronutrients and bladder cancer might also be due to collinearity among nutrients as a result of shared food sources . for instance , many fruits and vegetables rich in carotenoids are also good sources of vitamin c , potassium , and folate . to our knowledge , phosphorus has been investigated in relation to bladder cancer in only one other epidemiological study that reported no association following multivariate analyses . although we observed an approximate 50% reduction in the odds of bladder cancer associated with higher dietary intake of phosphorus , it was not statistically significant . given this ubiquitous micronutrient is an important physiological component of dna , rna , atp , and cell membranes , it may be worthy of further consideration . in our study , a higher intake of vitamin e related to a reduced risk of bladder cancer among heaviest smokers , a finding consistent with those reported in another american case control study . the reduced odds of bladder cancer we observed for the highest quartile of total carotenoids among the heaviest smokers also agrees with results presented in another case control studies [ 22 , 48 ] also reported increased risk of bladder cancer among smokers with the lower intakes of total and specific carotenoids , lutein , and zeaxanthin . borderline statistically significant interactions between the number of cigarettes smoked per day and intakes of thiamin and niacin may warrant further investigation . our findings indicate that higher intakes of vitamin d , thiamin , niacin , carotenoids , and possibly vitamin e may be beneficial to participants in the older age group . older individuals may have limited access to regular sunshine ( e.g. , living in institutions ) and therefore have to rely strongly on dietary sources of vitamin d and supplementation . bladder cancer is a disease that typically affects older people , and bioavailability of b - group vitamins may be compromised in this demographic by certain drugs ( e.g. , acid lowering agents ) . additionally , vitamin e , like carotenoids acts as an antioxidant and , as suggested by our results , could be more beneficial under conditions of the greatest oxidative stress such as smoking and aging . like most case control studies , potential for recall bias is a consideration in dietary estimation . however , as dietary factors are not commonly associated with bladder cancer etiology recall of dietary intake is likely to be non - differential between cases and controls . other potential limitations relating to measurement of dietary exposures include only one dietary collection period and the possibility of preclinical disease altering dietary intake . although there was only one collection period , dietary data were collected using a detailed and validated ffq . as for dietary change , a majority of the cases in our study were diagnosed with superficial bladder cancer , and it is considered unlikely that preclinical disease would alter dietary behavior . although we can not totally exclude the possibility of recall bias , dietary intake for major dietary components ; calories , total fat , alcohol , and coffee were comparable between cases and controls suggesting that it should be minimal . finally , controls were drawn from two sources department of transportation ( for those under 65 years ) and medicare ( for those 65 years and older ) . these are considered nearly complete sources of records in our population . among cases , over 95% report having a driver s license ( for those under 65 years ) as do over 95% ( of those 65 years and older ) report being enrolled in medicare ( data not shown ) . we examined the characteristics of controls younger than 65 years and over 65 years and found them to be comparable with respect to sex , smoking , and medical access variables ( i.e. , visit to a doctor in the past 2 years ) ; however , older subjects had less than a college education ( 40% of those under 65 years and 50% of those 65 years and older ) . however , level of education is not a strong risk factor for bladder cancer ( including in our own data ) and thus was not considered a potential confounder in our analysis . while multiple comparisons were made in this study , we attempted to address this issue by utilizing the fprp . statistically significant associations between higher intakes of carotenoids , vitamin d , thiamin , and niacin and risk of bladder cancer among the older individuals remained robust following this test . although it is worth noting that statistical power was low and determining appropriate prior probabilities is difficult . biologically plausible mechanisms of action and similar results reported from other studies provide additional support for some of our findings [ 8 , 20 , 27 ] . in conclusion , the effects of vitamin e , carotenoids , vitamin d , thiamin , and niacin in relation to the risk of developing bladder cancer may warrant further investigation . future studies should focus on optimal doses and combinations of these micronutrients particularly for high risk groups such as heavy smokers and older individuals . | objectivealthough the effect of fruit and vegetables on the risk of bladder cancer has been widely studied , little is known about their micronutrient components .
our aim was to investigate associations between minerals and vitamins and bladder cancer.methodsa case control study was conducted in new hampshire , usa .
dietary data were collected from 322 cases and 239 controls using a 121-item food frequency questionnaire .
odds ratios ( ors ) and 95% confidence intervals ( cis ) were calculated using logistic regression adjusting for sex , age , smoking characteristics , and energy intake.resultsthe ors ( 95% ci ) for highest quartile versus lowest quartile for total intake of vitamin e was 0.66 ( 0.361.20 ; p trend = 0.09 ) and 0.49 ( 0.211.17 ; p trend = 0.13 ) for dietary phosphorus .
the odds of bladder cancer for heavy smokers with the highest total intake of vitamin e , carotenoids , and niacin were 0.58 ( 0.340.99 ) , 0.62 ( 0.361.09 ) , and 0.66 ( 0.391.14 ) , respectively .
higher total intakes of carotenoids , vitamin d , thiamin , niacin , and vitamin e were inversely related to bladder cancer risk among older individuals.conclusionour findings suggest further investigation of the effect of vitamin e , carotenoids , vitamin d , thiamin , and niacin on bladder cancer risk may be warranted .
future studies should focus on high risk groups such as heavy smokers and older individuals . |
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over the past 15 years , u.s . has witnessed a general decline in overall rates of youth homicide . nonetheless , youth violence rates remain high in this country , with homicide being the third leading cause of death among persons aged 1024 years . youth violence can affect communities by substantially increasing the cost of health care , reducing productivity , and diminishing property values . in 2000 , it was estimated that the medical care and lost productivity costs associated with youth violence were more than $ 70 billion . youth violence has been linked to a variety of factors including individual , family , community , and societal characteristics . community - level risk can have negative influences even on youth who are not exposed to individual- or family - level risk factors . major community risk factors for violence include high density of alcohol outlets , community norms favorable toward violence , residential instability , transitions and mobility , low neighborhood attachment , social disorganization , the presence of gangs , and extreme economic deprivation [ 5 , 6 ] . although much research has been conducted on interventions to change the characteristics of individuals and families , less has focused on evaluating interventions and policies designed to change community economic conditions or characteristics of the physical environment . studies have been conducted on the impact of interventions on individual - level variables ; however , evaluations at the community level have been sparse . some research is emerging that highlights the promise of community- and policy - level strategies in preventing youth violence . for example , modifications to the built environment such as improvements to the pedestrian environment and architectural changes can contribute to relative reductions in 911 calls and crimes , possibly through increases in social capital and intolerance of criminal activity [ 7 , 8 ] . it is theorized that large - scale economic developments , such as sports and entertainment arenas and casinos , improve the living conditions , economics , public health , and overall wellbeing of area residents and may influence rates of violence within communities [ 9 , 10 ] . local government and business developers often suggest that building an arena or a casino in a neighborhood may provide potential societal benefits as a result of the construction and the business drawn in ; thus , many new arenas are placed in areas of needed economic growth . however , scientific research to support these ideas is scarce , and there is much debate about the true benefits of economic developments . research consistently suggests that economically disadvantaged neighborhoods have poorer public health outcomes , including higher rates of violent crime , chronic disease , and risky behavior , and that there is large heterogeneity with regard to these factors across neighborhoods within a city [ 11 , 12 ] . according to siegfried and zimbalist , independent work on the economic impact of stadiums and arenas has uniformly found that there is no statistically significant positive correlation between sports facility construction and economic development . whether casino development causes economic growth in surrounding neighborhoods is a complex question , and the literature is not consistent in its conclusions [ 9 , 10 ] . the few studies that have assessed economic growth looked at large geographic areas such as the county or state . for example , describes that after the licensing of casinos in monaco , nevada , and atlantic city , these cities grew dramatically economically and became destinations for tourism . also , few studies have looked at the effects of casinos on crime and delinquency , but , of those that have , the findings have been inconsistent [ 15 , 16 ] . there is a need for a more focused , community - level survey of resident perceptions regarding the effect of these major community development projects on perceived safety , violence , and economic benefits . a consistent literature suggests that the perception of crime is often different from actual crime . for instance , in a study conducted in australia , results suggested that people often exaggerated the risks of becoming a victim of crime . this was corroborated in two subsequent studies . in another study conducted in new zealand , crime in an individual 's own neighborhood influenced fear of crime , but crime occurring in neighboring communities had little effect on perceived safety . this difference in perception is referred to as the paradox of fear [ 20 , 21 ] . age , gender , and race have been demonstrated to affect the differences between the perception of crime and actual crime . for example , the elderly , women , and racial and ethnic minorities have been shown as having higher perceptions of crime than actual risk [ 23 , 24 ] . in the current study , we have a greater percent of elderly female minorities , explaining in part this discrepancy . when taken into context with other individual factors , this has been referred to as the vulnerability perspective . this idea emphasizes that fear is highest when individuals perceive themselves to be vulnerable . in a 2010 study , investigators examined resident 's perception of crime based on the neighborhood in which they live . the results suggested that perceived disorder of neighborhood structure , including social cohesion , was strongly associated with perception of crime even after controlling for race , age , and gender . in pittsburgh , pennsylvania , two large economic developments were recently constructed in two historically disadvantaged minority neighborhoods . in august 2009 , gambling was legalized in pittsburgh , and a casino was opened in the north side neighborhood . the consol energy center ( cec ) , an indoor sports and entertainment facility , was opened in the hill district neighborhood in august 2010 . these community - level changes provide a unique opportunity to study the potential effect of two different community economic development efforts to examine whether the economic benefits directly have an effect on perceptions and rates of community violence . specifically , the current study has three objectives : describing residents ' perceptions of the effect of the arena and casino on neighborhood violence , safety , and economic benefits.describing residents ' perceptions of change in neighborhood violence , safety , and economic benefits after the opening of the arena and casino.comparing the above residents ' perceptions with census and police data over the same time.results from this study will fill some of the existing gaps in the field around the relationship between community economic development efforts and community violence while highlighting some of the potential mechanisms through which they may have an effect ( e.g. , job availability for community residents ) . describing residents ' perceptions of the effect of the arena and casino on neighborhood violence , safety , and economic benefits . describing residents ' perceptions of change in neighborhood violence , safety , and economic benefits after the opening of the arena and casino . comparing the above residents ' perceptions with census and police data over the same time . we conducted a telephone survey in 2011 using a listed sample of randomly selected telephone numbers in each of six neighborhoods of pittsburgh ( figure 1 ) . the neighborhoods included the north side , where the casino was built in 2009 , as well as the hill district , where the entertainment arena was built in 2010 . one adjacent neighborhood for each of these communities was also included to assess whether any potential benefits spilled over into nearby areas . north oakland was the neighboring community for the hill district , and an area consisting of spring garden , fineview , spring hill - city view , and perry south ( hereafter collectively referred to as spring garden ) was the neighboring community for the north side . two additional neighborhoods were also examined and were intended to serve as comparisons to the economic development communities : squirrel hill is a neighborhood of high socioeconomic status ( ses ) and low violent crime rates , and homewood is a neighborhood of lower ses and higher levels of violent crime . interviews were conducted between july and december 2011 and lasted for an average of 25 minutes in length . survey questions gauged demographic variables , employment history , neighborhood factors , and relationships / interactions with both the casino and the arena . respondents were categorized by the neighborhood in which they resided based on self - report . in addition , respondents were asked to identify the closest intersection to validate neighborhood assignment . respondents were asked if they felt safe in their neighborhood both during the day and at night . respondents were asked whether , compared with 2006 , they thought there was more violent crime . employed respondents were identified as those who were employed for wages / salary , self - employed , student , or a combination of those ; unemployed respondents were identified as those who responded respondents were also asked if their household income had changed since 2006 , whether the rivers casino affected businesses in their neighborhood , and whether they thought either development affected employment or income in their neighborhood . the analyses were conducted using spss , version 21 , software ( spss inc . , we conducted descriptive analyses to examine the various measures of perceived violence and safety and economic benefit . pearson 's chi - square tests determined significant differences in proportions for all neighborhoods . if significant at the p < 0.05 level , we then ran pairwise comparisons of column proportions using the bonferroni correction . we only present comparisons for both the hill and the north side compared with each other and the other neighborhoods given that these were our a priori comparisons of interest . table 1 shows the participants ' demographic data . in general , the participants were older than the average age of the surveyed communities , with a mean of 64 15 years of age . the participants were primarily female ( 69.8% ) and white ( 62.1% ) , though this varied by neighborhood . two notable exceptions are the hill district and homewood , where around 90% of participants were identified as black ( see table 2 for census figures ) . in table 3 , we show statistically significant differences between the hill district and the north side . squirrel hill residents were significantly more likely to report feeling safe during the day compared with the hill and the north side ( 99.5% versus 90.6% and 92.4% resp . , the percentage of participants in squirrel hill who felt safe at night was higher ( 91.7% , p < 0.05 ) , while the percentage in homewood was lower ( 48.3% , p < 0.05 ) compared with both the hill and the north side . participants from the hill district , the north side , and spring garden agreed with the statement violence is common in my neighborhood at similar rates ( ranging from 32.1 to 42.9% ) while far fewer respondents from north oakland and squirrel hill ( 8.6% and 1.5% , resp . , p < 0.05 ) agreed with the statement compared with the hill and the north side , and 62.1% of participants from homewood agreed ( p < 0.05 ) . similarly , participants from north oakland and squirrel hill agreed with the statement compared with 2006 , there is more violent crime in my neighborhood now at the lowest rates ( 13.7% and 2.4% , resp . , p < 0.05 ) compared with the hill and the north side and participants from the remaining four neighborhoods agreed at rates ranging from 29.3 to 41.8% . squirrel hill had the least amount of recorded violence in 2011 ( 0.6 violent crimes per 1000 residents ) , followed by oakland ( 2.6 ) , spring garden ( 10.5 ) , hill district ( 12.1 ) , north side ( 15.1 ) , and homewood with the most amount of recorded violence ( 18.1 ) when looking at actual violent crime rates in these neighborhoods ( see figure 2 ) . all neighborhoods except spring garden experienced less violent crime in 2011 than in 2006 ; spring garden saw a slight increase ( + 0.4 ) . participants in the north side stated more often that the rivers casino has had a positive impact on their life ( 14.1% ) compared with 7.2% of participants from the north side who responded that the casino has had a negative impact . participants in squirrel hill were more likely to not report positive effects compared with the north side ( p < 0.05 ) . participants from both north oakland and squirrel hill were statistically much less likely to report an effect on economic issues compared with the north side ( p < 0.05 , table 3 ) . when asked , how has the rivers casino affected crime in your neighborhood ? , the only statistical difference was for squirrel hill , with no one reporting that the casino had a positive effect on crime in his or her neighborhood ( p < 0.05 ) . in summary , the north side appears to have benefitted more from the casino than any other neighborhoods , but this benefit was only reported by a minority of north side participants . this is reflected in responses to the question overall , how has the rivers casino affected your neighborhood ? the highest rate of responses indicating a positive effect was observed in the north side , at 24.5% , while the majority of participants in each neighborhood responded neither positively nor negatively the less disadvantaged neighborhoods , north oakland and squirrel hill , were the only neighborhoods with significant differences . when asked , how has the consol energy center impacted your life ? , the majority of participants in each neighborhood answered neither positively nor negatively ( 69.6% of participants from the hill district , where the arena is located , and around 80% of participants in all other neighborhoods ) . when asked , how has the consol energy center affected income in your neighborhood ? , the most common answer across all neighborhoods was neither positively nor negatively 31.9% of participants from the hill district reported that the consol energy center had a positive effect on employment in their neighborhood , and 24.6% of participants from the hill district reported that the consol energy center had a positive effect on local businesses in their neighborhood . participants from every other neighborhood reported statistically significantly lower proportions of positive effects on the economic questions compared to the hill ( p < 0.05 ) . in response to the question how has the consol energy center affected crime in your neighborhood ? , the majority of participants in each neighborhood responded neither positively nor negatively ( 68.6% of participants from the hill district ) , followed by the response do not know / unsure ( 18.8% of participants from the hill district ) . responses to the question how has the consol energy center affected violence in your neighborhood ? followed a similar pattern , with 68.6% of participants from the hill district responding neither positively nor negatively and 17.4% of participants from the hill district responding do not know / unsure . in summary , participants from the hill district reported benefitting the most from the consol energy center . when asked , overall , how has the consol energy center affected your neighborhood ? , 32.9% of participants from the hill district reported that the arena had a positive effect while 46.4% of respondents reported that the arena had neither a positive nor a negative effect , and 9.7% reported a negative effect . participants in all other neighborhoods reported statistically significantly lower levels of positive effects in comparison ( p < 0.05 ) . community economic development is one strategy that may lead to more resources and opportunities within neighborhoods . this , in turn , may result in reductions in community - level rates of violence . this study addressed the relationship between two large economic development efforts within the city of pittsburgh a casino and a sports arena on perceptions of economic opportunities and community safety . overall , we found that residents in neighborhoods with the large - scale economic developments reported more development in specific economic benefits than did residents from other neighborhoods . a large proportion of hill district respondents thought violent crime had increased since 2006 even though it actually decreased more in the hill district than in any other survey neighborhoods . in addition , hill district respondents felt less safe than north side respondents did , even though the north side experiences more crime than the hill district . if more crime in the north side is being committed by nonresidents in the entertainment districts than crime in the hill district being committed by nonresidents coming into the hill , the north side residents may actually be experiencing less violence near their homes than hill district residents . when participants ' perceptions are compared with the actual violent crime rates for their neighborhoods , they often matched . participants from squirrel hill were most likely to say they felt safe during the day or night and were least likely to believe that violence is common in their neighborhood while participants from homewood were least likely to say they felt safe during the day or night and were most likely to believe that violence is common in their neighborhood . accordingly , the 2011 violent crime rates indicate that squirrel hill experienced the least amount of violence ( 0.6 violent crimes per 1000 residents ) of the six neighborhoods survey while homewood experienced the most ( 18.1 violent crimes per 1000 residents ) . it should be noted , however , that although the majority of participants from homewood agreed that violence is common in their neighborhood and indeed it is thirty times more common in homewood than squirrel hill , the majority of participants did not feel unsafe , even at night . participants were also mostly correct in their perceptions about the change in violence since 2006 . the majority from every neighborhood did not believe that there was more violence now than in 2006 , and indeed violent crime rates have decreased in every neighborhood except spring garden , which saw a slight increase . interestingly , however , the rate of violent crime in spring garden in 2011 was at its lowest since 2006 due to a spike in 2007 , so it may have been difficult for participants in that neighborhood to differentiate between the slight increase in violence compared with 2006 and the decrease in violence seen almost every year since 2007 . even though homewood and the hill district had the greatest declines in violence from 2006 to 2011 of all survey neighborhoods , this was not evident in the participant responses in these neighborhoods when the perception of crime is taken into context with other individual factors , it has been referred to as the vulnerability perspective . this idea emphasizes that fear is highest when individuals perceive themselves to be vulnerable . in a 2010 study , investigators examined resident 's perception of crime based on the neighborhood in which they live . the results suggested that perceived disorder of neighborhood structure , including social cohesion , was strongly associated with perception of crime even after controlling for race , age , and gender . two major criminological theories on crime , the broken windows hypothesis and the collective efficacy perspective [ 29 , 30 ] , are related to both actual crime and the perception of crime . social cohesion measures mutual trust among residents and is thought to reduce community problems including fear of crime . previous studies have shown that concentrated disadvantage within communities , as measured by poverty , unemployment , and family disruption , is associated with fear of crime [ 26 , 29 , 31 ] . as in our study , low levels of social cohesion may lead to more crime [ 28 , 32 ] , which may in turn reduce cohesion among residents . there are likely differences in our results between perception and reality of crime because of individual characteristics , including age , gender , and race , as well as neighborhood context , which has been shown to affect individuals ' perception of crime . surprisingly , there has been a dearth of research examining whether casinos positively benefit community residents . the few studies that have assessed economic growth looked at large geographic areas , such as county or state , rather than areas directly surrounding the developments . an evaluation of the economic impact of casinos by eadington used historical perspective to demonstrate the potential economic benefits . the author cited monaco , nevada , and atlantic city as places that , before the commercialization of legalized gambling , were experiencing economic deterioration . however , after the licensing of casinos in these areas , they economically prospered and became destinations for tourism . other groups studied the causal impact of casino gambling profits and per capita income and found that there was no evidence for causation of casino profits impacting per capita income in two studies [ 10 , 35 ] . both evaluations were conducted at a state level where data were aggregated for all 11 states that allowed casino gambling on any level . our results begin to fill this gap in the literature by examining residents ' specific perceptions of the impact of area developments and providing a glimpse into how the developments affect individuals as well as communities . however , others theorize that economic growth often leaks outside of the community as profits and jobs are exported out of the community [ 9 , 10 ] . we found that the highest rate of respondents stating that the rivers casino has had a positive impact on their life was in the neighborhood where the casino is located ; a similar pattern was observed for the neighborhood in which the arena is located . additionally , the results of the economic benefit questions underlined these findings . residents of the neighborhoods in which the casino and arena are located reported the greatest positive effects on their income . there are a large number of studies that have assessed the effect of large - scale developments on violence . however , these are mostly related to casinos , and the results are varied . in a study by grinols and mustard , casino and noncasino counties property crime levels were evaluated for two years before the opening and five years following the opening . the results of the study showed that there was an increase in crime after the opening of a casino . further , park and stokowski compared four types of counties based on predominant type of recreation / tourism attraction offered , including casinos , and found that total arrest rate was highest in casino counties but this was not statistically significant . stokowski found that gaming counties in colorado had higher rates of property crimes but not violent crimes . research has also indicated that economic crimes and public order crimes increased in biloxi , ms , after introduction of casino gaming [ 15 , 37 ] , and disorderly conducted arrests increased after introduction of casino gaming in davenport , iowa . our results , while limited to residents ' perceptions of violence , further support the finding that violence increases after the opening of large - scale developments , in this case a casino . we found that residents in the neighborhoods with the developments reported greater changes in violence . the differences in results across studies are likely caused by differences in measures of violence and size of the geographic area studied . more research is needed in this area with particular emphasis on using consistent definitions , measures , and methodology . interestingly , in the grinols and mustard study , the increase in violent crime occurred most noticeably after a three - year lag . first , in the initial years following the opening of a casino , more community resources , such as funding for labor of police , are at their highest and this in turn reduces crime . additionally , the investigators cite data concerning the effects of addictive gamblers . our results do not contain data after 2 years for the casino and 1 year for the arena , but these findings provide important implications to consider in future research . there is a large amount of heterogeneity between communities which demonstrates the need to assess how casinos may affect different types of neighborhoods differently . for example , kang et al . studied residents ' perceptions of the impact of limited - stakes community - based casino gambling in colorado in 2006 . perceived positive economic impacts from the casino influenced residents ' support of the casino , while perceived negative social impacts did not . our sample consisted largely of older women , and our findings may not be representative of all age groups , particularly young men and women who may be more likely to visit , attend events at , and work at the casino and arena , likely due in part to only including landlines . further research should examine objective data in combination with subjective data regarding residents ' perceptions . this initial work is important in identifying how neighborhood residents perceive the large - scale economic developments in these neighborhoods . the potential benefits to a community are directly related to public health outcomes as well as public policy . more in - depth studies should be conducted to assess long - term effects as well as the social pathways through which these effects occur . | background .
emerging research highlights the promise of community- and policy - level strategies in preventing youth violence .
large - scale economic developments , such as sports and entertainment arenas and casinos , may improve the living conditions , economics , public health , and overall wellbeing of area residents and may influence rates of violence within communities .
objective . to assess the effect of community economic development efforts on neighborhood residents ' perceptions on violence , safety , and economic benefits . methods .
telephone survey in 2011 using a listed sample of randomly selected numbers in six pittsburgh neighborhoods .
descriptive analyses examined measures of perceived violence and safety and economic benefit .
responses were compared across neighborhoods using chi - square tests for multiple comparisons .
survey results were compared to census and police data .
results .
residents in neighborhoods with the large - scale economic developments reported more casino - specific and arena - specific economic benefits .
however , 42% of participants in the neighborhood with the entertainment arena felt there was an increase in crime , and 29% of respondents from the neighborhood with the casino felt there was an increase .
in contrast , crime decreased in both neighborhoods . conclusions .
large - scale economic developments have a direct influence on the perception of violence , despite actual violence rates . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the enlargement usually leads to lower urinary tract symptoms,1 irrespective of whether it is benign or malignant . with wide acceptance of serum prostate - specific antigen ( psa ) estimation,2 the ability to differentiate malignant from benign prostatic enlargement with or without symptoms before instituting definitive treatment has increased.3 however , an entirely new range of challenges have been encountered as a result of the poor sensitivity of serum total psa ( tpsa).35 the need to carry out prostate biopsy to confirm malignant prostate enlargement in cases of elevated serum tpsa comes to the fore.6 some of these prostate biopsies will be negative for malignant cells or identify low - volume , low - grade adenocarcinoma of uncertain clinical significance.7,8 the sensitivity of serum tpsa in detecting prostate cancer has been put at 25% in screening programs8 and as high as 99.6% in patients with symptoms.9 in nigeria , a serum tpsa value > 4.0 ng / ml has been widely accepted as an indication to exclude adenocarcinoma of the prostate through biopsy of the prostate,10 a cut - off value adopted from studies done mostly outside nigeria.1113 serum tpsa varies with body mass index , intravascular volume , prostate volume , benign and malignant diseases of the prostate . as a result , some studies have applied serum tpsa values of 3.0 ng / ml instead of > 4.0 ng / ml in the asian continent as a cut - off point for subsequent prostate biopsy.5,14 in nigeria , anunobi et al found that among patients with serum tpsa 4.049.9 ng / ml , core needle biopsy specimens revealed many benign prostates.15 clinical adenocarcinoma of the prostate varies between races16,17 and studies have shown that africa has a high prevalence of the disease.1,18,19 it is important that malignancy is excluded in all cases of symptomatic prostatic enlargement , so this study aims to relate the histologic findings of all prostate specimens obtained from core needle biopsy , open simple prostatectomy , and transurethral resection of the prostate to the respective serum tpsa values in an attempt to decipher the role of serum tpsa in the clinician s decision - making . this was a hospital - based retrospective study from april 2009 to march 2012 , ie , a period of 36 months . the case notes of patients who presented with and were evaluated for symptomatic prostate enlargement were retrieved and the relevant information contained therein was extracted for analysis . when biopsy was indicated , digitally guided transrectal extended sextant core needle biopsy of the prostate was offered due to the nonavailability of transrectal ultrasonography - guided biopsy . a disposable semiautomatic size 16 g or 18 g trucut biopsy needle was used for each prostate biopsy . the indication for biopsy was a suspicious digital rectal examination ( dre ) finding , suspicious prostate from abdominopelvic ultrasonography ( uss ) , or elevated serum tpsa ( > 4 ng / ml ) . in patients with a working diagnosis of benign prostatic hyperplasia ( bph ) requiring surgical management , simple retropubic prostatectomy , transvesical prostatectomy , or transurethral resection of the prostate was offered . in addition , the dre finding , serum tpsa result , uss assessment of the prostate , and the histologic report on the various specimens were extracted from each patient s case notes for analysis . the data analysis was done using statistical package for the social sciences version 20 software ( ibm corporation , armonk , ny , usa ) . the chi - square test was used for nonparametric tests ( yates correction for continuity was employed when necessary ) . the level of significance was set at a two - tailed p - value of < 0.05 . the number of patients in each age group and the average tpsa for the respective age groups are shown in table 1 . findings were benign in 90 ( 70.3% ) , and suspicious or suggestive of malignancy in 38 ( 29.7% ) , as shown in figure 1 . the average value was 24.3529.13 ng / ml , with a range of 0.4100 ng / ml . as shown in table 2 , 74.4% of tpsa values were > 4 ng / ml while 25.6% were 4 ng / ml . uss was done in 110 patients , with the results presented in figure 2 . in the remaining 108 patients , findings were benign in 72 ( 66.7% ) and suspicious in 36 ( 33.3% ) . adenocarcinoma was confirmed in 26 ( 56.5% ) , while 19 ( 41.3% ) showed benign features . a total of 23 patients underwent prostatectomy over the study period ; 19 ( 82.6% ) had open prostatectomy and four ( 17.4% ) had transurethral resection of the prostate . seven of the 23 patients who had prostatectomy underwent prostate biopsy due to serum tpsa > 4 ng / ml . the histology reports were bph . subsequent histology reports of prostatectomy specimens from these seven patients confirmed bph in six and adenocarcinoma ( gleason score 2 + 3 ) in one . table 4 shows the histologic report of the 23 prostatectomy specimens : 18 ( 78.3% ) were nodular hyperplasia ( bph ) , four ( 17.4% ) were chronic inflammatory cell infiltration on a background of nodular hyperplasia ( bph + prostatitis ) , and one ( 4.3% ) was adenocarcinoma ( gleason score 2 + 3 ) . thirty - nine patients underwent prostate biopsy due to suspicious prostates ( suspicious dre , suspicious ultrasound findings , or tpsa > 4 ng / ml ) , while 16 patients had simple prostatectomy without prior biopsy since there was no indication for biopsy , giving a total of 55 patients . of the 39 prostates that were biopsied , 26 were adenocarcinoma , one was high - grade prostatic intraepithelial neoplasia , and 12 were benign . thus , seven patients had prostate biopsy initially and prostatectomy at a later date , resulting in 62 histologic reports that were analyzed ( figure 3 ) . the histologic reports on all the prostate specimens were categorized for patient age , dre findings , serum tpsa values , and uss reports ( table 5 ) . there were nine histologic reports for patients with serum tpsa > 4 ng / ml and 10 ng / ml . the dre findings , uss findings , and duration of follow - up for these patients were analyzed ( table 6 ) . four with benign dre and uss findings had benign histology , while three adenocarcinoma cases had either suspicious dre or uss findings . findings were benign in 90 ( 70.3% ) , and suspicious or suggestive of malignancy in 38 ( 29.7% ) , as shown in figure 1 . the average value was 24.3529.13 ng / ml , with a range of 0.4100 ng / ml . as shown in table 2 , 74.4% of tpsa values were > 4 ng / ml while 25.6% were 4 ng / ml . in the remaining 108 patients , findings were benign in 72 ( 66.7% ) and suspicious in 36 ( 33.3% ) . adenocarcinoma was confirmed in 26 ( 56.5% ) , while 19 ( 41.3% ) showed benign features . a total of 23 patients underwent prostatectomy over the study period ; 19 ( 82.6% ) had open prostatectomy and four ( 17.4% ) had transurethral resection of the prostate . seven of the 23 patients who had prostatectomy underwent prostate biopsy due to serum tpsa > 4 ng / ml . the histology reports were bph . subsequent histology reports of prostatectomy specimens from these seven patients confirmed bph in six and adenocarcinoma ( gleason score 2 + 3 ) in one . table 4 shows the histologic report of the 23 prostatectomy specimens : 18 ( 78.3% ) were nodular hyperplasia ( bph ) , four ( 17.4% ) were chronic inflammatory cell infiltration on a background of nodular hyperplasia ( bph + prostatitis ) , and one ( 4.3% ) was adenocarcinoma ( gleason score 2 + 3 ) . thirty - nine patients underwent prostate biopsy due to suspicious prostates ( suspicious dre , suspicious ultrasound findings , or tpsa > 4 ng / ml ) , while 16 patients had simple prostatectomy without prior biopsy since there was no indication for biopsy , giving a total of 55 patients . of the 39 prostates that were biopsied , 26 were adenocarcinoma , one was high - grade prostatic intraepithelial neoplasia , and 12 were benign . thus , seven patients had prostate biopsy initially and prostatectomy at a later date , resulting in 62 histologic reports that were analyzed ( figure 3 ) . the histologic reports on all the prostate specimens were categorized for patient age , dre findings , serum tpsa values , and uss reports ( table 5 ) . there were nine histologic reports for patients with serum tpsa > 4 ng / ml and 10 ng / ml . the dre findings , uss findings , and duration of follow - up for these patients were analyzed ( table 6 ) . four with benign dre and uss findings had benign histology , while three adenocarcinoma cases had either suspicious dre or uss findings . case notes for 134 patients seen within the study period could be retrieved for analysis . all were for nigerians of mean age 69.979.50 ( 4594 ) years with a modal range of 6069 years ( table 1 ) . one hundred and twenty - eight dres of the prostate were performed , with 90 ( 70.3% ) and 38 ( 29.7% ) feeling benign and malignant , respectively ( figure 1 ) . twenty - four ( 85.7% ) of the 28 prostates that were histologically confirmed as adenocarcinoma of the prostate were suspicious or malignant on dre ( table 5 ) . a similar result was obtained by badmus et al1 in nigeria ( 92.1% ) , but not by ma and wan20 in the people s republic of china ( 31.25% ) or palmerola et al21 in the usa ( 44% ) . in contrast , of the 34 benign histology reports , four ( 11.8% ) had suspicious dre findings . the high sensitivity ( 85.7% ) , specificity ( 88.2% ) , positive predictive value ( 85.7% ) , and negative predictive value ( 88.2% ) of dre in this analysis ( table 7 ) may be due to late presentation , by which time locoregional features of the disease were well established . uss assessment was documented conclusively for 108 prostates , with 72 ( 66.7% ) showing benign features and 36 ( 33.3% ) showing malignant features ( figure 2 ) . table 5 shows that 18 of the 28 malignant prostates ( 69.2% ) had a suspicious ultrasonography report . in southwestern nigeria , in contrast , of the 34 histologically benign prostates , ten ( 30.3% ) were reported as suspicious for malignancy by ultrasonography . there was a significant positive correlation between age and serum tpsa ( r=0.318 , p<0.001 ; table 1 ) . a similar correlation was documented by mochtar et al for patients with a symptomatic benign prostate.22 tpsa values ranged from 0.4 to 100 ng / ml , with a mean of 24.3529.13 ng / ml and a median of 12.7 ng / ml . eighty - seven of these 117 tpsa values ( 74.4% ) were > 4 ng / ml and 30 ( 25.6% ) were 4 ng / ml ( table 2 ) . applying a serum tpsa of > 4 ng / ml , the sensitivity and specificity of tpsa for detecting prostate cancer were 96.3% and 18.2% , respectively , with a negative predictive value of 85.7% . these were similar to the findings of lokuhetty et al.9 therefore , in this study , using a serum tpsa > 4 ng / ml alone , 81.8% of benign prostates would have been subjected to unnecessary transrectal biopsy . however , applying a serum tpsa of > 10 ng / ml alone , with a sensitivity of 85.2% and a specificity of 33.3% , the negative predictive value decreased to 73.3% . similarly , from the study by abbiyesuku et al23 only 37% and 54.3% of histologically benign prostatic enlargements had tpsa < 4 g / l and < 10 thompson et al13 concluded that there is no cutpoint of psa with simultaneous high sensitivity and high specificity for monitoring healthy men for prostate cancer , but rather a continuum of prostate cancer risk at all psa values . the inference from the review by schrder and roobol24 is similar to the conclusion of thompson et al . in our study of men with symptomatic prostatic enlargement it may be argued that it is necessary to have followed - up the patients with tpsa > 4 ng / ml , but benign histological report , for longer periods to ensure they do not turn out to be cases of false negative histological reports . however , de - novo prostate cancer can also occur in the residual prostates after simple prostatectomy for benign prostate enlargement . among nigerian men with symptomatic prostate enlargement , serum tpsa > 4 ng / ml and 10 ng / ml may not be interpreted in isolation as an indication for prostate biopsy , since from this analysis 81.8% of benign prostate specimens had values > 4 ng / ml . however , with a negative predictive value of 85.7% for serum tpsa > 4 ng / ml ( table 7 ) , symptomatic patients with tpsa 4 ng / ml could be safely managed as benign prostate enlargement in the absence of suspicious dre and uss findings . applying serum tpsa > 10 ng / ml in isolation as a cut - off may appear to have reduced the number of benign prostates that would have been biopsied by 15.1% , but also increased the missed malignant prostates by 3.6% . the sensitivity of dre and uss in detecting suspicious features in symptomatic prostate enlargement should not be overlooked , especially in our community , where patients directly bear the cost of medical treatment . serum tpsa should be considered as an invaluable component of the triad ( dre , ultrasonography , psa ) in the management of patients with symptomatic prostate enlargement . dre and ultrasonography findings are important in deciding which prostate to biopsy when serum tpsa is between 4 ng / ml and 10 ng / ml ( table 6 ) . | backgroundprostatic enlargement is a common cause of bladder outlet obstruction in men in nigeria .
