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PMC3655293 | rarely the common hepatic artery ( cha ) crosses the intrapancreatic parenchyma at the pancreas head or neck , making it problematic to consider pancreatic head resection ( 1 ) .
in addition , organs such as esophagus , duodenum , common bile duct , or ureters can be compressed by blood vessels , causing symptoms like dysphagia , jaundice , or hydronephrosis . as far as we know , dilatation of main pancreatic duct by intrapancreatic - replaced cha has been not reported on the literature .
a 63-year - old male was admitted to the hospital for an elevation of hepatic enzymes and dark colored urine .
he had experienced fatigue and malaise . he had no abdominal pain and neither significant medical history nor family history .
based on the viral maker assay , he was diagnosed with acute hepatitis and possible hepatitis a infection . on routine abdominal ultrasonography to rule out other organic causes , main pancreatic duct dilatation was incidentally found .
the patient underwent contrast enhancement liver dynamic ct and mrcp for further characterization of duct dilatation and evaluation of liver .
liver dynamic ct consisting of four phase ( unenhanced , arterial [ 15 seconds ] , portal [ 40 seconds ] and delayed [ 3 minutes ] scan ) images were acquired with an 16-channel multi - detector raw ct ( mx8000 idt 16 ct scanner ; philips medical system , best , the netherlands ) .
in addition , mrcp images were acquired with a 3.0 tesla mr scanner ( achieva 3.0 t x - series , philips medical system , best , the netherlands ) .
on axial and coronal curved multiplanar reconstruction images using arterial phase scan , the cha was arising from the superior mesenteric artery ( sma ) and traveling through the pancreatic head parenchyma and across the main pancreatic duct , supero - posteriorly ( fig .
the upstream main pancreatic duct crossing the replaced cha shows diffuse tubular dilatation without abnormal wall thickening or enhancement . endoscopic retrograde cholangiopancreatography ( ercp ) shows abrupt transverse filling defect at pancreas head and stasis of the contrast medium during the ercp procedure ( fig .
routine mrcp images revealed a compression of main pancreatic duct by the replaced cha seen as a tubular signal void appearing as dark signal intensity at the corresponding area of the ercp , which is flow void on the cha , on both coronal t2 weighted image ( repetition time [ tr]/echo time [ te ] , 1728/80 ; section thickness 3 mm ) and maximum intensity projection of respiratory triggered 3-dimensional turbo spin - echo mrcp images ( tr / te , 1868/600 ) ( fig .
the main pancreatic duct was compressed and dilated by the intrapancreatic - replaced cha , resulted in prolonged stasis of the contrast medium in the upstream pancreatic duct noted on ercp .
however , this patient was asymptomatic and gave no evidence of history suggesting acute pancreatitis .
this finding posed a dilemma to the clinician and surgeon because the prolonged stasis of contrast medium in the dilated pancreatic duct is a potential cause of acute pancreatitis .
however , the clinician and surgeon decided upon close observation of patient status instead of the distal pancreatectomy because the high morbidity and mortality of surgical treatment was not beneficial to an asymptomatic patient .
according to the literature review , the proper hepatic artery originating from the sma occurs in 0.9 to 4.5% of individuals ( 2 ) .
the replaced cha courses along the portocaval space , supero - posterior margin of the pancreas , and enter the hepatoduodenal ligament .
the intrapancreatic - replaced cha is an incidental finding and usually asymptomatic and no problematic .
however , surgeons are challenged by the intrapancreatic - replaced cha or replaced right hepatic artery , especially in considering the pancreatic head resection .
preservation of the intrapancreatic - replaced cha is important and dissection from imbedded pancreatic parenchyma is sometimes difficult ( 1 , 3 ) .
common bile duct and ureters tend to be compressed by adjacent or aberrant origin arteries .
the development of the aberrant right subclavian artery is related to the degeneration of the entire right fourth aortic arch during the embryogenesis ( 4 , 5 ) .
the third portion of the duodenums is mechanical compression between the sma and abdominal aorta known as the sma syndrome .
sma syndrome occurs mainly through loss of mesenteric and retroperitoneal fat representing as a low aorto - mesenteric angle but anatomic factors like a short ligament of treitz or low origin of the sma , trauma , dietary disorders , postoperative state are also considered predisposing conditions ( 6 , 7 ) .
a unique case report of common bile duct obstruction by the postero - superior pancreatico - duodenal artery is similar to our case report ( 8) .
common bile duct obstruction by arterial compression results in bile stasis and is prone to form biliary stones .
in addition , ureter obstruction by vascular lesion is relatively common and either artery or vein can be the cause .
abdominal aortic aneurysm , iliac artery aneurysm , accessory renal arteries and other aberrant arteries are arterial causes of ureter obstruction .
normal venous structures such as accessory renal veins , iliac vein or gonadal veins can cause ureter compression and circumcaval ureter is a congenital anomaly causing ureter obstruction ( 9 , 10 ) . however , as far as we know , this is the first case report of compression of the main pancreatic duct by intrapancreatic - replaced cha . although prolonged stasis of the contrast medium was noted on ercp , the patient did not have any symptoms or signs of pancreatitis .
so the pancreatic duct compression by the intrapancreatic - replaced cha was an incidental finding but we pay attention to the findings of main pancreatic duct dilatation and stasis of the contrast medium that can predispose to acute pancreatitis . in summary ,
intrapancreatic - replaced cha is a very rare anatomic anomaly and a cause of pancreatic duct dilatation by aberrant course and compression of replaced cha . | we describe a unique case of a patient who presented with a linear , transverse , and incidentally - detected main pancreatic duct dilatation that was caused by the intrapancreatic - replaced common hepatic artery , detected on the mdct , mrcp and endoscopic retrograde cholangiopancreatography .
we believe this case to be the first of its kind reported in the literature . | INTRODUCTION
CASE REPORT
DISCUSSION |
PMC4366781 | endocrinology is the branch of medicine dealing with the endocrine system , its diseases , and its specific secretions called hormones .
indian journal of endocrinology and metabolism ( ijem ) , an official publication of endocrine society of india ( esi ) is a peer - reviewed indexed journal , online since 2007 .
the journal covers technical and clinical studies related to health , ethical and social issues in the field of diabetes , endocrinology and metabolism .
public health is the science and art of preventing disease , prolonging life and promoting health through the organized efforts and informed choices of society , organizations , public and private , communities and individuals .
it is the application of quantitative analysis and statistics to publications such as journal articles and their accompanying citation counts .
there are several bibliometric indicators like researcher 's bibliometric indicators ( number of publications or citations ) , university and department level bibliometric indicators and journal indicators .
while noncommunicable diseases ( ncds ) have become a major focus area for public health professionals , there is an equally important need to sensitize ncd clinicians to the public health importance of the diseases they are treating
. the current status of community health in ncd can be assessed by doing a bibliometric analysis of the leading ncd journals .
the present attempt reports the bibliometric analysis of ijem over a 4 years period ( 20112014 ) , to assess its coverage of community health .
bibliometric analysis for ijem has been done in the past for a 3-year period from 2007 to 2009 , but the current attempt reviews the contribution of ijem specifically to community health .
a total of four volumes ( 15 , 16 , 17 and 18 ) of ijem were analyzed .
the articles published were analyzed for each type of article ( editorial , original article , review article , brief communication , letter to editor , case reports , case reports with review of literature , guidelines and recommendations and two special sections introduced recently , that is , endocrine and gender and endocrine and arts ) under the different sub specialties of endocrinology and metabolism ( pituitary , parathyroid , thyroid , pancreas , adrenal , gonads and general endocrinology ) .
the volumes of ijem published were further searched and analyzed for the articles written from a community health perspective .
all articles were read independently by each author and classified according to their relevance to community health .
community health is an all - encompassing science , which includes virtually every aspect of medicine in its ambit .
, epidemiology , prevention per se , psychosocial health , nutrition ( including obesity and growth related articles ) , ncds and public health , knowledge , attitude and practices studies , recommendations and health programs , research and bibliometric analysis and heath economics . these community medicine related endocrinology articles were further divided as per the several sub specialties of endocrinology and metabolism ( pituitary , parathyroid , thyroid , pancreas , adrenal , gonads and general endocrinology ) .
over 4 years , ijem has published 4 volumes comprising of 22 issues and 10 supplements .
overall , these 32 issues include a total of 779 submissions , comprising of 48 editorials , 231 original articles , 148 reviews , 34 brief communications , 178 letters to editor , 97 case reports , 27 case reports with review of literature , 3 articles on endocrinology and gender and 5 pertaining to endocrinology and arts [ table 1 ] .
of these , 7 editorials , 41 original articles , 30 review articles , 12 brief communications , 20 letters to editor , 4 guidelines and 2 on endocrinology and gender directly or indirectly dealt with the community health perspective of endocrinology .
table 2 shows the year ( volume ) wise distribution of these community endocrinology articles .
table 3 shows the number of articles related to community endocrinology published under different contents of the journal .
table 4 gives the distribution of community health articles as per the various sub specialties of endocrinology .
2011 - 2014 number of articles published in ijem based on the various areas of community medicine w.e.f .
2011 - 2014 list of number of articles published in ijem of public health importance w.e.f .
2011 - 2014 as per type of article year wise fraction of community endocrinology articles in indian journal of endocrinology and metabolism distribution of community health articles as per the various sub specialities of endocrinology w.e.f .
2011 - 2014 if we calculate the percentages ( excluding case reports and case report with review of literature ) published from community health perspective out of the overall number in these 4 years , then community health related articles account for around 18% ( 116/655 ) .
13% , 15% , 17% , 13% of the articles in all the categories published in ijem in 2011 , 2012 , 2013 and 2014 respectively were community medicine related .
over the last few decades , ncds have emerged as the leading causes of death globally .
ncds place a huge burden on individuals and families , drain health resources , and threaten to derail the productivity , growth , and development of the nation .
the endocrine and metabolic diseases of obesity , diabetes mellitus ( dm ) , iodine deficiency ( i d ) disorders , vitamin d deficiency and osteoporosis are some of the major public health problems faced today .
for instance , the world prevalence of diabetes among adults ( aged 2079 years ) was 6.4% in 2010 and is expected to increase to 7.7% by 2030 .
defining the epidemiology of these conditions will provide clues to risk factors and identify areas of concern to help allocate community health resources .
jain et al . who did a retrospective analysis of all elderly patients following up at their closed community hospital in nagpur city of maharashtra observed a 30.42% of prevalence of dm .
the high prevalence of dm reported uniformly from across the region has inspired the editors to suggest the use of the term endemic rather than epidemic while describing dm .
endocrinologists are able to deal with secondary prevention of these silent killers exceptionally well , but india still remains one of the capitals of diabetes and cardiovascular diseases ( cvds ) .
there is a need for endocrinologists to look beyond just treating patients who come with the said disease to them ; it is required that they maintain a broader outlook towards various determinants of disease and address the problem from the biological , behavioral , psychosocial and community perspective .
the editors have also emphasized on the need for therapeutic patient education as a tool to achieve the desired therapeutic outcome they have also stressed upon patient empowerment and encourage inclusion of patient education as an integral part of all disease management .
there was found to be a significant improvement in the ( quality of life ) and a nonsignificant trend toward improvement in glycemic control in the group practicing the comprehensive yogic breathing program compared with the group that was following standard treatment alone .
it is suggested that yoga and related practices result in rapid gene expression alterations , which may be the basis for their longer - term health effects .
though existing risk factors such as smoking , dyslipidemia , hypertension , diabetes and obesity have been considered while calculating the risk among various ethnic groups , they can not fully explain excess risk in indians and other ethnic groups , indicating a search for alternative risk factors says gupta et al .
this highlights the imperative role of the community medicine specialists to cater to the unseen population ; to locate those cases who are at risk of developing these disorders .
for instance the public health implications of hypothyroidism were understood quite early in the evolution of thyroidology .
however even now , there are two major factors poverty and inability of consumers to assess the quality of salt they purchase that hinder iodized salt consumption in india , thereby putting a large number of people at risk of i d .
patients with hypothyroidism in india often remain undetected and untreated in spite of easy detection techniques and low cost treatment , impairing the work performance and economic productivity of indian people . in 2000 , a study by aiims showed that up to 90% of apparently healthy subjects were having hypovitaminosis d. early detection of vitamin d deficiency should be the priority of the government as well as the nongovernmental organizations involved in health .
there is also a need for the national food fortification program to improve the vitamin d status of the whole population .
vitamin d should be supplemented for vulnerable groups such as pregnant and lactating women , infants and elderly > 60 years as it is beneficial for both skeletal and nonskeletal health .
mithal and kalra et al . concluded that daily doses of 10002000 iu can be recommended in all antenatal women in south asia , without estimating serum 25(oh ) d levels .
seconds their conclusion that getting 25(oh ) d levels consistently above 75 nmol / l ( 30 ng / ml ) may require at least 15002000 iu / day of vitamin d. there were 8 articles on south asian guidelines for management of endocrine disorders in ramadan in the july august 2012 issue of ijem .
the holy month of ramadan is one of five main pillars of being a muslim .
the risk group approach is a commonly practiced managerial tool in public health and population medicine . using this approach ,
the concerns regarding fasting in diabetes , which by all counts is a public health problem of concern in current medical practice , can be taken care of .
this series of articles on ramadan management suggests the person centered , community centered ethos and international reach of ijem .
compiled the various national health programs running in the country in endocrinology and emphasized on the need for the program to maximize its effort in primary prevention or at least postponing the onset of these conditions .
most of the programs aim at primary prevention ( health education and specific protection ) and secondary prevention ( screening for early diagnosis of the disease and treatment ) .