malignant enlargements must be differentiated from benign enlargements for adequate treatment of each patient .
high serum total prostate - specific antigen ( tpsa ) levels suggest malignancy , but some of the biopsies done due to a serum tpsa value > 4 ng / ml would be negative for malignancy because of the low specificity of tpsa for prostate cancer .
this study aims to compare the histologic findings of all prostate specimens obtained from core needle biopsy , open simple prostatectomy , and transurethral resection of the prostate with the respective serum tpsa values in an attempt to decipher the role of serum tpsa in the management of these patients.methodsthe case notes of patients attended to from april 2009 to march 2012 were analyzed .
essentially , the age of the patient , findings on digital rectal examination , abdominopelvic ultrasonography report on the prostate , serum tpsa , and histology reports from biopsy or prostatectomy specimens as indicated were extracted for analysis.resultsthe relationship between age , findings on digital rectal examination , serum tpsa , abdominopelvic ultrasonography report , and histology are compared .
a statistically significant relationship existed between a malignant histology and age 65 years and older , suspicious findings on digital rectal examination , suspicious ultrasonography findings , and serum tpsa > 10 ng / ml , but not tpsa > 4 ng / ml.conclusionin nigerian patients with symptomatic prostate enlargement , serum tpsa should be seen as a continuum with increasing risk of prostate malignancy . |
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progression is diagnosed in 1 out of 4 patients after 3 years of radical treatment of prostate cancer . progression includes biochemical recurrence , local recurrence , distant metastases and death . elevated preoperative psa concentration , among other factors , is a strong predictor of progression after radical prostatectomy ( rp ) due to prostate cancer . however , this marker has some limitations . undoubtedly , a negative feature of psa concentration is the fact that it is subject to hemodilution . some authors claim that in overweight and obese patients psa concentration is lower , which is , in the first place , caused by the aforementioned phenomenon . this phenomenon is supposed to consist of the dissolution of psa mass in a large amount of plasma , finally resulting in lower psa concentration . every adult male is characterized by a quite invariable amount ( mass ) of this , secreted into the blood protein , depending on age , the size of prostate , the presence of cancer or other prostate diseases . however , standard psa determination means that psa mass is dissolved in plasma volume , which is mainly dependant on the degree of obesity . psa mass , as the absolute amount of psa protein secreted into circulation , may be quite easily computed on the basis of physiological formulas for estimated body surface ( ebs ) and plasma volume ( pv ) , and therefore is independent of the hemodilution phenomenon ( table 1 ) . in order to eliminate hemodilution , it was decided to evaluate the psa mass in patients with prostate cancer and compare its usefulness with psa concentration in prediction of cancer progression after rp . the study was approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki . all patients gave their informed consent prior to inclusion in the study . from 1994 until the end of 2007 , 206 radical retropubic prostatectomies in caucasian men suffering from prostate cancer were carried out in the department of urology in zabrze , medical university of silesia in katowice . the patients who underwent preoperative anti - androgen therapy , chemotherapy or radiotherapy were excluded from the study ( 29 patients ) , and 177 patients were qualified for inclusion . all patients were divided according to : bmi into 3 groups : i 45 patients with normal weight ( bmi < 25 ) , ii 95 overweight patients ( bmi 2529.9 ) , iii 37 obese patients ( bmi 30 ) . preoperative psa concentration into 3 groups : i 79 patients with psa < 10 ng / ml , ii 66 patients with psa 1019.9 ng / ml , iii 32 patients with psa 20 ng / ml . preoperative psa mass into 3 groups : i 71 patients with psa < 40 g , ii 78 patients with psa 4069.9 g and iii 28 patients with psa 70 g . preoperative data , such as age , height , weight , bmi , psa concentration ( immunoenzymatic elecsys test ; cobas 6000 hitachi ) , and postoperative data such as the extent of histopathologic differentiation of prostate tissue in gleason score , extracapsular extension ( pt3 ) , the presence of lymph nodes metastases and the presence of positive surgical margins . patients were under constant control in the hospital outpatient clinic , thanks to which data concerning progression ( biochemical recurrence , local recurrence , death ) were also collected , and the cancer - specific survival time was determined . the total volume of plasma and the psa mass were calculated on the basis of the formulas ( table 1 ) . all constant variables distributions were analyzed with regard to normality by means of kolmogorov - smirnov and lilliefors tests . by means of descriptive statistics the following characteristics were determined : mean or median , standard deviation and maximal and minimal values . in order to determine differences between the groups , where variables were of categorical character , -square test was used . in order to determine differences between a number of independent groups , where continuous variables have distribution other than normal , a multiple regression model was created to assess the correlation between bmi and psa concentration . covariates in the model included age , the extent of prostate cancer differentiation in gleason score , extracapsular extension ( pt3 ) and positive surgical margins . in order to evaluate and compare the odds ratio of biochemical recurrence together with the elevated psa concentration or mass , 2 models of logistic regression covariates in the models included age , bmi , the extent of prostate cancer differentiation in gleason score , nodal metastases , extracapsular extension ( pt3 ) and positive surgical margins . as both the concentration and the psa mass did not show normal distribution , logarithmic ( decimal ) transformation of data was performed . with the assistance of receiver operating characteristic ( roc ) , curves from 2 models of logistic regression were compared . cancer - specific survival of patients was evaluated by means of kaplan - meier analysis , while the significance of differences between them was evaluated by means of log - rank test . for all statistical tests , the study was approved by the appropriate ethics committee and has therefore been performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki . from 1994 until the end of 2007 , 206 radical retropubic prostatectomies in caucasian men suffering from prostate cancer were carried out in the department of urology in zabrze , medical university of silesia in katowice . the patients who underwent preoperative anti - androgen therapy , chemotherapy or radiotherapy were excluded from the study ( 29 patients ) , and 177 patients were qualified for inclusion . all patients were divided according to : bmi into 3 groups : i 45 patients with normal weight ( bmi < 25 ) , ii 95 overweight patients ( bmi 2529.9 ) , iii 37 obese patients ( bmi 30 ) . preoperative psa concentration into 3 groups : i 79 patients with psa < 10 ng / ml , ii 66 patients with psa 1019.9 ng / ml , iii 32 patients with psa 20 ng / ml . preoperative psa mass into 3 groups : i 71 patients with psa < 40 g , ii 78 patients with psa 4069.9 g and iii 28 patients with psa 70 g . preoperative data , such as age , height , weight , bmi , psa concentration ( immunoenzymatic elecsys test ; cobas 6000 hitachi ) , and postoperative data such as the extent of histopathologic differentiation of prostate tissue in gleason score , extracapsular extension ( pt3 ) , the presence of lymph nodes metastases and the presence of positive surgical margins . patients were under constant control in the hospital outpatient clinic , thanks to which data concerning progression ( biochemical recurrence , local recurrence , death ) were also collected , and the cancer - specific survival time was determined . the total volume of plasma and the psa mass were calculated on the basis of the formulas ( table 1 ) . all constant variables distributions were analyzed with regard to normality by means of kolmogorov - smirnov and lilliefors tests . by means of descriptive statistics the following characteristics were determined : mean or median , standard deviation and maximal and minimal values . in order to determine differences between the groups , where variables were of categorical character , -square test was used . in order to determine differences between a number of independent groups , where continuous variables have distribution other than normal a multiple regression model was created to assess the correlation between bmi and psa concentration . covariates in the model included age , the extent of prostate cancer differentiation in gleason score , extracapsular extension ( pt3 ) and positive surgical margins . in order to evaluate and compare the odds ratio of biochemical recurrence together with the elevated psa concentration or mass , covariates in the models included age , bmi , the extent of prostate cancer differentiation in gleason score , nodal metastases , extracapsular extension ( pt3 ) and positive surgical margins . as both the concentration and the psa mass did not show normal distribution , logarithmic ( decimal ) transformation of data was performed . with the assistance of receiver operating characteristic ( roc ) , curves from 2 models of logistic regression were compared . cancer - specific survival of patients was evaluated by means of kaplan - meier analysis , while the significance of differences between them was evaluated by means of log - rank test . for all statistical tests , the critical level of significance increasing values of psa mass and psa concentration had a statistically significant influence on the following features : extracapsular extension , presence of metastases in the surrounding lymph nodes , frequency of positive surgical margins , presence of biochemical and local recurrence and the rate of death ( table 4 ) . the research demonstrated that psa concentration and psa mass do not differ in bmi groups ( table 3 ) . differences in preoperative psa concentration between the 3 groups of patients are statistically insignificant ( p=0.28 ) . the model of multiple regression proved the lack of statistically significant correlation between preoperative psa concentration and bmi ( p=0.99 ) . in patients with increasing bmi , after excluding from the analysis patients with stage t3 , we observed a statistically significant negative correlation between bmi and psa concentration ( p=0.04 ) in multiple regression analysis . the study proved that the elevated preoperative value of psa mass ( p=0.02 ) is the factor that influences the cancer - specific survival of patients with prostate cancer after rp ( figure 1 ) . similarly higher values of psa concentration had crucial impact on cancer - specific survival after rp ( p=0.02 ) the odds ratio ( range ) of biochemical recurrence , with the psa mass increased 10 times , is equal to 10.9 ( p<0.001 for the whole model ) ( table 5 ) . the odds ratio of biochemical recurrence , with the psa concentration increased 10 times , is equal to 8.5 ( p<0.001 for the whole model ) . roc curves for 2 models with psa mass and psa concentration showed an area under curve ( auc ) of 0.74 and 0.69 , respectively , for biochemical recurrence after rp ( figure 2 ) . increasing values of psa mass and psa concentration had a statistically significant influence on the following features : extracapsular extension , presence of metastases in the surrounding lymph nodes , frequency of positive surgical margins , presence of biochemical and local recurrence and the rate of death ( table 4 ) . the research demonstrated that psa concentration and psa mass do not differ in bmi groups ( table 3 ) . differences in preoperative psa concentration between the 3 groups of patients are statistically insignificant ( p=0.28 ) . the total plasma volume is higher in obese patients ( p<0.001 ) . the model of multiple regression proved the lack of statistically significant correlation between preoperative psa concentration and bmi ( p=0.99 ) . in patients with increasing bmi , after excluding from the analysis patients with stage t3 , we observed a statistically significant negative correlation between bmi and psa concentration ( p=0.04 ) in multiple regression analysis . the study proved that the elevated preoperative value of psa mass ( p=0.02 ) is the factor that influences the cancer - specific survival of patients with prostate cancer after rp ( figure 1 ) . similarly higher values of psa concentration had crucial impact on cancer - specific survival after rp ( p=0.02 ) the odds ratio ( range ) of biochemical recurrence , with the psa mass increased 10 times , is equal to 10.9 ( p<0.001 for the whole model ) ( table 5 ) . the odds ratio of biochemical recurrence , with the psa concentration increased 10 times , is equal to 8.5 ( p<0.001 for the whole model ) . roc curves for 2 models with psa mass and psa concentration showed an area under curve ( auc ) of 0.74 and 0.69 , respectively , for biochemical recurrence after rp ( figure 2 ) . there are various theories concerning the influence of obesity on the natural development , diagnostics or progression after radical treatment of prostate cancer . the 5-times increased percentage of biochemical recurrence observed in afro - americans , compared to euro - americans , is sometimes explained by a 3-times more frequent presence of overweight or obesity among the former . its influence is definitely negative , including the following : difficulties in per rectum examination in obese patients , dishormonose [ 9 , 10 ] abnormal hormone concentrations , which induces the intensification of diagnostics and at the same time postpones proper treatment , comorbidities , which pushes the prostate diagnostics into the background and consequently patients suffer from more advanced forms of prostate cancer [ 1113 ] . the consequence of the aforesaid correlation may impact on prostate cancer diagnosis and evaluation of progression after its radical treatment . the authors who showed that obese patients are characterized by lower psa concentration refer to the phenomenon of hemodilution . the supporters of this theory claim that obesity is characterized by a larger amount of circulating blood , so , theoretically , the constant psa mass circulating in the organism would be dissolved in a large amount of plasma , resulting in a lower psa concentration . this phenomenon has already been described . however , our research did not show that the elevated bmi has a significant influence on the preoperative psa concentration in the whole study group . there are 2 hypotheses to explain the inconsistency : racial differences between the analyzed groups , the fact that obese patients in our group had more advanced disease ( pt3 ) . the following research has been done on a group of patients of caucasian race , while the aforesaid research has been frequently based on ethnically heterogeneous groups . the cause of differences between the outcomes can result from the polymorphism of the androgen receptor , which causes higher psa concentration in afro - americans , as well as statistically significant greater obesity in this group . that fact may have influenced the results of the aforementioned authors , and therefore the correlation between psa concentration and bmi was observed . the influence of ethnic differences can , of course , be dismissed by appropriate statistical manipulations ; nevertheless , it seems that research done on homogenous groups is characterized by greater statistical power . on the other hand , this surely influenced increased psa concentration in this group of patients , despite the hemodilution phenomenon . this fact may hinder the utilization of psa mass in practice , while its usefulness may be limited to organ - confined prostate cancers . however , we emphasize that analysis of roc curves indicate good predictive value of psa mass in the whole study group . in comparison of both parameters ( psa concentration and the psa mass ) it must be stressed that the probability of biochemical recurrence after rp is better predicted by psa mass , which surely results from the fact that the psa mass includes the element eliminating the phenomenon of hemodilution . despite the fact that both preoperative parameters equally well evaluate the progression after rp , the psa mass seems to be a little more sensitive parameter ( which is indicated by the difference in the odds ratio and auc ) . increased preoperative value of the psa mass is connected with : more frequent cancer diagnosis of pt3 prostate cancer , more frequent diagnosis of metastases in the surrounding lymph nodes , more frequent recognition of the positive surgical margin , shorter cancer - specific survival time , higher percentage of progression . the preoperative psa mass is a better predictor of biochemical recurrence after rp than is psa concentration . obese patients with organ - confined prostate cancer have lower psa concentration due to the hemodilution phenomenon . | summarybackgroundobese patients with prostate cancer may have lower preoperative psa concentration due to hemodilution .
lower psa concentration may falsely affect assessing the risk of progression after radical prostatectomy ( rp ) .
the aim of this study was to determine preoperative psa mass as the absolute amount of psa protein secreted into circulation , and evaluation of its usefulness in prediction of biochemical recurrence after rp.material/methods177 patients after rp due to prostate cancer were included in the study . on the basis of formulas ,
psa mass was calculated { psa mass [ g ] = ( weight [ kg])0.425 ( height [ cm])0.72 0.007184 1.670 psa concentration [ ng / ml]}. patients were divided into 3 groups according to increasing values of psa mass .
the following features were assessed and compared between these groups ( -square test ) : pathologic stage t3 , nodal metastases , positive surgical margins , biochemical and local recurrence and the rate of death .
cancer - specific survival was assessed depending on psa mass ( kaplan - meier curves with log rank test ) .
the usefulness of psa mass in prediction of biochemical recurrence was compared with psa concentration ( logistic regression with roc curves).resultspathologic stage t3 , nodal metastases , positive surgical margins and progression were more common in patients with higher levels of psa mass ( p<0.01 ) .
cancer - specific survival was significantly shorter in patients with elevated values of psa mass ( p=0.02 ) .
preoperative psa mass was a more sensitive predictor of biochemical recurrence than was psa concentration ( p=0.04).conclusionsthe preoperative psa mass is a better predictor of biochemical recurrence after rp than psa concentration . |
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the hospitals ' duty is beyond providing clinical and specialized services , and making plans for delivering health care services and promoting health is one of their important duties ( 1 ) . in each country , from 40% to 70% of health budget is absorbed by and spent in the hospitals and usually one to three percent of working population is employed in them ( 2 ) . on the other hand , the employees in hospitals suffer from high levels of job stress ( 3 ) . therefore , the plan of health promoting hospitals ( hph ) was launched more than a decade ago ( 45 ) . this plan refers to the fact that the hospital activities should be aimed not only at therapeutic and diagnostic activities ( 6 ) but also at disease prevention and health promotion , and the hospital services should be targeted to the needs of the people ( 7 ) . hence , developing a new strategy for hospital services and paying attention to the health promotion programs in the hospitals are essential in the present century , today , the hospitals are required which consider health promotion programs as a key service ( 89 ) . the issue of hph is paid more attention due to the increasing prevalence of lifestyle - related diseases and also chronic diseases , the changes in public expectations , the increasing number of chronic patients who need continuous support , and the number of hospital staff who daily exposure to psychological pressures and health risks ( 10 ) . on the other hand , health promotion is a concept that should be considered as a behavioral challenge ( 11 ) . in this context , and given the current problems in hospitals , 20 hospitals were selected officially by who from 11 european countries for a pilot project of ephp ( european pilot hospital project of hph ) in warsaw in april 1993 ( 12 ) and the international network of health promotion hospital was established ( 13 ) . these hospitals not only provide comprehensive and high quality medical and nursing services but also develop an organizational structure and culture assuming an active role for patients and staff in health promotion programs and actively cooperate with the community in health activities ( 2 ) . hospitals with the health promotion plan should take several measures to implement health promotion programs such as using organizational development processes , forming a joint committee of health promotion projects , providing regular reports by subgroups of the health promotion project , providing feedback , using public relations techniques , reporting and documentation systems ( 14 ) . in the model of hph , provided by the research and development center of hph in taiwan ( 11 ) , at first , the preliminary measures , including formation of a project implementation team and a health promoting hospital committee were taken and , then , the project planning and implementation were made . in many health promotion programs implemented in hospitals , other health promotion projects for implementing health promotion programs in the hospitals focus on managerial methods , sharing and using resources ( 17 ) , and collaboration with society organizations ( 18 ) . finally , many hospitals in the world have implemented the who hph program and have achieved cost - effectiveness and quality assurance of health services ( 1920 ) . however , only the peripheral levels of the health network system in iran play the role of prevention and hospitals are responsible for performing the traditional role of diagnosis and treatment and there is no clear plan to deliver health promotion services in the hospitals . although some of these services such as nutritional counseling are provided sporadically in some hospitals in iran , there has not been any defined structure for becoming a hph and very few studies ( 18 , 21 ) have been conducted in this field . therefore , this study aimed to determine factors affecting the establishment of a health promoting hospital in iran using factor analysis method . this study was done in 2013 , as an applied , cross - sectional and descriptive - analytical study in four steps , as follows : to develop a conceptual model of hph , the models of establishing and implementing health promotion programs , the executable models and the experiences of who - european pilot hospital project were reviewed and finally , the main dimensions of the model as a conceptual model were extracted . after determining the main dimensions of implementing health promotion project in hospitals , the validity and reliability of this questionnaire were approved using expert judgment and cronbach 's alpha ( 22 ) . first , four medical universities of medical sciences in four provinces of iran ( tehran , guilan , isfahan , shiraz ) were randomly selected using cluster sampling method . then , in the selected medical universities , a sample of 268 people was determined using the findings of pilot study and the following formula , assuming =0.1 , d= 0.1 and p = q= 0.5 , and was randomly selected among faculty members , managers and experts who had scientific and academic , administrative and managerial experiences in health promotion . n=(z2)(pq)d2
n=(1/64)2(pq)0/01s2=268 the measurement model was tested using confirmatory factor analysis ( cfa ) via amos graphics ( 18 ) . cfa is a structural equation modeling technique which is used to determine the model 's goodness of fit . in order to handle any missing data , evaluation of each model in cfa is based on considering a variety of fit indices . there are three categories of fit indices for model evaluation , including : 1 ) absolute fit indices , including gfa ( goodness of fit ) , agfi ( adjusted goodness of fit ) and rmsea ( root mean square error of approximation ) ; 2 ) comparative ( incremental ) fit indices , including nfi ( normed fit index ) and nnfi ( non - normed fit index ) ; and 3 . it has been suggested that researchers should report at least two indices from each category . if cfi , gfi , nfi , nnfi , ifi , rfi , and agfi are higher than 0.90 and rmsea and rmsri are less than 0.050 , the studied model will have desirable and appropriate fitness . to develop a conceptual model of hph , the models of establishing and implementing health promotion programs , the executable models and the experiences of who - european pilot hospital project were reviewed and finally , the main dimensions of the model as a conceptual model were extracted . after determining the main dimensions of implementing health promotion project in hospitals , a questionnaire was designed . the validity and reliability of this questionnaire were approved using expert judgment and cronbach 's alpha ( 22 ) . first , four medical universities of medical sciences in four provinces of iran ( tehran , guilan , isfahan , shiraz ) were randomly selected using cluster sampling method . then , in the selected medical universities , a sample of 268 people was determined using the findings of pilot study and the following formula , assuming =0.1 , d= 0.1 and p = q= 0.5 , and was randomly selected among faculty members , managers and experts who had scientific and academic , administrative and managerial experiences in health promotion . the measurement model was tested using confirmatory factor analysis ( cfa ) via amos graphics ( 18 ) . cfa is a structural equation modeling technique which is used to determine the model 's goodness of fit . in order to handle any missing data , evaluation of each model in cfa is based on considering a variety of fit indices . there are three categories of fit indices for model evaluation , including : 1 ) absolute fit indices , including gfa ( goodness of fit ) , agfi ( adjusted goodness of fit ) and rmsea ( root mean square error of approximation ) ; 2 ) comparative ( incremental ) fit indices , including nfi ( normed fit index ) and nnfi ( non - normed fit index ) ; and 3 . it has been suggested that researchers should report at least two indices from each category . if cfi , gfi , nfi , nnfi , ifi , rfi , and agfi are higher than 0.90 and rmsea and rmsri are less than 0.050 , the studied model will have desirable and appropriate fitness . most of the studied sample were managers ( 0.42 ) , male ( 62% ) , in the 4150 yr age group ( 46% ) , in the 2130 yr job experience group ( 48.2% ) , and had a master 's degrees ( 70% ) ( table 1 ) . six dimensions were determined as the dimensions of hph establishment which were as follows ( table 2 ) :
society and community assessment consisting of community needs assessment and epidemiological assessment ( i.e. the assessment of population health issues and disease patterns ) as its sub-dimensions.policy consisting of setting policies , implementing health promotion projects , attracting adequate funds and determining the related investors and sponsors for the implementation of health promotion projects , and mentioning the concept of health promotion in the hospital mission and goals as its sub-dimensions.management consisting of designing the projects and interventions required for a hph , creating and providing strategic leadership ( providing technical support for implementing projects ) , and developing strategies for implementing health promotion projects using data collected from society and policy dimensions.dissemination which means publishing health promotion programs and interventions and their results at international conferences and news media and arranging business meetings on health promotion in the hospital.technique . in order to implement a hph program , hospitals should have appropriate structures and techniques which the simplest one of them is the formation of a project team . another appropriate structure for implementing health promotion programs is a combination of project committees , project management , and project coordinator.evaluation . the purpose of this step is monitoring and evaluation of progress of each activity related to the implementation of health promotion programs . table 2:the means and standard deviations ( sds ) of hph model dimensionssocietypolicymanagementdisseminationtechniqueevaluation2.9.893.3.543.11.043.3.582.8.924.2.53 society and community assessment consisting of community needs assessment and epidemiological assessment ( i.e. the assessment of population health issues and disease patterns ) as its sub - dimensions . policy consisting of setting policies , implementing health promotion projects , attracting adequate funds and determining the related investors and sponsors for the implementation of health promotion projects , and mentioning the concept of health promotion in the hospital mission and goals as its sub - dimensions . management consisting of designing the projects and interventions required for a hph , creating and providing strategic leadership ( providing technical support for implementing projects ) , and developing strategies for implementing health promotion projects using data collected from society and policy dimensions . dissemination which means publishing health promotion programs and interventions and their results at international conferences and news media and arranging business meetings on health promotion in the hospital . technique . in order to implement a hph program , hospitals should have appropriate structures and techniques which the simplest one of them is the formation of a project team . another appropriate structure for implementing health promotion programs is a combination of project committees , project management , and project coordinator . the purpose of this step is monitoring and evaluation of progress of each activity related to the implementation of health promotion programs . the means and standard deviations ( sds ) of hph model dimensions among the dimensions of hph model , technique ( 2.80.92 ) and evaluation ( 4.20.53 ) had the highest and lowest means , respectively ( table 2 ) . moreover , society ( 0.97 ) and policy ( 0.74 ) had the highest regression coefficient ( effects ) and management had the lowest one ( fig . 1 ) . according to the results of model 's goodness of fit indices , all indices were within the acceptable range showing that a significant amount of variance had been assigned by the model . therefore , the conceptual model used in the present study was a valid and acceptable model ( table 3 ) . the hph model fit summary incremental index of fit / comparative fit index / chi - square /degree of freedom / parsimony normed of fit index / parsimony comparative fit index / root mean square error of approximation the results of the present study showed that the hph model had dimensions , including society , policy , management , dissemination , technique , and evaluation . in the current study , society with the highest regression weight ( 0.97 ) had the greatest impact on the health promotion in hospitals indicating the importance of this dimension ( 23 ) . community - based health care requires the society members ' involvement in improving their health . the reason for recognizing and understanding the community is that by collecting data from the assessment of community covered by the hospital , administrators and policy - makers can determine the health problems and preferences in the society . in an intervention program implemented in a hospital in south africa to move it towards a health promoting hospital , the needs assessment of staff , patients and their relatives was carried out at first , which is similar to the health and epidemiological needs assessment of the society groups in the present study ( 16 ) . the precede - proceed model is used to assess the perceptions of society members and to recognize the society 's health issues ( 24 ) , which is similar to the results of present study . the planned approach to community health focuses on the society 's facilities and possibilities assessment . in the present study , also , the assessment of society 's status has been one of the dimensions ( 24 ) . in some diseases such as cancer ( a specific disease ) , according to patients ' survival rate and the importance of increasing their longevity , it is necessary to determine the information needs of the patients ' health promotion ( 25 ) . in the current study , the toronto health promotion model ( 26 ) has studied the legal and political environment , stakeholders , population health needs , previous assessments , and the overall outlook for each project . in the present study , also , the political environment and its assessment has been paid attention . bangkok declaration ( 27 ) considers the existence of policy - making and participation as a central principle of national development in order to improve health and health equity . this can also be seen in the current study as the dimension of policy . for implementing hph programs , access to adequate budget ( 2831)and the health promotion policy ( 28 , 31 ) are important . using data obtained from the society and making the required policies , hospitals can develop and implement programs and interventions for health promotion ( the dimension of management ) . miseviciene and colleagues ( 32 ) have focused on the development of a strategic plan for implementing health promotion programs , which is similar to the dimension of management in the present study . in order to implement this dimension , providing strategic leadership ( providing technical support for implementing projects ) has been emphasized in the present study , which is similar to the results of lee and colleagues ' study ( 33 ) . in addition , johnson and baum ( 29 ) in their study concluded that top management support was important and necessary for implementing hph programs . to establish a hph , in fact , an efficient and effective hph is dependent on organizational and managerial prerequisites such as project management , etc . ( 35 ) , which is similar to the findings of the present study . in preston green hospital , the project management principles and appropriate organizational structure and processes have been used in order to achieve the objectives of health promotion ( 14 ) . in the present study , the effect of project implementation techniques on health promotion in the hospital with 0.53 loading factor was significant . the administrators of rudolf stiftung hospital have taken several measures to implement health promotion programs in their hospital such as employing the techniques of project implementation ( 23 ) , consistent with the results of present study . the results of other studies have supported the organizational structures as a prerequisite for establishing a comprehensive hph ( 36 ) , which confirm the results of current study . in the 2-year evaluation of the implementation and effects of a hph program in scotland , the existence of a framework and structure for implementing the health promotion in hospitals has been emphasized ( 37 ) . the hph coordinator is the minimum structure required for the membership of hospitals in the hph network ( 45 ) . the existence of a structure and framework for implementing health promotion programs in the present study has been referred to as technique . the health promotion program in hospitals in taiwan ( 15 ) has had four phases , including the formation of a project implementation team , formation of a health promotion committee , project planning and implementation , and evaluation of the effects of health promotion programs . the administrators of be jing hospital ( 28 ) have considered the dimensions of policy , reorientation of health care services , society , and health skills as the health promotion activities ( 28 ) which are similar to the dimensions of the current study conceptual model , as well as the design and implementation phases of health promotion in the proposed model of kouranie hospital ( 14 ) . in the present study , dissemination ( publishing health promotion programs and activities and their results at international conferences and news media and arranging business meetings on hph programs ) with a good loading factor ( 0.64 ) had a high impact on the implementation of health promotion programs in the hospital . the results of a study in taiwan have shown the similar results and have indicated that dissemination in the hph local and international conferences plays an important role in the establishment of a hph ( 38 ) . therefore , it can be said that if a hospital implement the health promotion programs well but is weak in the dissemination , it has not been able to introduce itself as a health promoting hospital . in a seven - dimension model developed by burke et al . ( 39 ) , which shows the needs for changing the organizational capacities to become a health promoting hospital , the development of mission and strategies , leadership and managerial activities have also been mentioned , which are similar to the results of the present study in which mentioning the concept of health promotion in the hospital mission and goals and developing strategies have been stated in the dimension of policy , and leadership and managerial activities in the dimension of management . like other studies , in the current study , the existence of planning and a specific technique and framework such as project management technique have been concluded as a dimension . some of obstacles to the implementation of health promotion programs include inefficient project management , the lack of communication , coordination and integration , and the lack of planning , guidelines , standards and frameworks for implementing health promotion programs ( 18 , 29 , 37 , 40 ) . all factors mentioned above have been stated in the present study as the dimensions of implementing health promotion programs in hospitals . the final dimension in the present study , the evaluation of health promotion programs and interventions , has been confirmed by the model of evaluating health promotion projects presented in the preston hospital ( 14 ) . although remarkable advances have been made in medical technologies , because of the increasing number of chronic patients who need permanent support and employees who are daily subjected to the psychological pressures , hospital managers and administrators should make plans to develop and implement health promotion programs and ensure healthy workplace in hospitals . the results of the present study showed that paying attention to six dimensions for establishing a hph is essential among which the most and least important ones were society and community assessment and management , respectively . it should be noted that considering all of these dimensions without making systematic planning ( 40 ) and organizational changes , providing health education for patients and determining factors affecting the establishment of a hph will not be effective after the researchers explained the purpose and procedures of the study to the participants , they consented to participate in the study . ethical issues ( including plagiarism , informed consent , double , etc . ) have been completely observed by the authors . | background : hospitals are the central entity of each health care system and health promoting hospitals ( hph ) was launched by who in 1988 .
however , there has not been any accurate and detailed model for establishing a hph in iran up to now .
therefore , this study aimed to determine factors affecting the establishment of a health promoting hospital in iran using factor analysis method.methods:this applied , cross - sectional and descriptive - analytical study was conducted in iran in four steps . confirmatory factor analysis ( cfa )
was used for determining factors affecting the establishment of a hph.results:society ( 0.97 ) and policy ( 0.74 ) had the highest regression weights ( effects ) and management had the lowest one.conclusion:community assessment was the most important dimension of proposed conceptual model for establishing a hph . |
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the research committee on intractable vasculitides , supported by the ministry of health , labour and welfare of japan , has conducted and promoted basic and clinical research on vasculitis since 1972 . we study 9 diseases : takayasu arteritis , temporal arteritis , polyarteritis nodosa , buerger disease , microscopic polyangiitis , granulomatosis with polyangiitis , eosinophilic granulomatosis with polyangiitis , antiphospholipid syndrome , and rheumatoid vasculitis . experts from several fields including nephrology , rheumatology , pulmonology , dermatology , cardiology , vascular surgery , pathology , epidemiology , and otorhinolaryngology work cooperatively . the present research committee on intractable vasculitides comprises 4 subcommittees under the direction of a principal investigator ( hirofumi makino):basic and pathological research subcommittee of vasculitis syndrome ( yasunori okada ) , clinical research subcommittee of small and medium - sized vessel vasculitis syndrome ( yoshihiro arimura ) , clinical research subcommittee of large - sized vessel vasculitis syndrome ( kazuo tanemoto ) , and international cooperation research subcommittee of vasculitis syndrome ( kazuo suzuki , shoichi fujimoto ) ( fig . crf case report form , anca antineutrophil cytoplasmic antibody , aav anca - associated vasculitis , dcvas diagnostic and classification criteria in vasculitis study , pexivas plasma exchange and glucocorticoid dosing in the treatment of anca - associated vasculitides , remit - jav - rpgn prospective cohort study of remission induction therapy in japanese patients with anca - associated vasculitides and rapidly progressive glomerulonephritis , co - remit - jav observational cohort study of remission maintenance therapy in japanese patients with anca - associated vasculitis , remit - jav prospective cohort study of remission induction therapy in japanese patients with anca - associated vasculitides overview of the tasks of the research committee on intractable vasculitides . crf case report form , anca antineutrophil cytoplasmic antibody , aav anca - associated vasculitis , dcvas diagnostic and classification criteria in vasculitis study , pexivas plasma exchange and glucocorticoid dosing in the treatment of anca - associated vasculitides , remit - jav - rpgn prospective cohort study of remission induction therapy in japanese patients with anca - associated vasculitides and rapidly progressive glomerulonephritis , co - remit - jav observational cohort study of remission maintenance therapy in japanese patients with anca - associated vasculitis , remit - jav prospective cohort study of remission induction therapy in japanese patients with anca - associated vasculitides since 2008 , we have conducted a retrospective cohort study elucidating risk factors associated with relapse in microscopic polyangiitis ( mpa ) patients and a nationwide epidemiologic study of eosinophilic granulomatosis with polyangiitis . to describe the current treatment status and evaluate the effectiveness of these treatments for japanese patients with all types of antineutrophil cytoplasmic antibodies ( anca)-associated vasculitides ( aav ) , we conducted a nationwide prospective cohort study of remission induction therapy in japanese patients with aav ( remit - jav ) . twenty - two university hospitals and referring hospitals participated in this study ; consecutive patients newly diagnosed with aav were enrolled from april 2009 to december 2010 . the criteria of primary systemic vasculitis proposed by the european medicines agency ( emea ) algorithm was employed for enrollment . this study was registered on the university hospital medical information network clinical trials registry ( umin000001648 ) . patients were evaluated at 3 , 6 , 12 , 18 , and 24 months and at relapse . the primary outcome measure was remission rate , and secondary outcome measures were survival rate , renal survival rate , and relapse . in total , 156 aav patients were enrolled ; all observations were completed by march 2013 . based on our retrospective study elucidating the risk factors for relapse in patients with myeloperoxidase ( mpo)-anca positive mpa , we are conducting an observational cohort study of remission maintenance therapy in japanese aav patients ( co - remit - jav ) ( umin000006373 ) . the study objective is to clarify the safety and efficacy of remission maintenance therapy in japanese aav patients . at present , 60 of 156 aav patients registered in remit - jav were extended to follow up every 6 months up to 48 months after the end of follow - up for remit - jav . the primary outcome measure is relapse rate , and secondary outcome measures are survival and renal survival rates . the observation stage will be completed by march 2015 ; data collection is currently in progress . after remit - jav , we conducted a nationwide , prospective cohort study of remission induction therapy in japanese patients with anca - associated vasculitides and rapidly progressive glomerulonephritis ( remit - jav - rpgn ) ( umin000005136 ) including 47 university hospitals and referring hospitals . enrollment of consecutive patients newly diagnosed with aav began in april 2011 and will continue till december 2013 . the primary and some secondary outcome measures are the same as those in remit - jav , but pathological analysis of renal involvement and radiological analysis of pulmonary involvement will be added . further , biological samples ( serum , urine , and total rna ) will be collected and offered to the basic and pathological research subcommittee for research for identifying candidate biomarkers . we also conducted a nationwide japanese prospective observational study on the current state and efficacy of therapeutics for large - vessel vasculitis ( umin000010414 ) . the subjects included patients newly diagnosed with takayasu arteritis and giant cell arteritis . the study objective was to clarify the current state and efficacy of therapeutics for large - vessel vasculitis in japan and to evaluate the utility of the current diagnostic criteria and classification for large - vessel vasculitis . the study began in november 2012 , and patients will be registered until march 2014 . the international cooperation research subcommittee is leading the effort to join some international collaborative clinical research studies : the diagnostic and classification criteria in vasculitis study ( dcvas ) ( nct01066208 ) , the plasma exchange and glucocorticoid dosing in the treatment of anca - associated vasculitis ( pexivas ) study ( nct00987389 ) , and a comparison study of phenotype and outcome in microscopic polyangiitis between europe and japan . a genome - wide association study in aav patients registered in the japanese clinical studies remit - jav and remit - jav - rpgn , and a prospective study of the severity - based treatment protocol for japanese patients with mpo - anca - associated vasculitis ( jmaav ) , is also in progress . | in japan , the research committee on intractable vasculitides , supported by the ministry of health , labour and welfare , has been promoting basic and clinical research on vasculitis since 1972 .
the present research committee on intractable vasculitides comprises 4 subcommittees under the direction of a principal investigator : basic and pathological research subcommittee , clinical research subcommittee of small and medium - sized vessel vasculitis , clinical research subcommittee of large - sized vessel vasculitis , and international cooperation research subcommittee .
since 2008 , 9 nationwide clinical studies for vasculitis have been conducted and 8 clinical and basic studies are in progress . |
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minipigs , or sometimes potbellied pigs , are strains of domestic pigs that are markedly smaller than farmyard varieties . for their low economic traits , the selection and cultivation of minipigs were far behind domestic farm pigs . because of anatomical and functional similarities to humans or because of availability of disease models , pigs begin to be used as research models in the field of skin , cardiovascular system , urinary system , and metabolic syndrome [ 13 ] , while the minipigs show a significant advantage over the domestic farm pigs for the reduced size which reduces the compound needs consequential prohibitive costs for the experiments and makes animals handling easier . the cultivation of miniature pigs began in the 1940s , and more than 10 strains had been used in biomedical researches till the 1980s . in europe , the gttingen minipig is the most popular breed used by pharmaceutical companies and contract research organizations . in the usa , strains of yucatan mini- and micropigs , sinclair minipigs , minnesota hormel minipigs , and hanford minipigs are widely used today . though these minipig strains are known as multispecies hybrid bred , their background information is clear and their hereditary stabilities are satisfied . china has rich resources of minipigs such as chinese wuzhishan ( wzs ) minipigs ( sus scrofa ) , guizhou minipigs , bama minipigs , and tibet minipigs , but the laboratory animal work is far behind the developed countries . compared with the strains used in developed countries , the chinese origin minipigs are formed naturally with high genetic homozygosity and stable phenotype , indicating that chinese origin minipigs are more ideal as laboratory animals for biomedical researches . wzs minipig is a special pig in china initially grazing in isolated tropical areas in hainan . it was firstly found and preserved in the 1980s ; after that , the laboratory wzs minipigs began to be cultivated and used in biomedical researches [ 7 , 8 ] . nowadays , one wzs minipigs colony for 20 generations of inbreeding has been established with a high inbreeding coefficient ( more than 0.965 ) [ 9 , 10 ] . here after more than ten years of phenotypic selection , downsizing cultivation , and microbiological standardization , we established an outbred wzs minipigs colony and highlighted its background data for its usefulness in modern laboratory animal science , especially for being suitable for long - term studies in which domestic swine breeds become too large . an outbred wzs minipigs colony with a core group ( 14 males and 30 females ) and an expanding group ( 20 males and 40 females ) was set as the objective . animals were bred in closed shelter with cooling water curtains and air supply fans in summer and infrared thermal equipment in winter . environmental temperatures were normally kept in 1829c and air exchanges were maintained at about 10 times per hour . body weight ( bw ) and body measurements were determined monthly up to 12 months and then at the interval of 6 months for bw . bw was taken by weighbridge and body measurements were made using the tailor 's tape measure . height - at - withers ( haw ) was measured as the straight - line distance from the withers to the base of the foot in a normal upright manner . heart girth ( hg ) , heart depth ( hd ) , and heart breadth ( hb ) represented the circumference , straight - line distance , and horizontal distance of the chest posterior to the scapula , respectively . shank girth ( sg ) showed the circumference of the lower parts of the foreleg . buttock - knee length ( bkl ) was measured from the most posterior point on either buttock to the anterior point of the knee . head length ( hl ) was measured from the distalmost dorsal point of the rhinarial pad to parietal . the reproductive performances of the colony were monitored continuously for at least 3 birth frequencies according to records for swine breeds ( gb 3038 - 82 ) , chinese national standards . reproductive behavior parameters include male first penis protruding time , male first ejaculation time , female puberty , and female gestation . litter performances include litter sizes , litter weights , and individual weights at birth , 21 d , and weaning ( 45 d ) . nineteen haematological indices were determined by automatic hematology analyzer ( sysmex xt-2000iv ) , and 12 blood biochemistry indices were determined by automatic biochemical analyzer ( hitachi 7020 ) . twenty - six commonly used microsatellite loci were employed to analyze the genetic diversity of the outbred wzs minipigs by multi - pcr as previous report . after that , the organ coefficients were counted , which were relative ( % of body weight ) organ weights . then the carcass properties were determined according to chinese technical regulation for testing of carcass traits in lean - type pigs ( ny / t825 - 2004 ) . variances of different time points in the same group were analyzed by one - way anova . all analyses were done by using sas software , version 8.01 ( sas institute , cary , north carolina ) . wzs minipigs have low body weight , slim body shape , and a small head with a long snout . each trotter ( foot ) has four hoofed toes with the two larger central toes bearing most of the weight . the dental formula of adult pigs is the same as domestic pigs , giving a total of 44 teeth . the most common appearance of wzs minipigs is a black or brown body with white abdomen and legs and a white triangle on its forehead . original wzs minipigs are normally active with sensitive response , while the temperament of outbred wzs minipigs tends to be gentle as domestic pigs . detailed data on temperament and behaviour of outbred wzs minipigs will be evaluated and compared with original and inbred wzs minipigs in future studies . the minipigs were weighed routinely at a month interval up to 12 months and then at the interval of 6 months . the body weights resemble gttingen minipigs , the famous and widely used minipigs [ 15 , 16 ] . the mean body weights of birth , 2 months old , 6 months old , and 12 months old are as follows : 0.57 0.11 kg , 4.54 1.66 kg , 12.11 2.99 kg , and 26.21 6.24 kg . the mean body weight of 6 months old is higher than that of wzs minipigs bred in original region ( 8.41 2.13 kg ) , but it is lower than that of wzs minipigs bred in beijing , nonoriginal region ( 13.43 3.27 kg ) . it is also lower than other chinese famous minipigs such as guizhou minipigs and banna mini- and micropigs . in figure 2 , as age increases , the body weights show a rapid increase at the first year ( 12 months ) and then increase slightly . body weight data were further analyzed by the following nonlinear theoretical models as described previously : logistic , gompertz , bertalanffy , and richards . though all the r of nonlinear theoretical models are above 0.99 , the gompertz growth model is more suitable for our research for the maximum body weight drawing from gompertz growth model is more practical ( figure 2 ) . according to the gompertz growth model , the maximum weight , inflection point of age , inflection point of body weight , and maximum daily increase of body weight are 45.82 kg , 230.11 d , 16.86 kg , and 0.0883 kg , respectively . fifteen indices of body measurements were carried out monthly up to 12 months and results were shown in table 2 . the mean values of body height , body length , chest depth , chest breadth , chest circumference , abdomen circumference , shank circumference , buttocks length , buttocks breadth , head length , forehead breadth , jaw width , caudal length , and caudal circumference of six - month - old wzs minipigs are as follows : 32.64 3.81 cm , 52.13 5.43 cm , 16.80 2.09 cm , 13.95 1.61 cm , 50.64 5.77 cm , 58.30 5.88 cm , 8.51 0.74 cm , 18.77 2.29 cm , 11.95 1.31 cm , 17.75 1.47 cm , 7.93 0.54 cm , 9.74 0.90 cm , 14.90 2.40 cm , and 5.10 0.68 cm . these data are a little higher than the values of wzs minipigs bred in original region and lower than the values of wzs minipigs bred in beijing . and they are also lower than chinese guizhou minipigs and banna mini- and micropigs . the mean body measurements values of 12-month - old wzs minipigs are as follows : 42.96 3.37 cm , 67.38 6.35 cm , 22.94 2.82 cm , 17.59 2.74 cm , 67.49 7.74 cm , 74.69 9.19 cm , 10.63 0.78 cm , 24.75 2.28 cm , 15.86 1.64 cm , 23.19 1.58 cm , 9.68 0.70 cm , 12.70 1.17 cm , 19.13 2.11 cm , and 6.72 0.72 cm . the body measurements changing curves are the same as that of body weight showing rapid to slight increase . but the increases of body measurements in 1st month are significantly sharper than other months . results show that wzs minipigs have the biological characteristics of small size and slow growing . the sexual mature time of males is earlier than that of females in wzs minipigs . ( n = 45 ) , and the first ejaculation time is 61.29 3.07 d ( n = 36 ) . for females , puberty , the age of females emerging first estrus , is 93.65 3.50 d. females in estrus will often assume the lordosis reflex , and the vulvar lips are swollen and red with a thin , mucous discharge . they also show some other signs of estrus including depressed appetite , restlessness , alertness , pacing , grunting , and chomping of the jaws . the period from the last end of estrus to this end , called estrus cycle , is 21.16 0.75 d for female wzs minipigs . and the behavioral estrus sustains 4.18 0.31 d. after fertilization , females will experience 113.0 2.33 d gestation till parturition . the overall litter characteristics of the present study are given in tables 4 and 5 , and the differences in litter performances are also shown in them . in table 4 , the effects on litter sizes caused by birth frequencies are illustrated . as birth frequencies increase , the total litter sizes at birth are increasing ; however , the survival numbers at birth , 21 d , and weaning ( about 45 d ) show no differences between each other . the mortality observed is comparable to values reported by others , showing that the larger the litter size the greater the mortality . the effects of birth frequencies on litter weights and individual weights , analyzed by the analysis of variance ( anova ) , are shown in table 5 . the litter weights show a small positive correlation with higher birth frequencies ( p > 0.05 ) , while these data provide no positive or negative correlations between birth frequencies and the individual weights at birth , 21 d , and weaning . hematology and blood biochemistry indices are important to animals for evaluation of their homeostasis and disease diagnosis . tables 6 and 7 present the background data of hematology and blood biochemistry for outbred wzs minipigs aged from 2 to 12 months and this range of ages represents the main range encountered in toxicological and biological studies [ 2 , 3 , 20 ] . in 19 haematological and 12 blood biochemical parameters , only eo , bun , chol , and tg show significant differences between male and female wzs minipigs . the main differences of haematological parameters between closed colony and inbred strain focus on indices of hemoglobin and blood platelet , and there were 6 different indices of biochemical parameters . twenty - six commonly used microsatellite loci were employed to analyze the genetic diversity of the outbred wzs minipigs by multi - pcr . results of 44 randomly selected animals showed that the mean allelic numbers per locus were 8.37 , the mean polymorphism information content ( pic ) was 0.7069 , and the mean heterozygosity ( h ) was 0.5478 . the mean allelic numbers per locus , mean pic , and mean h were significantly higher than those of inbred wzs minipigs reported elsewhere , which were 6.92 , 0.6042 , and 0.4478 , respectively . yao et al . had employed twenty - seven microsatellite markers recommended by the food and agriculture organization ( fao ) and the international society for animal genetics ( isag ) to analyze the genetic diversities of wzs minipigs bred in original region , hainan , china . that the outbred wzs minipigs are genetically stabilized breed with high heterozygosity and maintain the original characteristics . seventy - four healthy wzs minipigs ( 43 males and 31 females ) aged from 5 to 20 months were chosen to determine 6 organs weights and organ coefficients . student 's t - test is employed to analyze the differences of organ weights and organ coefficients between males and females of the same age range . only few organ weights of different aged animals show significant difference between males and females ( p < 0.05 ) ; they are lung weight of animals aged 59 months and kidney weight of animals aged 1015 months , but there was no significant difference for the organ coefficients ( p > 0.05 ) . furthermore , the effects of age factor on organ weights and organ coefficients were analyzed by anova . results of anova showed that organ weights increased significantly in accordance with the growths and increases of body weights ( p < 0.0001 ) ; the organ coefficients of heart , liver , lung , and stomach decreased significantly with the growth of animals ( p < 0.0001 ) , but the decreases of spleen and kidney coefficients were not significant ( p > 0.05 ) . sun and lu had reported some pork characteristics of wzs minipigs based on 3 young and 3 adult minipigs . in this study , we extensively determined the carcass properties of 11 adult wzs minipigs aged from 6 to 10 months . all detected carcass traits are shown in tables 9 and 10 . according to table 9 , there are no significant differences between males and females for all carcass properties . when compared to the same age range domestic pigs , all the traits of wzs minipigs the comparisons between wzs minipigs and tibet minipigs in china show that skin thickness , back fat thickness , bone percentage , and skin percentage of wzs minipigs are higher than those of tiber minipigs . besides the above traits , the eye muscle area of wzs minipigs is also higher than that of tibet minipigs for females . results of sensory characters in table 10 demonstrate that wzs minipigs are of perfect meat color , meat elasticity , and less marbling . in conclusion , we report the detailed biological characteristics of outbred wzs minipigs cultivated for more than ten years . series of background parameters have been detected out and proved to be stable in outbred wzs minipigs . these results indicate that outbred wzs minipigs are suitable laboratory animals and could be widely used in comparative medicine and bioresearches in future . | in order to meet the demands of experimental minipigs for biomedical researches , we have aimed at cultivating grazing chinese wuzhishan ( wzs ) minipigs and trying to make them useful and affordable since the 1990s . after more than ten years of captive cultivation following sound management practices and a rigorous selection program for fertility and litter size
, we established an outbred wzs minipigs colony with a core group ( 14 males and 30 females ) and an expanding group ( 20 males and 40 females ) . in 20102013 periods ,
extensive background data of this colony were recorded and analyzed .
this paper was written to provide pertinent information about outbred wzs minipigs for producers , users , and others concerned with wzs minipigs .
it contains physical characteristics , growth performance , productive performance , hematology and blood biochemistry , microsatellite analysis , organ coefficients , and carcass properties .
results show that wzs minipigs have characteristics of small body size , slow growth rate , long life cycle , high reproductive rate , and maintaining original genetic diversity .
all data present that outbred wzs minipigs are suitable laboratory animal and model animal . |
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a similar pattern is reported in many other parts of the world.24 the rise in the number of acute admissions imposes both a substantial organizational challenge to and a considerable financial strain on the health care system . stratification by admission type is used in studies of prognosis , in the surveillance of health care quality , and in the reimbursement of hospital costs between the hospitals and the state . valid data on the admission type are therefore pertinent from both an administrative and a research perspective . to our knowledge , only two national reports evaluating the admission type registered in the danish national patient registry ( dnpr ) exist.5,6 one was based on a random sample of 1094 hospital admissions in 1990.5 the evaluation covered medical , surgical , gynecological , orthopedic , and pediatric departments at a national level . more recently , a second report included a validation of the admission type across surgical and gynecology departments.6 this report estimated a 3% misclassification in admission type . the aim of the present study is to examine the validity of the registration of acute admissions among medical patients in the dnpr using medical record review as the reference standard . we conducted this cross - sectional validation study in the north denmark region , covering 580,000 inhabitants ( 10% of the total danish population ) . the danish population has unrestricted and unfettered access to tax - supported health care , guaranteeing equal access to treatment and hospital admission . we used the dnpr to identify a sample of 160 medical patients admitted to hospital in the north denmark region during 2009 . medical patients were defined as patients with an admission to any of the medical departments in the study area . in the dnpr , the admission type is assigned by a secretary upon admission and is either acute or nonacute . each year , the national board of health provides a guideline for correct registration . for the purpose of this study , oncology departments were not included because both surgical and nonsurgical cancer patients are referred to this department . the identified medical patients were admitted to15 different medical departments , of which nine were highly specialized departments . the dnpr contains information on all hospital admissions to nonpsychiatric hospitals since 1977 and all hospital contacts to emergency rooms and outpatient clinics since 1995.7 information on the admission type has been included in the dnpr since 1987 . besides the admission type , other information includes dates of referral , admission , and discharge , data on the hospital and each department , diagnostic codes , and surgical procedures . diagnostic codes include one principal diagnosis reflecting the main reason for hospital admission and up to 19 secondary discharge diagnoses coded according to the international classification of diseases ( icd ) , 8th revision until 1993 ( icd-8 ) , and the 10th revision ( icd-10 ) thereafter . each hospital electronically transfers administrative and clinical data from their patient administrative system to the dnpr . the dnpr is managed by the national board of health , and reporting to the dnpr is mandatory . based on the diagnosis related group system , information from the dnpr is extracted and used for the purpose of financial reimbursement between the hospitals and the state . the unique personal identification number assigned to all danish residents , as well as the hospital and department codes registered for the sampled medical inpatients , were used to retrieve the patients medical records for review . each medical record review was initiated with a verification of the patient s personal identification number and the date of hospital admission . all medical records were reviewed by the same physician ( bvh ) . through the review process , we confirmed the admissions to be acute if the attending physician used the word acute in the sentences concerning the type and reason for admission , or if it was stated that the admission was unscheduled . data on lifestyle factors are usually not available from administrative registries , but may be available from medical records . we therefore included data on smoking status , alcohol abuse ( more than 14 or 21 standard drinks in 1 week for women and men , respectively ) , and weight and height in order to calculate body mass index ( bmi ) from the medical records to demonstrate availability of these variables . we described the sample in terms of gender , age , smoking status , alcohol abuse , and bmi . age was described with the median age and the associated interquartile range ( iqr ) . the concordance between admission type in the dnpr and in the medical records was ascertained with estimates of the positive predictive value ( ppv ) , sensitivity , and specificity with corresponding 95% confidence intervals ( ci).8 we estimated 95% cis using jeffrey s method for a binomial proportion.9 data collected through the medical record review were used as the reference standard . to estimate the ppv of the registration of acute admissions in the dnpr , we computed the proportion of patients registered in the dnpr with an acute admission which was confirmed by medical record review ( ie , the numerator was the number of patients registered with an acute admission in both data sources , and the denominator was the number of patients registered with an acute admission in the dnpr ) . sensitivity was estimated with the numerator being the number of patients registered with an acute admission in both data sources , and the denominator being the total number of patients confirmed by medical record review to have an acute admission . the specificity was estimated with the numerator being the number of patients registered with a nonacute admission in both data sources , and the denominator being the total number of patients confirmed by medical record review to have a nonacute admission . furthermore , we estimated ppv , sensitivity , and specificity for each hospital . in a subsequent analysis , we restricted the analysis to the medical patients arriving through the emergency room to confirm whether the patients were registered with an acute admission when becoming an inpatient . sensitivity analysis using a different approach was conducted ; it included data from the dnpr on the date of referral and date of admission , as the dates are expected to be the same for acute admissions and different for nonacute admissions . data were analyzed with the statistical software package stata ( version 11 ; stata corp , college station , tx , usa ) . the study was approved by the danish data protection agency ( record number 2006 - 53 - 1396 ) . we conducted this cross - sectional validation study in the north denmark region , covering 580,000 inhabitants ( 10% of the total danish population ) . the danish population has unrestricted and unfettered access to tax - supported health care , guaranteeing equal access to treatment and hospital admission . we used the dnpr to identify a sample of 160 medical patients admitted to hospital in the north denmark region during 2009 . medical patients were defined as patients with an admission to any of the medical departments in the study area . in the dnpr , the admission type is assigned by a secretary upon admission and is either acute or nonacute . each year , the national board of health provides a guideline for correct registration . for the purpose of this study , oncology departments were not included because both surgical and nonsurgical cancer patients are referred to this department . the identified medical patients were admitted to15 different medical departments , of which nine were highly specialized departments . the dnpr contains information on all hospital admissions to nonpsychiatric hospitals since 1977 and all hospital contacts to emergency rooms and outpatient clinics since 1995.7 information on the admission type has been included in the dnpr since 1987 . besides the admission type , other information includes dates of referral , admission , and discharge , data on the hospital and each department , diagnostic codes , and surgical procedures . diagnostic codes include one principal diagnosis reflecting the main reason for hospital admission and up to 19 secondary discharge diagnoses coded according to the international classification of diseases ( icd ) , 8th revision until 1993 ( icd-8 ) , and the 10th revision ( icd-10 ) thereafter . each hospital electronically transfers administrative and clinical data from their patient administrative system to the dnpr . the dnpr is managed by the national board of health , and reporting to the dnpr is mandatory . based on the diagnosis related group system , information from the dnpr is extracted and used for the purpose of financial reimbursement between the hospitals and the state . the unique personal identification number assigned to all danish residents , as well as the hospital and department codes registered for the sampled medical inpatients , were used to retrieve the patients medical records for review . each medical record review was initiated with a verification of the patient s personal identification number and the date of hospital admission . all medical records were reviewed by the same physician ( bvh ) . through the review process , we confirmed the admissions to be acute if the attending physician used the word acute in the sentences concerning the type and reason for admission , or if it was stated that the admission was unscheduled . data on lifestyle factors are usually not available from administrative registries , but may be available from medical records . we therefore included data on smoking status , alcohol abuse ( more than 14 or 21 standard drinks in 1 week for women and men , respectively ) , and weight and height in order to calculate body mass index ( bmi ) from the medical records to demonstrate availability of these variables . we described the sample in terms of gender , age , smoking status , alcohol abuse , and bmi . age was described with the median age and the associated interquartile range ( iqr ) . the concordance between admission type in the dnpr and in the medical records was ascertained with estimates of the positive predictive value ( ppv ) , sensitivity , and specificity with corresponding 95% confidence intervals ( ci).8 we estimated 95% cis using jeffrey s method for a binomial proportion.9 data collected through the medical record review were used as the reference standard . to estimate the ppv of the registration of acute admissions in the dnpr , we computed the proportion of patients registered in the dnpr with an acute admission which was confirmed by medical record review ( ie , the numerator was the number of patients registered with an acute admission in both data sources , and the denominator was the number of patients registered with an acute admission in the dnpr ) . sensitivity was estimated with the numerator being the number of patients registered with an acute admission in both data sources , and the denominator being the total number of patients confirmed by medical record review to have an acute admission . the specificity was estimated with the numerator being the number of patients registered with a nonacute admission in both data sources , and the denominator being the total number of patients confirmed by medical record review to have a nonacute admission . furthermore , we estimated ppv , sensitivity , and specificity for each hospital . in a subsequent analysis , we restricted the analysis to the medical patients arriving through the emergency room to confirm whether the patients were registered with an acute admission when becoming an inpatient . sensitivity analysis using a different approach was conducted ; it included data from the dnpr on the date of referral and date of admission , as the dates are expected to be the same for acute admissions and different for nonacute admissions . data were analyzed with the statistical software package stata ( version 11 ; stata corp , college station , tx , usa ) . the study was approved by the danish data protection agency ( record number 2006 - 53 - 1396 ) . table 1 displays information on age , smoking , alcohol abuse , and bmi of the patients confirmed by the medical record review to be correctly registered with an acute or nonacute admission in the dnpr . the acute patients were slightly younger ( median age of 62 years [ iqr 4980 ] ) , on average , than the nonacute patients ( median age of 63 years [ iqr 5269 ] ) . among the acute patients , 49.6% were males , in contrast to 67.7% of the nonacute patients . the medical records lacked data on smoking status for only 13.4% of the acute patients and for 16.1% of the nonacute patients . data on weight and height in order to compute bmi was missing for 47.2% of the acute patients and for 35.4% of the nonacute patients . data on alcohol abuse were missing for 23.6% of the acute patients and for 51.6% of the nonacute patients ( table 1 ) . all 160 sampled inpatients had a registration of either acute or nonacute admission in the dnpr . in total , 128 ( 80.0% ) were registered as acute and 32 ( 20.0% ) were registered as nonacute admissions in the dnpr . in the review process , two medical records the final study population , on which the estimates were computed , included 158 medical inpatients , of which 127 were registered in the dnpr with an acute admission and 31 with a nonacute admission . of the 127 medical patients registered with an acute admission , we confirmed 124 to be an acute admission based on our review of the medical records . among the three nonconfirmed acute admissions , of the 31 medical patients registered with a nonacute admission in the dnpr , three should have been registered with an acute admission . the overall ppv of acute admissions was 97.6% ( 95% ci , 93.8%99.3% ) when using the medical record review as the reference standard . sensitivity was 97.6% ( 95% ci , 93.8%99.3% ) and specificity was 90.3% ( 95% ci , 76.4%97.2% ) ( table 2 ) . the analysis stratified by each hospital did not yield any major differences in results ( data not shown ) . when restricting the analysis to the 21 patients who visited the emergency room before admission , we confirmed that all 21 patients were correctly registered with an acute admission in the dnpr . among the six misclassified patients , one patient was transferred from another department during the course of the acute disease , which might explain the misclassification as a nonacute patient . no obvious explanations for the registration error of the remaining five misclassified patients could be found . the median age was 71 years for the misclassified acute patients ( ie , patients with a nonacute admission in the dnpr confirmed to be acute admissions through the medical record review ) compared with a median age of 62 years of the correctly classified acute patients . the characteristics of the misclassified nonacute patients ( ie , patients with an acute admission in the dnpr confirmed to be nonacute admissions through the medical record review ) were not different from those of the correctly classified patients . a sensitivity analysis comparing referral date and admission date registered in the dnpr confirmed the results from the review of the medical records except in one patient . this one patient was registered with an acute admission in the dnpr , but the medical record review showed that the patient had a scheduled admission . in the sensitivity analysis , the referral date was the same as the admission date , which confirmed the acute admission . table 1 displays information on age , smoking , alcohol abuse , and bmi of the patients confirmed by the medical record review to be correctly registered with an acute or nonacute admission in the dnpr . the acute patients were slightly younger ( median age of 62 years [ iqr 4980 ] ) , on average , than the nonacute patients ( median age of 63 years [ iqr 5269 ] ) . among the acute patients , 49.6% were males , in contrast to 67.7% of the nonacute patients . the medical records lacked data on smoking status for only 13.4% of the acute patients and for 16.1% of the nonacute patients . data on weight and height in order to compute bmi was missing for 47.2% of the acute patients and for 35.4% of the nonacute patients . data on alcohol abuse were missing for 23.6% of the acute patients and for 51.6% of the nonacute patients ( table 1 ) . all 160 sampled inpatients had a registration of either acute or nonacute admission in the dnpr . in total , 128 ( 80.0% ) were registered as acute and 32 ( 20.0% ) were registered as nonacute admissions in the dnpr . in the review process , two medical records could not be located and these admissions were therefore not included in the analysis . the final study population , on which the estimates were computed , included 158 medical inpatients , of which 127 were registered in the dnpr with an acute admission and 31 with a nonacute admission . of the 127 medical patients registered with an acute admission , we confirmed 124 to be an acute admission based on our review of the medical records . among the three nonconfirmed acute admissions , of the 31 medical patients registered with a nonacute admission in the dnpr , three should have been registered with an acute admission . the overall ppv of acute admissions was 97.6% ( 95% ci , 93.8%99.3% ) when using the medical record review as the reference standard . sensitivity was 97.6% ( 95% ci , 93.8%99.3% ) and specificity was 90.3% ( 95% ci , 76.4%97.2% ) ( table 2 ) . the analysis stratified by each hospital did not yield any major differences in results ( data not shown ) . when restricting the analysis to the 21 patients who visited the emergency room before admission , we confirmed that all 21 patients were correctly registered with an acute admission in the dnpr . among the six misclassified patients , one patient was transferred from another department during the course of the acute disease , which might explain the misclassification as a nonacute patient . no obvious explanations for the registration error of the remaining five misclassified patients could be found . the median age was 71 years for the misclassified acute patients ( ie , patients with a nonacute admission in the dnpr confirmed to be acute admissions through the medical record review ) compared with a median age of 62 years of the correctly classified acute patients . the characteristics of the misclassified nonacute patients ( ie , patients with an acute admission in the dnpr confirmed to be nonacute admissions through the medical record review ) were not different from those of the correctly classified patients . a sensitivity analysis comparing referral date and admission date registered in the dnpr confirmed the results from the review of the medical records except in one patient . this one patient was registered with an acute admission in the dnpr , but the medical record review showed that the patient had a scheduled admission . in the sensitivity analysis , the referral date was the same as the admission date , which confirmed the acute admission . in this validation study , we found a high ppv , sensitivity , and specificity of the registration of acute medical admissions in the dnpr . the variable for acute admissions is widely used , but to our knowledge no validation studies have previously been published . our study has several strengths , including the use of the large and virtually complete registry , the dnpr , which allows individual - level linkage to other databases.1012 in addition , we were able to ascertain both sensitivity and specificity , because our sample of medical inpatients included both acute and nonacute admissions , and the sample size was sufficient to provide reasonable precision of the estimates . first , the medical record reviewer was not blinded to the admission type registered in the dnpr , which might have led to results that were more concordant than they truly are . nonetheless , consistency in phrasing was high regarding admission type since the admission type and reason for admission were often stated in the first sentence of the admission note . third , no time trend in the reporting of acute and nonacute admissions is considered , as we only included data from 1 year . however , our results are quite similar to the findings in the previous report from 2004 , which might indicate no major changes in the intervening 5-year period . this might limit generalizability to other regions or counties , although the region includes 15 different medical departments , which are likely representative of other medical departments in the country . all 15 medical departments received direct referrals from general practitioners . despite these limitations , our findings were confirmed by a sensitivity analysis showing that virtually all acute patients were admitted the same day as they were referred to hospital , and that all nonacute patients had a referral date prior to the admission date . our results are comparable to the previous results in terms of the correct classification of acute admissions in 97% and 98.6% of cases.5,6 the latest validation of administrative data dates back to 2004 and includes only data from surgical and gynecology departments.6 the first report from 1990 included data on medical patients , but the results are reported in an overall analysis and missing data on admission type are considered correctly classified , which may cause an overestimation of the data quality.5 the results of the present study show that the admission type registered in the dnpr is valid . our study has important perspectives for future studies of acute medical admissions based on the dnpr . in conclusion , we found that the registration of acute admission among medical patients in the dnpr has a high validity . | backgroundin recent years , the number of acute hospital admissions has increased and this has imposed both organizational and financial strains on the health care system . consequently , it is of crucial importance that we have valid data on admission types in the administrative databases in order to provide data for health care planning and research.objectiveto examine the validity of registration of acute admissions among medical patients in the danish national patient registry ( dnpr ) using medical record reviews as the reference standard.methodswe used the nationwide dnpr to identify a sample of 160 medical patients admitted to a hospital in the north denmark region during 2009 .
data on admission type was obtained from the dnpr and confirmed by a medical record review .
we computed positive predictive values , sensitivity , and specificity including 95% confidence intervals ( ci ) using the medical record review as the reference standard.resultsamong the 160 medical inpatients identified in the dnpr , 128 were registered with an acute admission , and 32 were registered with a nonacute admission .
two medical records could not be located .
thus , the analyses included 158 medical patients . among the 127 patients registered with acute admission , 124
were confirmed to be correctly classified .
correspondingly , 28 of the 31 patients with a registered nonacute admission were confirmed to be correctly classified .
the overall positive predictive value of the acute admissions among medical patients was 97.6% ( 95% ci , 93.8%99.3% ) .