keeping in view that there are common preventable risk factors for cancer , diabetes , cvd and stroke , government of india initiated a national programme for prevention and control of cancers , diabetes , cardiovascular diseases and stroke ( npcdcs ) during 20102011 .
childhood obesity screening is done under school health programs , but for the follow - up care of obese children , no specific programs are available . for unseen syndromes such as congenital hypothyroidism , there is a felt need for the universal screening program .
all this can be achieved through consolidation of knowledge of endocrinologists and mobilization efforts of community medicine specialists .
it is important for both the endocrinologists and community medicine professionals to think prevention of obesity at all patient visits in clinics and in fields to incorporate relevant health education .
body mass index ( bmi ) charts and waist circumference should be incorporated in routine health records .
the children with bmi > 75 centile should be frequently monitored and life style interventions should be introduced .
the purpose should be to pool and integrate the expertise of both endocrinologists and community medicine specialists so that more efficient and comprehensive assessment and intervention services may be provided .
the role differentiation can be defined by the needs of the situation rather than by discipline - specific characteristics .
truly the 40 years experience of esi coupled with the energetic 15 years of ijem provides us with an academic cocktail with an idea of the past , current , and future trends in endocrine research .
analyzed the characteristics of all articles published by endocrinologists from india in pubmed indexed journals between 2001and 2012 and concluded that research productivity of endocrinology community is increasing during the last decade in pubmed indexed journals .
there is a further need for the similar kind of bibliometric analysis of the community medicine journals to analyze their contribution towards the field of endocrinology and metabolism .
this will help assess whether the upcoming field of ncd , especially diabetes and other metabolic disease , is being given adequate representation by community medicine fraternity .
much is said and written about how things need to be done at individual , community and government level if we wish to see a change .
working individually , however , in clinics and academic departments will not bear as much results as would the trans disciplinary approach .
a trans disciplinary approach requires team members to share roles and systematically cross discipline boundaries .
we must work to horizontally interconnect the fields of community medicine and endocrinology , so that we can consolidate the results of the articles published and convert it into some visible action in the field of endocrinology and metabolism .
we hope that this communication will herald the beginning of a new , much needed specialty : community endocrinology . | background : endocrine and metabolic diseases especially diabetes have become focus areas for public health professionals .
indian journal of endocrinology and metabolism ( ijem ) , a publication of endocrine society of india , is a peer - reviewed online journal , which covers technical and clinical studies related to health , ethical and social issues in field of diabetes , endocrinology and metabolism .
this bibliometric analysis assesses the journal from a community health perspective.materials and methods : every article published in ijem over a period of 4 years ( 20112014 ) was accessed to review coverage of community health in the field of endocrinology.results:seven editorials , 30 review articles , 41 original articles , 12 brief communications , 20 letter to editors , 4 articles on guidelines and 2 in the section endocrinology and gender directly or indirectly dealt with community health aspects of endocrinology .
together these amounted to 17% of all articles published through these 4 years .
there were 14 articles on general , 60 pertaining to pancreas and diabetes , 10 on thyroid , 7 on pituitary / adrenal / gonads , 21 on obesity and metabolism and 4 on parathyroid and bone ; all community medicine related.conclusion:community health is an integral part of the modern endocrinology diabetology and metabolism practice and it received adequate journal space during the last 4 years . the coverage is broad based involving all the major endocrine disorders . | I
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PMC411055 | current analytical techniques in biotechnology can potentially benefit from an integrated reduction in scale through lowered production and operating costs , and via the specific dynamics of flowing fluids occurring at the mico - scale , which enable the generation of accurate quantitative assays .
microfluidics combines multiple disciplines including biotechnology , microtechnology , physics , and analytical chemistry and has flourished as a research field .
the processes involved in biotechnology and microfluidics technologies take place on a very small scale ( microns to millimetres ) where some physical processes can become enhanced , e.g. osmotic movement , electrophoretic motility and surface interactions .
microfluidics technology has essentially taken advantage of the inherent properties of liquids and gases at the microscale and combined this with semiconductor technology in order to build singular devices using a streamlined manufacturing process .
in general , microfluidic devices can offer a number of advantages over more conventional systems , e.g. their compact size , disposable nature , increased utility and a prerequisite for reduced concentrations of sample reagents .
miniaturised assemblies can be designed to perform a wide range of tasks that range from detecting airborne toxins to analysing dna and protein sequences .
therefore , microfluidics systems provide a real potential for improving the efficiency of techniques applied in drug discovery and diagnostics . in order for microfluidic technology to interface with , and provide improvements for , current assaying techniques it needs to be adaptable .
typical devices include passive flow systems , such as the passive fluid control ( pfc ) micro fluid analysis system by biomicro systems .
pfc incorporates ' building block - like ' components into circuit designs in order to carry out sample processing , e.g. immobilisation , mixing , incubation .
essentially , pfc utilises hydrophobicity and ' passive valves ' ( a narrowing of capillaries ) to control the movement of small volumes of fluids ( < 1 l ) within a network of channels .
incorporation of active or passive pumps can also be used to control the movement of fluids in microfluidic systems , e.g. nanostream 's snap - n - flow system .
a further setup by gyros has integrated a cd element with the movement and control of nanolitre volumes . when the cd is set spinning centrifugal forces
are created allowing the device to be used to produce a controlled passage of samples through ' microfabricated units ' on the surface of the cd .
microfluidics systems capable of assaying ' unprocessed ' biological samples , e.g. blood , have been developed therefore eliminating the requirement for sample preparation , e.g. micronics .
micronics ' microflow system can be used to extract analytes directly from whole blood and other particulate suspensions ( 5200 l volumes ) .
the system utilises disposable ' lab cards ' , e.g. the activeh card can be used for sample preparation and isolation whereas the activet card can be used in immunoassays .
some specific microfluidic systems have been developed that are capable of a range of dna - type analyses .
a microfluidic integrated system , which minimises sample processing and handling , has been developed for pcr analysis . here
dna typing is achieved from whole blood samples using capillary microfluidics and capillary array electrophoresis , see figure 1 , whereby blood is used directly as the sample template for a pcr amplification analysis .
microfluidics technology has also illustrated a potential to be allied with the detection of very low numbers of dna molecules , i.e. potentially individual molecules .
have shown that the construction of fluidic channels of < 1 m enables the detection and relative proportions of mixtures of dna molecules to be measured .
in addition , using an electrical field to control the flow rates analysis times of only several milliseconds per dna molecule become achievable .
electrophoretic mobility shift assays for the detection of dna - protein interactions have also been carried out in a microfluidic chip environment .
some of the benefits achieved are reduced sample volumes , an avoidance of labelling procedures and decreased analysis times .
however , the technique generally relies on passive diffusion of the sample volume , containing the target analytes , towards the immobilised probe elements and this can result in long hybridisation times ( normally hours ) .
a method of accelerating the hybridisation time for dna arrays using plastic microfluidic chips , comprising networks of microfluidics channels plus an integrated pump , have been developed .
it has been shown that ' high initial hybridisation velocities ' can be attained and that equilibrium , in terms of bound versus free analyte , is quickly achieved and so negates the requirement for such long hybridisation events .
the assembly of arrays into microfluidic channels in order to improve the kinetics associated with hybridisation has also been shown by other researchers .
a low - density array , generated within microfluidics channels , has been used to detect gene fragments ( k - ras ) carrying a point mutation .
again it was found that microfluidics reduced the hybridisation time in this assay from hours , i.e. the time required in conventional static hybridisations , down to less than 1 minute .
an alternative method of reducing array hybridisation times based on cavitation microstreaming has also been shown .
essentially cavitation microstreaming involves the use of a sound field to induce the vibration of air - bubbles ( at a solid surface ) present within a fluid .
this ultimately causes a circulatory flow within the fluid and so mixing times become reduced from hours to seconds .
microfluidic technology has also been incorporated into the analysis of proteins / peptides . in particular , microfluidics can be linked with a mass spectrometric analysis of proteins or peptides .
thus , peptides can be adsorbed onto hydrophobic membranes , desalted , and through the use of microfluidics eluted in a controlled manner to allow the direct mass spectrometric analysis of picomole amounts of peptides by electrospray ionisation mass spectrometry procedures , figure 2 .
proteins are applied directly to a membrane , desalted and directed by microfluidic channel to mass spectrometric analysis .
the recently reported combinatorial peptidomics approach is also perfectly suited for use with integrated microfluidic systems and in principle allows identification of tryptic peptides directly from the crude proteolytic digest .
combinatorial peptidomics initially utilises peptidomics where a protein sample is proteolytically digested prior to assaying , and combines it with a combinatorial depletion of the digest ( peptide pool ) by chemical cross - linking via amino acid side chains to allow a subsequent profiling of the resulting sample , figure 3 .
sample solubilisation and protein purification are not necessary , since proteolyric digection may be carried on native cells / tissues ( dashed lines ) .
the amino acid filtering ( depletion ) step may be repeated using combinations of up to 6 amino acid " filters " , i.e. chemically reactive surfaces ( e.g. derivatised beads ) able to covalently cross - link particular amino - acids .
chemical depletion reduces the complexity of the peptide pool to a sufficient degree to make it compatible with direct ms detection .
this allows the amount of protein delivered to the membrane , the reaction temperature within the device and the reaction time to be directly controlled for optimal digestion .
thus , using microfluidics the sample can be supplied directly from upstream processing procedures , e.g. purification products from cell lysates .
the development of protein microarray methods [ 12 - 14 ] , analogous to dna microarray technologies , for protein / peptide analysis has the potential to hasten the discovery of proteins of pharmacological value .
as is the case with dna microarrays it is important that sample volumes required for analysis are low , the sensitivity of the assay is high ( particularly for low - abundance proteins ) , and hybridisation times are kept to a minimum in order to produce an efficient assay .
a system incorporating protein microarrays , fluorescent detection and integrated microfluidics using planar waveguide technology has been developed . in combination
these components enable quantitative measurements for protein profiling to be carried out with high sensitivity and also require shorter analysis times than static hybridisations .
finally , more novel uses for microfluidic technology at a cellular level include the handling of mammalian embryos , the manipulation of embryos and oocytes in assisted reproduction and even the isolation of motile spermatozoa .
it is evident that the inherent flexibility of microfluidic systems will allow them to permeate and advance the development of assays in multiple biological , chemical and physical disciplines .
thus , microfluidics should ultimately reduce the cost of running assays , decrease procedural times and limit the required concentration and hands - on manipulation of samples .
rb acknowledges the support from the research strategy fund , royal holloway univesity of london . | microfluidics enables biotechnological processes to proceed on a scale ( microns ) at which physical processes such as osmotic movement , electrophoretic - motility and surface interactions become enhanced . at the microscale sample volumes and assay times are reduced , and procedural costs are lowered .
the versatility of microfluidic devices allows interfacing with current methods and technologies .
microfluidics has been applied to dna analysis methods and shown to accelerate dna microarray assay hybridisation times .
the linking of microfluidics to protein analysis techologies , e.g. mass spectrometry , enables picomole amounts of peptide to be analysed within a controlled micro - environment .
the flexibility of microfluidics will facilitate its exploitation in assay development across multiple biotechnological disciplines . | Background
Commercial products/technologies
DNA applications
Protein applications
Future prospects
Acknowledgements |
PMC2661510 | birth history of the child was unremarkable . on initial examination , the size of the cornea looked smaller .
the child was referred for ocular ultrasound examination , with a clinical suspicion of retinoblastoma .
an ultrasound study was performed with a high - frequency transducer operating at 10 mhz ( l5 , ge medical systems , usa ) .
gray scale evaluation revealed an echogenic band in the posterior segment of both globes extending from the posterior surface of the lens capsule to the optic disc .
the axial length of the globes was 16 mm , which was suggestive of micro - ophthalmia .
hyperechoic foci were seen in both the posterior segments adjacent to the artery , suggestive of associated vitreous hemorrhage [ fig .
no calcification was seen . a 3-month - old infant who presented with bilateral leukokoria .
( a ) axial ultrasound image with color doppler shows a vessel running through the vitreous of both globes , from the posterior surface of the lens capsule to the optic disc ( arrowhead ) .
( b ) on pulsed doppler examination , the vessel shows arterial flow computed tomography ( ct ) of the orbits ( lightspeed , ge medical systems , usa ) revealed diffusely hyperdense vitreous in both globes , suggestive of hemorrhage [ fig . 2 ] .
subtle linear densities were seen in the vitreous of both globes , representing hyaloid artery in cloquet 's canal [ fig . 2 ] . a 3-month - old infant who presented with bilateral leukokoria .
axial ct image shows diffusely hyperdense attenuation of vitreous in both globes , suggesting hemorrhage .
subtle linear structures are also seen ( arrowheads ) , representing hyaloid artery in cloquet 's canal based on the above findings , the patient was diagnosed with bilateral phpv .
further investigation failed to reveal any history of similar disorder in the family of the patient .
the parents were advised to get him examined under general anesthesia , so that appropriate management could be decided . however , the parents were unwilling for a procedure under anesthesia and refused for the examination .
the primary vitreous is formed during the first month of development and contains branches of the hyaloid artery . this hyaloid artery begins to regress during the formation of the avascular secondary vitreous at 9 weeks . by the third month , the secondary vitreous , which ultimately forms the adult vitreous , fills most of the developing vitreous cavity .
the primary vitreous becomes condensed into a narrow band ( cloquet 's canal ) , running from the optic disc to the posterior aspect of the lens .
phpv is classified into three types : anterior , posterior , or a combination of the two .
bilateral leukokoria in a child may be due to a number of causes , of which retinoblastoma , congenital cataract , retinopathy of prematurity ( rop ) , severe intermediate uveitis , and retinal dysplasias are the most frequent .
bilateral phpv is rare . while analyzing patients presenting with phpv , pollard found two of 83 ( 2.4% ) cases to be bilateral , while haddad et al . found seven of 62 ( 11% ) cases to be bilateral .
persistent hyperplastic primary vitreous has a typical imaging appearance , which allows reliable differentiation from retinoblastoma . in phpv
, gray - scale ultrasonography shows an echogenic band extending from the posterior surface of the lens capsule to the optic disc .
doppler may show arterial flow within this band representing a persistent hyaloid artery [ fig . 1 ] .
ct findings of phpv are absence of calcification , increased density of the entire vitreous , tubular intravitreal density ( cloquet 's canal or nonattached retina ) [ fig .