sensitivity was 97.6% ( 95% ci , 93.8%99.3% ) and specificity was 90.3% ( 95% ci , 76.4%97.2%).conclusionthe registration of acute admission among medical patients in the dnpr has high validity . |
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hydroxychloroquine can induce adverse effects on the central nervous system , from irritability , nervousness , and emotional changes to true psychoses.doses greater than 6 mg / kg / day , pharmacokinetic interactions , a personal or family psychiatric history and the disease per se for which hcq is used can represent important favoring conditions.in susceptible individuals , hydroxychloroquine can exert a stimulant effect on the central nervous system in the absence of these favoring conditions , probably as a consequence of an individual s hypersensitivity . hydroxychloroquine ( hcq ) is a synthetic antimalarial drug derived from 4-aminoquinoline ; it has been used for several decades for the treatment of some rheumatic diseases such as rheumatoid arthritis ( ra ) . a dosage between 3 and 6 mg / bodyweight / day is considered therapeutic . in obese individuals , the dosage must be assessed considering the patient s ideal bodyweight [ 1 , 2 ] . hydroxychloroquine may potentially result in adverse effects on the central nervous system , mainly irritability , nervousness , emotional changes , nightmares , and even true psychoses [ 3 , 4 ] . we describe the case of an elderly person with elderly - onset rheumatoid arthritis ( ra ) , where the use of therapeutic doses of hcq was followed by the appearance of psychomotor agitation . edc was an 80-year - old caucasian woman affected with ra since 78 years of age . she was receiving pharmacologic treatment with methotrexate ( mtx 10 mg weekly subcutaneously ) , oral folate supplementation ( folic acid 5 mg , 24 h after mtx ) , and low doses of oral corticosteroids ( 6-methylprednisolone 8 mg / day in the first 2 weeks , then 4 mg / day for a further 2 weeks ; and then administered during exacerbations only for a short time ) . her comorbidities were : ( 1 ) familiar hypercholesterolemia ( she used pravastatin 20 mg after dinner ) ; ( 2 ) high blood pressure ( she used amlodipine 5 mg daily ) ; and ( 3 ) non - hemodynamically significant carotid atheromasia ( she used acetylsalicylic acid 100 mg at lunchtime ) . after 2 years , ra was stable with an overall minimal disease activity , with a 28-item disease activity score of 2.6 and a clinical disease activity index of 3.1 . the patient asked the rheumatologist to stop mtx because she complained of general malaise and fatigue , which significantly affected her quality of life . it was proposed to take one tablet of hcq / day ( hcq 200 mg ; 4 mg / kg / day ) ; her weight was 52 kg and her body mass index was 20.06 kg / m . ten days later she developed , while she was healthy , significant psychomotor agitation with marked physical and verbal violence towards her partner , including throwing objects at her partner . no disturbance in sleep and no anxiety , nervousness , or irritability had emerged prior to the onset of her psychomotor agitation . it was necessary to transport edc to the hospital s emergency room in her city , where she was administered half a vial of intramuscular promazine ( equal to 25 mg ) . the agitation ceased in less than 1 h and there were no relapses or sequelae . brain magnetic resonance imaging plus contrast medium was performed ; it was negative for vascular lesions or neoplastic diseases and only a mild age - related atrophy was found . because no other potential triggers were identified , hcq administration was interrupted . at a consultation 2 week later , we did not attribute the agitation to hcq use and advised to introduce it again . the patients was treated with promazine drops ( 15 drops equal to 30 mg ) and hcq was stopped again the agitation disappeared . the administration of a placebo did not cause the onset of agitation to the patient . when we applied the naranjo scale to our patient , she had a score of 9 and this authorized the diagnosis of a defined ( and not random ) adverse drug reaction . one year later , the patient presented with neither cognitive impairment nor psychosis , with no further episodes of agitation . the patient s cognition , in particular , was assessed using the mini - mental state examination by folstein et al . in the italian version validated by magni et al . ; her score was equal to 26 . at present , she is taking pravastatin , amlodipine , and acetylsalicylic acid with small doses of corticosteroids ( 4 mg of 6-methylprednisolone or 5 mg of prednisone ) as needed . the ra is stable and well controlled ( 28-item disease activity score of 2.8 ; clinical disease activity index score of 3.1 ) . it is more polar , less lipophilic , and has more difficulty in diffusing across cell membranes compared with chloroquine . near - complete absorption following an oral dose occurs within 24 h and is relatively unaffected by concomitant ingestion of food . variability in the extent of absorption leads to differences in steady - state hcq concentrations among patients , potentially contributing to the variability in response observed in clinical practice . hydroxychloroquine has a large volume of distribution owing to extensive sequestration of the drug by tissues . in particular , a plasma volume of distribution up to 44,257 l for hcq has been reported [ 12 , 13 ] . drug disposition proceeds in three phases : distribution from blood to tissues , equilibration between blood and tissues , and release from tissues back into blood [ 11 , 13 , 14 ] . these phases have half - lives of 38 , 40216 h , and 3060 days , respectively . the most commonly quoted median value for the terminal elimination half - life is 40 days . metabolism of hcq occurs by dealkylation in the liver ; the two most important metabolites are desethyl chloroquine and bisdesethyl chloroquine , both of which have pharmacologic activity and are thought to be approximately as toxic as the parent compounds . thirty to 79% of an oral dose of hcq is metabolized and 2170% is excreted without metabolism . at steady state , cytochrome p450 3a4 inhibitors such as ketoconazole , cimetidine , and ciprofloxacin may increase the half - lives of hcq [ 16 , 17 , 18 ] . the possible hcq - induced neuropsychiatric side effects depend on its ability to cross the blood brain barrier . in the brain , hcq can have a tissue concentration 1020 times higher than a plasma concentration [ 2 , 14 ] . for example , the induction of a cholinergic imbalance with acetylcholine reduction , probably mediated by prostaglandin e and interleukin-1 ; the accumulation of metabolic and toxic wastes as a result of lysosome dysfunction , where hcq accumulates ; the down - regulation of p - glycoprotein at the level of the blood brain barrier . finally , the possibility that it may be an idiosyncratic or bizarre - type adverse drug reaction must be considered . other antimalarial drugs such as chloroquine and mefloquine can cause neuropsychiatric side effects [ 2024 ] . there are only a few case reports of psychosis due to hcq [ 3 , 18 , 25 , 26 ] . our patient used a dose of hcq equal to 4 mg / bodyweight / day ; this dosage is within the therapeutic range . none of the three molecules taken from our patient was within the categories of drugs causing interaction with hcq . psychomotor agitation can not be considered a manifestation of elderly - onset ra ; neurological and psychiatric manifestations in the course of ra mostly depend on vasculitis , opportunistic infections , and accelerated atherosclerosis , all conditions excluded in our patient . therefore , the ra was ( at the time of the introduction of hcq ) in a minimal activity and typically there is a close relationship between disease activity and ra extra - articular manifestations . our patient was not receiving corticosteroid treatment and this aspect needs to be highlighted . in other case reports regarding the relationship between neuropsychiatric manifestations and therapy with hcq , some important biases were present , such as important pharmacokinetic interactions ( 18 ) , a potential effect of the rheumatic disease on the neuropsychiatric manifestations ( 3 ) , and a personal and/or family history of psychiatric disorders ( 25 , 26 ) . furthermore , the bodyweight ( real or ideal ) of the patient was not always reported or properly assessed in other case reports . these side effects usually appear with high doses of the drug ( > 6 mg / bodyweight / day ) or in the presence of favoring elements ( pharmacokinetic interactions , personal and family psychiatric history , the disease for which hcq is used ) . to our knowledge , the possibility that hcq may cause psychomotor agitation in the absence of such favoring elements has not been reported . the disappearance of agitation after targeted pharmacologic intervention and hcq interruption , its reonset following reintroduction of the drug and the score of naranjo s algorithm are surely linked to the existence of a causal relationship between hcq and psychomotor agitation . furthermore , 1 year after the discontinuation of hcq , our patient presented with no further episodes of psychomotor agitation or other psychotic manifestations and she had good cognitive performance . we can hypothesize that in susceptible people , for unknown reasons , hcq might exert a stimulant effect on the central nervous system resulting in agitation onset that disappears on drug interruption and is responsive to sedative treatments . the neuropsychiatric manifestations described in our patient could be considered a bizarre - type adverse drug reaction linked to an individual s hypersensitivity . ciro manzo , pietro gareri , and alberto castagna declare they have no conflicts of interest directly relevant to the content of this case report . | we describe the case of an elderly woman with elderly - onset rheumatoid arthritis , where the use of 4 mg / kg / day of hydroxychloroquine ( hcq ) was followed by the onset of psychomotor agitation with marked physical and verbal violence towards her partner , including throwing objects at her partner .
no disturbance in sleep and no anxiety , nervousness , or irritability had emerged before the onset of her psychomotor agitation .
the disappearance of agitation following targeted pharmacologic intervention and hcq interruption , its re - onset after reintroduction of the drug , and the high score ( 9 ) of naranjo s algorithm are surely linked to the existence of a causal relationship between hcq and psychomotor agitation .
hcq may produce undesirable effects on the central nervous system , mainly irritability , nervousness , emotional changes , and nightmares . to the best of our knowledge , there are only a few case reports of psychosis due to hcq .
no favoring condition such as pharmacokinetic interactions or a personal and family psychiatric history was present in our patient .
the neuropsychiatric manifestations we observed could be considered a bizarre - type adverse drug reaction linked to an individual s hypersensitivity . |
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approximately 1,000 type 2 diabetic patients who were at least 18 years of age receiving regular follow - up at the outpatient clinics of taipei veterans general hospital were invited to participate in this study . patients with cardiovascular disease , malignancy , pregnancy , and acute intercurrent illness were excluded . seven hundred and seventy - seven participants provided written informed consent and subsequently received examinations for serum creatinine , urine albumin excretion , blood pressure , fasting blood glucose , hba1c , serum cholesterol , triglyceride , and eye fundus photographs ( supplementary fig . after exclusion of subjects with baseline advanced diabetic retinopathy that meant proliferative diabetic retinopathy , postlaser photocoagulation , or vitrectomy and those who had missing egfr or urine albumin - to - creatinine ratio ( uacr ) data , 579 subjects had data for both egfr and uacr . among the 579 subjects , 92 patients with egfr < 30 ml / min/1.73 m and/or uacr > 300 mg / g cr were excluded . moderate renal impairment was defined as baseline egfr 3059.9 ml / min/1.73 m , as well as normoalbuminuria as baseline uacr < 30 mg / g cr and microalbuminuria as baseline uacr 30299.9 mg / g cr . finally , a total of 487 subjects were enrolled in the study and categorized into four groups , including egfr 60 ml / min/1.73 m and normoalbuminuria ( group 1 ) , moderate renal impairment and normoalbuminuria ( group 2 ) , egfr 60 ml / min/1.73 m and microalbuminuria ( group 3 ) , and moderate renal impairment and microalbuminuria ( group 4 ) . the study was approved by the institutional review board of the taipei veterans general hospital . blood pressure was taken twice by using an electric blood pressure monitor in the sitting position after 10 min of rest . based on medical records , information about duration of diabetes and prescribed medication was obtained . venipuncture was performed , and a fasting serum sample was collected for the measurement of diabetic profiles , serum creatinine , and other biochemical tests . egfr was calculated by computer using the modification of diet in renal disease ( mdrd ) equation ( 9 ) . urinary albumin excretion was determined as the uacr from the mean of two spot urine samples on two consecutive mornings . color photographs of the retinas were taken according to the methodology of the accord ( action to control cardiovascular risk in diabetes ) eye study ( 10 ) , which was slightly modified from the early treatment diabetic retinopathy study ( etdrs ) ( 11 ) . macula- and disc - centered views were taken at an angle of 45 with a fundus camera after pharmacological mydriasis . the fundus photographs were evaluated by trained graders , who were unaware of the medical conditions , on the basis of the photographic standards defined for the accord eye study ( 10 ) . the etdrs severity scale has 17 steps , ranging from no retinopathy in either eye ( step 1 ) to high - risk proliferative retinopathy in both eyes ( step 17 ) . we combined the severity of retinopathy into four categories : absent ( steps 1 3 ) , mild to moderate nonproliferative diabetic retinopathy ( npdr ) ( steps 47 ) , severe npdr ( steps 811 ) , and advanced diabetic retinopathy ( pdr , postlaser photocoagulation , or vitrectomy ) ( step 12 or above ) . hba1c was measured using high - performance liquid chromatography ( hplc ) instruments ( hlc-723 ghb iiis ; tosoh , tokyo , japan ) with a reference range of 4.25.8% . the interassay between - batch coefficient of variation ( cv ) was < 2.0% at a1c levels between 4.4 and 8.2% . urinary albumin concentration was measured by rate nephelometry ( immage immunochemistry system ; beckman coulter , brea , ca ) . the interassay cv was 7.5% at urine albumin of 0.56 mg / dl and 2.0% at 3.19 mg / dl , respectively . urinary creatinine was measured using a hitachi 7600 automatic analyzer ( hitachi , tokyo , japan ) . the primary outcomes were composite retinal outcome and progression of nephropathy ( renal outcome ) . these subjects received repeated eye fundus photographs or were examined by ophthalmologists during follow - up . the composite retinal outcome was defined as an increase of at least three steps on the etdrs severity scale or development of advanced diabetic retinopathy . advanced diabetic retinopathy was defined as development of proliferative diabetic retinopathy , retinopathy treated with laser photocoagulation , or vitrectomy . progressive loss of renal function ( renal outcome ) was defined as egfr decrease 15 ml / min/1.73 m and final egfr < 60 ml / min/1.73 m. the secondary outcomes were cardiovascular events and all - cause mortality . cardiovascular events were defined as admission owing to angina , myocardial infarction , heart failure , acute coronary syndrome , and cerebrovascular accident . two research physicians who did not know the egfr and uacr of these patients verified all information . subsequently , a medical expert in the field reviewed all events coded by the research physicians and verified that all coding rules had been applied correctly . when discrepancies between the medical expert and research physicians occurred , the expert s judgment was considered final . egfr and uacr were expressed as median ( interquartile range ) and analyzed by nonparametric test . hazard ratios ( hrs ) of incidence and 95% cis were calculated by multivariable cox regression models . in comparison between egfr 3059.9 and 60 ml / min/1.73 m ( reference ) , the results were adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , insulin use , ace inhibitors or angiotensin ii receptor blockers ( arbs ) , and uacr . in comparison between uacr 30299.9 and < 30 mg / g ( reference ) , the results were adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , insulin use , ace inhibitors or arbs , and egfr . for the comparison between patients of groups 2 and 3 , the cox model was adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , and use of insulin , ace inhibitors , or arbs . a p value of < 0.05 was considered statistically significant . blood pressure was taken twice by using an electric blood pressure monitor in the sitting position after 10 min of rest . based on medical records , information about duration of diabetes and prescribed medication was obtained . venipuncture was performed , and a fasting serum sample was collected for the measurement of diabetic profiles , serum creatinine , and other biochemical tests . egfr was calculated by computer using the modification of diet in renal disease ( mdrd ) equation ( 9 ) . urinary albumin excretion was determined as the uacr from the mean of two spot urine samples on two consecutive mornings . color photographs of the retinas were taken according to the methodology of the accord ( action to control cardiovascular risk in diabetes ) eye study ( 10 ) , which was slightly modified from the early treatment diabetic retinopathy study ( etdrs ) ( 11 ) . macula- and disc - centered views were taken at an angle of 45 with a fundus camera after pharmacological mydriasis . the fundus photographs were evaluated by trained graders , who were unaware of the medical conditions , on the basis of the photographic standards defined for the accord eye study ( 10 ) . the etdrs severity scale has 17 steps , ranging from no retinopathy in either eye ( step 1 ) to high - risk proliferative retinopathy in both eyes ( step 17 ) . we combined the severity of retinopathy into four categories : absent ( steps 1 3 ) , mild to moderate nonproliferative diabetic retinopathy ( npdr ) ( steps 47 ) , severe npdr ( steps 811 ) , and advanced diabetic retinopathy ( pdr , postlaser photocoagulation , or vitrectomy ) ( step 12 or above ) . hba1c was measured using high - performance liquid chromatography ( hplc ) instruments ( hlc-723 ghb iiis ; tosoh , tokyo , japan ) with a reference range of 4.25.8% . the interassay between - batch coefficient of variation ( cv ) was < 2.0% at a1c levels between 4.4 and 8.2% . urinary albumin concentration was measured by rate nephelometry ( immage immunochemistry system ; beckman coulter , brea , ca ) . the interassay cv was 7.5% at urine albumin of 0.56 mg / dl and 2.0% at 3.19 mg / dl , respectively . urinary creatinine was measured using a hitachi 7600 automatic analyzer ( hitachi , tokyo , japan ) . the primary outcomes were composite retinal outcome and progression of nephropathy ( renal outcome ) . these subjects received repeated eye fundus photographs or were examined by ophthalmologists during follow - up . the composite retinal outcome was defined as an increase of at least three steps on the etdrs severity scale or development of advanced diabetic retinopathy . advanced diabetic retinopathy was defined as development of proliferative diabetic retinopathy , retinopathy treated with laser photocoagulation , or vitrectomy . progressive loss of renal function ( renal outcome ) was defined as egfr decrease 15 ml / min/1.73 m and final egfr < 60 ml / min/1.73 m. the secondary outcomes were cardiovascular events and all - cause mortality . cardiovascular events were defined as admission owing to angina , myocardial infarction , heart failure , acute coronary syndrome , and cerebrovascular accident . two research physicians who did not know the egfr and uacr of these patients verified all information . subsequently , a medical expert in the field reviewed all events coded by the research physicians and verified that all coding rules had been applied correctly . when discrepancies between the medical expert and research physicians occurred , the expert s judgment was considered final . egfr and uacr were expressed as median ( interquartile range ) and analyzed by nonparametric test . hazard ratios ( hrs ) of incidence and 95% cis were calculated by multivariable cox regression models . in comparison between egfr 3059.9 and 60 ml / min/1.73 m ( reference ) , the results were adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , insulin use , ace inhibitors or angiotensin ii receptor blockers ( arbs ) , and uacr . in comparison between uacr 30299.9 and < 30 mg / g ( reference ) , the results were adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , insulin use , ace inhibitors or arbs , and egfr . for the comparison between patients of groups 2 and 3 , the cox model was adjusted for age , sex , systolic blood pressure , bmi , hba1c , total cholesterol , triglyceride , duration of diabetes , history of hypertension , history of smoking , and use of insulin , ace inhibitors , or arbs the clinical characteristics of 487 participants are summarized in table 1 according to the egfr and uacr at baseline . patients with moderate renal impairment and normoalbuminuria were older and had a lower proportion of receiving insulin therapy than those with microalbuminuria and egfr 60 ml / min/1.73 m. characteristics of participants at baseline by egfr and urine albumin excretion during the follow - up of 6.6 1.2 years ( median 7.6 years ) , 16.5% of the subjects noted the development or progression of diabetic retinopathy ( composite retinal outcome ) , 5.4% of the subjects developed advanced diabetic retinopathy , 19.5% of subjects had progressive loss of renal function , 12.9% of subjects suffered from cardiovascular events , and 17.0% of subjects died . kaplan - meier analysis curves for end points of composite retinal outcome , renal outcome , cardiovascular events , and all - cause mortality ( fig . d ) showed that patients with microalbuminuria ( groups 3 and 4 ) were at a higher risk for development and progression of diabetic retinopathy than were those with normoalbuminuria ( groups 1 and 2 ) . cox proportional hazards model was then used to examine the relative risks of retinal , renal outcome , cardiovascular events , and all - cause mortality in relation to microalbuminuria and moderate renal impairment , as seen in tables 2 and 3 . the hr of composite retinal outcome ( table 2 ) showed significant difference between normoalbuminuria and microalbuminuria after multivariate adjustment ( hr 4.18 [ 95% ci 1.859.42 ] , p = 0.001 ) but no difference between egfr 60 ml / min/1.73 m and moderate renal impairment ( 0.73 [ 0.311.76 ] , p = 0.487 ) . the result was similar when the outcome changed to development of advanced diabetic retinopathy , even though this occurred little . as for the renal outcome , cardiovascular events , and all - cause mortality , the hrs were not statistically different between normoalbuminuria and microalbuminuria or between egfr 60 ml / min/1.73 m and moderate renal impairment . the hr of composite retinal outcome ( table 3 ) was significantly higher in group 3 subjects than that of group 2 subjects after adjustment for multiple confounders ( 3.34 [ 1.0410.70 ] , p = 0.043 ) . there was a trend that group 3 subjects had a higher risk for development of advanced diabetic retinopathy , progressive loss of renal function , cardiovascular events , and all - cause mortality compared with group 2 subjects ( table 3 ) , but statistical significance was not reached . when adding 92 individuals with egfr < 30 ml / min/1.73 m and albumin - to - creatinine ratio > 300 mg / g cr in the cox proportional hazards model , the data were quite similar to the previous results and showed that microalbuminuria had greater risk in the progression of retinopathy than moderately reduced egfr ( data not shown ) . cumulative incidence of composite retinal outcome ( a ) , renal outcome ( b ) , cardiovascular events ( c ) , and all - cause mortality ( d ) of four groups of subjects . , group 1 ; , group 2 ; , group 3 ; , group 4 . hrs for retinal and renal outcomes , cardiovascular events , and all - cause mortality in the cox proportional hazards models hrs for retinal and renal outcomes , cardiovascular events , and all - cause mortality in the cox proportional hazards models this is the first study , to our knowledge , to compare the effect of microalbuminuria and moderate renal dysfunction in diabetic retinopathy in type 2 diabetic patients . in the follow - up of 7.6 years , our data revealed that the risks for renal outcome , cardiovascular events , and all - cause mortality were not significantly different between the patients of groups 2 and 3 . however , microalbuminuria had a more predictive impact on the composite retinal outcome compared with patients with moderate renal impairment . intriguingly , our study showed that microalbuminuria significantly increases the risk for development and progression of diabetic retinopathy in type 2 diabetic patients even after adjustment for duration of diabetes , one of the most important predictors of diabetic retinopathy , and other comorbid conditions ( 12 ) . diabetic retinopathy and nephropathy seem to progress in a parallel manner . this may be because diabetic retinopathy shares similar pathophysiologic features with diabetic nephropathy . the classic clinical course of nephropathy in type 1 diabetes is the development of microalbuminuria , followed by macroalbuminuria and then by loss of gfr ( 13 ) . in contrast , kidney disease in type 2 diabetes is more heterogeneous than that in type 1 diabetes . albuminuria was absent in one - third of type 2 diabetic patients with chronic renal insufficiency according to the third national health and nutrition survey ( 14 ) . in these patients , normoalbuminuria may be due to using blockers of the renin - angiotensin system or aggressive antihypertensive therapy ( 14,15 ) . in fact , patients with diabetes are also susceptible to nondiabetic renal disease ( ndrd ) ( 1618 ) . since microalbuminuria is a pathophysiologic surrogate of the underlying diabetic nephropathy , a decrease in gfr with normoalbuminuria in such patients sometimes may be due to superimposed nondiabetic renal disease or accelerated aging of the kidney . therefore , in the current study , there is a significantly increased risk of development and progression of diabetic retinopathy for subjects with microalbuminuria and presumably through its influence on alterations in the microvasculature of retina and kidneys . increased urinary albumin excretion and reduced gfr both have been demonstrated to be an independent risk for progressive kidney failure , cardiovascular events , and mortality in patients with chronic kidney disease ( ckd ) ( 1929 ) . therefore , it is pivotal to combine egfr and albuminuria for more precise assessment of the clinical outcomes . recently , the american diabetes association recommended that in addition to level of egfr , the presence of abnormal urine albumin excretion may be useful in the staging of diabetic nephropathy ( 30 ) . previous data have shown the increased risks for renal and cardiovascular outcomes and mortality in patients with albuminuria and stage 1 or 2 ckd than in those with stage 3 ckd without proteinuria , although not all the studies have compared statistical significance ( 21,23,25,27 ) . the results showed that patients with microalbuminuria and egfr 60 ml / min/1.73 m have a tendency to increased risks for progressive loss of renal function , cardiovascular events , and all - cause mortality compared with those with moderate renal impairment and normoalbuminuria . although statistical significance was not reached , the trend corroborates the previous findings . some limitations in the current study the overall sample size became rather small when we stratified these patients into four groups . the numbers of renal events were low because most subjects were in the early stage of diabetic nephropathy . for patients with early diabetic nephropathy however , as our study had a longer observation period for the study end points , this may compensate for these two shortcomings . in summary , our data show that microalbuminuria is a more useful biomarker than moderately decreased egfr in predicting retinal outcome of type 2 diabetic patients . patients with microalbuminuria have a higher risk of the development and progression of diabetic retinopathy even when egfr is still > 60 ml / min/1.73 m. accordingly , we emphasize the importance of regular follow - up of uacr besides egfr in type 2 diabetic patients , and changes in uacr and egfr should also be identified during follow - up . once microalbuminuria occurs , diabetic retinopathy should be more aggressively assessed in these patients even if egfr is > 60 ml / min/1.73 m. | objectivethe current study aimed to investigate whether microalbuminuria or moderately decreased glomerular filtration rate ( gfr ) is a better predictor for the development and progression of retinopathy in type 2 diabetic patients.research design and methodstype 2 diabetic patients without cardiovascular diseases , malignancy , pregnancy , and acute intercurrent illness were enrolled between 1 august 2001 and 31 december 2002 .
all participants provided their detailed medical history and underwent an eye fundus examination .
they were followed up in outpatient clinics , and serum creatinine , urinary albumin - to - creatinine ratio ( uacr ) , and retinal photographs were followed up annually until 31 december 2009 .
the primary outcomes were development and progression of diabetic retinopathy and nephropathy .
the secondary outcomes were cardiovascular events and all - cause mortality.resultsamong 487 participants , 81 subjects had normoalbuminuria and moderate renal impairment ( baseline egfr 3059.9 ml / min/1.73 m2 ) , and 106 subjects had microalbuminuria and baseline egfr 60 ml / min/1.73 m2 .
patients with microalbuminuria and egfr 60 ml / min/1.73 m2 had a significantly greater risk for development and progression of diabetic retinopathy ( hr 3.34 [ 95% ci 1.0410.70 ] ) compared with those with moderate renal impairment and normoalbuminuria after multivariate adjustment . risks for renal outcome , cardiovascular events , and all - cause mortality were not significantly different between the two groups.conclusionsmicroalbuminuria has a greater impact on predicting the development and progression of diabetic retinopathy compared with moderate decline in gfr among type 2 diabetic patients . |
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primary immune thrombocytopenia , formerly known as idiopathic thrombocytopenic purpura ( itp ) , is an autoimmune hemorrhagic disorder characterized by isolated thrombocytopenia ( peripheral blood platelet count < 100 10/l ) in the absence of other causes or disorders that may be associated with thrombocytopenia . the annual incidence of itp has been reported to range from 16 cases per million per year to as high as 39 cases per million per year with women being more commonly affected than men . childhood itp , on the other hand , has a reported incidence of between 22 per million children per year and 53 per million children per year . the clinical features of itp in adults are different from the clinical features seen in childhood . itp in childhood mostly presents itself in an acute form in the age group of 210 years with a peak incidence at 2 to 4 years of age . the disease is usually self - limiting , mostly occurring after a viral infection or immunization . itp seen in adults generally has no conceivable precipitating factor , persists for longer periods and rarely resolves spontaneously . only when other causes of thrombocytopenia are ruled out , a diagnosis of itp is made . these causes may include nutritional deficiency anemia , congenital or hereditary thrombocytopenia , von willebrand disease , disorders with reduction in the number of megakaryocytes such as leukemia and aplastic anemia , human immunodeficiency virus infection , medications and certain infections . it has also been associated with immunodeficiency and autoimmune disorders . according to the american society of hematology ( ash ) , the diagnosis of itp is based on the patient s history and physical examination , as well as a complete blood count and examination of the peripheral blood smear . recent researches regarding pathogenesis of itp point out to a complicated picture involving increased platelet destruction through platelet autoantibodies and impaired platelet production wherein cytotoxic t - cells have also been implicated in the destruction of platelets . manifestations of itp range from no clinical symptoms to severe bleeding which may be fatal in certain cases . the characteristic clinical indicators of itp include easy bruising of the skin , mucocutaneous lesions such as petechiae and ecchymosis , prolonged bleeding on injury , epistaxis , gastrointestinal bleeding , hematuria and bleeding from the gums . it is important for a dentist to be aware of the clinical presentation of itp as it may lead to not only successful management of the patient , but also to formation of a provisional diagnosis of the condition in previously undetected cases . a case of itp with oral findings requiring periodontal management is presented here to emphasize that a vigilant approach is required to deliver dental care appropriately and successfully for the patient . a 19-year - old indian female reported to the department of periodontics at govt dental college , rohtak with a chief complaint of spontaneous gingival bleeding . discoloration and bruising of the lips were strikingly noticed even before a formal clinical examination had begun . medical history revealed that she was diagnosed with itp three years ago after an unexplained episode of high - grade fever . she was treated with oral corticosteroids for a few months which caused only a transient and unstable improvement of the platelet count . a year later , she underwent splenectomy , which maintained her platelet count at approximately 45,000/mm without treatment . intraoral examination revealed poor oral hygiene , spontaneous gingival bleeding and an edematous , shiny and somewhat fiery red in color gingiva ( fig 1 ) . submucosal ecchymosis at the tip of the tongue as well discoloration on the palate was noticed ( fig 2 , 3 ) . intra - oral periapical radiograph of the lower anterior region depicted mild horizontal bone loss . on questioning about oral hygiene habits , the patient revealed that she had stopped brushing from 45 months before , as she was afraid that it would further increase oral bleeding . apart from oral signs and symptoms , the patient also disclosed experiencing unusually heavy menses in the past few months and also frequent episodes of skin discolorations even with very mild trauma . a complete hemogram was carried out which revealed a platelet count of 18,000/mm , hemoglobin concentration of 11.5g / dl and a total leucocyte count of 10000/mm . taking into consideration the low platelet count and history as obtained , the patient was referred to the department of hematology regarding suitability of dental treatment at the time . periodontal therapy was initiated with debridement , supra and sub - gingival scaling and root planing . post scaling , the patient was put on tranexamic acid mouthwash twice daily for 3 days . she was motivated and taught to practice good oral hygiene habits which included regular brushing of the teeth with a soft - bristle toothbrush and 0.2% chlorhexidine mouthrinses . two weeks later , the inflammation had subsided and spontaneous bleeding was no longer evident ( fig 4 ) . she reported back for follow up after 6 months , at which she was in a state of good periodontal health ( fig 5 ) . the manifestations of itp are rather nonpeculiar for the disorder and arise as a direct consequence to platelet deficiency . spontaneous bleeding associated with thrombocytopenia most often involves small vessels and the common sites of such hemorrhage are the skin and mucous membrane of the gastrointestinal tract and genitourinary tract with manifestations as purpura ( petechiae , ecchymosis ) , epistaxis , menorrhagia , hematuria , malena or bleeding from the gums . although not very common , intracranial hemorrhage is considered the biggest threat to patients with a severely depressed platelet count . to the best of our knowledge , only 12 cases have been reported in dental literature [ 1020 ] , with the earliest report published in the early 1970s . while some reports dealt with dental patients with previously diagnosed itp , others reported cases which were diagnosed and suitably managed in dental setup . the latter further emphasized the need for dental practitioners to be aware of the findings in such disorders to identify undetected cases of itp . another case report described a case of undiagnosed hiv infection with associated thrombocytopenic purpura . refractory itp has also been suggested as an absolute contraindication to any surgical procedures if the platelet count is too low . finally , it should further be emphasized that there is only one report in the literature similar to ours dealing with the periodontal management of a patient with itp . lack of such case reports also means that fewer dentists may be aware of the findings to look for in such cases . first , it will aid the dental practitioner in efficiently managing such cases and secondly , a good knowledge of presenting symptoms may even help in diagnosing previously unidentified cases . the patient may complain of frequent bruising , gingival bleeding , nose bleeds , heavy menses or bloody stools . a complete blood count reveals a decrease in the number of platelets while examination of peripheral blood smear shows platelets of normal appearance . bone marrow testing may show normal to increased megakaryocytes with appearances varying from normal to immature with large , non - lobulated , single nuclei . the bleeding time is prolonged , but prothrombin time ( pt ) and partial thromboplastin time ( ptt ) are normal . a plaque present adjacent to the gingiva causes inflammation of the gingival tissues leading to bleeding from the gums . the risk of bleeding from these inflamed and hyperemic gingival tissues is even greater in patients with bleeding tendencies . thus , it is vital to maintain periodontal health in such patients to prevent any unwanted complications . besides , if periodontal health is ignored , the condition may worsen and further lead to alveolar bone loss and tooth mobility ultimately giving rise to a situation warranting extraction , a procedure which is complicated in patients with itp . the best measure to avoid any such undesirable sequelae is professional removal of the plaque in early stages resulting in resolution of inflammation . mucocutaneous lesions such as petechiae , ecchymosis and hematomas are most often encountered by the dental surgeon . frequently traumatized areas in and around the oral cavity such as the lips , border regions of the tongue and palate are among the most commonly involved . the case presented here only had oral signs at the time of reporting to the dental establishment and this emphasizes the necessity that the dentist should know about oral manifestations of various bleeding disorders because they often warrant special considerations . the entire dental procedure and possible complications should be thoroughly discussed with the hematologist who can then suggest suitable modifications required at pre - surgical , surgical and post - surgical phases to ensure successful delivery of dental care . professional cleaning can be safely accomplished at the dental establishment if the platelet count is above 50,000/mm . antifibrinolytic mouthwashes , such as those containing epsilon aminocaproic acid and tranexamic acid may also be used to advantage in cases where the bleeding problem is anticipated . in case periodontal surgery is indicated , it is important to ensure that the tissues are handled as atraumatically as possible and are least invaded with minimal flap extension and least possible practicable flap elevation . conventional measures of achieving hemostasis like application of pressure packs and dressings may be sufficient in suitably selected candidates . commercially available hemostatic agents like thrombin and oxidized cellulose are also of value in cases with bleeding disorders . as a general guideline , while routine dental surgical procedures may be performed in cases with a platelet count as low as 50,000/mm , a minimum level of 75,000/ mm has been suggested for other major surgeries . in case the platelet concentration is low and the surgical procedure can not be avoided , transfusion may be required to raise the platelets to an acceptable level . infiltration and intraligamentary anesthesia is preferable to regional blocks which should be avoided , especially if the platelet count is below 30,000/mm . itp patients referred to dental offices should be asked about any prescribed medications being currently used . these drugs may act by preventing the production of autoantibodies as well as destruction of platelets in the reticuloendothelial system by suppression of the defective immune response . while routine dental procedures may not result in cortisol response delayed wound healing and decreased resistance to secondary infections may also complicate the postsurgical healing response . prophylactic antibiotic therapy may thus be indicated in appropriate cases along with strict adherence to standard aseptic procedures . it should also be borne in mind that since itp is primarily a platelet disorder , any medication such as aspirin should be avoided . in addition to addressing the chief complaint of the patient , preventive dental procedures like improvement in oral hygiene habits , more frequent dental visits and regular professional cleaning should be initiated . the goal of the treatment plan should be to prevent the progress of dental diseases at the earliest stage possible to avoid any subsequent requirement of more complicated procedures . most of the patients with itp can be managed safely in a dental establishment . however , while managing such cases , it is important to take into account both the severity of the disorder and availability of a procedure suitable in the given scenario . for this , it is imperative that a physician , preferably a hematologist should be involved for treatment planning to suit the needs of the patient . successful management of itp patients should also involve preventive dental care through patient education and oral hygiene motivation to avoid the need for any more aggressive therapy at a later stage . | primary immune thrombocytopenia is an acquired bleeding disorder with no clinically apparent cause of thrombocytopenia .