2 ] , decubitus positioning showing a gravitational effect on fluid - fluid level , micro - ophthalmia , enhancement of abnormal intravitreal tissue , and small or irregular lens . magnetic resonance imaging ( mri ) findings of phpv consist of a tubular structure , representing the hyaloid vessel ; a funnel - shaped retinal detachment , with the subretinal fluid hyperintense on both t1- and t2-weighted images ; fluid - fluid level due to the presence of hemorrhage in the subretinal space ; a retrolental mass ; micro - ophthalmia , and vitreous hemorrhage .
phpv can be differentiated from retinoblastoma by the absence of a calcified mass , artery running through cloquet 's canal , and typical signal characteristics of retinoblastoma on mri , i.e. , hyperintense on t1-weighted images and hypointense on t2-weighted images .
a patent hyaloid artery , as noted in this case , is not a feature of vitreoretinal dysplasias .
differentiation from advanced retinopathy of prematurity ( rop ) can be difficult on imaging alone .
history of a premature , low birth weight infant undergoing prolonged supplemental oxygen therapy helps to distinguish it from bilateral phpv .
complications of phpv include rupture of the lens capsule , cataract formation , intraocular hemorrhage , secondary glaucoma , tractional retinal folds , and subsequent phthisis bulbi .
the friability of the hyaloid vasculature predisposes to vitreous hemorrhage , which was noted in both globes in the present case .
this entity , although rare , should be considered in the differential diagnosis of retinoblastoma while evaluating bilateral leukokoria . | a case of bilateral persistent hyperplastic primary vitreous ( phpv ) in a 3-month - old male infant , who had bilateral leukokoria , is presented .
the child was referred for imaging with a clinical suspicion of retinoblastoma .
gray - scale ultrasound evaluation revealed an echogenic band in the posterior segment of both globes , extending from the posterior surface of the lens capsule to the optic disc .
doppler examination revealed the presence of arterial flow in the band in both globes .
associated echogenic hemorrhage was also seen , which was confirmed by computed tomography .
most cases of phpv are sporadic and unilateral , and bilateral phpv is rare .
the imaging features in this case suggest the diagnosis of bilateral phpv and differentiate it from retinoblastoma .
this entity , although infrequent , should be considered in the differential diagnosis while evaluating bilateral leukokoria . | Case Report
Discussion |
PMC540046 | chicken ( gallus gallus ) is an important model organism for biomedical research , the study of embryology and development ( 1,2 ) , aging ( 3 ) , quantitative trait loci ( qtl ) analysis ( 4 ) , in addition to being a major food source . through the comprehensive study of sequence polymorphisms in the chicken
, significant progress can be made in understanding the phenotypic differences between individuals / strains and the evolution of populations . towards this end ,
the beijing genomics institute ( bgi ) led an international team of scientists from china , usa , uk , sweden , the netherlands and germany to identify and characterize extensive dna sequence variation throughout the chicken genome .
sequence variation within the genomes of three different breeds of domesticated chickens [ a male broiler ( cornish ) from roslin institute , a female layer ( white leghorn ) from swedish university of agricultural sciences and a female silkie from the chinese agricultural university in beijing ] , was identified by direct comparison with the genome sequence of the red jungle fowl ( rjf ) , assembled by the washington university genome sequencing center ( wugsc ) , as a reference chicken genome sequence ( chicken genome consortium , 2004 ) . from these comparisons
3.1 million unique , high - quality sequence variants were identified , primarily single nucleotide polymorphisms ( snps ) . in order to facilitate the use of this information
, we created the chicken variation database ( chickvd ) , an integrated information system for the storage , retrieval , visualization and analysis of chicken dna sequence variation . to enhance the discovery of relationships between sequence variation and genes , we mapped each variant onto the rjf reference genome sequence in the context of gene annotations and other relevant features , such as genetic markers and qtls .
therefore the chickvd database , provides both a powerful information resource and an analysis workbench for applications in biological research , medicine and agriculture .
the primary purpose of the chicken snp project was to discover and map extensive sequence variation in the chicken genome , to increase the density of markers on the genetic map , and to facilitate studies on the genetic basis of phenotypic traits .
we compared the sequence reads from the three chicken breeds to the 6.6x rjf reference genome sequence ( chicken genome consortium , 2004 ) .
for each of the breeds , we sampled 25% of the genome ( a million sequence reads from each breed ) using automated capillary sequencers ( amersham megabace 1000 ) . to minimize sequencing errors
, we used the phred quality score ( q - value ) ( 5,6 ) to set minimum thresholds . for base substitutions , we used conservative thresholds of > q25 at the variant site and > q20 for the two flanking 5 bp regions .
even more stringent thresholds of q30 and q25 were used to score insertion deletions ( indels ) . using a genome - wide blastn search , we were able to determine the sequence position on the rjf genome assembly and eliminate confusion between paralogs .
a subset of polymorphisms was confirmed by pcr - based resequencing from the lines in which they were initially detected . to display each sequence variant in the context of its physical relationship to the nearest gene or other relevant features , we employed a number of methods to identify these target sequences .
these included annotations within public databases , homology searches , prediction programs and information on experimentally derived genes .
a non - redundant dataset of genes / cdnas was integrated within chickvd , including ensembl gene annotations ( 7 ) , genbank genes with
complete cds ( 8) , full - length cdnas ( 9 ) ( http://pheasant.gsf.de/department/dt40/dt40transcript.html ) and chicken orthologs of human disease genes ( c. webber and c. p. ponting , unpublished data ) .
we used blat ( 10 ) to map these sequences using the rjf reference genome , sim4 ( 11 ) to determine the detailed exon
intron boundaries and then classified coding snps into synonymous or non - synonymous substitutions . to associate the location of sequence variants with genetically mapped phenotypic traits
, we included information on mapped qtls , using data provided by a number of collaborating institutions ( roslin institute , department of medical biochemistry and microbiology of uppsala university , usda - ars avian disease and oncology laboratory , animal breeding and genetics group of wageningen university ) and public resources , such as chickace ( https://acedb.asg.wur.nl/ ) .
chickvd contains 3.1 million dna sequence variants ; 2.8 million are categorized as snps and 0.3 million are indels .
follow - up experiments indicate that over 90% of these snps are true snps , over 70% are common snps that segregate in many chicken breeds and the mean nucleotide diversity is estimated to be 5 snp / kb ( international chicken polymorphism map consortium , 2004 ) . for the convenience of data display ,
all types of dna sequence variation ( substitutions , insertions or deletions ) are referred to as
snps. snp information is detailed in a snp report , including type , allelic differences , flanking sequences and pcr - primer designs , location , associated genes and quality scores of sequence reads , functional site in the chicken genome ( e.g. coding region , intron , untranslated regions ) , and details of any predicted or known functional outcomes ( e.g. codon and deduced amino acid changes ) . for additional information to the potential user , 1.5 million contig - covered ( ctgcov )
regions from the broiler , layer and silkie are aligned to the rjf reference genome and 2.5 million raw sequence traces are available within chickvd . for queries about gene - associated polymorphisms ,
gene report , including gene structure , functional classification , gene ontology ( 12 ) and interpro ( 13 ) annotations , gene - associated snps , and nucleotide / protein sequences . for chicken orthologs of human disease genes , hypertext links to the given ensembl files of human disease genes and omim entries of disease descriptions ( 14 )
a collection of 606 qtls for a wide range of traits are integrated and cross - referenced to markers , genetic maps , genes and snps mapped onto the same chromosomal region , and to pubmed ( 15 ) for literature sources .
chickvd also hosts a collection of 884 references , which focus on chicken sequence variation , qtl study and basic chicken biology .
a summary of chickvd content is shown in table 1 and all data are freely available from our ftp site ( http://chicken.genomics.org.cn/chicken/jsp/download.jsp ) .
all data housed in chickvd are uniquely mapped onto the rjf reference genome and graphically represented in mapview , an efficient visualization tool initially developed in our rice information system ( bgi - ris ) ( 16 ) that allows users to browse snps in genomic and functional context .
mapview is composed of four types of subviewers in hierarchical architecture , namely chroview , geneview , snpview and traceview ( figure 1 ) .
chroview is based on the reference sequence of rjf with qtls marked along chromosomes , and displays density and statistics of genes and snps .
chroview also allows users to center the map onto a specific chromosome location and make options to expand for more detailed views of genes / cdnas , snps via geneview and snpview , respectively .
a factual report for each element contained in the visualization system is displayed automatically on demand .
q - values for each nucleotide and a position - location function facilitate further investigation of the snps .
the chickvd online search tool is the entry point for querying snps and other data types in the database .
the results can be further restricted to a specific chicken breed , a certain snp type , a threshold of quality score or to a specific snp functional class , such as a coding non - synonymous snp .
blast - based snp search compares a user - supplied sequence against the flanking sequences that immediately surround the sequence variant .
advanced search interfaces for other data types ( genes / cdnas , qtls , sequence traces and references ) are also provided . for sequence searches ,
a key development is the implementation of seqgetter , a search tool that allows extraction of all variations residing within genomic domains , as defined by flanking elements that can be other variants , chromosomal positions , genes , or even specific coding sequences , introns or regulatory domains .
chickvd consists of three hardware components , a world wide web server , a database server and a sequence analysis / homology search server .
the system is based on an oracle9i relational database , and the front end consists of a set of jsp scripts running on tomcat web server .
java applets are applied for traceview functions . to handle the large amount of complex data
, we developed the standard sets of snp - centric and qtl - centric xml formats that lay the foundation for our research work and allow chickvd to accommodate the fast - accumulating data and to integrate new data types when they arise .
chickvd will also be updated for each new chicken genome update , including annotations and more qtls , markers and references , as soon as they become available .
so in the near future , it will be possible for users to retrieve data from different versions , trace up and locate changes of a given entity between different versions .
new versions of chickvd will integrate allele frequencies of each variation site from ongoing projects with collaborating institutions . to enhance data utility , our development efforts will extend data structures and establish systems to support haplotype information that is expected to become the principal functional unit for chicken genetics .
chickvd continues to make enhancements to user interfaces , improve the functionality of searches and data representation , and evolve the database infrastructure and data model to ensure data quality and consistency .
a side - by - side comparative map viewer will make comparative analysis of the snp map and chicken genes easier .
we welcome comments and suggestions from the chicken research community to make chickvd a user - friendly knowledge resource .
this project was funded by the chinese academy of sciences , commission for economy planning , ministry of science and technology ( 2002aa104250 , 2002aa234011 , 2001aa231061 , 2001aa231011 , 2001aa231101 and 2004aa231050 ) , danish national research foundation ( danish platform for integrative biology ) and the china national grid ( 2002aa104250 ) . | working in parallel with the efforts to sequence the chicken ( gallus gallus ) genome , the beijing genomics institute led an international team of scientists from china , usa , uk , sweden , the netherlands and germany to map extensive dna sequence variation throughout the chicken genome by sampling dna from domestic breeds . using the red jungle fowl genome sequence as a reference , we identified 3.1 million non - redundant dna sequence variants . to facilitate the application of our data to avian genetics and to provide a foundation for functional and evolutionary studies
, we created the chicken variation database ( chickvd ) .
a graphical mapview shows variants mapped onto the chicken genome in the context of gene annotations and other features , including genetic markers , trait loci , cdnas , chicken orthologs of human disease genes and raw sequence traces .
chickvd also stores information on quantitative trait loci using data from collaborating institutions and public resources .
our data can be queried by search engine and homology - based blast searches .
chickvd is publicly accessible at http://chicken.genomics.org.cn . | INTRODUCTION
DATA SOURCE
DATA CONTENT
DATABASE USAGE AND ACCESS
SYSTEM DESIGN AND IMPLEMENTATION
FUTURE DEVELOPMENTS
ACKNOWLEDGEMENTS |
PMC3325619 | an asymptomatic 22-year - old female was referred to our clinic with a diagnosis of fundus abnormality left eye ( os ) detected upon preoperative examination prior to refractive surgery .
dilated fundus examination os revealed an old , localized retinal detachment with a demarcation line and lattice degeneration , with a retinal hole in the inferior temporal periphery .
pigmentary changes were conspicuous , with a convex margin at the attached retina posterior to the demarcation line ( fig .
ultrasound biomicroscopic ( ubm 840 ; zeiss - humphrey , jena , germany ) examination of the retinal periphery near the causative retinal hole revealed vitreous fibers both anterior and posterior to the retinal hole ( fig . 1b and 1c ) .
no change in retinal lesions was observed over a follow - up period of two years .
a 58-year - old male was referred to our clinic with a diagnosis of fundus abnormality od .
dilated fundus examination od revealed a pigmented peripheral retinal lesion with convex margin at the 6-o'clock position of the peripheral retina .
optical coherence tomographic ( oct ; cirrus , carl zeiss meditec , dublin , ca , usa ) examination revealed that the presumed epiretinal membrane was actually a thickened posterior hyaloid membrane .
it was also determined that partial posterior vitreous detachment ( pvd ) had developed only on the temporal side of the fovea ( fig . 2b and 2c ) .
the patient underwent a pars plana vitrectomy to relieve macular edema due to vitreous traction .
an asymptomatic 38-year - old male was referred to our clinic with a diagnosis of retinal break od , which was incidentally detected when the patient underwent a scleral buckling operation in the fellow eye .
dilated fundus examination od revealed a pigment - stippled peripheral retinal lesion with a convex margin from 7-o'clock to 11-o'clock .
a two disc - diameter sized horseshoe tear with a rolled edge was noted at the 8-o'clock position of the peripheral retina , within the srrrd lesion .
the retinal tear was covered and incompletely sealed by a thin translucent vascularized membrane ( fig .
ubm examination showed vitreous fibers and a membranous structure overlying the retinal tear ( fig . 3b and 3c ) .
an asymptomatic 22-year - old female was referred to our clinic with a diagnosis of fundus abnormality left eye ( os ) detected upon preoperative examination prior to refractive surgery .
dilated fundus examination os revealed an old , localized retinal detachment with a demarcation line and lattice degeneration , with a retinal hole in the inferior temporal periphery .
pigmentary changes were conspicuous , with a convex margin at the attached retina posterior to the demarcation line ( fig .
ultrasound biomicroscopic ( ubm 840 ; zeiss - humphrey , jena , germany ) examination of the retinal periphery near the causative retinal hole revealed vitreous fibers both anterior and posterior to the retinal hole ( fig . 1b and 1c ) .
no change in retinal lesions was observed over a follow - up period of two years .
a 58-year - old male was referred to our clinic with a diagnosis of fundus abnormality od .
dilated fundus examination od revealed a pigmented peripheral retinal lesion with convex margin at the 6-o'clock position of the peripheral retina .
optical coherence tomographic ( oct ; cirrus , carl zeiss meditec , dublin , ca , usa ) examination revealed that the presumed epiretinal membrane was actually a thickened posterior hyaloid membrane .
it was also determined that partial posterior vitreous detachment ( pvd ) had developed only on the temporal side of the fovea ( fig . 2b and 2c ) .
the patient underwent a pars plana vitrectomy to relieve macular edema due to vitreous traction .
an asymptomatic 38-year - old male was referred to our clinic with a diagnosis of retinal break od , which was incidentally detected when the patient underwent a scleral buckling operation in the fellow eye .
dilated fundus examination od revealed a pigment - stippled peripheral retinal lesion with a convex margin from 7-o'clock to 11-o'clock .
a two disc - diameter sized horseshoe tear with a rolled edge was noted at the 8-o'clock position of the peripheral retina , within the srrrd lesion .
the retinal tear was covered and incompletely sealed by a thin translucent vascularized membrane ( fig .
ubm examination showed vitreous fibers and a membranous structure overlying the retinal tear ( fig . 3b and 3c ) .
findings for all three patients were consistent with srrrd , including retinal pigmentary alterations with convex margin of the retinal lesion .
interestingly , ubm examination in case 1 revealed vitreous fibers attached to the retinal hole at both the posterior and anterior margins .
oct in case 2 demonstrated that a pvd developed only at the horizontal meridian , suggesting vitreous attachment at the optic disc and elsewhere .
thus , it seems most likely that pvd was not present in the inferior retinal periphery , where the srrrd was observed .
retinal detachments may develop in the absence of a pvd , as was noted in the retinal detachment due to atrophic holes within lattice degeneration .
this finding has been shown to occur in younger patients with myopia . however , the development of retinal detachment in the absence of a pvd is apparently a rare event .
based on our observations , we suspect that the developmental mechanisms of srrrd are as follows . initially ,
both focal vitreoretinal adhesion and vitreoretinal traction induce a retinal break in the peripheral retina in an eye without a complete pvd ( as in cases 2 and 3 ) .
this is followed by an influx of liquefied vitreous into the retinal break , constituting the development of retinal detachment .
however , the direction of vitreoretinal traction changes to parallel the elevated retinal surface ( as in case 1 ) .
eventually , vitreoretinal traction is relieved and the retinal break is closed by vitreous fibers running parallel to the retinal plane .
this is especially likely if there is vitreous attachment at the posterior border of the retinal break , as in case 1 . as with the interruption of fluid currents through the subretinal space
, srrrd develops , and the thin membrane proliferates over the retinal break ( case 3 ) .
proliferative membrane sealing of the retinal break as a form of wound healing response contributes to the induction and/or maintenance of retinal reattachment .
we suspect the proliferative membrane results from fibroglial and retinal pigment epithelial hyperplasia . in our current report of three cases with srrrd , retinal breaks causing retinal detachment
it is worthy to note that the localization of the retinal break in the eyes that developed srrrd is apparently a rare event . despite an extensive search ,
a retinal break was not identified in any of the 15 cases in a previous series .
results of our current study lend indirect support to the prior contention that srrrd preferentially develops in cases of retinal detachment with a small retinal break .
identification of a small causative retinal hole would be difficult once development of srrrd is complete , due to the bridging of thin fibrous tissue over the small retinal hole .
in addition , diffuse retinal pigment atrophy and sub- and/or an epiretinal membrane within the extent of srrrd could hinder detection of a small retinal hole . in conclusion , we determined that the incidence of srrrd appears closely related to the absence of a pvd near the retinal break , as well as to membrane proliferation over the retinal break . | this retrospective observational case series on eyes from three patients was done to elucidate the developmental mechanism of spontaneous reattachment of rhegmatogenous retinal detachment ( srrrd ) .
the study eyes of each patients showed evidence of retinal break and diffuse retinal pigmentary change .
ultrasound biomicroscopic examination revealed vitreous fibers attached to the area around the retinal break .
posterior vitreous attachment was confirmed in each eye .
a thin fibrovascular membrane incompletely sealing the retinal break was noted in one case .
we suggest that the vitreous attachment around the retinal break and the sealing of the break with adjacent vitreous fibers seem to be involved in the developmental mechanism of srrrd . | Case Reports
Case 1
Case 2
Case 3
Discussion |
PMC3996432 | cardiac rehabilitation has been known to play a key role in the enhancement of exercise
ability , secondary prevention , and reduction in the mortality rate of patients with coronary
syndrome through comprehensive programs including exercise prescription , nutrition
counseling , drug therapy , and weight control1 , 2 .
however , an improper rehabilitation program
may result in negative outcomes , especially in patients with cardiac problems .
therefore ,
the programs should be closely monitored and controlled to prevent undesirable side - effects .
establishing the proper exercise intensity unique to a patient is one of the major steps in
designing an appropriate cardiac rehabilitation program .
the exercise intensity
is established based on the maximal heart rate or maximal oxygen consumption
( vo2peak ) obtained during an exercise stress test4 . although karvonen s formula ( 220 age ) , or the formula suggested
by aha ( american heart association ) { 206.9 ( 0.67 age ) } , are the most widely used formula
for establishing exercise intensity5 , 6 , they are not recommended for patients with
cardiovascular disorders .
an exercise stress test is recommended to establish the maximal
heart rate , since medications such as beta blockers taken by patients are known to
considerably lower heart rates , not only at rest but also during exercise7 , 8 .
the exercise stress test is usually conducted on a treadmill or ergometer to establish the
appropriate exercise intensity based on the percent heart rate reserve ( % hrr)4 .
the exercise intensities obtained from
either treadmill or ergometer exercise have been interchangeably applied to prescribe
exercise programs for patients with acs .
it was reported that vo2max obtained during tm was 6% to 25% higher
than vo2max obtained during ce , along with increased maximal heart rate9,10,11,12,13,14 .
the maximum exercise capacity could not be achieved due to comparatively lower cardiac
outputs and early fatigue tendencies in the lower limbs during ce11 , 15 .
in addition ,
the maximal sbp measured during exercise stress tests was higher during ce than during
tm10 , 16 . when the exercise intensity established during a tm stress test is
applied to ce , despite the physiological differences
reed17 reported a higher rate pressure production ( rpp ) during ce than
during tm , due to higher blood pressure responses with targeted heart rates established at
low- ( 40% ) and mid - intensity exercise ( 60% ) .
angina pectoris was seen in patients with
stable angina who experienced chest pain during exercise at certain values of rpp18 , 19 .
thus , when exercise is conducted on both a treadmill and an
ergometer with identical target heart rates , a higher exercise intensity may be achieved
during ce than during tm .
moreover , the cardiac burden may be significantly increased at
higher exercise intensities , especially at 85% hrr or greater .
in particular , rpp , which
measures the hemodynamic stress of the heart , may significantly increase to detriment of the
treatment effects and exercise performance , and further induce cardiovascular symptoms .
despite such significant differences in ce and tm , many guidelines for
exercise prescription
do not clearly distinguish the characteristics and risks involved in the two exercise types .
therefore , in order to elucidate the differences and risks involved in applying the
established exercise intensity to two different exercise types , this study calculated two
commonly used exercise intensities using a treadmill graded exercise test performed by
patients with acs . then , we applied the treadmill graded exercise test - derived exercise
intensities of 60% and 85% hrr to both tm and ce to assess the differences in the
hemodynamic responses , rate of perceived exertion ( rpe ) , and the number of cardiac events
between the two exercise types . the results may be used to improve exercise prescription
guidelines for patients with acs .
the subjects of this study were patients who were hospitalized for acs , who received
percutaneous transluminal coronary angioplasty ( ptca ) and drug therapy , and participated in
a hospital - based cardiac rehabilitation program from may 2009 to april 2011 .
the subjects
were recruited on a voluntary basis . after explaining the purpose and procedure of the
study , the subjects volunteered and gave their written consent before participating in the
study .
the study design and procedures were approved by the institutional review board ( irb )
of inje university sanggae baik hospital .
the study was performed according to the
guidelines set by the ethics committee20 .
the patients with severe arrhythmia , heart failure with 40% or less ejection fraction in the
left ventricle , exercise - induced high blood pressure , an interrupted exercise stress test
due to abnormal cardiovascular response , changes in administered drugs during the study
period , or chronic obstructive pulmonary or musculoskeletal disease contraindications to the
exercise stress test were excluded from this study .
although no premature termination was
observed during the exercise stress test , 16 out of 118 patients were excluded from the
study due to reasons such as difficulty adapting to ce or the prescribed exercise loads .
of
the 102 patients who completed this study , 38 patients were diagnosed as having unstable
angina ( 37.3% ) and 64 were diagnosed as having acute myocardial infarction ( 62.7% ) .
the
patient characteristics such as age , sex , height , weight , body mass index ( bmi ) ,
complications , administered drugs , heart rate ( hr ) , blood pressure ( bp ) , rpp , and
vo2max are described in table
1table 1 .
demographic characteristics of the subjects ( n=102)variablesmeansd / frequencyage ( y)56.69.7 ( m ) / 57.83.6 ( f)sex ( male / female)93 ( 91.2% ) / 9 ( 8.8%)height ( cm)167.0.6.0 ( m ) / 154.69.3 ( f)weight ( kg)69.68.9 ( m ) / 61.711.3 ( f)bmi ( kg / m)25.03.1 ( m ) / 25.73.1 ( f)diseaseua38 ( 37.3%)mi64 ( 62.7%)complicationshypertension56 ( 54.9%)diabetes mellitus24 ( 23.5%)dyslipidemia24 ( 24.5%)drugsaspirin102 ( 100%)plavix98 ( 96.1%)beta - blocker37 ( 36.3%)ccb9 ( 8.8%)carvedilol26 ( 25.5%)acei51 ( 50%)arb10 ( 9.8%)nitrate63 ( 61.8%)diuretics10 ( 9.8%)statin95 ( 93.1%)hrrest ( beat / min)72.512.6sbprest ( mm hg)117.316.9dbprest ( mm hg)78.510.0rpprest8486.81784.7 vo2max ( o2 ml / kg / min)28.36.9hr max ( beat / min)138.218.4sbp max ( mm hg)174.928.4dbp max ( mm hg)78.510.0rpp max24359.4 5771.5values are meansd , bmi : body mass index , mi : myocardial infarction , ccb : calcium
channel blocker , acei : angiotensin converting enzyme inhibitor , arb : angiotensin ii
receptor blocker , hr : heart rate , sbp : systolic blood pressure , dbp : diastolic blood
pressure , rpp : rate pressure product .
values are meansd , bmi : body mass index , mi : myocardial infarction , ccb : calcium
channel blocker , acei : angiotensin converting enzyme inhibitor , arb : angiotensin ii
receptor blocker , hr : heart rate , sbp : systolic blood pressure , dbp : diastolic blood
pressure , rpp : rate pressure product symptom - limited graded exercise tests ( gxt ) were conducted with a modified bruce protocol
within a week of discharge .
the tests were conducted to determine the appropriate exercise
intensities prior to cardiac rehabilitation exercise on a treadmill and cycle ergometer .
peak oxygen consumption ( vo2peak ) and the highest value of vo2 were
measured during the gxt .
a 12-channel real - time
electrocardiograph ( q4500 , quinton instrument co. , boston , usa ) , a gas analyzer ( qmc
( quinton metabolic cart ) , quinton instrument co. , boston , usa ) , an automated blood pressure
and pulsation measurement device ( model 412 , quinton instrument co. , boston , usa ) , and a
treadmill ( medtrack st 55 , quinton instrument co. , boston , usa ) were used for the exercise
stress tests . heart rate ( hr ) , blood pressure ( bp ) , and vo2 at rest and during
maximal exercise were recorded .
the borg scale ( 620 ) was also used to measure the rate of
perceived exertion ( rpe ) .
s
formula,{(maximal heart rate resting heart rate % intensity ) + resting heart rate } based
on the results obtained during the exercise stress test6 .