clinical indicators of itp include easy bruising of the skin , prolonged bleeding on injury , mucocutaneous lesions such as petechiae and ecchymosis , epistaxis , gastrointestinal bleeding , hematuria and bleeding from the gums .
it is important for a dentist to be aware of the clinical manifestations of itp as it may not only lead to successful management of the patient , but in some cases it may even lead to formation of a provisional diagnosis of the condition in previously undetected cases . however , very few cases of itp have been reported in dental practice making it difficult for a dentist to identify the disorder when a patient suffering from itp reports for dental treatment . a case report of a female patient with itp
is thus described with emphasis on the importance of periodontal health in such patients to prevent consequent unwanted sequelae .
it is followed by discussion of oral manifestations of the disorder and dental management of such patients . |
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snake bite remains a public health problem in many countries even though ; it is difficult to be precise about the actual number of cases . it is estimated that the true incidence of snake envenomation could exceed 5 million per year . reflects variations in health reporting accuracy as well as the diversity of economic and ecological conditions . accurate records to determine the exact epidemiology or even mortality of snake bite cases are generally unavailable . hospital records fall far short of the actual number , owing to dependence on traditional healers and practitioners of witchcraft , especially in developing countries . it has been reported that in most developing countries , up to 80% of individuals bitten by snakes first consult traditional practitioners before visiting a medical centre [ 3 , 4 ] . owing to the delay , several victims die during transit to the hospital . envenoming by snakes such as e. ocellatus and naja n. nigricollis is responsible for several clinical complications of severe systemic and local pathology . for example , e. ocellatus leads to inflammation ( such as swelling , blistering , and necrosis ) and haemorrhages due to both metalloproteases and ecarin ( an enzyme that activates prothrombin ) . on the other hand envenoming by naja n. nigricollis induced clinical complications different from that caused by e. ocellatus . these include local necrosis , haemorrhage , complement depletion , and respiratory arrest or paralysis [ 5 , 8 ] . moreover , the venom of the naja n. nigricollis consists of phospholipase a2 ( an anticoagulant enzyme which inhibits the prothrombinase complex by its binding to coagulation factor xa ) [ 9 , 10 ] and cardiotoxin . furthermore , in some cases envenoming by naja n. nigricollis can induce corneal ulceration and anterior uveitis [ 12 , 13 ] . although an intravenous administration of antivenom , prepared from igg of venom - immunised horses or sheep , is an effective treatment for systemic envenoming , the clinical consensus is that antivenom is of limited effectiveness against the effects of local envenoming that develop rapidly after a bite . such effects include severe pain , oedema , localized haemorrhage , and necrosis which often results in permanent scarring and deformity . the ineffectiveness of antivenom in treating local envenoming has been attributed to the rapid activity of the toxins and the inability of antivenom igg to cross the blood / tissue barrier [ 17 , 18 ] . despite their smaller size , f(ab2)2 and fab fragments of igg are also ineffective against the local effects of envenoming , whether administered by intravenous or intramuscular routes [ 19 , 20 ] . research to develop a treatment for local envenoming is therefore a clinical priority and has focused on the application of natural or synthetic inhibitors of snake venom potent molecules . the use of plant remedies to treat snakebite victims in rural areas and poor communities in the developing countries is a common practice . the natives who are predominantly rural farmers come in contact with snakes during their farming engagements . due to high cost of hospital treatment and unavailability of antivenoms , most often the rural people find it more convenient to consult native doctors who are acclaimed for curing snakebite patients . anecdotal evidence abounds to indicate that plant remedies used by the native doctors are effective , and there appears to be a high rate of survival among snakebite patients advanced clinical stages of venom toxicity . the present study aims to study the anti - snake venom activities of a local plant , hibiscus aethiopicus l. which is brought over 200 years ago from africa and regrown in bani - hushash region , sana'a yemen . this plant was found to be used by traditional healers in bani - hushash east of sana'a to treat patients bitten by snakes and/or scorpions . although hibiscus aethiopicus l. has long been used as a medicinal plant by traditional healers , the validity of the claims made for this plant has not been tested scientifically . in other words hibiscus aethiopicus like many other important plant species , has not been investigated adequately in terms of its anti - snake venom activities . we report in this study and for the first time , how significant this folk tradition medicine is ( i.e. , hibiscus aethiopicus ) to neutralized snake venom activity , of a typical and highly poisonous snakes such as the west african e. ocellatus snake . the whole plant of hibiscus aethiopicus was collected with assistance of a traditional healer , from bani - hushaiesh . a. wadieh , department of botany , naser college , in lahj governorate , university of aden , republic of yemen . the dried sample was then pulverized and stored in plastic bags . the air - dried and pulverized plant material ( 200 g ) was extracted with 250 ml water by using a shaking water - bath at 70c for 2 hours . the obtained water extract was filtered and evaporated using a rotary evaporator and freeze dryer to give the crude dried extract . the venom of naja n. nigricollis was purchased from sigma aldrich ltd ( category no . ethical approval for this study was obtained from the relevant ethics and research committee of the university of science and technology , sana'a , yemen . all experiments that involve animals were performed according to ethical standards . the effects of hibiscus aethiopicus extract and venoms ( e. ocellatus and naja n. nigricollis ) on cultured c2c12 myoblast cells were investigated separately following the tetrazolium salt ( mtt ) assay method . cells were seeded in 96 well microtitre plates ( 10 cells per well in 100 l medium ) and allowed to attach and reach log phase of growth ( 24 h ) . various concentrations of hibiscus aethiopicus extract ( 75 , 150 , and 300 g / ml ) with or without venom ( 15 g / ml of e. ocellatus , 30 g / ml naja n. nigricollis ) were added to each well in 100 l medium . the cells were incubated the cells at 37c 18 h. ten microliters ( 10 l ) of mtt ( 5 mg / ml ) was added to each well and plates were incubated for 4 h , at 37c , after which the medium was aspirated from the wells and a volume of 150 l dmso was added per well to solubilise the cells . the microplates were shaken for 2 minutes at 400 rpm on a microplate shaker . the optical density was measured at 570 nm using a dynex mrx plate reader ( mtx lab systems ) . one - day - old fertile eggs obtained from a local hatchery were incubated till day 4 at 38c . the eggs were cracked on day 4 into clingfilm hammocks following a standard method [ 21 , 25 ] and incubated further till day 6 . 2 ) were impregnated with a standard hemorrhagic dose ( shd ) of e. ocellatus venom ( 3 g/1.5 l ) alone or venom and various concentrations ( 2.5 , 5.0 , 7.5 , and 10 g/1.5 l ) of hibiscus aethiopicus extract . the discs were placed on the yolk sac membrane over a major bilateral vein and left for 3 h for hemorrhagic corona to form . control experiments were performed with the buffered saline solution used to prepare the extract and venom solutions . the minimum concentration required to abolish haemorrhage was recorded as the minimum effective neutralizing dose ( mend ) . oral and intraperitoneal routes were performed to evaluate the acute toxicity and the cumulative effect safety protection was obtained from the relevant ethics and research committee of university of science and technology , sana'a , yemen . two different species of animals rabbits ( group - a ) and guinea - pigs ( group - b ) , were used in the toxicity profile . animals were obtained from a well - known animal keeper , nuccom , sana'a , republic of yemen . group - a included ten rabbits weighed between 800 g and 1300 g. group - b included ten guinea - pigs weighed between 300 g and 900 g. both rabbits and guinea - pigs were given different dosages to investigate the lethal dosage as illustrated in tables 2 and 3 . the extract solution was given orally using anesthesia at variable dosages to reach the optimum of 16 g / kg ( a lethal dosage according to the international standardisation for the classification of substances ) for each animal group . the animals were observed for behaviour change continuously for a period of two weeks after such administration . observation was conducted hourly at day 1 , and during the following days , observation was conducted 46 times per day . subsequently , blood samples for biochemical assays , alanine aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , complete blood count ( cbc ) , and gamma glutamyl transpeptidase ( ggt ) were collected . at the end of the observation period , animals were sacrificed and dissected for adverse effects if any based on histopathology examination of their eyes , liver , lung , and spleen . intra - peritoneal ( i.p ) acute toxicity test was performed instead of the oral route . a total of 35 guinea pigs of both sexes distributed randomly into five groups and being treated i.p . with increasing doses ( 250 , 500 , 750 , 1000 , 1300 , and 1600 mg / kg ) of hibiscus aethiopicus extract . animals ( i.e. , guinea pigs ) were observed regularly over a period of 24 h for signs of acute toxicity and death . seventy eight adult guinea pigs of both sexes ( 500600 g ) were divided into four groups . group 1 ( of 3 guinea pigs ) was injected with naja n. nigricollis venom ( 2.5 mg / kg ) alone . group 2 was divided into twelve equal subgroups ( g2.1g2.12 ) of three guinea pigs each . a mixture of naja n. nigricollis venom and hibiscus aethiopicus extract ( 50 , 75 , 100 , 200 , 300 , 400 , 500 , 600 , 700 , 800 , 900 , and 1000 mg / kg ) accordingly after both venom and extract were incubated in a test tube for 30 minutes . groups 3 and 4 were similar to groups 1 and 2 except that e. ocellatus venom ( 5 mg / kg ) was used instead of naja n. nigricollis . all animals were observed over the 24 hours . at the end of the observation period , animals were sacrificed and their skins were dissected to examine the neutralisation efficacy of the hibiscus aethiopicus extract . sixty adult guinea pigs of both sexes ( 500600 g ) were divided into four equal groups . group 1 ( of 5 guinea pigs ) was injected with naja n. nigricollis venom ( 2.5 mg / kg , i.p . ) . group 2 was divided into five equal subgroups ( g2.1g2.5 ) of five guinea pigs each . all of the subgroups were injected i.p . with the same dose ( 2.5 mg / kg , i.p . ) of naja n. nigricollis venom 30 min after hibiscus aethiopicus extract was administered orally at different concentrations ( 100 , 200 , 300 , 400 , and 1000 mg / kg ) by gastric incubation . groups 3 and 4 were similar to groups 1 and 2 except that e. ocellatus venom ( 5 mg / kg ) was used instead of naja n. nigricollis . the hibiscus aethiopicus extract alone had no effect on the viability of c2c12 muscle cells , but it significantly ( p < .05 ) protected muscle cells against the toxic effects of e. ocellatus venom ( 30 g / ml ) at all concentrations of the extract tested ( 55 , 150 , and 300 g / ml ) ( figure 1 ) . the maximum ( 67% ) protective effect of the extract was exhibited with extract at 75 g / ml . the extract significantly ( p < .001 ) inhibited the cytotoxic effects of e. ocellatus venom only at 300 g / ml ( figure 1 ) . on the other hand the hibiscus aethiopicus extract showed no effect to protect the muscle cells against the toxic effects of naja n. nigricollis ( data not shown ) . the hibiscus aethiopicus totally ( 100% ) blocked the haemorrhagic activity of e. ocellatus in the egg embryo at 5 g/1.5 l ( 3.3 mg / ml ) of extract ( table 1 ) . the mend is 5 g/1.5 l . all animals ( rabbits and guinea pigs ) were alive after the two weeks of given the lethal dosage of 16 g / kg . no abnormal behaviour was observed of both groups of rabbits and guinea - pigs during the observation period . the rabbits and guinea pigs showed normal body weight increase during the two weeks period . biochemical analysis showed normal range of alt , ast , cbc , and ggt ( table 2 ) . inspection of the eyes , liver , lung , and spleen ( after scarification and dissection ) showed no extraordinary signs . the results when compared to a general acute toxicity index were normal and no acute toxicity was observed . guinea pigs dosed intraperitoneally with hibiscus aethiopicus extract were initially dull with significantly reduced movement for 1020 minutes . however , neither death nor signs of toxicity were observed even at the highest dose ( 1600 mg / kg ) tested . all guinea pigs injected with e. ocellatus venom ( 5 mg / kg ) alone died . however all guinea pigs injected with both e. ocellatus venom and the plant extract ( at a concentration of 400 to 1000 mg / kg ) survived ( table 3 ) . however , all guinea pigs treated with naja n. nigricollis venom alone ( 2.5 mg / kg ) and/or venom with the water extract ( at a concentration of 400 to 1000 mg / kg ) died ( table 3 ) . there was no significant difference between the time of death in both the treated and control groups ( table 3 ) . mg / kg alone group 1 induced acute haemorrhage and died ( figure 2(a ) ) . in contrast all guinea pigs treated with venom and the plant extract group 2 at a concentration between 400 and 1000 mg / kg survived and showed no signs of acute haemorrhage ( figure 2(b ) ) . moreover , all guinea pigs treated with venom and the plant extract below 400 mg / kg died . however , all of the guinea pigs treated with naja n. nigricollis venom 2.5 mg / kg group 3 alone and venom with the plant extract group 4 died . although , all guinea pigs treated with a mixture of naja n. nigricollis venom and the extract after preincubation died , the time of death was significantly ( p < .05 ) increased from 0.95 h to 5.57 h in the group treated with the mixture of extract and venom in comparison with the control . there was no significant difference between the time of death in the treated and control groups . because natural products of higher plants may give a new source of medication , there are many research groups that are now engaged in medicinal plants research not only for the discovery for new drugs but possibly for discovering compounds with novel mechanisms of action that can stimulate new fields of research . furthermore and considering the high cost of conventional antivenoms and the significant percentage ( 80% ) of patients who react adversely to them [ 26 , 27 ] , a systematic investigation of plant - based remedies for snake bite is justified . however , many of the reported studies lack detailed scientific investigation , which is needed in the development of medicinal agents from plants [ 28 , 30 ] . research to develop a treatment for local envenoming is a clinical priority and has focused on the application of natural or synthetic inhibitors of snake venom metalloproteinases ( svmps ) and understanding the pathological role of svmp - activated proinflammatory cytokines [ 3234 ] . in this study we have used one of the main traditional herbal plants called hibiscus aethiopicus which is used as phytotherapy practiced by a large proportion of the yemen population for the treatment of several clinical complications such as physical , physiological , mental , and social ailments as well as snake envenomation . in comparison to other plants that were adequately investigated worldwide there is little scientific research done to investigate generally the plants of yemen which are used in herbal medicine . however , no scientific reports were found for hibiscus aethiopicus and its capacity to neutralized snake venom(s ) . therefore , this study represents the first report about hibiscus aethiopicus and its uses as antivenom agent . to examine such neutralisation efficacy , two venoms from two different snake species ( e. ocellatus and naja n. nigricollis ) were used . prior to such examination , the toxicity effect of the hibiscus aethiopicus extract was examined on animals using in vivo assays based on oral and intra - peritoneal administration routes . the results of the oral route when compared to a general acute toxicity index showed normal with no extraordinary syndromes as well as no acute toxicity . however , both routes showed that no death was recorded even at the highest tested dose ( 16 mg / kg ) . however , results from the intraperitoneally route showed initially dull with significantly reduced movement of the guinea pigs for about 1020 minutes . in the subsequent experiments we used the hibiscus aethiopicus extract to assess its efficacy to neutralise the haemorrhagic activity of e. ocellatus and naja n. nigricollis venoms using an in vivo minimum haemorrhagic dose ( mhd ) assay utilised to preclinically assess new antivenoms . it was interesting to note that results of the evaluation assays of anti - snake venom activity showed that hibiscus aethiopicus induces significant neutralisation capacity against venom of e. ocellatus compared to that of naja n. nigricollis venom which showed no antivenom activity at all , as all guinea pigs treated with a preincubation mixture of naja n. nigricollis venom and the extract died . there was no significant difference between the time of death in the treated and control groups . the hibiscus aethiopicus extract significantly blocked many of the toxic effects of e. ocellatus venom in vitro . remarkably , the extract could not block the neurotoxic activities of naja n. nigricollis ( results not shown ) venom on chicken blastodermal cells ( cbcs ) muscle preparation whether the former was added three minutes before or after the venom when the venom 's effects on the preparation are normally irreversible by three times washout of the venom . although the component(s ) of the plant extract responsible for the antivenom activity observed in the present study has not yet been identified to verify the complete absence or present of tannins , particularly low molecular weight polyphenolic ( epi - gallocatechin , epi - catechin etc . ) and a long - chain ester of transferulic acid , we presume that it is unlikely that the antivenom activity is due to polyphenolic components . moreover , in comparison with the antivenom activity of the extract on the e. ocellatus venom to that of naja n. nigricollis , it can be observed that it is not possible to be due to the extract acting through a mechanistic intervention rather than a direct physical interaction with the venom in vitro . this is similar to the mode of action of many polyphenolic compounds found in plant extracts . this prospect was confirmed partially by the extract protective effects of plant extract when they are preincubated with e. ocellatus venom before administration to the biological assay . therefore , to validate the above speculation(s ) future work is necessary to isolate and examine such component(s ) . we have clearly shown that the extract is very effective against the activity of e. ocellatus venom in the circulatory system . these findings reflect that the extract of hibiscus aethiopicus plant may contain an endogenous inhibitor of venom - induced haemorrhage . this obviously would need further investigations . despite these protective effects of the plant extract of the hibiscus aethiopicus in vitro , the extract did protect animals challenged with lethal doses of the e. ocellatus venom when the extract and venom were administered independently . moreover , the extract did not protect animals challenged with lethal doses of the naja n. nigricollis venom when extract and venoms were administered independently . however , haemorrhage induction was significantly reduced ( correlated with the increased of the extract concentration ) with an increase in the survival time of the guinea pigs treated with a mixture of the extract and the e. ocellatus venom after 30 min of preincubation . in addition , none of the guinea pigs treated with a mixture of naja n. nigricollis venom and the extract hibiscus aethiopicus survived . possibly the plant extract could be effective against viper snake that is , naja n. nigricollis , venom activity in vivo if the experiment model is modified to stimulate actual life experience . in order to obtain more positive results two possible considerations are suggested for future work ; first the dose of venom should be adjusted to achieve 75% instead of 100% mortality in the control animal to ensure that they are not challenged with very high dose of venom beyond the dose that any snake can inject into its victim . a very high dose may not give enough time for the extract to induce its antivenom effect . the other suggestion is to give repeated doses of the extract at various time intervals ( include an administration of the extract immediately after venom injection ) , which approximate to what obtains when humans are treated for snakebite . treatment is normally continued until clinical signs of the envenomation disappear . as venom toxicity within the same species varies and very hard to standardize , the doses that we used in this study to induce lethal effects by both e. ocellatus and naja for instance , ode and asuzu ( 2006 ) used different venom concentrations ( to that we used ) of 10 mg / kg and 6 mg / kg of e. ocellatus and naja n. nigricollis , respectively , to induce lethal effects . moreover , from our previous work with e. ocellatus [ 3638 ] , for example , we have showed that venom variation occurred both locally and from region to region within the same species . and broadle reported that snakes of the naja n. nigricollis species have variation in their neurotoxin content both qualitative and quantitative . first we have to perform a fractionation(s ) assay of the hibiscus aethiopicus extract , so that the effective component(s ) responsible for the inhibitory effect can be identified . subsequently , this will allow us to perform the classical toxicity and neutralisation assays including the lethal dose ( ld50 ) of the venom as well as to calculate the effective dose ( ed50 ) of the extract . | the objective of the study is to investigate whether the hibiscus aethiopicus l. plant has neutralization activity against venoms of two clinically important snakes .
the h. aethiopicus was dried and extracted with water .
different assays were performed to evaluate the plant 's acute toxicity and its anti - snake venom activities .
the results showed that h. aethiopicus extract alone had no effect on the viability of c2c12 muscle cells , but significantly ( p < .05 ) protected muscle cells against the toxic effects of e. ocellatus venom at 55 , 150 , and 300 g / ml .
the maximum protective effect of the extract was exhibited at 75 g / ml .
the extract significantly ( p < .001 ) inhibited the cytotoxic effects of e. ocellatus venom at 300 g / ml . all rabbits ( n = 10 ) and guinea pigs ( n = 10 ) were alive after the two weeks of given the lethal dosage 16 g / kg of the h. aethiopicus extract herbal solution .
no abnormal behaviour was observed of both groups of animals .
all guinea pigs ( n = 3 ) treated with venoms alone ( 5 mg / kg ) died .
however , all guinea pigs ( n = 21 ) treated with venom ( 5 mg / kg ) and the extract ( 400 to 1000 mg / kg ) survived . guinea pigs
( n = 3 ) treated with naja n. nigricollis venom alone ( 2.5 mg / kg ) and guinea pigs ( n = 21 ) venom with the extract ( 400 to 1000 mg / kg ) died .
the h. aethiopicus completely ( 100% ) blocked the haemorrhagic activity of e. ocellatus in the egg embryo at 3.3 mg / ml of extract .
these findings suggest that h. aethiopicus may contain an endogenous inhibitor of venom - induced haemorrhage . |
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primary fallopian tube carcinoma ( pftc ) is a rare neoplasm , accounting for 0.14%-1.8% of all gynecological malignancies . however , recent data suggest that the true incidence of pftc has been substantially underestimated ; this conclusion is based on compelling evidence that papillary serous carcinoma , the most common subtype of epithelial ovarian carcinoma ( eoc ) , actually arises from the epithelial lining of the fallopian tube . the mean age at diagnosis of pftc is 64 years , and incidence peaks at 70 - 74 years . total abdominal hysterectomy with bilateral salpingooophorectomy and infracolic omentectomy , appendectomy , peritoneal washing , and peritoneal biopsy constitute the primary treatment of choice for pftc ; inclusion of pelvic and para - aortic lymphadenectomy has been controversial . klein et al . reported that routine retroperitoneal sampling and node dissection are necessary for proper staging , whereas other authors have reported that systemic retroperitoneal lymphadenectomy is preferable due to the strong tendency of the tumor to spread in the lymphatic system . in addition , the therapeutic value of a systematic complete lymphadenectomy remains controversial , with a paucity of clinical data to support its use . stage , patient age , and residual tumor after initial surgery are consistently important prognostic factors . in addition , according to some reports , a closed fimbriated end of the fallopian tube , positive peritoneal cytology , lesion site within the tube ( fimbrial vs. non - fimbrial ) , human epidermal growth factor receptor 2/nu - positive expression , p53 alteration , elevated pretreatment cancer antigen 125 ( ca-125 ) , and lymphovascular space involvement are also important prognostic factors [ 2 - 6 ] . interest in elevated neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) as markers of systemic inflammatory response ( sir ) in various clinical circumstances is increasing . in recent clinical studies , these ratios have been investigated as advanced - stage predictors or prognostic factors in different types of gynecologic cancer [ 7 - 14 ] . in addition , preoperative thrombocytosis has been associated with adverse outcomes from endometrial and ovarian cancers . however , no study investigating the predictive value of nlr , plr , or thrombocytosis in overall survival ( os ) and disease - free survival of patients with pftc has been reported . therefore , we retrospectively reviewed the clinical characteristics , management , and outcomes of 91 patients with pftc who were treated in six gynecological oncology departments . in addition , this study sought to determine whether 1 ) nlr , plr , or thrombocytosis plays a role in disease - free survival and os of pftc and 2 ) performance of para - aortic lymphadenectomy is essential for patients with pftc . the databases of six gynecological oncology departments from turkey , including izmir tepecik education and research hospital , izmir ege university school of medicine , eskisehir osmangazi university school of medicine , ankara university school of medicine , antalya akdeniz university school of medicine , and adana cukurova university school of medicine were reviewed for identification of patients with complete information and pathologically proven pftc who underwent surgical staging between january 1 , 1996 , and december 31 , 2011 . this study was conducted in accordance with the ethical standards of the declaration of helsinki and was approved by the local ethics committees of izmir tepecik education and research hospital . cases were identified according to the pftc diagnostic criteria established by hu et al . and modified by sedlis . the following clinical data were collected from patient medical , surgical , pathological , and chemotherapy reports : demographic characteristics , presenting symptoms , serum ca-125 level , preoperative complete blood cell count ( cbc ) , date and type of surgical procedure , presence or absence of residual tumor after surgery , number of excised and positive lymph nodes , presence or absence of ascites , tumor pathological characteristics ( grade and size ) , type of first - line chemotherapy , date of recurrence , treatment after recurrence , date of last medical examination , and date of death . patients with any inflammatory signs or conditions , hematological disease , or other malignancies that might affect the cbc results , smokers , those who used corticosteroids or -agonists , and patients with missing data including preoperative cbc and surgico - pathological notes , were excluded . international federation of gynecology and obstetrics ( figo ) staging according to the 2009 revised classification system was used . cbc was measured within one month preoperatively using a coulter lh 750 device ( beckman coulter , brea , ca ) or a sysmex xe-2100 device ( toa medical electronics , kobe , japan ) at the six centers . when more than one cbc result was available , the result from the date closest to staging surgery was recorded for statistical analysis . the nlr was defined as the absolute neutrophil count divided by the absolute lymphocyte count , and the plr was defined as the absolute platelet count divided by the lymphocyte count . patients were classified as not having been staged if only a unilateral salpingo - oophorectomy , total hysterectomy with unilateral or bilateral salpingo - oophorectomy with or without omentectomy was performed . partial staging was defined as pelvic washing , peritoneal biopsy , omentectomy , bilateral pelvic lymph node dissection with a unilateral salpingo - oophorectomy , or total hysterectomy with unilateral or bilateral salpingo - oophorectomy . complete staging was defined as pelvic washing , peritoneal biopsy , omentectomy , bilateral pelvic and para - aortic lymph node dissection with a unilateral salpingo - oophorectomy , or total abdominal hysterectomy with unilateral or bilateral salpingo - oophorectomy . optimal debulking was defined as a procedure that left a maximum residual tumor < 1 cm in diameter . patients returned for a follow - up evaluation every three months for the first 2 years , every 6 months for the next 3 years , and annually thereafter . computed tomography or magnetic resonance imaging the survival analysis was based on the kaplan - meier method , and the results were compared using the log - rank test . progressionfree survival ( pfs ) was defined as the time from the date of primary surgery to detection of recurrence or the latest observation . os was defined as the time from the date of primary surgery to death or the latest observation . the chi - square test and student s t test for unpaired data were used for the statistical analysis . cox regression analysis was used to determine factors affecting survival , and results are presented as hazard ratios ( hrs ) with 95% confidence intervals ( cis ) . the two most common preoperative diagnoses were ovarian carcinoma ( 42.9% ) and adnexal mass of unknown nature ( 29.7% ) . the median follow - up period was 34 months ( range , 14 to 78 months ) . thirty - four patients ( 37.4% ) had figo stage i disease , 10 ( 10.9% ) had stage ii , 43 ( 47.3% ) had stage iii , and four ( 4.4% ) had stage iv disease . nine patients ( 9.9% ) had no surgical staging , 34 ( 37.4% ) had partial staging , and 48 ( 52.7% ) had complete staging . four unstaged patients who refused reoperation were treated based on high - risk features such as grade 3 disease or tumor rupture . we studied nlr and plr at different levels to determine the optimal cut - off point for prediction of disease stage ( early vs. advanced ) and primary surgical outcome ( optimal vs. suboptimal ) . the receiver operating characteristics ( roc ) curve indicated that nlr > 2.7 and plr > 233.3 was optimum for prediction of advanced stage ( stage iii / iv ) and suboptimal surgery ( table 3 ) . the mean and median pfs were 34.2 months and 32 months ( range , 9 to 76 months ) , respectively . results of univariate analysis indicated that advanced figo stages iii and iv , suboptimal surgery , and pretreatment nlr > 2.7 ( fig . 1a and c ) were adverse prognostic factors for pfs . results of multivariate analysis showed that advanced figo stage and suboptimal surgery were independent prognostic factors for pfs . the 5-year os rate was 63.7% , and the mean and median survival durations were 38.9 months and 35 months ( range , 14 to 78 months ) , respectively . results of univariate and multivariate analyses for os are shown in table 5 . in univariate analysis of survival rates , pretreatment ca-125 35 u / ml , presence of ascites , advanced figo stage , suboptimal surgery , pretreatment nlr > 2.7 ( fig . 1d ) , pretreatment platelet count 400,000 cells / mm , surgical staging type ( none vs. partial vs. complete ) , and histological subtype ( serous vs. non - serous ) showed significant association with poor os . in contrast , multivariate analysis of os showed significant association of advanced figo stage , suboptimal surgery , pretreatment nlr > 2.7 , and surgical staging type ( none vs. partial vs. complete ) with poor os . of of these , 48 ( 58.6% ) had complete staging , and 34 ( 41.4% ) had incomplete staging . the mean and median pfs tended to be better in the complete staging group ( 38.1 months and 34.0 months [ range , 9 to 76 months ] , respectively ) than in the partial staging group ( 27.8 months and 24.0 months [ range , 11 to 76 months ] ) , however , the difference was not significant ( hr , 1.7 ; 95% ci , 0.9 to 3.2 ; p=0.09 ) ( fig complete staging showed significant association with longer os , with mean and median os rates of 42.0 months and 35.5 months ( range , 22 to 78 months ) , respectively , vs. 33.5 months and 27.5 months ( range , 14 to 76 months ) , respectively , in the partial staging group ( hr , 3.1 ; 95% ci , 1.4 to 5.7 ; p=0.002 ) ( fig . the current study retrospectively analyzed patients with pftc who were treated at six gynecologic oncology centers in turkey . our aim was to describe the demographic and clinical characteristics of pftc and to identify variables affecting pfs and os in patients with this disease . it is not routinely suspected in women with a pelvic mass . in our study , none of the patients had a definitive preoperative diagnosis of pftc . in the literature , the mean age of patients with pftc is 55 years , similar to that in our patient population . reported that pftc is diagnosed at an earlier stage than ovarian cancer , possibly as a result of abdominal pain resulting from tubal distention and intermittent serosanguineous discharge . the 5-year os rate for all stages of pftc varies from 22% to 81.7% [ 20 - 22 ] . several studies have reported various prognostic factors for predicting the outcome of pftc , including age , stage , cell type , elevated ca-125 , and residual tumor after surgery . reported that in a univariate analysis , pretreatment serum ca-125 values were prognostic with respect to both pfs and os . they also showed that the side where the tumor was located was associated with pfs . however , multivariate analyses were not performed in these two studies . in the current study , advanced figo stage , presence of a residual tumor > 1 cm after surgery , and surgical staging type ( none vs. partial vs. complete ) showed significant association with poor os in both univariate and multivariate analyses . location and grade of tumor prognostic factors other than age and elevated ca-125 can only be assessed during or after surgery based on the pathological features of the cancer . do nlr , plr , and thrombocytosis play a role in disease - free and os in patients with pftc ? an increase in neutrophil count , a slight increase in platelet count , and a decline in lymphocytes are systemic alterations that are part of the inflammatory response to the growth , progression , and spread of tumors . in this respect , interest in nlr and plr has shown a recent increase , and they have been studied as sir markers in non - gynecologic and gynecologic cancers , with results suggesting that a preoperative increase in nlr and/or plr is significantly associated with nodal metastasis and advanced stage and is a significant prognostic factor [ 8 - 14 ] . indeed , it has been suggested that these ratios , which can be assessed using noninvasive , simple , and inexpensive methods and can be easily calculated from the cbc , should be included in the routine assessment of all patients with cancer . more recently , thrombocytosis was reported as an independent predictor of increased risk of recurrence in 587 women who underwent staging surgery for eoc . only four studies have analyzed pretreatment nlr and/or plr values in patients with eoc [ 11 - 14 ] , and the value of these ratios as prognostic factors in patients with pftc has not been validated . a preoperative nlr cut - off point > 2.6 distinguished ovarian cancer from benign conditions and predicted adverse outcomes , and a similar ratio was associated with advanced - stage disease or suboptimal ovarian surgery . plr was superior to nlr for prediction of poor survival in two ovarian cancer studies . first , asher et al . showed that plr > 300 was superior to nlr > 4.0 for prediction of poor survival and was a novel independent prognostic marker for eoc . later , it was demonstrated that plr > 200 had the potential to predict advanced - stage disease or suboptimal surgery and was a better prognostic indicator for survival of patients with eoc than nlr of 2.6 . in our study , results of the roc curve analysis showed that the optimum cut - off value for prediction of advanced stage ( stage iii / iv ) and suboptimal surgery in patients with pftc was 2.7 for nlr , with 85.1% sensitivity and 75.0% specificity for advanced stage ; suboptimal surgery and a plr value of 233.3 showed 77.9% sensitivity and 84.3% specificity , with 87.2% sensitivity and 63.4% specificity for advanced stage and 81.3% sensitivity and 71.8% specificity for suboptimal surgery . in a univariate cox survival analysis , these ratios and a pretreatment platelet count 400,000 cells / mm showed significant association with poor os . however , in the multivariate cox survival analysis , nlr > 2.7 was also a significant prognostic marker , along with stage and postoperative residual disease . our results are consistent with those of previous studies and support the growing notion that the inflammatory process is linked to adverse outcomes in pelvic serous tumors . is performance of a para - aortic lymphadenectomy essential for pftc ? due to its rarity , optimal management for patients with pftc has not been well defined , and there is a tendency to treat this malignancy like ovarian cancer . the need for complete surgical staging , including lymph node sampling or dissection , is controversial . these procedures may be performed in different ways by different surgeons in many centers ; some highlight early extensive lymphadenectomy , some perform only pelvic lymphadenectomy , and others favor lymph node sampling or do not always perform lymphadenectomy . in our study , in univariate and multivariate analyses , type of staging surgery ( no staging , partial , or complete ) showed significant association with os . koo et al . reported that para - aortic lymphadenectomy is an important procedure in patients with pftc and suggested that para - aortic nodes are the sentinel lymph nodes in these patients . in our analysis of patients treated with pelvic lymphadenectomy with or without para - aortic lymphadenectomy ( partial vs. complete staging ) , we found that the complete staging group showed significantly longer os compared with the partial staging group . the mean and median pfs tended to be better in the complete staging group than in the partial staging group , but the difference was not significant . potential limitations of this study include its retrospective nature , the use of two different devices for blood sample analysis for calculation of nlr , plr , and platelet counts , and the evaluation of data without preoperative c - reactive protein results because this test is not performed routinely at the six centers . despite these limitations , the availability of good follow - up data and one of the largest pftc cases series should increase the validity of results and mitigate the weaknesses . the following conclusions can be drawn from our data . first , in univariate analysis , only pretreatment nlr > 2.7 . however , in univariate analysis , pretreatment nlr > 2.7 , pretreatment plr > 233.3 , and pretreatment platelet count 400,000 cells / mm showed significant association with poor os . nlr and plr are inexpensive and easy tests to perform , and they provide useful prognostic information . finally , patients who underwent complete staging surgery for pftc had longer pfs and os . | purposethe purpose of this study is to evaluate the prognostic role of preoperative neutrophil to lymphocyte ratio ( nlr ) and platelet to lymphocyte ratio ( plr ) and the need for para - aortic lymphadectomy in patients with primary fallopian tube carcinoma ( pftc).materials and methodsninety - one patients with a diagnosis of pftc were identified through the gynecologic oncology service database of six academic centers .
clinicopathological , surgical , and complete blood count data were collected.resultsin univariate analysis , advanced stage , suboptimal surgery , and nlr > 2.7 were significant prognostic factors for progression - free survival , whereas in multivariate analysis , only advanced stage and suboptimal surgery were significant .
in addition , in univariate analysis , cancer antigen 125 35 u / ml , ascites , advanced stage , suboptimal surgery , nlr > 2.7 , plr > 233.3 , platelet count 400,000 cells / mm3 , staging type , and histological subtype were significant prognostic factors for overall survival ( os ) ; however , in multivariate analysis , only advanced stage , suboptimal surgery , nlr > 2.7 , and staging type were significant .
inclusion of pelvic and para - aortic lymphadenectomy in surgery showed significant association with longer os , with a mean and median os of 42.0 months and 35.5 months ( range , 22 to 78 months ) , respectively , vs. 33.5 months and 27.5 months ( range , 14 to 76 months ) , respectively , for patients who underwent surgery without para - aortic lymphadenectomy ( hazard ratio , 3.1 ; 95% confidence interval , 1.4 to 5.7 ; p=0.002).conclusionnlr ( in both univariate and multivariate analysis ) and plr ( only in univariate analysis ) were prognostic factors in pftc .
nlr and plr are inexpensive and easy tests to perform .
in addition , patients with pftc who underwent bilateral pelvic and para - aortic lymphadenectomy had longer os . |
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castleman 's disease ( cd ) is a relatively rare lymphoproliferative disorder with excessive inflammatory features . castleman 's disease has been histopathologically subclassified into three types , that is , hyaline - vascular ( hv ) , plasma - cell ( pc ) , and mixed types , while the disease has been also subclassified into two entities from the perspective of their clinical presentations , that is , unicentric cd and multicentric cd ( mcd ) . in general , while the former is a localized disease , is often asymptomatic , and is curable by surgical excision of the involved lymph node(s ) , the latter with systemic lymphadenopathy is frequently accompanied by various systemic manifestations , such as pyrexia , fatigue , organomegaly , and systemic fluid retention . in addition , cd has been etiologically subcategorized into two subtypes based upon the positive- and negative- associations with human immunodeficiency virus ( hiv ) infection . while most hiv - associated cd patients are positive for human herpes virus ( hhv)-8 , hhv-8 has been shown to be associated with 4050% of hiv - negative cd patients . recently , the newly proposed classification distinguished disease subtypes of mcd based on hhv-8 infection status , which is more closely associated with pathogenesis and response to treatments . . 1 defined hiv - negative and hhv-8-negative mcd as idiopathic mcd ( imcd ) , and they suggested that one or more underlying pathologic conditions , that is , systemic inflammatory disease , paraneoplastic syndrome and/or a non - hhv-8 viral infection , might drive imcd with hypercytokinemia . nevertheless , the precise pathophysiology of mcd remains not to be fully defined , however , the deregulated interplay of various inflammatory soluble factors has been considered to contribute to the development and progression of mcd . in particular , interleukin-6 ( il-6 ) has been shown to act as a key player in the pathogenesis of mcd 2 . indeed , while anti - hhv therapy and rituximab with or without chemotherapy are required for the treatment of hiv - positive mcd 3 , treatments using corticosteroids and/or the anti - il-6 targeting therapies , that is , tocilizumab ( an anti - il-6 receptor antibody ) , or siltuximab ( an anti - il-6 antibody ) , have been highly efficacious against hiv - negative mcd 4,5 . recent studies have proposed a new disease entity of tafro syndrome as a rare variant of cd which is characterized by concomitant thrombocytopenia , anasarca , myelofibrosis , renal dysfunction and organomegaly , in addition to the typical clinical / histological features of mcd 6 . while hiv or hhv-8 infection has no relevant association with tafro syndrome 7,8 , elevation of serum il-6 it has been also reported that the complete remission rates following therapies with corticosteroids and/or il-6-targeting strategies are lower in tafro syndrome than in classical mcd 8 , suggesting the possible involvement of various proinflammatory cytokines other than il-6 in tafro syndrome . thalidomide is an immunomodulatory therapeutic arsenal against plasma cell malignancies , especially for multiple myeloma , wherein il-6 plays a crucial role in disease activity . thalidomide has been shown to be potent in inhibiting tumor necrosis factor ( tnf)- , il-1 , il-12 , and vegf in addition to il-6 , and can stimulate t cells via its interaction with cereblon 10,11 . it has been also demonstrated that thalidomide is capable of decreasing il-6 levels , lowering c - reactive protein , and , thereby , inducing remission in mcd . in this article , we report a case of a patient with tafro syndrome who was successfully treated with thalidomide , in addition to corticosteroid and tocilizumab . although the initial therapy with prednisolone ( psl ) and tocilizumab only achieved partial remission and failed to resolve the ascites , the addition of thalidomide efficiently resolved the intractable ascites . a 56-year - old man was admitted to our hospital complaining of dyspnea , abdominal distension , pyrexia , and systemic lymphadenopathy . computed tomography revealed the presence of mild hepatosplenomegaly , pleural effusion , pericardial effusion , massive ascites , and systemic lymphadenopathy . in addition , blood examination showed anemia ( hemoglobin 8.1 g / dl ) , increased white blood cells ( 12.7 10/l containing 89% of neutrophils ) and thrombocytopenia ( 76.0 10/l ) , while serological testing showed elevated c - reactive protein ( crp ) ( 11.7 mg / dl ( normal range ; < 0.3 ) ) and alkaline phosphatase ( alp ) ( 1007 iu / l ( normal range ; 80260 ) ) , hypoalbuminemia of 2.0 g / dl ( normal range ; 4.15.1 ) , renal dysfunction ( serum creatinine 1.43 mg / dl ( normal range ; 0.61.1 ) and positivity for anti - nuclear antibody ( discrete - speckled type ( 1280 ) and speckled type ( 80 ) ( normal range ; < 40 ) ) . the serum il-6 was increased to 8.1 pg / ml ( normal range ; 4.0 pg / ml ) , and the plasma vascular endothelial cell growth factor ( vegf ) level was elevated to 244 pg / ml ( normal range ; < 115 pg / ml ) . albumin concentrations in the pleural effusion and ascites were increased to 2.4 g / dl and 3.0 g / dl , respectively , indicating increased vessel permeability . bone marrow examination showed normal cellularity with slight megakaryocyte hyperplasia , but not plasmacytosis or myelofibrosis . biopsy of the right inguinal lymph node revealed the histological diagnosis of pc - type mcd ( fig.1 ) , while the in situ hybridization of hhv-8 was negative in the lymph node specimen . he was diagnosed as having tafro syndrome based on the pathological findings , laboratory tests and the clinical features . the initial treatment with 1 mg / kg of psl failed to resolve the series of systemic symptoms . however , the additional therapy of four doses of 4 mg / kg tocilizumab given every other week ameliorated the systemic symptoms , including lymphadenopathy , or pleural effusion , and improved the laboratory data , including anemia , thrombocytopenia , hypoalbuminemia , and crp elevation persisted . prednisolone was tapered off during this period ; however , massive ascites was refractory to the combination therapy with tocilizumab and psl ( fig.2a ) . during this time period , the serum levels of il-6 and vegf were further elevated to 188 pg / ml and 996 pg / ml , respectively ( fig.3 ) , which suggested the need for additional therapeutic intervention against the proinflammatory cytokines that might promote vessel hyperpermeability and cause intractable ascites even under treatment with tocilizumab plus psl therapy . supported by the previous reports demonstrating the inhibitory effect of thalidomide on proinflammatory cytokines , including vegf , in patients with refractory ascites and pericardial effusion along with the angiogenic effects 12,13 , thalidomide was initiated at 100 mg / day , every other week with the administration of tocilizumab because of the lack of any promising therapeutic strategy for the sublethal disease condition . before starting thalidomide therapy , the use of thalidomide for this patient with tafro was approved by the institutional ethical committee , and the patient was fully given informed consent . his abdominal distention gradually subsided and complete resolution of the ascites was confirmed by ct after approximately 2 months of thalidomide therapy ( fig.2b ) . although thalidomide was discontinued due to arthralgia and muscle cramps after 4 months of treatment , there has been no recurrence of ascites developed up to 2 years after the continuance of tocilizumab therapy for 2 years . hematoxylin and eosin ( he ) staining showed that the biopsied lymph node comprised slightly - indistinct large hyperplastic follicles with expanded mantle zones . cd20 immunostaining indicated that the follicle comprised b lymphocytes ( c ) . computed tomography ( ct ) scan images . abdominal ct scan views prior ( a ) and following ( b ) thalidomide treatment . the massive ascites at the earlier stage ( a ) was successfully resolved after 2 months of treatment with thalidomide ( b ) . treatment course and serum interleukin-6 ( il-6 ) and plasma vascular endothelial cell growth factor ( vegf ) levels . the baseline serum il-6 and plasma vegf levels were 8.1 and 244 pg / ml , respectively , at diagnosis . these levels increased further to 188 and 1680 pg / ml after the initiation of tocilizumab therapy , and decreased gradually following the addition of thalidomide ( thal ) therapy . there are two major issues to be discussed in association with the clinical course of the present case . first , it is worthwhile to discuss the possible mechanism underlying the tocilizumab - refractory ascites despite the dramatic efficacy of tocilizumab for systemic symptoms other than ascites . when we looked back at the serum il-6 level and the plasma vegf level , the data showed prominent increases of both cytokines after the initiation of tocilizumab treatment ( fig.3 ) . one possible explanation for the increase in il-6 was that blockade of the il-6 receptor by tocilizumab treatment stimulated the production of il-6 via a feedback effect 14 . since it has also been suggested that the il-6 signaling was pivotal in the promotion of vegf production 15 , the plasma vegf level was expected to decrease following blockade of the il-6 receptor by tocilizumab 16,17 . however , in our case , the plasma level of vegf continued to increase even after treatment with tocilizumab , and the ascites remained the only symptom that was refractory to tocilizumab , in contrast to other symptoms . it has been shown that several other proinflammatory cytokines , such as il-1 , tnf- , and thrombin , can induce vegf synthesis in peritoneal mesothelial cells 18 . thus , it is possible that the local peritoneal inflammation associated with tafro syndrome and the increased vegf synthesis might be induced not simply by il-6 , but also by other , as yet undetermined , proinflammatory cytokines that promote vegf production in our case . the second issue to be debated is the mechanism of action of thalidomide on the ascites in our case . thalidomide has been shown to exhibit a preventive effect on angiogenesis through inhibition of vegf . therefore , if blockade of vegf was the central mechanism in resolving the tocilizumab - refractory ascites by thalidomide treatment , one could expect that the reduction in plasma vegf would precede the improvement of ascites . however , in our case , the plasma vegf level remained high with thalidomide therapy , despite the improvement of tocilizumab - resistant ascites , and eventually decreased after the improvement of ascites in our case . considering the multifaceted immunomodulatory effects of thalidomide , that is , the inhibitory effect on basic fibroblast growth factor-2 and tnf- , in addition to il-6 and vegf 1921 , we speculate that the overexpression / hyperactivation of various cytokines , other than il-6 or vegf , was involved in tafro syndrome - associated ascites , and that thalidomide resolved the tocilizumab - refractory ascites not by the inhibition of the il-6/vegf axis but by the inhibition of , as yet undetermined , proinflammatory soluble factors that function as the upstream mediators for vegf production . thus , it is possible that the coincident decline of both il-6 and vegf was not requisite for the improvement of ascites but represented the resultant phenomena after the improvement of peritoneal inflammation in our case . while this report is the first to demonstrate the therapeutic effect of thalidomide for tafro syndrome , the effect of thalidomide on mcd has been reported in several cases ( table1 ) 13,2227 . in those series , the pc type was dominant histologically , and thalidomide was generally effective in both inducing and maintaining remission . furthermore , thalidomide is effective for plasma cell dyscrasias , such as multiple myeloma , through its immunomodulatory effects . thus , it is possible that thalidomide is also effective for tafro syndrome , which is a subtype of mcd . indeed , while there has been no report of thalidomide treatment in tafro syndrome , other than our case , one mcd case with thrombocytopenia and anasarca reported by lee et al . 13 seemed to at least partially meet the criteria of tafro syndrome , and was induced in partial remission by thalidomide therapy . on the other hand , tafro syndrome has been treated with various types of immunosuppressive therapies , including calcineurin inhibitors , corticosteroids , and tocilizumab ( table2 ) 79,28,29 . while those immunosuppressive therapies have been largely effective for tafro syndrome , caution should be noted for the complication of severe infections . since the standard therapy has not been established so far , thalidomide can be one of therapeutic options for tafro syndrome . review of thalidomide therapy for multicentric castleman 's disease and tafro syndrome n , number of patients reported ; pc , plasma - cell type ; hv , hyaline - vascular type ; hiv , human immunodeficiency virus infection ; hhv-8 , human herpes virus-8 involvement ; tx . , therapy ; cs ; corticosteroid(s ) , chop , cyclophosphamide ( cy ) , adriamycin , vincristine and prednisolone ; vp-16 , etoposide ; rit , rituximab ; ivig , intravenous immunoglobulin therapy ; csa , cyclosporine a ; toc , tocilizumab ; pr , partial remission ; cr , complete remission ; pd , progressive disease ; thal , thalidomide ; len , lenalidomide ; na , not available . review of reported cases with tafro syndrome il-6 , interleukin-6 ; vegf , vascular endothelial cell growth factor ; mcd , multicentric castleman 's disease ; cmv , cytomegalovirus . in conclusion , we report a case of tafro syndrome that was successfully treated with thalidomide in addition to tocilizumab , and this case report suggests the possible therapeutic application of thalidomide for tafro syndrome for which a standard treatment strategy has not been established to date . as a novel disease concept , it is urgently needed to clarify the pathophysiology of tafro syndrome , so that we will be able to develop more rationalistic treatment strategy for tafro syndrome which possibly contains agents directed against both il-6 signaling and vegf . | key clinical messagetafro syndrome have been proposed as a rare variant of castleman 's disease .
this article reports a case of a 56-year - old man with tafro syndrome who was successfully treated with thalidomide in spite of the refractoriness to prednisolone and tocilizumab .
thalidomide may be one of the treatment options for tafro syndrome . |
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depression and anxiety are common mental health disorders ; however , the risks for depression and anxiety are not distributed equally . women are twice as likely as men to experience depression and generalized anxiety disorder due to a variety of biological and social factors [ 1 , 2 ] . women 's hormonal fluctuations associated with menstruation , pregnancy , and childbirth may increase their vulnerability to mental distress . social factors such as poverty , race , and neighborhood also increase women 's risk for depression and anxiety . low - income women 's risk of depression is almost double that of their nonpoor counterparts [ 2 , 3 ] . poverty contributes to daily worries about basic needs and limits the ability to engage in recreational activities . poor women are more likely to experience financial troubles , relationship problems , poor health , and unemployment which can contribute to the onset of depression [ 35 ] . poverty may combine with a lack of social support to make low - income women vulnerable to anxiety and depressive disorders . for women , racial differences in risk for depression and anxiety vary widely . in some studies , african american women have higher rates of depression and anxiety compared to white women , while other data demonstrate that african american women have lower rates [ 2 , 711 ] . lower rates of anxiety and depression among african american women may be attributed to psychosocial resources , emotional resilience , social support , and ethnic identity . higher rates in african american women are generally attributed to greater levels of poverty , poor health , and stigma about mental health care [ 1214 ] . anxiety disorders among african american women are particularly understudied , which contributes to uncertainty about its risk factors and rates in this population . neighborhood disadvantage can obstruct the creation of bonds among residents , reducing social support and social control that serve as a protective factor against psychological and economic stressors . by increasing daily hassles and reducing protective factors , poor neighborhoods increase an individuals ' vulnerability to depression when negative events occur . women who live in poor neighborhoods have fewer opportunities for employment , education , recreational activities , and positive social interactions which can cause or exacerbate depression or anxiety . african american women are more likely to live in poor neighborhoods than their white counterparts . to date , few studies have been able to compare rates of depression and anxiety in african american and white women while examining both individual and neighborhood characteristics . we used a unique cohort of african american and white women from the pathways to adulthood , a follow - up of a longitudinal cohort of inner city women and their children in baltimore , md , usa . our aims were to ( 1 ) examine what individual and neighborhood socioeconomic factors are associated with depression and anxiety and ( 2 ) determine whether these relationships varied between african american and white women . the johns hopkins perinatal collaborative study in conjunction with the pathways to adulthood follow - up was a retrospective study of three generations of families initially living in the inner city baltimore . the perinatal collaborative study enrolled 2307 inner - city women ( referred to throughout as first - generation mothers ( g-1s ) ) who were selected at random at the time of their first prenatal visit to a public obstetric clinic at johns hopkins hospital between 1959 and 1965 . the study later collected data on the 2694 children of these first - generation mothers who were born between 1960 and 1965 ( children referred to as second generation ( g-2s ) throughout ) . these children were initially studied prospectively with data gathered between birth and 8 years of age regarding their neurologic and cognitive development , health , behavior , and family , and neighborhood socioeconomic characteristics . from 1992 to 1994 , the pathways to adulthood study collected additional information from the 1758 g-2s ( then aged 27 to 33 ) about their lives from age 9 to present . follow - up data included information on education , employment , family composition , health , health care usage , and income . in addition to these data that were collected directly from the g-2 individuals , data regarding the neighborhood characteristics of each g-2 at birth , at 11 - 12 years of age , at 16 - 17 years of age , and at age of follow - up ( ages 2733 ) were obtained through census data . for a full description of sample population and methods , see . our final sample ( n = 989 g2 females ) included the 75% of female respondents who provided information ( in - person or via telephone ) for the pathways to adulthood study and had information on the depression or anxiety measures . the general health questionnaire ( ghq-28 ) is a self - administered screening questionnaire aimed to detect probable psychiatric disorders in primary care settings . the threshold for a case ( individuals who probably need further evaluation ) is 4 - 5 depending on the population . for greater specificity , we defined a case as a ghq score greater than or equal to 5 . the ghq-28 has four subscales : somatic , anxiety , social dysfunction , and depression and that each contains seven questions . the subscales provide individual diagnostic or profile information ; however , they have no diagnostic thresholds . for our study , a ghq depression subscale score greater than or equal to 4 was defined as severe depression and a ghq anxiety subscale score greater than or equal to 4 was defined as anxiety . we examined individual characteristics ( including demographic age , race , number of children , and marital status ) and health - related characteristics ( current drinking status and self - reported health rating ) . although we had two measures for income , self - reported total household income and personal income , the high rate of missing values in both measures precluded their use in the analysis . we treated assets as a continuous variable ranging from 0 to 6 , with one point given for a yes response when asked about each of six assets ( current personal checking account ; current ira or pension , own house or condo , car , truck , or motorcycle ownership , credit or charge account , and current savings account ) . after factor analysis showed home ownership to be the most robust measure , we included homeownership in our model . each respondent 's address at the time of the interview was linked to data from the appropriate census tract . we used cross - tabulations to compare all categorical variables by race , neighborhood , and weight status . we used chi - square statistics as the corresponding measure of heterogeneity . for continuous variables , we determined summary measures ( mean and standard error ) for each sub - group . next , we used linear regression to examine the multivariate associations between our outcomes depression and anxiety subscale scores and the covariates of interest . during this process , we included those in which we had substantive a priori interest based on prior literature and included those that were of at least borderline statistical significance during forward stepwise selection ( p < 0.10 ) . for multivariate analysis , we used linear regression clustered on census tract while controlling for marital status , race , age at first child , number of children , years of education , homeownership , neighborhood poverty , and percent african american in the neighborhood . the point estimate is the same as in nonclustered linear regression ; only the standard error is corrected . all analyses were done using intercooled stata ( version 11.0 ; stata corporation , college station , tex , usa ) . a summary of baseline sociodemographic , health , and neighborhood characteristics of the cohort is presented in table 1 . white women scored significantly higher than african american women on total ghq score ( 4.42 versus 3.12 ) , mean depression score ( 0.57 versus 0.25 ) , and mean anxiety score ( 1.80 versus 1.22 ) , all p < 0.05 . although household income was not significantly different between white and african american women ( $ 35,638 versus $ 31,807 , p = 0.18 ) , they differed on many other socioeconomic indicators . compared to their white counterparts , african american women had more years of education ( 12.87 versus 10.89 for whites ) , were more likely to have completed college ( 14% versus 3% ) , and had higher personal income ( p < 0.05 ) . white women had a higher mean number of assets than their african american counterparts ( 3.17 versus 2.48 , p < 0.05 ) , and african american and white women did not differ on the number of children or age of first child ( p > 0.05 ) . however , white women were significantly more likely to be married ( 59% versus 27% , p < 0.05 ) , which may , in part , account for the differences in both assets and household income . african american and white women also substantially differed by current neighborhood characteristics ( table 1 ) . white women lived in neighborhoods with an average household income that was significantly higher than african americans ( $ 37,578 versus $ 28,655 ) . white women were less likely to live in poor neighborhoods ( 25% versus 58% ) or to report a concern with crime ( 25% versus 36% ) , all p < 0.05 . the neighborhoods in which the african american study participants lived had a significantly higher proportion of african americans than their white counterparts . on average , white women lived in neighborhoods that were 8% african american , while african women lived in neighborhoods that were predominantly african american ( 72% ) . approximately 30% of the sample had a ghq score greater than or equal to 5 ( table 2 ) . they had fewer years of education ( 12 versus 12.7 ) , fewer assets ( 2.2 versus 2.8 ) , lower family income ( $ 29,790 versus $ 33,505 ) , and personal income ( $ 12,520 versus $ 16,487 ) , all p values < 0.05 . women with a greater ghq score had a higher bmi ( 27.3 versus 26.1 ) , were more likely to be smokers ( 52% versus 42% ) , and were less likely to report good or excellent health ( 32.1 versus 65.2 ) , all p values < 0.05 . overall , over half the population in both groups lived in poor neighborhoods that were predominately african american , and a significant proportion had concerns about crime within their neighborhoods . in multivariate logistic models , only white race and poor self - rated health were associated with a higher ghq score . women with good or excellent health were 75% less likely to have a ghq score greater than 4 ( or 0.25 , 95% ci 0.170.36 ) . african american women were almost 50% less likely than their white counterparts to have a positive ghq score ( or 0.56 , 95% ci 0.320.99 ) . women with higher depression scale ratings ( 4 ) differed significantly from their less depressed counterparts ( table 3 ) . more depressed women had fewer years of education ( 10.9 versus 12.6 ) and were less likely to have a college degree ( 0% versus 12% ) , all p < 0.05 . depressed women had lower assets ( 0.8 versus 2.7 ) , lower household income ( $ 21,300 versus $ 32 , 633 ) , and lower personal income ( $ 8,591 versus $ 15,482 ) , all p < 0.05 . depressed women were less likely to be married ( 8% versus 34% ) and had a lower age at first child 's birth ( 18.7 versus 21.2 ) , all p < 0.05 . they had a higher bmi ( 29 versus 26 ) , were more likely to be drinkers ( 64% versus 44% ) , and less likely to report good health ( 24% versus 57% ) , all p < 0.05 . depressed women lived in neighborhoods with lower median household incomes ( $ 24,594 versus $ 30,045 , p < 0.05 ) . however , they did not differ significantly from their counterparts on other neighborhood characteristics such as percent african american , neighborhood poverty , and neighborhood crime . women with higher anxiety ratings ( 4 ) differed significantly from their less anxious counterparts ( table 4 ) . women with a high anxiety score were less likely to be african american ( 76% versus 83.6% ) , had fewer years of education ( 11.9 versus 12.6 ) , and had more children ( 1.72 versus 1.47 ) , all p < 0.05 . in addition , more anxious women had fewer assets ( 1.9 versus 2.76 ) , lower family income ( $ 26,325 versus 33,654 ) , and lower personal income ( $ 12,336 versus 15,917 ) , all p < 0.05 . more anxious women were more likely to be smokers ( 53% versus 43% ) and less likely to report very good or excellent health ( 34% versus 60% ) , all p < 0.05 . table 5 shows the factors associated with score in ghq depression domain . decreased self - rated health , lower age at first child , being unmarried , and white race were significantly associated with a higher depression score . in multivariable analysis , women who were african american ( b = 0.44 ) , were married ( b = 0.21 ) , had older age at first child ( b = 0.02 ) , or reported good or excellent health had lower ghq depression scores , all p < 0.05 . a 30-year - old white unmarried woman who had her first child at age 16 would have an average depression score of 0.95 ( 95% ci 0.711.20 ) , while an african american woman under the same circumstances would have an average score of 0.48 ( 95% ci 0.330.62 ) . a 30-year - old woman who was married and had her first child at 21 would have a depression score of 0.62 ( 95% ci 0.420.82 ) if she were white and 0.13 ( 95% ci 0.020.29 ) if she were african american african american women ( b = 0.77 ) , those with older age at first child ( b = 0.06 ) , and those who reported good or excellent health ( b = 0.99 ) had lower anxiety scores , all p < 0.05 . the average white woman in our sample had an anxiety score of 1.72 ( 95% ci 1.292.14 ) if she reported good or excellent self - rated health and 2.7 ( 95% ci 2.273.14 ) if she did not . the average african american woman in our sample had an anxiety score of 0.81 ( 95% ci 0.491.13 ) if she reported good or excellent health and 1.8 ( 95% ci 1.452.15 ) if she did not . in our study african american women had significantly lower mean depression scores compared to white women . our work is consistent with a number of studies that have shown that african americans have lower rates of depression than their white counterparts [ 2 , 711 ] . although african american women live with greater stress than white women , their coping strategies may make them less vulnerable to the impact of chronic strains [ 25 , 25 ] . additionally , our study indicated african american women had significantly lower anxiety scores than white women . very little research has focused on the rates of generalized anxiety disorder among african american women and white women . a large portion of anxiety research focuses on specific population subgroups ( e.g. , postmenopausal women , mothers of preterm infants ) and subcategories of anxiety ( e.g. , women with hiv , social phobia , etc . ) . consensus has not yet been reached on african american prevalence and presentation of anxiety disorders [ 15 , 26 ] . our study is one of the few to report a decreased risk of both depression and anxiety for african american women compared to white women . for african american and white women in similar circumstances , african american women consistently had lower rates of both depression and anxiety . however due to historic and ongoing socioeconomic disparities , african american women experience significantly more challenging life experiences than their white counterparts ( i.e. , discrimination , neighborhood poverty , and low income ) [ 27 , 28 ] . unmarried women and women who had children at a younger age had higher depression scores . this is consistent with the literature that shows that adolescent mothers have an increased rate of depression and anxiety due to stressful life events [ 29 , 30 ] . prior work also shows that marriage provides economic , social , and psychological support , which contributes to a person 's well - being . these social supports and structured routine can encourage healthy behaviors and discourage harmful ones [ 8 , 31 , 32 ] . health status was also associated with depression and anxiety scores . poor health can increase daily hassles and reduce pleasure , increasing one 's risk for mental distress [ 5 , 13 ] . conversely , depression and anxiety can increase one 's perception of poor health and can worsen outcomes for preexisting conditions . determining causality is beyond the scope of this study . however , simply knowing what factors are associated with greater psychological distress can still lead to better efforts to screen for depression and to target mental health services . the ghq-28 was developed to determine people in a general population who need further evaluation for mental health disorders . the threshold for a case , those who probably need further evaluation , is 4 - 5 , depending on the population . we used similar cut - offs for the depression and anxiety scale of greater than or equal to 4 to determine diagnosis . the pathways to adulthood data had a limited geographic focus , children born in the johns hopkins hospital catchment area . this group had a higher proportion of african americans and higher poverty rates than the usa as a whole . our findings suggest racial differences in anxiety and depression may be due , in part , to differences in socioeconomic status or neighborhood environment . one strength of this study is the use of census data for aggregate neighborhood measures . using a different data source to determine individual and group measures avoids one source of error because the study population may not be a representative sample of the population . an additional limitation , also faced by other researchers studying neighborhood , was characterizing the neighborhood environment solely through some proxy measures based on census data . despite the wealth of census information , other variables that may be more directly associated with depression or anxiety asset mapping may allow a fuller characterization of neighborhood and a deeper understanding of what factors confer health risks . in this cross - sectional study of african american and white women with similar rates of poverty , we sought to quantify the association between measures of anxiety and depression and individual and neighborhood socioeconomic status and to determine whether these relationships varied by race . at similar levels of disadvantage , white women had higher scores for depression and anxiety . we found an increased risk for depression in unmarried women and those with younger motherhood . women with lower reported self - rated health had higher scores for both depression and anxiety . neither neighborhood poverty nor racial composition was in and of itself a predictor of greater risk for anxiety or depression ; however , the significant risk factors cluster in disadvantaged neighborhoods . our work highlights the importance of universal screening for depression or anxiety with more in - depth surveillance based on risk factors rather than on racial classification . | background .
we examined factors associated with depression and anxiety in a cohort of low - income baltimore women .
methods .
we used pathways to adulthood data , a cohort of adults aged 27 to 33 who were born in baltimore between 1960 and 1965 .
our outcomes were a score of > 4 on the general health questionnaire ( ghq-28 ) across the depression or anxiety domains .
linear regression clustered on census tract was used for multivariate analysis .
results . in multivariable analyses , unmarried women , white women , those with lower self - rated health , and younger mothers had higher depression scores .
only lower self - rated health and white race were associated with a higher anxiety score .
neither neighborhood poverty nor racial composition was a predictor for anxiety or depression ; however , the significant risk factors cluster in disadvantaged neighborhoods . conclusion .