the cardiac rehabilitation program was composed of 10 minutes warm up ( stretching ) , 24
minutes of main exercise ( treadmill or cycle ergometer ) , and 10 minutes of cool down ( 3
minutes of slow walking and stretching)22 .
the main exercise was divided into two 12-minute exercise bouts
either on a treadmill ( quinton med - track sr 60 , quinton instrument co. , boston , usa ) or a
fixed cycle ergometer ( quinton corival 400 , quinton instrument co. , boston , usa ) .
the
participants took a three - minute rest by calmly walking on a flat floor between the two
12-minute exercise bouts .
the exercise sessions were conducted every other day for two
weeks , in a total of six sessions .
the cardiac rehabilitation exercise program was conducted alternatively on a treadmill and
a cycle ergometer with a gradual increase in the exercise intensity . on the first , third ,
and fifth sessions
, the treadmill exercise intensity was gradually increased to 60% hrr , 70%
hrr , and 85% hrr , respectively . on the second , fourth , and sixth sessions , the cycle
ergometer intensity
was gradually increased to 60% hrr , 70% hrr , and 85% hrr ,
respectively .
hr , sbp , dbp , rpp , rpe , and cardiac events were assessed two minutes prior to the end of
all the first exercise bouts .
an ecg telemetry system ( quinton instrument co. , boston , usa )
and a mercury blood pressure unit with a stethoscope were used to measure hr and bp at the
chest height of the patients .
the devices were also used to monitor for clinical signs of
myocardial ischemia , or arrhythmia .
rpe was
measured on the borg scale23 , and
possible chest pain was expressed using an angina pectoris scale24 .
the measurements obtained during tm and ce at 60% and 85%
hrr were used for comparison .
the measurements were performed by the same investigators to
maintain the measurement accuracy and impartiality .
flow chart of the study procedure descriptive statistics were used to calculate the patients characteristics .
the
measurements were evaluated using one - way repeated - measures anova with the exercise
intensities of 60% hrr , and 85% hrr during ce and tm as the independent variables .
the measurement
differences between patients without cardiac event during ce ( ce - ne ) and tm ( tm - ne ) and in
patients with cardiac events during ce ( ce - e ) were analyzed using one - way anova .
after
determining the significance of the difference by anova , a post - hoc multiple comparison was
performed using the scheffe test .
fisher s exact test was performed to assess the
significance of differences between the proportions of the two exercise types .
all statistical analyses were performed
using spss for windows , version 14 ( spss inc . ,
medium and high exercise intensities of 60% and 85% hrr were calculated for the cardiac
rehabilitation exercise based on the results obtained during gxt using karvonen s formula .
the hemodynamic
responses , rpe , and cardiac events were measured to determine the differences between the
exercise types and to evaluate the significance of the differences in patients with acs .
hemodynamic responses ( n=102 ) to treadmill and cycle ergometer exercise at
60%hrr and 85%hrr60%hrr - tm60%hrr - ce85%hrr - tm85%hrr - cehr
( beats / min)111.913.0 110.713.8127.416.4128.214.4sbpmax ( mm hg)135.218.8 155.822.6151.620.0177.123.6%sbpmax78.210.0%89.911.3%87.710.9%102.212.0%dbpmax ( mm hg)77.18.9 83.711.579.28.988.711.3%dbpmax101.716.3%110.118.1%104.415.8%116.817.8%rppmax152273187175163641195744014228534782%rppmax63.48.6%73.09.5%81.410.1%95.011.1%rpemax11.11.512.41.313.01.215.31.2hr : heart rate , sbp : systolic blood pressure ( mmhg ) , dbp : diastolic blood pressure
( mmhg ) , rpp : rate pressure product , rpe : rate of perceived exertion .
these values were obtained by dividing
the maximal sbp , dbp , and rpp values obtained at 60% hrr and 85% hrr during ce and tm
by sbpmax ( 17528 mmhg ) , dbpmax ( 7713 mmhg ) , and
rppmax ( 243595771 ) obtained during treadmill gxt , respectively , and
multiplying each result by 100%.*=significantly different from the previous trial , p < 0.001 shows the maximal hemodynamic responses of hr , sbp , dbp , as well as rpp and
rpe at the 60% and 85% exercise intensities during tm and ce .
the maximal sbp , dbp , and rpp
values were divided by the maximal sbp , dbp , and rpp values obtained during treadmill gxt
and multiplied by 100% to obtain percentages for comparison .
hr at the exercise intensities
of 60% and 85% were identical ( tm vs. ce = 111.913.0 bpm vs. 110.713.8 bpm at 60% hrr ; tm
vs. ce = 127.416.4 bpm vs. 128.214.4 bpm at 85% hrr .
hr : heart rate , sbp : systolic blood pressure ( mmhg ) , dbp : diastolic blood pressure
( mmhg ) , rpp : rate pressure product , rpe : rate of perceived exertion .
these values were obtained by dividing
the maximal sbp , dbp , and rpp values obtained at 60% hrr and 85% hrr during ce and tm
by sbpmax ( 17528 mmhg ) , dbpmax ( 7713 mmhg ) , and
rppmax ( 243595771 ) obtained during treadmill gxt , respectively , and
multiplying each result by 100% . * = significantly different from the previous trial , p < 0.001 for sbp expressed as a percentage of maximal sbp , % sbpmax ( 89.911.3% ) of 60%
hrr during ce was significantly higher than % sbpmax ( 78.210.0% ) of 60% hrr during tm .
% sbpmax ( 102.212.0% ) of 85% hrr during ce was also significantly higher than % sbpmax
( 87.710.9% ) of 85% hrr during tm ( p < 0.001 ) , and % sbpmax of 85% hrr during
ce showed the highest value .
% rppmax
( 73.09.5% ) of 60% hrr during ce was significantly higher than % rppmax ( 63.48.6% ) of 60%
hrr during tm , and % rppmax ( 95.011.1% ) of 85% hrr during ce was also significantly higher
than % rppmax ( 81.410.1% ) of 85% hrr during tm ( p < 0.001 ) .
in addition , all measurements of dbp and rpe at the exercise intensities of 60% hrr and 85%
hrr were significantly higher during ce than during tm ( p < 0.001 ) . in order to confirm the increased risk of ce ,
the patients were continuously monitored and
recorded for cardiac events during all the exercise sessions ( table 3table 3 .
blood pressure and rpe responses of the patients with and without cardiac - events
at the two different exercise intensities cycle ergometer and treadmill
exercise60%hrr85%hrrtm - ne ( n=101)ce - ne ( n=72)ce - e ( n=29)tm ( n=101)ce - ne ( n=72)ce - e ( n=29)%sbpmax78.09.888.310.793.411.587.610.999.29.9110.413.7%rppmax63.38.471.48.876.19.981.410.192.19.1101.912.7rpemax11.01.412.31.212.71.613.01.114.60.816.90.5tm - ne = patients without cardiac event during treadmill exercise , ce - ne = patients
without cardiac event during cycle ergometer exercise , ce - e = patients with cardiac
events during cycle ergometer exercise . the tm - e group was omitted from the comparison
since only one patient showed a cardiac event during treadmill exercise .
sbp : systolic
blood pressure , rpp : rate pressure product , rpe : rate of perceived exertion .
% sbpmax and % rppmax were calculated by dividing the maximal
sbp and rpp obtained at 60%hrr and 85%hrr during ce and tm by sbpmax
( 17528 mmhg ) and rppmax ( 243595771 ) obtained during treadmill gxt ,
respectively , and multiplying each result by 100% . = significantly different from
60%hrr - tm , p < 0.05 ,
= significantly different from 85%hrr - tm , p < 0.05 , * =
significantly different from 85%hrr - ce - ne , p < 0.05 . ) . of
the 102 patients , 29 patients ( 28.4% of the patients ; ce - e : patients with
cardiac events during cycle ergometer exercise ) showed cardiac events at 85% hrr during ce .
% sbpmax and % rppmax of 60% hrr and 85% hrr during tm for the 29
patients with cardiac events and the 72 patients without cardiac events were calculated for
comparison ( table 3 ) .
significantly higher
maximal % sbp , % rpp , and rpe were shown at 60% hrr and 85% hrr during ce than during tm in
the patients with ( ce - e ) compared to those without ( ce - ne ) cardiac events .
% sbpmax and % rppmax showed non - significant differences in both ce - e
and ce - ne at 60% hrr during ce .
however , the patients without cardiac event ( ce - ne ) at 85%
hrr during ce had significantly higher % sbpmax and % rppmax
( p<0.001 ) .
furthermore , % sbpmax and % rppmax of ce - e were
110.413.7% , and 101.912.7% , respectively , exceeding the maximal sbp and rpp obtained in
the treadmill gxt .
although rpemax for ce - ne ( 14.60.8 ) was significantly greater
than rpemax of tm ( 13.01.1 ) , rpemax for ce - e was much greater
( 16.90.5 ) with a difficulty level of very very difficult
tm - ne = patients without cardiac event during treadmill exercise , ce - ne = patients
without cardiac event during cycle ergometer exercise , ce - e = patients with cardiac
events during cycle ergometer exercise .
the tm - e group was omitted from the comparison
since only one patient showed a cardiac event during treadmill exercise .
sbp : systolic
blood pressure , rpp : rate pressure product , rpe : rate of perceived exertion .
% sbpmax and % rppmax were calculated by dividing the maximal
sbp and rpp obtained at 60%hrr and 85%hrr during ce and tm by sbpmax
( 17528 mmhg ) and rppmax ( 243595771 ) obtained during treadmill gxt ,
respectively , and multiplying each result by 100% . = significantly different from
60%hrr - tm , p < 0.05 ,
= significantly different from 85%hrr - tm , p < 0.05 , * =
significantly different from 85%hrr - ce - ne , p < 0.05
total cardiac events and rpe responses ( n=102 ) at the different exercise
intensities of the different exercise types60% hrr60% hrr85% hrr85% hrrtmcetmcecardiac eventschest pain ( 2 ) + rpe ( 17)0011chest pain ( 2 ) + rpe ( 15)0003intermittent pvc + rpe ( 17)0001bigeminy pvc + rpe ( 17)0001af + rpe ( 17)0001st depression without chest pain0001total cardiac events0018rpe responserpe 1700021chest pain scale ( 2 ) : mild chest pain .
rpe : rate of perceived exertion , pvc :
premature ventricle contraction , af : atrial fibrillation , st segment depression : 1 mm
( horizontal ) , * = significantly different , p < 0.01 shows the number of patients who were interrupted during exercise due to the
occurrence of cardiac events .
one patient at 85% hrr during tm and one patient at 85% hrr
during ce complained of rpe of 17 ( very hard ) with some chest pain .
three patients at 85%
hrr during ce complained of rpe of 15 ( hard ) with some chest pain . at 85% hrr during ce ,
one
patient complained of rpe of 17 with intermittent pvcs ( premature ventricular contractions ) ,
one patient complained of rpe of 17 with bigeminy pvc , one patient complained of rpe of 17
with atrial fibrillation , and one patient showed st segment depression ( 1 mm , horizontal ) .
chest pain or arrhythmia occurred between 8 to 12 minutes after the initiation of the first
exercise session .
twenty - one patients complained of rpe of 17 without an event at the 85%
hrr during ce .
all the relative hemodynamic values of the two exercise types at 85%hrr
showed significant differences ( p<0.01 ) .
cardiac events were not observed at 70% hrr
during tm , but 4 patients terminated the exercise sessions during ce .
rpe : rate of perceived exertion , pvc :
premature ventricle contraction , af : atrial fibrillation , st segment depression : 1 mm
( horizontal ) , * = significantly different , p < 0.01
the hemodynamic responses , rpe , and cardiac events during two common exercise types
conducted at 60% and 85% hrr were assessed in patients with acs .
although early
rehabilitation exercise is helpful for improving health status , the exercise mode should be
carefully monitored .
patient - specific exercise intensity has been traditionally obtained through a treadmill
exercise graded test for both treadmill and cycle ergometer exercises .
rpp was significantly higher during ce than during tm at intensities of 60% hrr and 85% hrr
due to higher blood pressure .
rpp measured during 60% hrr - ce was close to 90% of the maximal
rpp , and rpp during 85% hrr - ce exceeded the maximal rpp ( 102.212.0% ) .
various cardiac
events were induced when rpp exceeded the maximal rpp during 85% hrr - ce .
there have been
many studies of the physiological characteristics of the exercise stress test done on a
treadmill and cycle ergometer .
the major differences in characteristics of ce , compared to
tm , have been reported to be increased maximal heart rate and vo2max9,10,11,12,13,14 .
moreover , higher blood pressures were also induced during ce than during tm6 , 16 .
because of these physiological differences , application of exercise intensity based on the
maximal heart rates obtained from either exercise type may expose cardiac patients to risk
of various clinical problems . in this study ,
both rpp and rpe were significantly higher during ce than during tm at
60%hrr and 85%hrr due to elevation of blood pressure .
in addition , rpp during 60% hrr - ce was
close to 90% of the maximal rpp , and rpp during 85%hrr - ce exceeded the maximal rpp .
this
significant increase in rpp was also caused by elevated blood pressure during ce .
although
ce is mostly aerobic exercise , it is a combined form of exercise with some anaerobic factors
in it19 .
the sbp of professional cyclists may increase to 200 mmhg during a maximal ce stress
test19 , and their heart rates can be
maintained close to the maximal hr for the duration of a race24 .
the average bp of endurance runners has been reported to be around
175/69 mmhg during exercise25 , while the
average heart rate of resistance exercise athletes has been reported to be between 102 and
170 bpm , with maximal bp of 480/350 mmhg26 .
it was reported that the athletes specializing in cycling or
rowing not only have bigger internal cardiac chambers , but have thicker myocardial walls due
to the differences in hemodynamic responses compared to other athletes19 .
moreover , endurance runners generally have larger
internal cardiac diameters , and resistance exercise athletes have thicker cardiac chamber
walls .
generally , ce requires active performance in resistive pedaling with a greater range of
motion and higher angle , while tm is performed under a condition which is relatively less
active , and has a fixed grade and speed . for these reasons ,
increase in blood pressure
during ce may be caused by greater resistance to blood flow in the exercising muscles27 , and larger tension within the lower limb
muscles due to the increase in exercise intensity28 .
reed reported that both the systolic and diastolic phases of females with sedentary
lifestyles were higher during ce than during tm at exercise intensities of 40% hrr and 60%
hrr17 .
these results indicate that rpp
can be increase more during ce than during tm at identical exercise intensities .
rpp can be easily measured by multiplying the mean heart rate by the systolic blood
pressure obtained during an exercise stress test29 ,
30 .
rpp is a reliable indicator of
myocardial perfusion demand not only for patients with coronary arterial diseases , but also
for healthy individuals18 .
many patients
with chronic stable angina tend to experience chest pain at a certain rpp18 , 31 .
furthermore , an exercise which
increases rpp in patients with aortic aneurysm may lead to aneurysm rupture33 .
the beta blockers used to decrease the
ischemic threshold , or prevent arrhythmia or aortic aneurysm rupture are also used to reduce
rpp32 , 33 . in this study ,
the 102 subjects who completed the cardiac rehabilitation exercise program
on the cycle ergometer exceeded maximal rpp during 85% hrr - ce . in terms of cardiac events ,
ce induced 8 cases of cardiac events which included light chest pain ( 4 cases ) , arrhythmia
( 3 cases ) compared to just one case of chest pain during tm .
twenty - one patients complained
of strenuous exertion with pre 17 or over during cycle ergometer exercise at 85%hrr ( table 4 ) .
a number of patients complained during performance of ce and terminated
exercise ( 70% hrr exercise was excluded ) .
although cardiac events were not observed , 16 out
of 118 patients who were excluded from the study groups complained of leg pain or
interrupted exercise sessions due to excessive fatigue during ce11 , 15 .
therefore ,
additional recommendations for cardiac rehabilitation guidelines may be needed , explaining
the differences in exercise intensity between tm and ce along with selective guidelines for
patients with cardiovascular diseases , aneurysm , or exercise - induced high blood
pressure .
first , it was difficult to reach target heart rates
within the given time of 12 minutes by control of the exercise intensity .
the pedaling speed
as well as the power ( watts ) was adjusted for each patient in consideration of his / her
physical condition .
in addition , it was difficult to balance or adjust the angle of incline
as well as the speed on the treadmill during tm .
differences in adjustments may have
resulted in minor differences in exercise intensities among patients . | [ purpose ] this study investigated the effects of mid , and high exercise intensities on
hemodynamic responses and cardiac events during two exercise types of treadmill exercise
( tm ) and cycle ergometer exercises ( ce ) in patients with acute coronary syndrome ( acs ) .
[ subjects ] patients who had percutaneous coronary intervention ( pci ) for acs and were
participating in cardiac rehabilitation program were included .
[ methods ] the patients were
assessed for hemodynamic responses , cardiac events , and rate of perceived exertion ( rpe )
with target heart rates of 60% and 85% heart rate reserve ( hrr ) during tm and ce .
[ results ] maximum systolic blood pressure ( sbp ) , diastolic blood pressure ( dbp ) , rpe , and
rate pressure product ( rpp ) measured during ce were significantly higher than their values
in tm at the same exercise intensities . the highest sbp was shown at 85% hrr during ce .
sbpmax to sbpmax ratios obtained during the graded exercise test
( gxt ) showed that all % sbpmax were significantly greater in ce than in tm at
the same exercise intensities .
out of 102 patients , cardiac events occurred in 8 at 85%
hrr during ce , and 1 at 85% hrr during tm .
patients with cardiac events ( ce - e ) had
significantly higher % sbp , % rpp , and rpe at 85% hrr than those without events ( ce - ne )
during ce .
[ conclusion ] prescribing exercise based on the intensity obtained in a
treadmill gxt may expose patients to cardiovascular complications such as higher rpp ,
higher exercise intensity , and cardiac events during ce . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC5170853 | i thought long and hard whether i could avoid talking about family and personal life and just share the excitement of being a scientist and how science continues to sustain us all .
but so many people , especially younger scientists , want to know , and always ask , how did you do it ?
a woman from iran , a middle eastern country and essentially muslim , now considered backward and misguided , if not downright scary , traveling very young and alone to the united states , finishing college and graduate school together with having children in your first year of graduate school and during your second postdoc year , going against a number of entrenched dogmas , and yet succeeding against many odds and obstacles , and all the while on soft money ? first , a few facts : iran used to be called persia , which confuses many in the united states , who also do nt know that iranians are not arabs and before the seventh century were zoroastrians
one of the first monotheistic religions before christ and the name has been iran for thousands of years it means the land of aryans .
it was alexander the great who brought his army to the capital of iran in the province of pars ( hence persia ) and burned the persepolis , the palace of the kings of iran .
photo credit : shoey sindel ( [email protected] ) , berkeley , ca , 2016 mina bissell ( wearing hat ) , her husband monty , and their children and grandchildren .
so , when people ask , how ? ! , the answer i give is : badly !
almost no one does it perfectly or even well , but the trick is to choose what you love to do and persist ! i persisted .
but i believe it is precisely because of my background in iran that i could persist in the era of mad men in the 1960s and 1970s , even in the face of some amazingly bad behavior from a number of men in charge .
my daughter and son are well educated , are spreading good in ways other than typical academia , and have happy families .
my father was the first of 10 children , a lawyer with a phd from france .
three of my five uncles were judges , prosecutors , and attorneys , and one was a medical professor who spoke seven languages fluently and was also an accomplished poet ! almost 100 years ago , all my aunts were college educated , and one had a phd from the sorbonne in french literature .
one was a principal of a school , and three were not professionals , but all were socially and politically involved , and two of their daughters were still playing in repertory theater despite being married .
i never saw one of them with even a scarf over her head , let alone what is referred to as a
hijab , despite the fact my grandfather was a highly respected ayatollah ( not a mullah ) and a descendant of at least five generations of ayatollahs !
first sons were supposed to go to divinity school , become well educated in all forms of religion , and follow in their fathers footsteps .
but my father not only had zero intention of joining the clergy , he had no use for religion .
yet he and my grandfather were great friends ; they looked upon one another as equals .
my grandfather was a scholar , like many in those days , an amazingly educated and thoughtful man .
he despised intolerance , and one of his closest friends and advisers was a well - educated iranian , mr .
zoroastrians , muslims , jews , christians , armenians , and many other religions were well integrated in iranian society and had been since the times of cyrus the great , and the shahabi family remained very close friends of our family and helped my mother for a year when my father became very ill with typhoid fever , in those days a deadly disease .
my grandfather was one of the reasons i ended up in united states rather than england , which was where my father wanted me to go for my education .
my father believed all religions are sources of much exploitation , wars , and misery .
he held either we were reasoning humans , understood our responsibilities to our fellow humans , including our families and ourselves , or we were nt !
( as did his father , and now best exemplified by the magnificent current pope ) , most did more harm than good .
he would debate my sister and me across the lunch table and would tell us we could join any profession we wanted as long as we could maintain our integrity .
he was a fantastic orator and was known to defend people from all levels of society if they were victims of injustice or victims of the regime in charge at that moment !
this meant helping both the shah s enemies and the shah s supporters , even if they were on opposite sides !
in retrospect , my father was one of my early heroes and the reason i have lived my life the way i have .
one of my mentoring points to mothers of sons and young men : if fathers believe in their daughters and have high expectations for both sexes , many of their daughters will succeed in having productive and satisfying lives . on the maternal side , there was less interest in scholarly pursuits and no interest in politics , but one aunt was a u.s .- educated medical doctor and the chair of immunology at tehran university , and the other was in public health ; one uncle was in government , and the other was a professor of mathematics in the united states .
the only member of this extended family who had not finished college was my mother .
her father was assassinated at the age of 27 in the ghajar dynasty court , and despite being loved by all , including her two daughters , she felt inadequate and wanting in holding her ground intellectually , and at times was deeply unhappy .
she found it degrading to be financially dependent on my father but was too proud to ask her mother for money .
she drilled my sister and me to pursue higher education and become independent no matter what .
i think , even without reading virginia woolf s a room of one s own ( 1921 ) ( a book to read by both sexes , even though it was written in the 1920s ) , she had arrived at the same conclusion : for a woman to succeed , she must have a room of her own and 500 pounds a year
hence , one of my other key mentoring refrains is : there is huge dignity in work and earning your own living .
i was born with what could be called a photographic memory for written and spoken words , a trait that surprised some grown - ups , who kept testing me for their amusement .
this made me uncomfortable and separated me from my many cousins and friends in school .
however , i lost quite a bit of this gift at age 8 , being a bit of a tomboy , climbing trees and engaging in sports and other unseemly behavior for girls in those days .
while jumping over a high bar set between two columns with nothing but hard ground underneath ( a contrivance of one of my boy cousins who was 15 years old ) , i fell hard and was bedridden for months but survived and recovered against all odds .
the result was essentially complete loss of hearing in my left ear and loss of most of my photographic memory . despite the injury , i felt better integrated after recovery !
persians have a saying that i often still repeat : enemy can bring good , if goodness is willing ! my mother , who i adored , never seemed to be interested in books and politics but understood quite early that i would be able to amount to something when i grew up ; she also understood how easily i would get hurt and how easily i cried when there was any kind of perceived injustice or unkindness , and she felt i needed some protection .
( i used to be teased mercilessly by my sister , who i looked up to and loved and who is still my best friend , and cousins alike , who would make me cry by making fun of my crying ! )
i liked nothing more than hiding and reading books , and she tried to help me become more integrated socially , including sending me to a ballet class ( although i was a bit too old at 10 ) .
i loved it even if i never could do the split no matter how hard i tried ! nevertheless , it actually allowed me to become more at ease , and made me more open , socially and otherwise .
when talking about the significance of form and function in my studies , i often show slides of dancers and quote yeats s among school children , as i did in a ted talk : how can you tell the dancer from the dance ? if you decide to have kids , try harder with them if they are loners , and do nt be a loner yourself ; there is wisdom in group activities , in scientific collaboration and team sports : we are born to be social creatures , and almost all of us have inherited the capacity for empathy from our ancestors and fellow creatures ( de waal , 2013 ) .
i loved school ; it was effortless , and i was a top student in my primary and secondary schools for 12 years . in the last year , in the countrywide exams , i became the top student in the country .
there are scores of children all over the world who could and will do many things better than i did in all areas if they have the opportunity .
i received a medal and award from the shah of iran ( there are a couple of pictures of me with the shah , with me getting the medal and a book ) , and my fellowship for going abroad was not from a college in the united states but was earned by taking an exam for a special award given to five students in science , technology , engineering , and mathematics ( stem ) fields in that year in iran .
my father felt the united states was too young a country to educate women , but i wanted to go to the united states , and as i said earlier , my grandfather the ayatollah haeri mazandarani explained to my father , she has earned it , she is good , and she deserves to go wherever she wants ! my father consented .
i applied to a few universities in the united states by sending applications to the american friends of the middle east ( afme)a branch of the american government where we had to send my application for a u.s .
when the head of afme saw my application , he called my father ( who spoke reasonable english and , of course , french ) and asked for me to come see him .
he suggested i should go to bryn mawr , one of the seven sister colleges in the united states ( analogous to the seven ivy league schools , which then accepted only male students ) , since to his mind and later to my mind
i knew nothing about the place , and luckily i was never asked to take the college entrance exam ! at this point , my english was quite elementary , but they let me in ! after a short stay in new york city to take a perfunctory english course , arranged with the help of my uncle , the math professor , i spent an enchanting two years at bryn mawr .
but i did not enjoy english and arabic at all in high school , partly because , apparently along with the hearing loss in my left ear , i had also damaged the nomic gland behind the left frontal lobe and had difficulty even then remembering names , places , or anything to do with the left side of the brain that i had to memorize .
but i liked literature in iran , and fell in love with english literature , because i love well - written words .
to this day , she is my favorite teacher and at age 93 is one of my most beloved friends .
i debated between majoring in english literature or chemistry , and the latter won out , mainly for practical reasons .
i will always remain grateful to the head of the afme , the gentleman who cared enough and took the time to meet up with my father and me , and showed me the way .
i never learned his name , since he died shortly after i had left for the united states , and i also found out much later from american newspapers that the afme was a front for the cia and behind a coup that toppled mossadegh , then the most popular , beloved , and democratically elected prime minister iran had ever had .
for the young : politics matter in a democratic society . get involved if you want to keep democracy alive please vote ! if you want to understand the times we live in now , please read and understand the history of different regions more deeply , particularly that of the middle east . in my sophomore year in college , in short order , i met a graduate student from harvard university who was iranian and was getting a phd in political science and economics ; he proposed and i said yes .
i declared chemistry as my major , moved as a junior to radcliffe / harvard university , won the medal from the american institute of chemists as a junior , married in my senior year , and ended up being a vanishing
cliffie , since i received my bachelor s degree in chemistry as one of the inaugural members of the integrated harvard class along with people like david botstein .
my husband was still a graduate student , so i opted to stay in the area to get a phd in bacterial genetics from harvard medical school .
this was eons ago , and at that time , there were 200 men and three women in the harvard medical school class that year , and the entire place had one woman faculty member . in the entering class in bacteriology , there were three men and three women ( the latter all cliffies ! ) . despite this early balance , after the first year
, all three men left they found hms to be too ingrown , too arrogant toward outsiders , and too wanting in humanity .