our work highlights the importance of universal screening for depression or anxiety with more in - depth surveillance based on risk factors rather than on race . |
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the idea of site - selective artificial ribonucleases which are capable of cleaving any particular rna sequence in vitro and in vivo , is a very attractive approach as , apart from being useful tools in molecular biology , these chemical ribonucleases are anticipated to be helpful for target validation , and even for the development of potential antiviral or anticancer therapeutics . the idealized site - selective artificial ribonuclease would be a compound that is easily synthesized , chemically stable , targeted to any chosen rna sequence , and highly efficient in cleaving of the phosphodiester bonds . the oligonucleotide - based artificial ribonucleases seem to meet the majority of these criteria due to the fact that they can be directed to almost any desired rna region in a site specific - manner by controlling the sequence of the oligonucleotide recognition part . virtually all types of the reactive groups , which are known to catalyze rna scission , have been exploited for design and preparation of artificial ribonucleases [ 13 ] . conjugates of oligonucleotides and rna cleaving groups can be synthesized using two main approaches : incorporation of a catalytic structure in the oligonucleotide in the course of standard synthesis or by postsynthetic derivatization of an unprotected oligonucleotide . the first approach often involves the automated oligonucleotide synthesis , when phosphoramidites of suitably protected nucleosides and catalytic groups are used as building blocks . monomeric building blocks containing various metal complexes [ 58 ] or imidazole - based structures [ 916 ] have been previously proposed for the synthesis of the oligonucleotide - based artificial ribonucleases . the successive synthesis of oligonucleotide and catalytic structures of conjugates on the same solid - phase support is a promising variation of this approach [ 1719 ] . however , this approach is inappropriate for the majority of the potential users of the conjugates due to the necessity to synthesize the unique phosphoramidite derivatives of the corresponding catalytic structures in each particular case , which creates the problems associated with the choice of appropriate protection groups and the deprotection procedure . various rna - cleaving catalysts have been tethered to the 5-terminus of oligonucleotides . however , the available synthetic methods , except for a few examples , allow the incorporation of only a single functional moiety per phosphoramidite unit . generally , 5-tethering can be implemented in two ways either by direct incorporation of a suitably protected and activated constructs into the 5-terminus at the end of standard solid phase synthesis or by postsynthetic derivatization . in the former case , synthesis of highly modified oligonucleotides may become somewhat problematic because increase in a number of incorporations is expected to substantially decrease the overall yield of the conjugated oligonucleotides . alternatively , a precursor molecule can first be formed by reacting the 5-oh group of an assembled oligonucleotide with a heterobifunctional reagent ( first modifier ) bearing both the phosphoramidite moiety and a reactive group . the reactive group of the precursor oligonucleotide is then postsynthetically derivatized with an appropriate functional additive ( second modifier ) [ 21 , 22 ] . this precursor strategy has the obvious advantage that the same parent compound can be used to synthesize a number of differently tethered products . the first generation of oligonucleotide - based artificial ribonucleases , which was prepared using the precursor technique , has recently been reported to display high cleaving activity [ 23 , 24 ] . the precursor approach seems to be advantageous for the synthesis of libraries of oligonucleotide conjugates containing different functional groups . however , the cleavage activity of these artificial ribonucleases so far has been inferior to the natural counterparts in terms of targeting and/or cleavage efficiency . we anticipate that the high level of efficiency of rna cleavage by these oligonucleotide - based artificial ribonucleases can only be achieved if the optimal arrangement of all groups involved in the binding and catalysis is accomplished . it might be possible to improve on this by iterative design of the binding and cleavage domains by taking into account the optimal structures of the cleaving groups and their mutual spatial orientations against the rna target . the structural organisation of artificial ribonucleases and the dynamic behavior of their cleaving constructs seem to be the key factors contributing to the cleavage activity of these compounds . several years ago , we reported a new type of oligonucleotide - based arnases containing bis - imidazole cleaving constructs , which were prepared by precursor technique . it was demonstrated that these highly active bis - imidazole cleaving constructs were extremely flexible and the cleavage of the phosphodiester bond seemed to be a random event , which happened with the high efficiency every time , when these catalytic groups approached the scissile phosphodiester bond [ 23 , 24 ] . in the present paper , we use the developed precursor approach to synthesize the scaffolds of arnases with the systematically varied structure of the cleaving constructs bearing multiple imidazole residues in the catalytic part . we demonstrate here that some of these conjugates exhibit remarkably high cleavage activity within the trna conjugate hybrid and identify possible location(s ) of cleaving group(s ) relative to the scissile c63-a64 site . we also show , here , that the cleaving activity of the conjugates is governed by the intrinsic properties of the catalytic constructs , but not the detailed structure of the dna : rna hybrid , which was the same in all experiments . all buffers used in the experiments were prepared using milliq water , contained 0.1 mm edta , and were filtered through filters with pore size 0.22 m ( millipore ) . oligonucleotides tggtgcgaattctg ( a ) and gatcgaacacaggacct ( b ) were synthesized on asm-700 dna synthesizer ( biosset , russia ) by standard solid - phase phosphoramidite procedure . g. keith ( institut de biologie molculaire et cellulaire du cnrs , strasbourg , france ) . methoxyoxalamido ( mox ) modifiers m used for conjugate synthesis were prepared as described in [ 21 , 24 ] . deoxyribooligonucleotide b ( gatcgaacacaggacct ) was synthesized using standard solid - phase phosphoramidite chemistry , except that the dc - phosphoramidite was replaced with the dc - phosphoramidite . at the end of the automatic synthesis , the core 17-mer was coupled with one of the modifiers m ( type 1 , type 2 , type 3 , or type 4 ; see figure 1 ) in 0.2 m in acetonitrile for 15 min . the prepared oligonucleotide mox precursors were then functionalized with 2 m solution of histamine in dimethylformamide for 3 hrs at 20c at constant shaking followed by deprotection with ammonia to produce the oligonucleotide conjugates containing multiple - imidazole catalytic structures . the conjugates were purified by electrophoresis in 16% polyacrylamide/8 m urea gel under denaturing conditions . the purified conjugates were analyzed by esi and/or maldi ms ( calculated and experimental values of molecular masses are listed in figure 1 ) . in the cases when maldi - ms analysis did not produce the expected mass spectra due to the conjugate fragmentation upon analysis , the conjugates were analyzed by electrophoresis under denaturing conditions ( figure 1(c ) ) and by hplc ( primary data not shown ) . conjugates were designated as b - im(n / m ) , where b - im corresponds to the oligonucleotide b and shows that i m are in catalytic part , n is the number of imidazole residues ( from 2 to 32 ) , and m corresponds to the type of anchor group ( 1 , 2 , 3 , or 4 ; see figure 1(a ) ) . specific radioactivity of the [ p]-trna was 5 10 cpm / pmole . standard reaction mixture ( 10 l ) contained imidazole buffer ( 50 mm imidazole buffer , ph 7.0 , 200 mm kcl , 0.1 mm edta , 100 g / ml total trna from escherichia coli as carrier ) , 5 10 m [ 3-p]-trna , and one of oligonucleotide conjugates b - im(n / m ) at concentrations ranging from 5 10 to 5 10 m ( as indicated in the legends in the figures ) . reactions were carried out at 37c and were quenched by precipitation of trna and trna fragments with 150 l of 2% lithium perchlorate solution in acetone . rna was collected by centrifugation and dissolved in loading buffer ( 6 m urea , 0.025% bromophenol blue , 0.025% xylene cyanol ) . trna cleavage products were analyzed by electrophoresis in 12% polyacrylamide/8 m urea gel . to identify cleavage sites , an imidazole ladder and a g - ladder produced by partial trna cleavage with rnase t1 were run in parallel . to obtain quantitative data , the gel was dried and analyzed using molecular imager fx ( bio - rad ) . the total extent of rna cleavage was determined as a ratio of radioactivity found in the trna fragments to total radioactivity applied on the gel lane . to measure the kinetics of the trna cleavage with the oligonucleotide conjugates b - im(n / m ) , the reaction mixture of 100 l was prepared containing imidazole buffer , 5 10 m 3-[p]-trna and one of the oligonucleotide conjugates b - im(n / m ) at different concentrations ( from 5 10 to 5 10 m ) . an aliquot ( 10 l ) of the reaction mixture was withdrawn and frozen for lately assay . reaction aliquots were defrosted and immediately precipitated with 150 l 2% lithium perchlorate in acetone . after that , the reaction mixtures were assayed as described above . in some experiments , imidazole buffer was replaced by hepes - buffer ( 50 mm hepes - koh , ph 7.0 ; 0.2 m kcl , 0.1 mm edta , 100 g / ml rna - carrier ) , or cacodylate buffer ( 50 mm cacodylic acid - koh , ph 7.0 ; 0.2 m kcl , 0.1 mm edta , 100 g / ml rna - carrier ) or tris - hcl - buffer ( 50 mm tris - hcl , ph 7.0 ; 0.2 m kcl , 0.1 mm edta , 100 g / ml rna - carrier ) . the experiments where imidazole buffer was replaced by other buffers are indicated in text and figure legends . in the competition experiments , to a reaction mixtures ( 10 l ) containing imidazole buffer , 5 10 m 3-[p]-trna oligonucleotide a ( complementary to sequence 62 - 76 of the trna ) or oligonucleotide b was added up to concentration 5 10 m. the mixtures were incubated for 10 min at 37c , then one of oligonucleotide conjugates b - im(n / m ) at concentration 5 10 m was added and the mixtures were incubated at 37c for different times a gel - mobility shift assay of trna binding with oligonucleotide b and conjugates b - im(4/2 ) was performed as follows . [ 3-p ] trna ( 5 10 m ) was incubated at 37c during 30 min in reaction mixture ( 10 l ) containing hybridization buffer ( 50 mm tris - hci , ph 7.0 , 200 mm kci , 0.1 mm edta ) and oligonucleotide b or conjugate b - im(4/2 ) ranging in concentration from 1 10 to 5 10 m. after incubation , 8 l of loading buffer ( 50% glycerol , 0.025% bromophenol blue , 0.025 xylene cyanol ) was added to each probe and probes were immediately applied onto running native 10% page with 100 mm tris - borate , ph 8.3 as running buffer preequilibrated at 4c for 3 h. electrophoresis was performed at 4c for 5 h at 450 v. to obtain quantitative data , the gel was dried and analyzed using molecular imager fx ( bio - rad ) . molecular modelling was performed using sybyl 6.6 ( tripos inc . ) on a silicon graphics o2 workstation as described in . all calculations ( mm / md ) were carried out in vacuo using kollman - all force field parameters and a distance - dependent dielectric constant equal to 4 . the building of the model molecules ( see figure 1 ) was carried out in three stages . first , an a - like dna : rna hybrid was built using biopolymer / sybyl 6.6 . second , the catalytic fragments im(4/1 ) , im(4/2a ) , and im(4/2b ) were built using sketch / sybyl 6.6 . charge distributions for these constructs were calculated using a semiempirical mo program , mopac , after geometric optimization . in the last step , each the corresponding cleaving fragment was attached to the dna : rna hybrid via the terminal 5-phosphate group of the g1 nucleotide residue . for each molecule , several different starting structures were created , differing in the conformation and location of the cleaving group , ranging from in to out extreme positions relative to the dna : rna hybrid . the starting structures of each molecule were subjected to minimization to a gradient convergence of 0.05 kcal / mol , considering the dna : rna hybrid as a solid body . each starting structure was individually subjected to a 12 ps md run using simulated annealing as follows . the system was heated at 600 k during 2 ps followed by a gradual cooling down to 100 k over 10 ps . a final minimization to convergence criteria was performed resulting in the respective final conformations for each type of molecule . s and f symbols before the conjugate name indicate the starting and final conformation , respectively . active conformations were obtained for each model molecule ( o1-im4/1 , o2-im4/1 o1-im4/2a , o2-im4/2a , o1-im4/2b , and o2-im4/2b , where type1 shows the anchor group 1 ) using a similar simulating annealing protocol . in these molecules , the cleaving groups were deliberately located in the most favorable position for successful cleavage at the c63-a64 site . namely , the distance between n1 of one of the imidazole residues and 2 oh group of c63 was ~3 and the distance between n1 of the other imidazole residue and the oxygen of the phosphate group , connecting c63 and a64 , was around 3 [ 26 , 27 ] . the rna cleaving oligonucleotide conjugates have been prepared in two - step procedure described in [ 21 , 22 ] . in the first step , the precursor 17-mer gatcgaacacaggacct ( b ) was built up with multiple methoxyoxalamido ( mox ) modifiers of different type ( see supplementary material available online at doi:10.4061/2011/748632 for the structures of all conjugates synthesized in this study ) . in the second step , the prepared oligonucleotide - mox precursors were functionalized with histamine to yield after deprotection step the oligonucleotide conjugates b - im(n / m ) , bearing from 4 to 32 histamine residues at the 5-end . figure 1 provides structures of oligonucleotide conjugates b - im(4/n ) and b - im(8/1 ) studied in this work . the conjugates hereafter were referred to as b - im(n / m ) , where b - im designates the oligonucleotide - imidazole conjugates , n shows the number of imidazole residues , and m indicates the type of anchor group . in the cases , when two or three conjugates contain equal number of imidazole residues and the same anchor group , the conjugate number is shown in the brackets . the distance between oligonucleotide and imidazole residues of the cleaving part the thymidine nucleoside , presenting in the structure of conjugate , was supposed to be involved in base pairing with rna or might be stacked on heteroduplex contributing to the duplex stability . in the conjugate type 3 , an anchor group was chemical construct mimicking phosphodiester bond and used as a rule as nonnucleotide insertion in oligonucleotide . type 4 anchor group was used in the conjugates 1013 to increase the number of branches , bearing imidazole residues . for this purpose and to increase the linker length and flexibility , the anchor groups of types 1 and 2 were incorporated also into the linker structures ( see figure 1-sm , conjugates 69 and 1113 ) . moreover , the length and structure of linkers , connecting imidazole residues and anchor , were distinguished sufficiently among the compounds ( table 1 and figure 1-sm ) . conjugates containing equal number of imidazole residues , for example , b - im(4/m ) or b - im(8/m ) , differ not only in anchor groups , but also in a linker length . in this conjugate , four imidazole residues were placed nonsymmetrically in respect to the phosphate groups forming the anchor , which connected the oligonucleotide b and the cleaving construct . the identity of the conjugates was confirmed by mass - spectrometry ; ( see figure 1 ) . in addition , the purity of the conjugates was confirmed by electrophoresis in 12% paam/8 m urea gel ( figure 1(c ) ) . it is seen that the purity of the conjugates is close to 95% . only in the case of conjugates 2 , 5 , 10 , and 16 , yeast trna was chosen deliberately as a target to assess cleavage activity of the conjugates . the rationale behind this was that other oligonucleotide - based arnases including three prepared using precursor approach had been tested using this target [ 1 , 12 , 1416 , 28 ] . figure 2(a ) represents cloverleaf structure of yeast trna showing the target site for the conjugates b - r(n / m ) . the recognition element of the studied arnases was represented by the 17-mer oligonucleotide b , which was complementary to the sequence 44 - 60 and was designed to deliver the imidazole residues to the sequence cacag in the t - arm of the trna , which is known to be highly sensitive towards cleavage [ 2932 ] . it was previously shown that oligonucleotide b could efficiently bind to this sequence in the trna , which resulted in unfolding of entire tc hairpin , whereas the aminoacceptor and anticodon stems of the trna were only slightly affected by the oligonucleotide binding [ 3335 ] . the conjugates b - im(n / m ) were designed to catalyze rna cleavage via transesterification reaction . to compare the ribonuclease activities of the conjugates , we used the conditions of single reaction turnover ( [ b - im(n / m)]>[rna ] ) . the oligonucleotide part of the conjugate provides efficient hybridization with target sequence in trna and formation of the stable heteroduplex ( kd = 7 m 1 m ; v. petyuk , unpublished data ) . we believe that under the experimental conditions the rate and efficiency of site - selective cleavage of the target is affected only by the catalytic part of the conjugate . site - selective cleavage of [ 3-p]trna by the conjugates was carried out at 37c in 50 mm imidazole buffer . the reaction was initiated by addition of the conjugate b - im(n / m ) to the reaction mixtures . localization of cleavage sites was performed by comparison of products obtained upon cleavage of trna by the conjugates with the products of random rna cleavage with rnase t1 and 2 m imidazole buffer , ph 7.0 [ 25 , 36 ] . figure 2(b ) shows typical results of the analysis of trna cleavage by the conjugates b - im(n / m ) at 37c under standard conditions . no measurable spontaneous degradation of trna under the experimental conditions in the absence of reagents and in the presence of unmodified parent oligonucleotide b occurred ( figure 2(a ) , lanes c1 and c2 ) . the conjugates cleave trna with high rate mostly at the phosphodiester bonds c63-a64 and a64-g65 . the extents of trna cleavage at these bonds are about 70% and 20% , respectively . moreover , some minor cleavage at the phosphodiester bonds c61-a62 and a62-c63 within the target sequence is observed ( figure 2(a ) , lanes 110 ) . the ratio between the products of trna cleavage at these phosphodiester bonds located in close proximity to each other in the target sequence seems to correspond to steric accessibility of these bonds to cleavage by the conjugates b - im(n / m ) . in the case of conjugate b - im(4/2a ) ( similar cleavages were observed for the conjugates with 16 , 24 , and 32 imidazole residues ) , slight cleavages are observed at u8-a9 and c75-a76 phosphodiester bonds ( less then 3% of the total extent of trna cleavage ) , known as well as c61-a62 and c63-a64 bonds in the yeast trna to be very sensitive toward cleavage by various agents [ 2932 ] . the bond u8-a9 is located in the proximity to sequence c61-g65 if tertiary structure of trna is taken into account . thus , one may assume that the observed cleavage of this bond occurs in complementary complex formed by conjugate b - im(n / m ) with trna and is the result of flexibility of rna - cleaving group of the conjugates . besides , we can assume that initial cleavage of phosphodiester bond c63-a64 produces a nick in trna molecule that may increase the flexibility of the complex trna - conjugate b - im(n / m ) and thus makes phosphodiester bonds c64-g65 and u8-a9 available for catalyst 's function groups . the slight cleavage observed at c75-a76 site apparently is the result of spontaneous trna hydrolysis in the duplex with the conjugate . competition experiments were used to prove that trna cleavage occurs in trna - conjugate complex . trna cleavage by conjugate b - im(4/1 ) was performed in the presence of oligonucleotide inhibitors of two types : parent oligonucleotide b and oligonucleotide a complementary to the sequence 61 - 75 ( figure 2(b ) , lanes 1719 ) . as expected , the cleavage of trna by conjugate b - im(4/1 ) is considerably decreased in the presence of oligonucleotides b ( primary data not shown ) and is completely abolished in the presence of oligonucleotide a , which protects target sequence by duplex formation . it is worth noting that in the presence of oligonucleotides b , the cleavage of trna is suppressed in all sites due to competitive binding , whereas oligonucleotides a inhibit trna cleavage only at the target sequence failing to change reaction rate at other sites . these results indicate the structural specificity of cleavage and stress the fact that the cleavage occurs only at a single - stranded target sequence . figure 2(c ) shows concentration dependencies of the cleavage reaction for conjugates b - im(4/1 ) ( 3 ) and im(24/4 + 2 ) ( 11 ) ( curves 1 and 2 , resp . ) and binding of the conjugate b - im(4/2a ) and parent oligonucleotide b with trna ( curves 3 and 4 , resp . ) . furthermore , the binding affinity of the parent oligonucleotide and the conjugate studied under identical conditions are found to be similar ( figure 2(c ) ) . therefore , the conjugation of bulky imidazole - containing reactive groups to the oligonucleotide does not affect the hybridization process . to estimate the influence of buffer on efficiency of site - selective rna cleavage , we compared trna cleavage by the conjugate b - im(4/2 ) in different buffer solutions ( figure 2(d ) ) , taken 50 mm imidazole buffer , ph 7.0 as a standard conditions . the rate of site - selective cleavage is affected by replacement of 50 mm imidazole buffer , ph 7.0 , by 50 mm tris - hcl buffer or 50 mm cacodylate buffer at ph 7.0 by the factor of 1.5 indicating that imidazole itself may contribute to cleavage reaction . at longer incubation time , this difference became insignificant and does not exceed estimated experimental error . one explanation could be that sulfonic acid of hepes [ 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid ] may interact with protonated imidazole residues of the conjugates and inactivate it . previously , we also observed twofold decrease of the rate of rna cleavage by oligonucleotide conjugate bearing two imidazole residues in 50 mm hepes - koh buffer , whereas the other buffers only slightly affected the rate of rna cleavage . in figure 2 , ( a ) represents cloverleaf structure of yeast trna and target sequences for oligonucleotide b - based artificial ribonucleases b - im(n / m ) . bold letters show target sequence for oligonucleotide - competitor a. arrows indicate the sites of site - selective trna cleavage by the conjugates ; ( b ) represents cleavage of trna with oligonucleotide conjugates b - im(4/m ) . cleavage reactions were performed at 37c in 50 mm imidazole buffer , ph 7.0 , containing 200 mm kcl , 0.2 mm edta , 100 g / ml rna carrier , 5 10 m [ 3-p]trna and 1 10 m one of the conjugates b - im(4/1 ) , b - im(4/2a ) , and b - im(4/2b ) . lanes c1 and c2 are trna incubated in the reaction buffer for 8 h in the absence and in the presence of oligonucleotide b ( 5 10 m ) , respectively . trna cleavage by conjugate b - im(4/1 ) ( 3 ) for 0.5 , 1 , 3 , 5 , and 8 h ( lanes 15 , resp . ) , by conjugate b - im(4/2a ) ( 4a ) for 0.5 , 1 , 3 , 5 , and 8 h ( lanes 610 ) , by the conjugate b - im(4/2b ) ( 4b ) for 0.5 , 1 , 3 , 7 , 10 , and 18 h ( lanes 1116 ) ; by the conjugate b - im(4/1 ) ( 3 ) in the presence of oligonucleotide a ( 1 10 ) for 0.5 , 1 , and 5 h ( lanes 1719 ) . concentration of the conjugates in the reaction mixtures was 1 10 m. reactions were quenched and analyzed as described in the experimental part ; ( c ) represents concentration dependencies of the trna cleavage by the conjugates b - im(4/1 ) ( 3 ) and b - im(24/2 ) ( 12 ) ( curves 1 and 2 , resp . ) and complex formation with the conjugate b - im(4/2a ) ( 4a ) ( curve 3 ) and parent oligonucleotide b ( curve 4 ) . the reaction mixtures were incubated for 2 h under the conditions described above ; ( d ) represents the influence of the buffer nature on the rate of trna cleavage by the conjugate b - im(4/2a ) ( 4a ) . m ) was incubated with 2 m of ( 4a ) , in 50 mm imidazole buffer , ph 7.0 ( i m ) or 50 mm cacodylate buffer , ph 7.0 ( cac ) or 50 mm tris - hcl buffer , ph 7.0 ( tris ) or 50 mm hepes buffer , ph 7.0 ( hepes ) containing 0.2 m kcl , 0.2 mm edta , and 0.1 mg / ml rna carrier .
figure 3(a ) displays the kinetics of trna cleavage with conjugates under the study taken at conjugate concentration 10 m . one of the most effective conjugate is b - im(4/1 ) , as complete site - selective cleavage of the target by this conjugate is achieved within 2 - 3 hours . similar kinetics are displayed by the conjugate b - im(8/1 ) ( 6 ) and the b - im(24/4 + 2 ) ( 12 ) . the conjugates b - im(4/2a ) ( 4a ) and b - im(8/2 ) ( 7 ) catalyze rna cleavage less efficiently as compared with b - im(4/1 ) or b - im(8/1 ) . cleavage efficiency of the conjugate b - im(4/3 ) ( 5 ) was essentially poor . figure 3(b ) shows kinetic curves of trna cleavage by conjugate b - im(4/1 ) obtained at different concentrations of the conjugate . the character of kinetic curves evidences the bimolecular reaction : the rate of rna cleavage is increased with the increasing of the conjugate concentrations . at concentration of conjugate b - im(4/1 ) of 10 m , which provides its almost quantitative binding to trna , the half - life of trna is about 1 h ( figure 3(b ) , curve 10 m ) . when concentration of conjugate b - im(4/1 ) is increased up to 50 m , the half - life of trna is counted by minutes ( figure 3(b ) , curve it should be noted that the site - selective cleavage of trna proceeds much slower than the binding of the parent oligonucleotide b to trna under similar conditions [ 3335 ] . at concentration of the conjugate equal to or twice as higher as concentration of the trna , the curves reveal lag period ( figure 3(b ) , curves 0.5 and 1 m , resp . ) indicating that the process of site - selective rna cleavage includes several consecutive steps occurring with comparable rates . another explanation of this could be that the reaction has more complex character and goes through some stages other than binding of the conjugate and cleavage , which results in sigmoid - like kinetic curve . for conjugate b - im(4/1 ) , we calculated the association rate and cleavage rate constants from the data shown in figure 3(b ) , assuming that cleavage of the trna occurs within the complex with the conjugate . in this case , hybridization step is the second - order reaction and cleavage stage is intramolecular first - order reaction . it turns out that cleavage pe se occurs 50 times faster than the binding of the conjugate with the complementary sequence within rna target ( association rate constant and cleavage rate constant are ( 0.074 0.002 ) m s and ( 3.4 0.55)10 s , resp . ) . in other word , binding of the conjugate with trna is the rate - limiting stage of site - selective rna cleavage . the conjugates display different cleavage activities ( table 1 ) ranging from very high cleavage rate ( 1/21 h in the case of b - im(4/1 ) ) to very low ( 1/2 value was not achieved under the experimental conditions in the case of b - im(4/3 ) ) . remarkable cleaving activity of the conjugates 3 , 6 , and 12 as well as moderate cleavage activity of the conjugates 4a , 8 , 10 , 11 , and 13 and poor efficiency of the conjugate 5 are likely resulted from the differences in the structure of the catalytic part of the conjugates rather than due to different hybridization properties and the cleavage reaction pe se . the conjugates under the study contain identical oligonucleotide recognition element and display similar cleavage specificity with only minor differences in the cleavage pattern . therefore , neither hybridization nor sensitivity of the phosphodiester bonds toward the cleavage could explain the differences in the catalytic properties of the conjugates . even the catalytic parts of the conjugates in several cases contain the equal number of imidazole residues . table 1 summarizes data on the cleavage activity and structure of the conjugates under the study ( linker length in total number of simple c c , c o bonds between 5-end of oligonucleotide b and imidazole residues , type of anchor group , and reaction halftimes ) . it is seen that hydrolytic activity of compounds is in a good agreement with the length of their linkers ( table 1 ) . amongst the conjugates bearing four imidazole residues , the highest cleavage activity is observed for the conjugate b - im(4/1 ) , which has the linkers of 41 simple bonds . the conjugates b - im(4/2a ) with the linkers of 29 chemical bonds exhibit 3-fold lower cleavage activity , whereas b - im(4/3 ) ( 18/24 bonds ) cleaves trna with lowest efficiency . thus , stepwise shortening of a linker part of conjugate b - im(4/m ) from 41 to 18/24 simple bonds results in decreasing of cleavage efficiency from 50% per hour to 1% per hour . as it is seen from table 1 , only the conjugates containing the linkers of 40 simple bonds are capable of efficiently catalyzing cleavage of trna in a site - selective manner . thus , we can conclude that a long flexible linker of 40 or more simple c c , c n , or p o bonds is required for efficient site - selective cleavage of rna target . figure 4 display structure - activity correlations found for the conjugates b - im(n / m ) . it is seen that cleavage activity of the conjugates b - im(n/1 ) depends on the number of imidazole groups in the catalytic part ( figure 4(a ) ) . the conjugates bearing 4 and 8 imidazole residues exhibit the highest cleavage activity , while increasing of the number of imidazole groups inhibits cleavage . it is worth pointing out that sigmoidicity of kinetic curves taken under identical conditions is increased with increasing of the number of imidazoles in the conjugate ( primary data not shown ) . it is likely that cleavage activity of these conjugates is affected by a steric interference between imidazoles , thus leading to a loss of cleavage rate . therefore , four ( and/or eight ) imidazole residues in the catalytic part of the conjugate seem to be sufficient for efficient rna cleavage . from the data shown in figure 4(b ) , it is seen that ribonuclease activity of the conjugates depends on type of the anchor group used for attachment of parent oligonucleotide to rna - cleaving construct . the conjugates with type 1 anchor group ( b - im(n/1 ) ) , cyclohexyl moiety ( figure 1(a ) ) , display higher cleavage activity than conjugates with the anchor group in a form of thymidine residue ( type 2 , figure 1 ) and especially than conjugates with a nonnucleoside anchor group ( type 3 , figure 1 ) ( table 1 ) . the difference between cleavage activity of the conjugates with type 2 and type 4 anchor groups does not exceed experimental error , but is reliably lower than that for conjugates with type 1 anchor group . thus , the structure and flexibility of the linker part , the number of imidazole residues in the catalytic part , and the type of anchor group affect cleavage activity of the conjugates and play a role in the optimal positioning of the catalyst in respect to scissile phosphodiester bond . the main goal of the molecular modelling was to clarify the following issues . is there any preferable orientation(s ) of the cleaving group(s ) in the vicinity of the cleavage site , which could explain the hydrolytic activity ( or its absence ) of each conjugate ? is there any evidence of interaction(s ) between the cleaving constructions and the dna : rna hybrid , which could stabilize preferable orientation(s ) of these groups near the cleavage site ? what is the correlation between the structure of the cleaving constructs and the linker groups and their hydrolytic activity ? it is evident from the biochemical assays that hydrolytic activity within the heteroduplex trna -conjugate is governed by the intrinsic properties of the cleaving constructs , but not the detailed structure of the dna - rna hybrid , which was the same in all experiments . therefore , the main priority was to identify possible location(s ) of the cleaving group(s ) relatively to the cleavage site rather than to determine the detailed conformation of the hybrid ( which would be impossible in the absence of relevant nmr / x - ray data ) . it has been shown earlier that hybridization of long oligonucleotides to tc - loop [ 33 , 38 ] or to the 3-acceptor stem [ 3335 ] of trna induced a local unfolding of the trna secondary structure . therefore , we assumed that the hybrid part of the molecules under the study can be represented to a first approximation by a double - stranded dna : rna duplex , which would form primarily an a - like conformation , according to physicochemical investigations [ 3941 ] . in our molecular modeling studies , we used a shortened model for the heteroduplex , comprising the target rna sequence ( corresponding to the a44-u69 fragment of trna ) and the oligodeoxyribonucleotide conjugate complementary to this rna region ( figure 2(a ) ) . based on this hybrid duplex , three model molecules were created ( m - im(4/1 ) , m - im(4/2a ) , and m - im(4/2b ) ) by attachment of the respective cleaving constructs to the 5 terminal phosphate group of the g nucleotide residue ( see figures 1 and 2 ) . at this stage , in the absence of the detailed structural data for the hybridized heteroduplex , it was reasonable to treat the hybrid as a solid structure whilst allowing cleaving constructs to possess a full conformational flexibility during the calculations . for each molecule m - im(4/1 ) , m - im(4/2a ) , and m - im(4/2b ) , several starting structures were created differing in the spatial orientation of the cleaving construct(s ) , ranging from in to out extremes ( see section 2 ) . this increased the conformational space searched for each model molecule . all starting structures were subjected to independent simulating annealing ( see section 2 ) giving rise to the respective final structures ( m - im(4/1 ) : f1-im(4/1)f9-im(4/1 ) ; m - im(4/2a ) : f1-im(4/2a)f9-im(4/2a ) ; m - im(4/2b ) : f1-im(4/2b)f9-im(4/2b ) . both structural parameters and energy values of each final structure were analyzed and compared with those of active conformations ( o1-im(4/1 ) , o2-im(4/1 ) , o1-im(4/2a ) , o2-im(4/2a ) , o1-im(4/2b ) , and o2-im(4/2b ) ) , which had deliberately preorganized cleaving centers according to [ 20 , 26 , 27 , 42 , 43 ] ( see section 2 ) ( table 2 ) . molecules containing four imidazole residues m - im(4/1 ) , m - im(4/2a ) , and m - im(4/2b ) , having two bis - imidazole cleaving constructs , differ in the geometrical properties of their linker groups ( see figure 1 ) . the linker group of m - im(4/1 ) possess a cyclohexyl fragment connecting the cleaving moieties with the oligonucleotide . in m - im(4/2a ) , the cyclohexyl fragment is replaced by a deoxyribothymidine fragment . m - im(4/2b ) is a shorter analogue of m - im(4/2a ) . for each molecule , nine final structures plus two optimal conformations were obtained as a result of the simulated annealing calculations . the data on the distances between n1 atoms of imidazole rings and target site as well as energy values are presented in table 2 . for m - im(4/1 ) ( table 2 ) , the most energetically favorable conformations ( o1-im(4/1 ) , o2-im(4/1 ) , and f4-im(4/1 ) ) are characterized by the location of at least two imidazole groups close to the target site ( 37 ) . in the most cases , m - im(4/2a ) conformers have at least one imidazole group located near the cleavage site , which suggests a high probability for cleaving groups to form a preorganized active conformation . these data are in agreement with the biochemical assays on high hydrolytic activity seen for b - im(4/2a ) conjugate , showing that this compound reaches the maximum activity ( 49.8% ) found for artificial ribonucleases . figure 5 represents the fragments of the f4-im(4/1 ) structure , as the most energetically favorable conformation of m - im(4/1 ) , and shows the probable orientation of the cleaving groups . in the case of m - im(4/2a ) , most of the final structures obtained in the simulated annealing experiments are characterized by a distant location of cleaving groups from the target site ( table 2 ) . for example , the lowest - energy structures f1-im(4/2a ) , f3-im(4/2a ) , and f8-im(4/2a ) have very unfavorable orientations of imidazole cleaving groups in terms of hydrolytic activity ( see figure 6 showing f1-im(4/2a ) final structure as an example ) . the possible reason of this could be the presence of a deoxyribothymidine fragment linking the oligonucleotide and the cleaving construct . according to our data , the thymidine base is involved in the dna : rna helix structure in a semistacking interaction with the neighboring nucleotide residue dg . the thymidine base matches comfortably the duplex structure , although no additional hydrogen bonding with the opposite strand was detected ( for instance , with the a ) . this interaction could control the overall conformation of the rest of the cleaving construct and possibly restrict the conformational freedom of this group , resulting in a decreased probability to render an active conformation . this could explain the relatively low hydrolytic activity of b - im(4/2a ) ( 16.6% ) compared to b - im(4/1 ) . even more pronounced decrease in hydrolytic activity ( down to 57% ) was observed for conjugate b - im(4/2b ) , a structural analogue of b - im(4/2a ) with a shortened linker group . data obtained in biochemical assays are consistent with results from molecular modeling for m - im(4/2b ) ( table 2 ) , showing that the most energetically favorable conformations ( for instance , f2-im(4/2b ) , f3-im(4/2b ) , and f5-im(4/2b ) ) have a distant location of the cleaving groups relative to the target site ( figure 7 shows f5-im(4/2b ) ) . moreover , only one of two bis - imidazole groups can occupy a position , favorable for cleaving activity . this decreases the statistical probability of reaching an active conformation for m - im(4/2b ) . generally , most of the final structures of m - im(4/2b ) are characterized by long distances between imidazole rings and target atoms . analysis of the structural parameters showed that the length of the linker group is not enough to provide a good chance to reach the target site . this possibly contributes to the restricted conformational freedom caused by thymidine interactions with the rna : dna duplex structure ( see above for m - im(4/2a ) ) . taken together , these explain the lowest hydrolytic activity for b - im(4/2b ) between the artificial ribonucleases . analysis of final structures resulting from the simulating annealing experiments shows a remarkable ability of all cleaving constructs to form multiple hydrogen bonds that generally can be represented by two types ( scheme 1 ) . the first type is the intermolecular interactions between cleaving constructs and the rna target , which are formed by oxygen atoms of the rna phosphate backbone and either ( i ) by imidazole rings of the cleaving groups or ( ii ) by protons of the linker amido groups ( see scheme 1 ) . these types of hydrogen bonding seem to be in favor of the hydrolytic activity of compounds due to their ability to stabilize the location of cleaving groups , close to the rna target . the second type of possible hydrogen bonding ( scheme 1 ) refers to intramolecular interactions within cleaving constructs themselves and involves interactions between imidazole rings and amido groups , carbonyl groups and phosphate groups of the linker . this type of hydrogen bond interaction also includes intralinker bridges formed by amido and carbonyl / phosphate groups . intramolecular hydrogen bonds seem to compete with favorable intermolecular interactions , preventing the imidazole groups from appropriate contacts with the rna backbone , which may result in a decrease of their hydrolytic activity . based on these results , we propose several suggestions on how to improve further the cleaving potential of artificial ribonucleases . first , it is important to avoid the use of any carbonyl / phosphate groups within the linker in order to decrease the possibility of intramolecular hydrogen bonding . secondly , it could be advantageous to introduce polycationic groups within the linker , which might increase possible contacts of cleaving groups with negatively charged phosphate backbones of target rna . finally , the insertion of a suitable hydrophobic / intercalating groups within the linker might also increase chances for these groups to be located in the vicinity of the rna target due to additional stacking / hydrophobic interactions with different ribonucleotide environments . in the current research , we studied oligonucleotide - based artificial ribonucleases containing multiple imidazole residues in the catalytic part of the arnases with systematically varied structure of cleaving constructs . all the conjugates contain the same addressing oligonucleotide which provides efficient and almost quantitative binding of the conjugates to the target sequence within the trna . this allows comparing different cleaving constructs in terms of site specificity and efficiency of rna cleavage . obtained results let us conclude that the efficiency of site - selective rna cleavage is governed by a number of dynamic parameters among which the most important is the flexibility of a linker between the catalytic imidazole residues and the addressing oligonucleotide . the other factors affecting the efficiency of site - selective rna cleavage are the number of imidazole residues in the catalytic part , the type of anchor group , connecting linker structure and the oligonucleotide , and the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide . we found that four imidazole residues located in the catalytic part of the conjugate can efficiently catalysed cleavage of phosphodiester bond in the case when they are located in the close proximity of rna sugar - phosphate backbone , while increasing of the number of imidazole groups inhibits cleavage , possibly due to steric interference between the imidazole residues . molecular modeling carried out to explain differences in ribonuclease activity of the conjugates showed that preferable orientation(s ) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker . the inclusion of deoxyribothymidine as an anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site due to a stacking interaction with the neighbouring nucleotide residue . remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct , which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond . the other very important factor affecting the efficiency of site - selective rna cleavage is sensitivity of phosphodiester bonds of rna toward cleavage . different catalytic structures display some sequence preference . on the other hand , phosphodiester bonds in different rna sequence display different sensitivities to cleaving agents [ 3 , 4448 ] . these factors should be taken into account when selecting target sites within specific rnas . in our experiments , the cleaving groups were targeted to caca sequence of trna , known to be of particular sensitivity to cleavage by different agents . so , the ribonuclease activity of the conjugates was compared under favourable conditions . on the other hand , binding of oligonucleotide conjugates to target rna is followed by rna structure rearrangements that can bring the target site in favorable or unfavorable positions toward the catalytic groups [ 32 , 49 , 50 ] . thus , optimisation of the structure of catalytic groups of oligonucleotide conjugates and identification of optimal structures of rna for targeting with artificial ribonucleases may facilitate the creation of highly specific artificial ribonucleases of a new generation . results of the studies performed by the authors revealed the problems to be solved in order to develop the highly efficient rna cleaving oligonucleotide conjugates . a few important factors determining efficacy of rna cleavage remain to be investigated so that the oligonucleotide conjugates take advantage of the known mechanisms used by natural enzymes for achieving high reaction rate . | design of site - selective artificial ribonucleases ( arnases ) is one of the most challenging tasks in rna targeting . here , we designed and studied oligonucleotide - based arnases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs .
we demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part , the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide , and the type of anchor group , connecting linker structure and the oligonucleotide .
molecular modeling of the most active arnases showed that preferable orientation(s ) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker .
the inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site , presumably due to a stacking interaction with the neighbouring nucleotide residue . altogether
the obtained results show that dynamics factors play an important role in site - specific rna cleavage .
remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct , which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond . |
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glutamate is the major excitatory neurotransmitter in the mammalian central nervous system ( cns ) 1 . it has the potential to be involved in the pathogenesis of many cns diseases either due to excessive release , reduced uptake or alteration of receptor function 2 . neuronal excitotoxicity usually refers to injury and death of neurons arising from prolonged exposure to glutamate and associated excessive influx of ions into the cell . the resulting calcium overload is particularly neurotoxic , leading to the activation of enzymes that degrade proteins , membranes and nucleic acids 3 . glutamate is released from damaged axons and glia under hypoxic / ischemic conditions 4 and glutamate receptor - mediated excitotoxicity has been described as a predominant mechanism of hypoxic injury to the developing cerebral white matter 5 - 8 . in the cns , the conversion of glutamate to glutamine by glutamine synthetase ( gs ; ec 6.3.1.2 ) , that takes place within the astrocytes , represents a key mechanism in the regulation of excitatory neurotransmission under normal conditions as well as in injured brain 9 . thus gs is involved in modulation of the turnover of glutamate through the glutamate - glutamine cycle 10 . reactive oxygen species ( ros ) are free radicals that are normal products of oxygen metabolism and are produced in excess during the course of ischemia / reperfusion through a variety of mechanism . intracellular ros are capable of inducing damage and , in severe cases , cell death through mitochondrial alterations leading to the release of cytochrome c 11 - 12 , through activation of the jnk pathway 13 or by activation of nuclear factor - kb ( nf - kb ) transcription factors 14 . the ability to control ros is thus critical in neurodegenerative diseases , because neuronal damage occurs when the generation of nitric oxide ( no ) , a versatile molecule in signaling processes and unspecific immune defense , is intertwined with synthesis , catabolism and transport of arginine which thus ultimately participates in the regulation of a fine - tuned balance between normal and pathophysiological consequences of no production 16 . the exact mechanisms contributing to increased production of no in anoxia are not well established . no is synthesized from arginine by nitric oxide synthase ( nos ; ec 1.14.13.39 ) , and the citrulline generated as a by - product can be recycled to arginine by successive actions of argininosuccinate synthetase ( as ; ec 6.3.4.5 ) and argininosuccinate lyase ( al ; ec 4.3.2.1 ) via the citrulline - no cycle 18 . arginine in brain is also utilized by arginase ( ec 3.5.3.1 ) for production of ornithine . co - induction of as , cationic amino acid transporter-2 , and nos in activated murine microglial cells 19 and co - induction of inducible nos and arginine recycling enzymes in cytokine - stimulated pc12 cells and high output production of no were reported 18 . in our earlier study we reported the increased activities of nos , as and al in kainic acid ( ka ) mediated excitotoxicity in rat brain 20 . thus it is hypothesized that the citulline - no cycle enzyme activities are increased to facilitate high and continuous production of no and increased no may decrease the activity of gs and increase the oxidative stress in anoxia / reperfusion induced excitotoxicity . global hypobaric hypoxia ( anoxia ) is associated with many physiological and pathological conditions such as pulmonary and cardiac diseases , high altitude pathophysiology , obstructive sleep apnea , depressurization accidents and also during incidents involving anesthesia . to understand the role of citrulline - no cycle enzymes , gs and the oxidative status in anoxia and reperfusion , nos , as , al , gs and arginase activities along with the concentration of no as nitrate /nitrite ( nox ) , lipid peroxidation products as thiobarbituric acid reactive substances ( tbars ) and total antioxidant status ( tas ) were estimated in cerebral cortex ( cc ) , cerebellum ( cb ) and brain stem ( bs ) of rats subjected to anoxia ( hypobaric hypoxia ) and reperfusion ( reoxygenation ) . the animals had free access to food and water . animal ethics committee and research committee of universiti sains malaysia , health campus , kubang kerian , malaysia , approved the experimental design . the animals were divided into control , anoxia ( global hypobaric hypoxia ) and reperfusion ( reoxygenation ) groups ( n=6 rats / group ) . in the anoxia group of animals , the rats were placed in a desiccator whose outlet was connected to a vacuum pump and the air removed producing hypobaric conditions . about 4 - 5 min after the exposure of rats to hypobaric condition , the rats became lethargic and motionless . at this juncture , the brains were quickly removed and the different regions ( cc , cb and bs ) were separated according to the procedure described by sadasivudu and lajtha 22 . each of the brain regions was weighed and used for the preparation of homogenates in 0.05 m phosphate buffer ph 7.3 . in the reperfusion group ( re- oxygenated ) , the animals were subjected to anoxia as described for anoxia group once and after removal of animals from desiccator , they were allowed to stay at normal conditions and were given normal diet for 5 days and decapitated and the different brain regions ( cc , cb and bs ) were used for study . it was reported by ananth et al 23 that 5 days showed most severe damage in a 1 - 21 days study after induction of excitotoxicity and hence that period ( 5 days ) was chosen for the reperfusion group . enzyme assays : total nos activity ( all isoforms of nos : nnos , inos & enos ) was estimated by the method of yui et al 24 as described by swamy et al 25 , in which the stable end products , nox , were estimated using the nitric oxide synthase assay kit from calbiochem ( catalogue number 482702 ) . as , al activities were estimated by the modified method of levin 26 as described by swamy et al 25 . arginase activity was assayed according to the method of herzfeld and raper 27 as described by swamy et al 24 . gs activity was assayed by the method rowe et al 28 as described by sadasivudu et al 29 . estimations of no , tbars and tas : no was estimated as nox by griess reaction after conversion of nitrate to nitrite by nitrate reductase , as described by swamy et al 24 using the commercially available nitric oxide assay kit from cayman chemical company ( catalogue number 780001 ; anna arbor , machigan , usa ) . lipid peroxidation was determined by the method of chatterjee et al 30 by estimating tbars . statistical analysis : results were reported as mean standard deviation ( sd ) from 6 animals for each parameter calculated . statistical analysis of results was done by one - way analysis of variance ( anova ) followed by post hoc analysis using bonferroni 's test , using the spss software ( version 12.0.1 ) to determine the statistical significance of difference in values between the control , anoxia and reperfusion groups . the activity of nos ( figure 1 ) was increased significantly in all the three brain regions indicating increased production of no in anoxia . in reperfusion group the activities of nos was increased when compared to control , however it was decreased when compared to anoxia in all the brain regions tested . in anoxia group the activity may be attributed to inos and nnos and increased activity may be mainly by inos due to expected inflammation after anoxia . the figure 2 shows activities of as , al and arginase in the study . as and al activities increased in all the three brain regions significantly in anoxia suggesting an increased utilization of citrulline for the production of arginine in anoxia . in reperfusion group the activities of these enzymes were increased when compared to control , however they were decreased when compared to anoxia in all the brain regions tested . the activity of arginase ( figure 2 ) showed no significant change , indicating there was no increased utilization of arginine by this enzyme in anoxia . however in reperfusion group arginase activity was significantly increased and that may be responsible to curtail the supply of arginine for no production in reperfusion . the gs activity ( figure 3 ) was decreased in all the tree brain regions in anoxia and showed further decrease in reperfusion group compared to control . in anoxia the possible decrease of gs may be due to the proposed modification of this enzyme by no 32 - 33 . in reperfusion group may be a cumulative effect of many factors such as down regulation of enzyme production and increased clearance along with the modulation by no . the figure 4 shows the concentration of nox , tas and tbars in this study . the concentration of nox and tbars increased significantly in all the brain regions tested in anoxia compared to control . in reperfusion group the concentration of nox and tbars increased significantly when compared to control , however they were decreased when compared to anoxia in all the brain regions tested . the pattern observed for the increase in concentration of nox in the three different brain regions was similar to that of increased nos activity in anoxia and reperfusion groups . concentration of tas ( figure 4 ) decreased significantly in all the brain regions tested in anoxia compared to control . in reperfusion groups the decrease in tas and increase in tbars levels confirms that , there is an increased oxidative stress in anoxia and reperfusion . under physiological conditions , most of the glutamate in the cns localizes to presynaptic vesicles 34 and returns there rapidly after being released during depolarizing events . however , pathological situations such as hypoxia and ischemia can lead to excessive release of glutamate and its accumulation in the extracellular space , which initiates the pathway of neuronal death known as excitotoxicity 35 - 36 . neuronal excitation involving the excitatory glutamate receptors is recognized as an important underlying mechanism in neurodegenerative disorders 37 . in neurons , no synthesis is stimulated by ca - influx , which is induced by activation of glutamate receptors , preferentially nmda receptor 38 . the literature findings implicate neuronal no generation in the pathogenesis of both direct and secondary excitotoxic neuronal injuries in vivo . excitotoxicity , oxidative stress and apoptosis comprise major routs of hypoxic - ischemic neuronal death . each route is likely activated and propagated through selective transmembrane and intracellular signaling system 39 . one of the events triggered by excessive glutamate release and relevant to excitotoxicity is the production of no 40 . no synthesis is activated cerebrovascular diseases by release of glutamate combined with inhibition of glutamate removal , which leads to nmda receptor over activation and excess ca influx 41 . the increased activity of nos and the increased formation of no in brain in anoxia as observed by increase in nox concentration in this study support the earlier findings of no involvement in pathophysiology of hypobaric hypoxia in brain 42 - 43 . in this study the increased activity of nos in anoxia may represent predominantly the nnos form . in inflammatory conditions microglia and astroglia are capable of expressing inos 44 . following induction inos is known to produce large amounts of no over prolonged periods of time , in response to many stimuli such as inflammation 45 . the activity of nos in reperfusion group may be attributed to inos and nnos and the relative contribution to the increased activity in reperfusion may be mainly the inos form and some contribution by nnos , as reported that nnos expression in degenerating neurons observed through increased nnos immunoreactivity at 5days after induction of excitotoxicity 23 . increased formation of no by stimulated activity of nos depends upon a continuous supply of arginine at the site of synthesis . arginine is a semi essential amino acid and in cns , its availability depends upon : ( a ) uptake from the circulating arginine and ( b ) recycling of citrulline to arginine by the actions of as and al . l - arginine is transported into synaptosomes 46 , neurons 47 and astroglia 48 by the y+ cat system . the cat , a sodium - independent transporter , also functions as a carrier for l - ornithine in addition to l - arginine 49 . activities of as and al were elevated in all the brain regions of rats subjected to anoxia . however the possibility of induction can not be ruled out along with the modification of enzyme with unknown mechanism . further , our observation of higher activities of nos , as , and al in cerebellum suggests a higher flux of the citrulline - no cycle in cerebellum . the functional significance of such a high flux of citrulline - no cycle in cerebellum needs further clarification . our results on the activities of as and al suggest that the citrulline - no cycle plays a significant role in ensuring adequate supply of arginine for the increased production of no observed in anoxia . a similar increase of nos , as and al activities along with increased production of no in ka mediated excitotoxicity apart from no synthesis l - arginine may also serve as a substrate for glutamate formation and may also provide increased substrate for arginase 50 . no significant changes in the activity of arginase in the anoxic group indicate that there is no enhanced utilization of arginine by this enzyme in anoxia . however the observed increase of arginase in reperfusion group may favor decreased production of no in this condition . both nos and arginase use arginine as a common substrate , and arginase may down - regulate no production by competing with nos for arginine 51 , thus may be reducing the effects of no in reperfusion group . the mechanism of increased activity of arginase in reperfusion is not known and may be up regulated with the supply of oxygen . the glutamine synthetase activity is present in all parts of brain and it is equally high in cerebral cortex , cerebellum and hippocampus 33 , 52 . modulation of gs activity in brain therefore important and its impairment or saturation may have pathological consequences 53 . the decreased activity of gs observed in this study indicates the probable inhibition of gs by no in anoxia . the exact mechanism of inhibition of gs by no is not known , but it is thought to be as a covalent modification as a result of nitrosylation or nitration of tyrosin in gs 31 - 32 , 54 . it is proposed that the inhibition of gs by no may provide prolonged availability of glutamate causing excitotoxicity in hypobaric hypoxia . similar decrease of gs activity in ka mediated excitotoxicity was reported 20 . in view of these observations the pharmacological agents who can increase gs may prove beneficial in the treatment of neurological disorders involving excitotoxicity as a result of anoxia . the decreased activity of gs in reperfusion group compared to anoxia may be a cumulative of other factors , including a down regulation of gs production and an increased clearance of this enzyme apart from modulation of gs by no . activity of the gs could be involved in the regulation of concentration of glutamate in the extra - cellular space of neurons with other systems , astroglial glutamate transporter - i ( glt-1 ) , and also microglial antiporter for cystine and glutamate which may release glutamate when the demand of glutathione synthesis was increased by oxidative stress 55 . from our study , the increased concentration of tbars and decreased concentration of tas supports the oxidative stress in anoxia . in this study the results of citrulline - no cycle enzymes and nox , tas and tbars indicating the trend of normalization of these parameters with the supply of oxygen in reperfusion group . the increased activity of arginase in reperfusion group may be beneficial by the way of competing nos for arginine . in a recent study using gs inhibitor , it is showed a reduced concentration of glutamine and glutamate in brain due to gs inhibition 56 . the sustained decreased activity of gs in reperfusion group compared to anoxia may be a cumulative of other factors , including a down regulation of gs production and an increased clearance of this enzyme apart from modulation of gs activity by no . the prolonged decreased gs activity may be providing benefit in reducing glutamine and glutamate concentration because the glutamine produced in the glial cells enters the neuron and converted to glutamate by glutaminase , which is described as glutamate - glutamine cycle 10 , 56 . hence the decreased gs and increased arginase activities may be protective in reperfusion group . in conclusion , this study clearly demonstrated the increased formation of no and supports the involvement of no in the pathophysiology of anoxia ( hypobaric hypoxia ) and reperfusion damage in brain . the increased activities of as and al indicate the effective recycling of citrulline to arginine and suggest a functional role to citrulline -no cycle enzymes in anoxia . the decreased activity of gs in the brain regions indicate the modulation of its activity by no and favors the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia . the increased concentration of tbars and decreased concentration of tas supports the oxidative stress in anoxia and reperfusion and suggests a possible role for anti oxidants in preventing neurodegeneration in anoxia and reperfusion injuries to brain . the increased arginase and sustained decrease of gs activity in reperfusion group are likely to be protective . | nitric oxide is postulated to be involved in the pathophysiology of neurological disorders due to hypoxia/ anoxia in brain due to increased release of glutamate and activation of n - methyl - d - aspartate receptors .
reactive oxygen species have been implicated in pathophysiology of many neurological disorders and in brain function . to understand their role in anoxia ( hypobaric hypoxia ) and reperfusion ( reoxygenation ) , the nitric oxide synthase , argininosuccinate synthetase , argininosuccinate lyase ,
glutamine synthetase and arginase activities along with the concentration of nitrate /nitrite , thiobarbituric acid reactive substances and total antioxidant status were estimated in cerebral cortex , cerebellum and brain stem of rats subjected to anoxia and reperfusion .
the results of this study clearly demonstrated the increased production of nitric oxide by increased activity of nitric oxide synthase .
the increased activities of argininosuccinate synthetase and argininosuccinate lyase suggest the increased and effective recycling of citrulline to arginine in anoxia , making nitric oxide production more effective and contributing to its toxic effects .
the decreased activity of glutamine synthetase may favor the prolonged availability of glutamic acid causing excitotoxicity leading to neuronal damage in anoxia .
the increased formation of thiobarbituric acid reactive substances and decreased total antioxidant status indicate the presence of oxidative stress in anoxia and reperfusion .
the increased arginase and sustained decrease of gs activity in reperfusion group likely to be protective . |
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cellulose , one of the most abundant renewable biopolymer , is a linear insoluble polysaccharide of -d - glucose ( ~100 to 20,000 residues ) joined by -1,4-glucosidic bond . as a major component of plant cell wall , cellulose degradation provides several benefits considered from environmental and bio - energy issues [ 1 , 2 ] . decomposition of plant litter is also an important process to release nutrients into the soil . in nature , cellulose can be hydrolysed by enzymatic means , the enzymes mainly being derived from a wide range of microbes . cellulase enzymes are generally composed of a multienzyme system including cellobiohydrolase ( cbh , e.c . the cbh and eg enzymes function cooperatively and synergistically , and depolymerise cellulose to cellobiose and oligosaccharides . the cbh enzymes are known as the key component of cellulase system that display an exo - type of digesting biopolymers , and the major product of their action on cellulose is cellobiose . based on amino acid sequence similarity , the cbh enzymes can be grouped into glycoside hydrolase families : gh6 , gh7 , and gh48 . of these , only the gh7 type is thought to be exclusively of fungal origin and this family includes the cbhi and egi enzymes from both ascomycetes and basidiomycetes . several fungi can utilize cellulose as an energy source by secreting a complex of cellulase enzymes . these enzymes are of great interest considered from the viewpoint of industrial and environmental application . the fungal cbh in particular is of great interest as potent cellulolytic fungi are capable of producing two different forms of the cbh enzymes : cbhi and cbhii . it should be noted however that the cbhi is the principal component of the cellulase enzymes as reported in trichoderma reesei and in volvariella volvacea . considered a key role of fungi in degrading plant biomass in ecosystems , a programme was initiated to investigate the nature of the cellulolytic system with an emphasis on the cbhi of some basidiomycetes . in this paper , we report the presence of a gene encoding the cellobiohydrolase enzyme from auricularia fuscosuccinea , pleurotus giganteus , p. eryngii , p. ostreatus , and p. sajor - caju . the fungal strains used in this study were a. fuscosuccinea mg01 , p. eryngii mg02 , p. giganteus mflu100154 , p. ostreatus mg03 , and p. sajor - caju mg04 . the genomic dna was extracted from the fungal mycelium using a modified cetyltrimethyl ammonium bromide ( ctab ) protocol . fifty mg of fungal mycelium were collected and resuspended in 500 l of the extraction buffer ( 200 mm tris - hcl [ ph 8.5 ] , 250 mm nacl , 25 mm ethylenediaminetetraacetic acid [ ph 8.0 ] , 0.5% sds ) . three hundred l of phenol and 150 l of chloroform were added and the cell suspension was mixed by inverting . the supernatant was transferred to a new microfuge tube and 5 l of 10 mg / ml rnasea was added ; the reaction mixture was incubated at 37 for 10 min . the treated dna was precipitated by adding 250 l of isopropanol by incubating at -20 overnight . the dna was then pelleted by centrifugation at 13,000 rpm for 15 min at 4. two hundred fifty l of ice - cold ethanol was then added and the mixture was mixed by inverting and centrifuged at high speed ( 13,000 rpm ) for 5 min . approximately 100 ng of the genomic dna was used as a template for pcr amplification . a pcr reaction was set up in a final volume of 25 l containing 1 mm mgcl2 , pcr buffer , each dntps at 200 m , 20 pmol of the ib ( 5'-tgygaytcicartgyccicgiga-3 ' ) and the ic ( 5'-gartciagccaiagcatrttigc-3 ' ) primers and 1 u of taq dna polymerase . the pcr amplification was carried out in a t - gradient thermocycler ( biometra , gottingen , germany ) using the following incubation cycles : 94 for 3 min , 52 for 1 min , and 72 for 1 min followed by 30 cycles of 94 for 30 sec , 52 for 30 sec , and 72 for 1 min , with a final extension at 72 for 10 min . after completion of the pcr , 10 l of the samples was electrophoresed on a 0.8% agarose gel to determine whether the dna of interest was amplified . the amplified products were then purified using the nucleospin extract ii column ( macherey - nagel inc . , dren , germany ) and cloned using the pgem - t easy vector systems ( promega , madison , wi , usa ) . the recombinant plasmids were transformed into the competent escherichia coli dh5 cells prepared in accordance with the protocol of sambrook et al . . the cloned cbhi genes were then sequenced by shanghai sangon biological engineering technology & services co. , ltd . the nucleotide sequence data obtained were deposited in the genbank database with the following accession nos . : jq717249 for a. fuscosuccinea mg01 , jq717250 for p. giganteus mflu100154 , jq717251 for pleurotus eryngii mg02 , jq717252 for p. sajor - caju mg04 , and jq717253 for p. ostreatus mg03 . these dna sequences were then analysed using the clustal w and blast programmes [ 13 , 14 ] . to obtain more information , the cbhi genes were also deduced after alignment against the known cbhi mrna sequences ( p. ostreatus sp . ' florida ' [ am262873.1 and am262871.1 ] , flammulina velutipes fv-1 [ ab540999.1 ] , and irpex lacteus [ ab019376.1 ] ) . the fungal strains used in this study were a. fuscosuccinea mg01 , p. eryngii mg02 , p. giganteus mflu100154 , p. ostreatus mg03 , and p. sajor - caju mg04 . the genomic dna was extracted from the fungal mycelium using a modified cetyltrimethyl ammonium bromide ( ctab ) protocol . fifty mg of fungal mycelium were collected and resuspended in 500 l of the extraction buffer ( 200 mm tris - hcl [ ph 8.5 ] , 250 mm nacl , 25 mm ethylenediaminetetraacetic acid [ ph 8.0 ] , 0.5% sds ) . three hundred l of phenol and 150 l of chloroform were added and the cell suspension was mixed by inverting . the supernatant was transferred to a new microfuge tube and 5 l of 10 mg / ml rnasea was added ; the reaction mixture was incubated at 37 for 10 min . the treated dna was precipitated by adding 250 l of isopropanol by incubating at -20 overnight . the dna was then pelleted by centrifugation at 13,000 rpm for 15 min at 4. two hundred fifty l of ice - cold ethanol was then added and the mixture was mixed by inverting and centrifuged at high speed ( 13,000 rpm ) for 5 min . approximately 100 ng of the genomic dna was used as a template for pcr amplification . a pcr reaction was set up in a final volume of 25 l containing 1 mm mgcl2 , pcr buffer , each dntps at 200 m , 20 pmol of the ib ( 5'-tgygaytcicartgyccicgiga-3 ' ) and the ic ( 5'-gartciagccaiagcatrttigc-3 ' ) primers and 1 u of taq dna polymerase . the pcr amplification was carried out in a t - gradient thermocycler ( biometra , gottingen , germany ) using the following incubation cycles : 94 for 3 min , 52 for 1 min , and 72 for 1 min followed by 30 cycles of 94 for 30 sec , 52 for 30 sec , and 72 for 1 min , with a final extension at 72 for 10 min . after completion of the pcr , 10 l of the samples was electrophoresed on a 0.8% agarose gel to determine whether the dna of interest was amplified . the amplified products were then purified using the nucleospin extract ii column ( macherey - nagel inc . , dren , germany ) and cloned using the pgem - t easy vector systems ( promega , madison , wi , usa ) . the recombinant plasmids were transformed into the competent escherichia coli dh5 cells prepared in accordance with the protocol of sambrook et al . . the cloned cbhi genes were then sequenced by shanghai sangon biological engineering technology & services co. , ltd . the nucleotide sequence data obtained were deposited in the genbank database with the following accession nos . : jq717249 for a. fuscosuccinea mg01 , jq717250 for p. giganteus mflu100154 , jq717251 for pleurotus eryngii mg02 , jq717252 for p. sajor - caju mg04 , and jq717253 for p. ostreatus mg03 . these dna sequences were then analysed using the clustal w and blast programmes [ 13 , 14 ] . to obtain more information , the cbhi genes were also deduced after alignment against the known cbhi mrna sequences ( p. ostreatus sp . ' class i cellobiohydrolases ( also known as cbhi or family 7 glycosyl hydrolases ) are the principal enzymes of the cellulase complexes . in this work , we aimed to extend previous knowledge which has described the presence of the cbhi gene in mushrooms [ 10 , 15 - 17 ] . initially , the genomic dna samples were extracted from the mushroom mycelia ( fig . 1 ) and thus used as template for pcr . it was found that a partial sequence of the cbhi genes ( ~750 bp ) were amplified successfully using the ib and ic primers ( fig . it was confirmed , by a pcr method , that the cbhi gene was successfully cloned ( data not shown ) and these amplicons were subjected to nucleotide sequencing . each dna sequence obtained was subsequently submitted as a query in a blastn search of the genbank database . this homology search was performed resulting that all dna sequences were closely related to those of the cbhi gene family of either p. ostreatus or f. velutipes ( table 1 ) . the similarity of these mushroom cbhi genes was determined using the clustal w software . based on this analysis , their similarity was very high ranging from 71 to 99% ( data not shown ) . the translation of these cbhi genes ( excluding introns ) into the cbhi proteins was also performed using the translate tool ( http://web.expasy.org/translate/ ) . the analysis revealed five putative amino acid sequences similar to the cellobiosidase of p. ostreatus sp . ' it is evident that these mushroom cbhi proteins showed high homology with the enzymes belonging to glycoside hydrolase family 7 . there are several studies on fungal cellulolytic systems but these are limited to a few key species such as trichoderma reseei and p. chrysosporium . the cellulase activity has also been reported in some mushrooms including agaricus bisporus , lentinula edodes , and v. volvacea . our data provide additional information on the presence of the cbhi genes in several mushrooms including the recently recollected species p. giganteus . for example , the cbhi gene of p. ostreatus mg03 ( in this study ) was distinct although it showed high homology to the cbhi - ii of p. ostreatus sp . this finding is in agreement with previous work in which multiple forms of the cbhi gene have been reported [ 20 , 21 ] . however , the existence of different forms of the cbhi gene may be advantageous considered from their ability in protein expression when exposed to different environmental conditions . in summary , we successfully cloned the genes encoding the cbhi proteins from five mushrooms . this present work was undertaken with an expectation that future research will be explored including the expression of the cbhi gene as well as the factors affecting the cbhi activity . | genes encoding the cellobiohydrolase enzyme ( cbhi ) , designated as cbhi , were isolated from the basidiomycetes auricularia fuscosuccinea , pleurotus giganteus , p. eryngii , p. ostreatus , and p. sajor - caju .
initially , the fungal genomic dna was extracted using a modified cetyltrimethyl ammonium bromide ( ctab ) protocol and used as a dna template .
the cbhi genes were then amplified and cloned using the pgem - t easy vector systems .
the sizes of these pcr amplicons were between 700~800 bp .
the dna sequences obtained were similar showing high identity to the cbhi gene family .
these cbhi genes were partial consisting of three coding regions and two introns .
the deduced amino acid sequences exhibited significant similarity to those of fungal cbhi enzymes belonging to glycosyl hydrolase family 7 . |
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we analyzed ahi signs and symptoms in 90 patients given a diagnosis of ahi during 20072014 . as part of this confidential hiv testing program , routine , individual donation , hiv nucleic acid amplification testing ( nat ) has been provided to all rapid antibody negative participants since june 2007 ( samples for nat are obtained at the time of rapid antibody testing ) ( 7,10,11 ) . ahi was defined as having a negative or indeterminate hiv antibody test result in the presence of detectable hiv-1 rna , corresponding to fiebig stages i ii , with a mean estimated date of infection within the previous 10 days ( 95% ci 714 days ) ( 12 ) . dates of infection were estimated for all recently infected patients using previously published criteria on the basis of serologic and virologic test results ( 13 ) . at each patient s first visit after documentation of ahi diagnosis ( median 4 days , interquartile range [ iqr ] 36 days after ahi testing ) , we obtained blood samples for cd4 and viral load testing and collected detailed information regarding occurrence , duration , and start and stop dates for 11 signs and symptoms associated with ahi ( 5,14 ) . in addition , patients who participated during 20072011 were asked if they had sought medical attention for any signs or symptoms . typical ahi ( i.e. , > 2 signs / symptoms ) was defined according to criteria described by braun et al . ( 14 ) . for statistical analysis , , chicago , il , usa ) was used . for analysis on signs or symptoms compatible with ahi , signs or symptoms that started > 5 days before the estimated date of infection ( i.e. , before the 714 day 95% ci ) were excluded . the university of california san diego s human research protections program approved the study protocol , consent process , and all study - related procedures . all 90 participants were male and self - identified as men who have sex with men ( msm ) . median number of male partners reported for the previous 12 months was 20 ( iqr 1431 ) . a total of 72 ( 80% ) patients had signs or symptoms associated with ahi that occurred within 2 weeks before undergoing nat ; of these 72 patients , 47 ( 52% of the study population ) had ongoing signs or symptoms , while signs or symptoms had resolved by the time of testing for 25 ( 28% of the study population ) . twelve ( 13% ) reported signs or symptoms starting after testing , while 6 ( 7% ) reported the absence of signs or symptoms ( table 1 ) . a total of 66 patients ( 73% of the study population ) reported headache , pharyngitis , or myalgia occurring during the 14 days before ahi testing . * ahi , acute hiv infection ; iqr , interquartile range ; nat , nucleic acid amplification testing ; ns , not significant . most frequently observed signs or symptoms that occurred during the 14 days before nat or were ongoing at the time of nat were fatigue ( 53 persons , 59% of the study population ) , fever ( 51 , 57% ) , myalgia ( 48 , 53% ) , headache ( 41 , 46% ) , night sweats ( 35 , 39% ) , pharyngitis ( 32 , 36% ) , and gastrointestinal symptoms ( 29 , 32% ) . overall , 69 patients ( 77% ) reported signs or symptoms that met criteria of compatibility with ahi ( table 2 ) . onset of signs or symptoms compatible with ahi occurred at a median of 5 days ( iqr 08 , range 4 to 15 days ) after the estimated date of infection . neither viral load nor cd4 count correlated with duration or actual number of signs or symptoms . * ahi , acute hiv infection ; gi , gastrointestinal ; iqr , interquartile range ; nat , nucleic acid amplification testing ; ns , not significant . defined as having started < 4 days before estimated date of infection or after the estimated date of infection . data on whether a patient sought medical attention because of signs or symptoms were available for 42 ( 47% ) of 90 patients ; of these , 12 ( 29% ) reported that they sought medical attention because of their signs or symptoms and 30 ( 71% ) did not . significantly higher viral loads were observed for those who sought medical attention compared with those who did not ( median 6.1 [ iqr 5.76.7 ] log copies / ml vs. 4.7 [ iqr 3.45.5 ] log copies / ml ; p<0.01 ) . overall , 70 ( 78% ) of the 90 patients fulfilled criteria for having typical ahi and 20 ( 22% ) did not ( of the latter , 14 had only 1 sign or symptom , and 6 were asymptomatic ) . patients with typical ahi had significantly higher viral loads compared with patients without ( p<0.01 ) . a total of 61 ( 85% ) of 72 patients with signs or symptoms before nat testing fulfilled criteria for having typical ahi . in addition , 40 ( 85% ) of 47 patients who had ongoing signs or symptoms at the time of ahi testing fulfilled criteria for having typical ahi at that time . we characterized signs or symptoms relative to the date of ahi diagnosis among persons seeking hiv testing in a program offering universal ahi screening . two findings are notable : 1 ) 52% of participants reported ongoing signs or symptoms at the time of ahi testing , and 2 ) 80% reported signs or symptoms occurring within 2 weeks before undergoing testing . these findings may have major clinical implications for community - based settings that restrict ahi testing to persons with ongoing signs or symptoms . this practice may be relatively insensitive in settings where msm undergo hiv screening frequently ( 11 ) . our results show that expansion of ahi screening to include those with signs or symptoms during the 2 weeks before the test may increase the yield of ahi diagnoses by more than half . although our results may allow for estimation of sensitivity of signs and symptoms for ahi in persons seeking hiv testing , specificity of signs and symptoms remains unknown ( in this study , signs and symptoms were not assessed in those who tested negative , and no control group was available ) . estimates on frequency of signs and symptoms in hiv - negative persons ( i.e. , specificity ) ranged widely in previous studies . although in one study a specificity of 65% was estimated for influenza illness like symptoms ( 15 ) , specificities ranging from 38% to 91% for recent symptoms were estimated in another study ( 5 ) . limitations of those studies include the fact that exact time frames for occurrence of signs or symptoms ( e.g. , ongoing at the time of testing or occurring within the last 14 days ) have not been evaluated , which makes comparison of results difficult . therefore , our results may not be applicable to populations other than msm , although previous studies have reported that clinical features of ahi may not differ by sex and age of patients ( 4 ) . in summary , hiv diagnostic testing strategies that limit ahi testing to patients with ongoing signs or symptoms in contrast , hiv nat provided for msm who report signs or symptoms during the preceding 2 weeks ( representing 80% of ahi diagnoses ) may increase the yield of ahi diagnoses by more than half . | we analyzed signs and symptoms in 90 patients diagnosed with acute hiv infection in a community - based program that offered universal hiv-1 nucleic acid amplification testing .
forty - seven ( 52% ) patients reported ongoing signs or symptoms at the time of testing .
another 25 ( 28% ) reported signs or symptoms that had occurred during the 14 days before testing . |
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