i had chosen to do my thesis with a progressive italian professor , luigi gorini , or rather , he chose me after i answered a difficult physics question in a high - resolution microscopy class !
he was already almost 70 years old but very active and a real wiz in bacterial genetics .
but he still felt i should quit , because i was pregnantwhat would your mother say ? my mother ( and father ) called quickly from iran to make sure i would not be quitting
so much for the iranian parents who more than 50 years ago were more understanding than most western ones .
when my daughter was two - and - a - half years old , i left my husband a nice man , but somewhat incompatible with me as a life partner .
it was a hard decision with a baby , but i felt i could raise her better by myself .
i met my future husband , monty bissell , arguing over a centrifuge he was a medical student who was doing a year of research in the bacteriology department and learning to play the cello , not necessarily in that order !
we married a year later and will be celebrating our 50th anniversary this coming july .
i think he liked me because i was the size of his cello and talked back ! and
i liked him because he was an english major in college , wrote beautifully written phrases , and towered over me ! deep down , he was also a poet and romantic .
more so now than early on ; we had some aspect of our science in common but had entirely different styles in running labs .
monty became the head of the liver center at university of california san francisco ( ucsf ) and later the chief of the gi department and was admired not only for his science but also because he was successful in hiring terrific faculty .
i was so proud that quite a few were outstanding women and that almost all his fellows loved him as a mentor .
of course it is not easy to live with a guy who is a perfectionist in everything he does ( he even has perfect pitch ! ) , but making any marriage work is like science ; it takes patience and perseverance , originality and empathy , and the willingness to talk frankly with each other .
but when it lasts by choice , it can be worth the effort , sometimes it even may feel like drinking a good aged wine . but get out fast if the other side is not capable or worth hard work and often makes your life miserable
. alone does not mean lonely , and it often is the preferred course another reason for having your own career .
based on the existing literature , a model is presented that postulates a dynamic reciprocity between the extracellular matrix ( ecm ) on the one hand and the cytoskeleton and the nuclear matrix on the other hand . the ecm is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with the mrna and the interaction of the chromatin with the nuclear matrix .
this , in turn , would affect the ecm , which would affect the cell , which
journal of theoretical biology , 1982 based on the existing literature , a model is presented that postulates a dynamic reciprocity between the extracellular matrix ( ecm ) on the one hand and the cytoskeleton and the nuclear matrix on the other hand .
the ecm is postulated to exert physical and chemical influences on the geometry and the biochemistry of the cell via transmembrane receptors so as to alter the pattern of gene expression by changing the association of the cytoskeleton with the mrna and the interaction of the chromatin with the nuclear matrix .
this , in turn , would affect the ecm , which would affect the cell , which
, journal of theoretical biology , 1982 when i wrote the above article , even my close colleagues who were my friends asked what i was smoking !
i had no intention of just postulating ; my students and fellows ( and often many undergraduates ) and i worked long and hard for more than three decades , essentially on one organ , the mammary gland , to unravel how the extracellular matrix ( ecm ) may talk to the nucleus and vice versa .
the majority of the critics have come around , and the number of papers in the area of tumor microenvironment ( tme ) has increased logarithmically .
the model i proposed not the name , but the concept of ecm regulating gene expression at many levels and the roles of basement membrane and tissue architecture is here to stay .
dozens of our own papers have shown the significance and mechanism of interaction between the ecm and cytoskeleton and the nuclear matrix and chromatin .
we showed in the early 1990s that the promoters of milk protein genes contained ecm - response elements and that loss of interaction of laminin 111 with integrins leads to loss of milk function and induction of apoptosis . in the presence of laminin - rich gels
( a 20% pure laminin 111 and rat - tail collagen gel can substitute for matrigel ) and certain oncogenic pathways , inhibitors can revert the malignant cells to a normal phenotype , despite the tumorigenic genome .
together with a number of outstanding collaborators , including zena werb ( ucsf ) , ole william petersen ( copenhagen ) , cathy park ( ucsf ) , and more recently david lyden ( weill cornell medical college ) , we have explored many aspects of mammary gland morphogenesis , function , and homeostasis and breast cancer and exosomes , and have dared challenge other paradigms .
there are , however , more than 30 interviews and write - ups listed on my website ( www2.lbl.gov/lbl-programs/lifesciences/bisselllab/main.html ) , a few of which i note in the reference list ( vaughan , 2002 ; abbott , 2003 ; flintoft , 2003 ; friedrich , 2003 ; graebner , 2003 ; bissell and devine , 2005 ; bonetta , 2005 ; novak , 2005 ; pon , 2005 ; cohen , 2006 ; klein , 2006 ; mason , 2006 ; schuldt , 2006 ; arry , 2007 ; blow , 2007 ; mervis , 2007 ; platoni , 2007 , 2008 ; shekhar , 2007 ; wong , 2007 ; beishon , 2008 ; marx , 2008 ; fleischman , 2009 ; kolata , 2009 ; lako and daher , 2009 ; short , 2009 ; yarris , 2009 ; hayden , 2010 ; zagorski , 2010 ; claiborn , 2011 ; bissell , 2015 ) .
there are also scores of write - ups on our publications and press releases in the lawrence berkeley national laboratory archives ( today at berkeley lab , https://today.lbl.gov ) , and then there are more than 400 publications from our laboratory ( also on the website ) .
do you have any regrets ? but of course ! even though i look at the bright side more often , and i am blessed with much , it takes extreme indifference and arrogance not to have regrets .
so i would like to leave you with a sentiment that has resonated with me deeply .
it is in a tiny , tiny book , may be even still on the web .
it is the text of a convocation address to a graduating class at syracuse university given by george saunders .
it brought tears to my eyes , and i envied him for putting it so well .
here s something i know to be true , although it s a little corny , and i do nt know what to do with it : what i regret most in my life are failures of kindness | i thought long and hard whether i could avoid talking about family and personal life , and just share the excitement of being a scientist and how science continues to sustain us all .
but so many people , especially younger scientists , want to know and always ask how did you do it ?
a woman from iran , a middle eastern country and essentially muslim , now considered backwards and misguided if not downright scary , traveling very young and alone to the united states , finishing college and graduate school together with having children , first - year graduate school and second - year post doc
years ago , going against a number of entrenched dogmas , and yet succeeding against many odds and obstacles , and all the while on soft money ?
below is my personal narrative answering some of these questions . | WHAT ARE THE QUESTIONS TO ANSWER AND WHY?
NOW FOR SOMETHING COMPLETELY DIFFERENT: FAMILY BACKGROUND!
COULD THERE BE ONE OR MORE LESSONS HERE FOR ALL OF US, ESPECIALLY OUR POLITICIANS?!
CHILDHOOD
SCHOOL YEARS AND MAKING A FAMILY
ADVENTURES IN VIROLOGY AND CELL AND CANCER BIOLOGY: DISCOVERING THAT HALF THE SECRET OF THE CELL IS OUTSIDE THE CELL! |
PMC4714299 | in most winters , influenza causes substantial morbidity and mortality [ 1 , 2 ] , with
widely variable clinical manifestations ranging from subclinical infection to fatal illness
.
although many persons become infected ,
seroprevalence studies indicate that only a minority make use of health services . nevertheless , those that do create considerable
pressure on both primary- and secondary - care services [ 5 , 6 ] . the risk of serious illness involving
hospital admission or death is thought to be higher in persons with chronic illness , but the total impact of influenza on health services
comes at least as much from otherwise healthy persons . in the uk
this impact is concentrated in a comparatively short epidemic period
of around 8 weeks , creating surge pressures on
health services , commonly in midwinter , when the impact of other respiratory viral
infections is also highest .
policies to prevent and treat influenza need to accommodate the varying threat from
different virus strain types , changes in severity of epidemics over time , advances in
vaccines , the ageing population , increasing population expectancies from healthcare and
changes in healthcare delivery .
these factors interact to influence the cost effectiveness
of medical intervention , and create the need to update burden assessments on a continuing
basis using measurements from several seasons .
most estimates of the influenza burden employ modelling methods in which the identification
of the influenza epidemic period is determined from available virology data and the impact
measured in the commensurate changes in the chosen outcome [ 1115 ] . in some modelling
studies , influenza virus data have been used exclusively , but the confounding effect of
other viruses has prompted the inclusion of data for other viruses , especially respiratory
syncytial virus ( rsv ) . in a few studies ,
this study aims to provide a comprehensive analysis of the burden of persons
consulting a general practitioner ( gp ) for a cause attributable to influenza over many
seasons , to inform cost - benefit analyses of vaccination and treatment interventions .
we used
consultation data from the clinical practice research datalink ( cprd ) and virology data from
public health england ( phe ) in a linear regression model to estimate influenza - attributable
consultations and antibiotic prescriptions over 14 seasons , while controlling for rsv and
other seasonal variables , and stratifying the analyses by age , comorbidity risk and
influenza vaccination status .
viral surveillance time - series regression models were used to estimate the number of
persons consulting a gp for influenza - attributable respiratory disease in england in each
season ( july in the index year through june the following year ) , from 1995 to 2009 ,
stratifying by age , risk status and vaccination status ( www.clinicaltrials.gov : nct01520935 ) .
seasons after july 2009 were not included as
we were concerned to establish the normal or average seasonal burden of influenza , which
was disturbed by the pandemic experience in summer 2009 . at the midpoint of the study ( january 2001 ) the monitored population was around 37
million .
influenza reports are based on
regular returns of virus swab - positive tests to phe , mainly from community - based
investigation of persons with influenza - like illness .
rsv reports are mainly confirmed
infections in young children admitted to hospital with respiratory infections .
all analyses of cprd were restricted to english practices to match the coverage of
virology data .
a gp episode was counted as the first in a series of consultations for a particular
diagnosis / diagnostic group .
a minimum of 28 days was required following any previous
consultation for that same diagnosis / diagnostic group before defining a new episode .
diagnostic read codes were combined into respiratory diagnoses consistent with recognized
icd-10 groupings ( table 1 ) ( see supplementary
material ) .
we undertook a preliminary search of records to find codes that increased
during influenza active periods .
our respiratory disease broadly defined outcome
included all disorders in the icd-10 respiratory disease chapter , otitis media , as well as
selected presenting symptom codes we found were used by some gps in lieu of specifying a
clinical diagnosis .
a negative control outcome ( urinary tract infection ) with no seasonal
pattern and association with influenza was included to control for the possibility of bias
in model attribution over the entire study period .
table 1.outcomes : general practitioner consultations ( cprd)*outcomeicd-10 codes
respiratory diseases
respiratory disease broadly
defined : resp dis + symptoms + sepsisj0099 , r0506 , b34 , a4041 , p36 respiratory diseasej0099 acute upper respiratory diseasej00 , j0206 pneumonia and influenzaj0918 bronchitis / bronchiolitisj2022 , j40 influenza - like illnessj0911 , b34
non - respiratory diseases
otitis mediah6566 , h70 urinary tract infectionn39
drug prescriptions
antibiotics ( broad spectrum penicillins ,
macrolides , tetracyclines )
risk factor definitions
chronic heart disease ; renal ; diabetes
( diagnosis / monitoring ) ; liver ; stroke / tia ; ms - cns ; copd_no_asthma ; asthma ;
immuno ; immuno_suppressant treatment ; anti - asthmatic treatment*any clinical practice research datalink ( cprd ) gp episode , including office
visits , home visits , telephone consults and other types , for subjects registered
with research quality data in cprd .
cprd diagnostic data are coded using read
codes selected to correspond to icd-10 definitions .
outcomes : general practitioner consultations ( cprd ) * any clinical practice research datalink ( cprd ) gp episode , including office
visits , home visits , telephone consults and other types , for subjects registered
with research quality data in cprd .
cprd diagnostic data are coded using read
codes selected to correspond to icd-10 definitions .
time - series of numbers of prescriptions of broad spectrum penicillin , tetracycline and
macrolide antibiotics , which are widely prescribed for acute respiratory infections , were
also examined as an outcome category .
the cprd dataset contains unique patient identifiers , allowing patients to be followed
longitudinally .
since almost all influenza vaccination during the study period was
undertaken by gps and recorded using specific codes , we were able to check the vaccination
status for each person in each season .
vaccine coverage in the cprd population was used to
estimate the vaccination coverage in each age - risk stratum .
we also externally validated
the cprd vaccination coverage estimates against national data on vaccination coverage
.
we classified each patient as high or
low risk by the prior occurrence of diagnoses that would prioritize the patient to
receive influenza vaccination by reference to the accumulated historical records in the
database ( table 1 ) .
the cprd covers a population which is representative of england in age and gender
distribution and is thus a suitable population - denominated database for the assessment of
vaccination status and national patterns of comorbidity in age- and gender - specific groups
.
we found excellent agreement between cprd
and independent data sources for the uk with respect to the population age structure , the
prevalence of various risk factors , and influenza vaccination coverage ; for example , cprd
data yielded an average vaccination rate of 71% for persons aged 65 years for the years
20012008 , in close agreement with phe estimates .
the 2001 uk population ( office of national statistics ) was thus used to weight the english cprd population by age to
reflect the uk profile .
weekly
time - series of the number of specimens positive for influenza a , influenza b and rsv were
generated using phe virology surveillance data .
weekly time - series for multiple
influenza - related health outcomes ( table 1 ) were
generated from the cprd for age groups < 5 , 517 , 1849 , 5064 , and 65 years
( for
selected analyses age groups 6574 and 75 were examined separately ) and low-/high - risk
groups .
a multiple linear regression model was applied to each age group to associate gp
episodes to influenza a or b , while controlling for rsv and unspecified seasonal factors
( fig .
major changes in virus detection
methodology were undertaken by phe in 2001 ; thus , to avoid bias the model separated the
periods before and after this date : where , y is the incidence ( rate ) of outcome definition
for each time period t ( weeks ) , rsv and influenza are the proportion of
laboratory isolates during t , sin and cos are harmonic functions of
t , and the remaining terms track other types of secular trends in the
data .
the time period t used in the model is a running index of weeks
starting 1 october 1997 and ending 31 march 2009 .
1.attribution modelling showing excess gp episodes attributable to influenza a and b
and respiratory syncytial virus ( rsv ) in children aged 517 years .
attribution modelling showing excess gp episodes attributable to influenza a and b
and respiratory syncytial virus ( rsv ) in children aged 517 years .
p1ainfluenza a(pre - july
2001 ) + p1binfluenza
a(post - july 2001 ) are influenza terms pre- and post - july , 2001 . the rsv terms control for
rsv epidemics . influenza a , influenza b and rsv are observed counts of positive tests from
the phe labbase dataset .
outcome and pathogen series were tri - mean - smoothed , i.e. the smoothed value of
x at week t is
( xt1 + xt + xt+1)/3 .
the week numbers ( t ) were calculated
as iso 8601 v - weeks . the weekly attribution to each virus
was computed as the product of
the observed value of the explanatory variable ( i.e. number of positive virology samples )
and the corresponding regression coefficient
( p1ap2b ) ,
and the weekly estimates summed to produce the seasonal estimates .
the variability of
seasonal estimates was assessed using standard deviations but limited to all - age
estimates .
this standard deviation represents the variability of the attributable burden
between seasons and not the uncertainty of the individual seasonal estimates .
results were
expressed as numbers of persons consulting a gp ( gp episodes ) and as the mean seasonal
( from september to mid - may ) rates of persons consulting a gp per 100 000 population .
outside pandemics , influenza is exclusively a winter illness and thus summertime data
were excluded from the analysis .
previous evidence suggests that winter increases in the
incidence of rsv - associated outcomes ( e.g. acute bronchitis ) in adults follow those in
children by 23 weeks .
therefore , for age
groups aged 18 years , we lagged the rsv time - series by 2 weeks and achieved an improved
model fit ; we did not lag the influenza time - series as doing so did not substantially
alter model fit or influenza burden attributions .
the time - series of prescriptions of
antibiotics was modelled in the same way as the health outcome time - series to estimate
antibiotic prescribing attributable to influenza .
the model did not attribute any positive
influenza burden to the control outcome ( urinary tract infection ) .
the protocol was approved by the independent scientific advisory committee of the
clinical practice research datalink ( cprd , formerly the general practice research
database ) .
the onset of the influenza epidemic period usually followed rsv , and influenza a usually
preceded influenza b ( illustrated for the 517 years age group in fig .
the goodness - of - fit was assessed using adjusted r. the model
fit in most strata was very good , with adjusted r between
075 and 086 for the respiratory broad outcome ( supplementary table s1 ) .
moreover , we
found a substantial lift in the adjusted r value when
introducing the virology terms into the base secular model .
we did not adjust the model
form ( by including or excluding terms ) individually for each outcome , age , and risk
stratum , but applied the given form across all strata . in a mean season there were an estimated 857 996 gp episodes in persons of all ages ( 15%
of the population ) for influenza - attributable respiratory disease ( broadly defined ) , of
which around 75% ( 649 219 ) were for influenza a ( table
2 ) .
there was wide inter - seasonal variability in influenza a- and b - attributable
gp episodes , compared to a more stable consulting rate for rsv ( fig .
2 ) . in the seasons studied the number of gp episodes for an
influenza - attributable respiratory disease ranged between 174 305 ( 20022003 ) and
1 340 937 ( 19951996 ) for influenza a , and between 2075 ( 19971998 ) and 646 457
( 19961997 ) for influenza b. the highest rates of gp episodes for influenza a - attributable
respiratory disease were observed in children aged < 5 years ( table 2 ) .
the highest seasonal incidence rates for influenza b were
observed in those aged 517 years .
2.seasonal rates ( with 95% confidence intervals ) of gp episodes for respiratory
disease ( broadly defined ) attributable to influenza a , influenza b and respiratory
syncytial virus ( rsv ) .
table 2.persons consulting a general practitioner in a mean season ( number and rate/100 000
population ) attributable to influenza in the uk , by age ( 19952009)influenza ainfluenza boutcomeage , yrn ( s.d.)rate ( s.d.)n ( s.d.)rate ( s.d.)respiratory diseasebroadly
defined0484 806243618 781539517120 151121798 3809961849234 47689975 1602885064110 768107618 38617965102 5131140681076all ages649 219 ( 366 681)1105 ( 624)208 777 ( 218 110)355 ( 371)bronchitis andbronchiolitis0410 0272880051717 07817310 058102184964 17924618 77472506450 6404926603646563 621707373542all ages210 607 ( 137 270)359 ( 234)38 120 ( 46 417)65 ( 79)influenza - like illness0421 955631426012251733 15333619 103193184991 20235025 95499506438 0453696100596525 589284211023all ages209 154 ( 146 553)356 ( 250)55 969 ( 61 902)95 ( 105)pneumonia andinfluenza04892325612473651719 991202897891184973 27328120 18177506432 3943154521446525 722286215824all ages161 228 ( 123 129)274 ( 210)36 541 ( 43 993)62 ( 75)otitis media0418 202523658918951719 09319315 0411521849881634292411506487891052106515823153all ages44 534 ( 25 065)76 ( 43)24 243 ( 28 284)41 ( 48)antibioticprescriptions0449 759142912 13034851769 00869959 4276021849159 65061254 858210506493 80691115 2861486588 172980554762all ages461 839 ( 262 785)786 ( 447)142 203 ( 162 168)242 ( 276)n , number of episodes ;
seasonal rates ( with 95% confidence intervals ) of gp episodes for respiratory
disease ( broadly defined ) attributable to influenza a , influenza b and respiratory
syncytial virus ( rsv ) .
persons consulting a general practitioner in a mean season ( number and rate/100 000
population ) attributable to influenza in the uk , by age ( 19952009 ) n , number of episodes ; s.d . , standard deviation .
the rate of gp episodes for influenza a - attributable respiratory disease tended to
increase after age 50 years , whereas gp episodes for influenza b - attributable respiratory
disease decreased with age ( table 2 ) . in the 75
years age group , the rate of gp episodes for respiratory disease was 1255/100 000 for
influenza a and 93/100 000 for influenza b. in a mean season , the rate of gp episodes for influenza a - attributable bronchitis was
higher in adults than in children , whereas the rate of gp episodes for influenza - like
illness was higher in children than adults ( table
2 ) .
the rates of influenza a - attributable gp episodes for pneumonia and influenza
were higher in adults than in children . in those aged
75 years , the mean seasonal rate of
gp episodes for pneumonia and influenza was 309/100 000 for influenza a , and 24/100 000
for influenza b. influenza a and b contributed to 68 777 gp episodes for otitis media per season , with the
highest number in children ( table 2 ) . around one
third of the gp episodes for otitis media were attributed to influenza b. there were 604 042 prescriptions for antibiotics attributed to influenza - associated
respiratory disease ( as measured in the broad respiratory outcome ) per season across all
age groups ( table 2 ) .
individuals with comorbid conditions that put them at high risk of severe influenza
infection were more likely to consult a gp for an influenza - attributable respiratory
disease than individuals at low risk ( table 3 ) .
the exceptions were children aged < 5 years for influenza a , and adults aged 5064
years for influenza b. the average seasonal rates of influenza a - attributable gp episodes
in the unvaccinated high comorbid risk group were 179- , 156- and 183-fold higher than
in the low comorbid risk group for older adults aged 5064 , 6574 and 75 years ,
respectively .
trends for influenza b were similar for the 6574 and 75 years age groups
( 174- and 297-fold higher rates in high comorbid risk group , respectively ) .
table 3.persons consulting a general practitioner in a mean season ( rate/100 000
population ) for outcomes attributed to influenza a and b by comorbid risk statusinfluenza ainfluenza boutcomeage , yrlow risk ( s.d.)high risk ( s.d.)low risk ( s.d.)high risk ( s.d.)respiratory broadly defined042482171352874551711661562953131518498311454286309506494815341921416588914396798all ages1024 ( 569)1464 ( 920)374 ( 390)289 ( 299)bronchitis and bronchiolitis04279417095517148335911811849212523699850644117786565655169343160all ages275 ( 179)718 ( 496)62 ( 74)82 ( 103)influenza - like illness0461582812114951731646518625218493305159712150643394826055652423372126all ages339 ( 231)435 ( 341)99 ( 109)81 ( 91)pneumonia and influenza042483623544517189295881101849264425759750642854254541652263621832all ages251 ( 187)383 ( 316)63 ( 76)58 ( 72)s.d . , standard deviation . persons consulting a general practitioner in a mean season ( rate/100 000
population ) for outcomes attributed to influenza a and b by comorbid risk status s.d . , standard deviation .
higher rates of gp episodes were observed in all age groups when comorbid risk factors
were present than when they were absent for the other outcomes studied . between 2000 and 2008
influenza vaccine coverage in the cprd population aged 65 years
ranged between 64% and 76% , which was similar to coverage estimated by phe . in each season studied the rate of influenza
a - attributable gp episodes in the high comorbid risk group aged 1849 years was 06- to
089-fold lower in vaccinated compared to unvaccinated individuals .
the rate ratio of
influenza a - attributable gp episodes in the high comorbid risk group in vaccinated over
unvaccinated individuals was between 074 and 076 in those aged 5064 years , and
081085 in those aged 65 years .
similarly , in these age groups the rate of gp episodes was lower in vaccinated
vs. unvaccinated persons at low risk : the rate ratio of influenza - a
attributable gp episodes in the low comorbid risk group in vaccinated over unvaccinated
was 0781 in those aged 1849 years , 079092 in those aged 5064 years , and 076098
in those aged 65 years .
a wide spectrum of diseases that resulted in a gp episode was attributable to influenza a
and b ( fig .
3 ) . the relative proportions
attributable to influenza a and b varied with the gp - reported diagnoses , highlighting the
importance of influenza b in the 517 years age group ; especially so since influenza b
seasons are less frequent than influenza a. fig .
3.the percentage of the estimated burdens for several outcomes attributed to
influenza a and influenza b by five age groups ( mean across seasons ) .
the total
number of gp episodes for each outcome was : respiratory disease ( broadly defined )
( n = 857 996 ) , bronchitis and bronchiolitis
( n = 248 727 ) , influenza - like illness
( n = 265 123 ) , pneumonia and influenza
( n = 197 769 ) , otitis media ( n = 68 777 ) ,
antibiotic use ( n = 604 042 ) .
the percentage of the estimated burdens for several outcomes attributed to
influenza a and influenza b by five age groups ( mean across seasons ) .
the total
number of gp episodes for each outcome was : respiratory disease ( broadly defined )
( n = 857 996 ) , bronchitis and bronchiolitis
( n = 248 727 ) , influenza - like illness
( n = 265 123 ) , pneumonia and influenza
( n = 197 769 ) , otitis media ( n = 68 777 ) ,
antibiotic use ( n = 604 042 ) .
we used the cprd in the uk , a promising but as yet underutilized nationally representative
database for influenza burden assessment , to estimate the age - specific influenza burden in
primary care .
we employed traditional methods
used for hospitalization and mortality studies , and applied them to time - series of gp
episodes for a diverse range of outcomes forming part of the burden of influenza , such as
comorbidity risk status and antibiotic prescriptions , after taking account of the
potentially confounding effects of rsv . in a mean season , around 15% of the uk population
were estimated to present to gps with diverse respiratory syndromes attributable to
influenza a or b. the estimated mean seasonal burden of influenza a was much larger than
influenza b in all age groups and for most respiratory outcomes .
approximately 07% of
children aged < 5 years were estimated to consult a gp for influenza - related otitis
media in a mean season , and nearly 2% of all children received antibiotic prescriptions
which were attributable to influenza . compared to the relatively stable disease burden
attributable to rsv in each season , the influenza burden was characterized by marked
inter - seasonal variability according to epidemic activity .
that used the cprd data
source to study gp office visits examined only a single season ( 20022003 ) . in that study ,
this is higher
than our estimate for that season of 330 763 gp episodes , but closer to our mean seasonal
estimate encompassing 14 seasons . as well as the inherent instability of estimates produced
using a single season , the model used by pitman et al .
reported two alternative estimates obtained by
models with and without a bacterial pathogen series included in the explanatory variables .
the bacterial series ,
characterized by a dominant sinusoidal pattern , likely affected the influenza burden
estimate similarly to our use of cyclic components .
estimates of the number of persons infected with influenza in an average season vary
widely , but when measured as persons consulting with clinical illness they are mostly less
than 5% of the population , depending on the diagnostic outcomes measured [ 8 , 9 , 21 , 22 ] .
our
findings are consistent with a simpler analysis conducted in a database of the royal college
of general practitioners , which estimated an influenza burden affecting 21% of the
population of england and wales in seasons 19891999 .
we observed that in persons with comorbid conditions that placed them in a high risk group
and in all persons aged < 65 years , there were fewer gp episodes for
influenza - attributable respiratory disease in vaccinated than non - vaccinated persons .
this
finding is difficult to interpret because our study was not designed to measure vaccine
effectiveness , which would require a different study design .
such studies would require an
analysis of the propensity to consult , which differs between vaccinated and non - vaccinated
persons , and between those with high and those with low comorbidity risk .
population - based studies of the influenza burden relying on individual laboratory - confirmed
cases alone are not practical , due to the lack of standard testing , the expense of
large - scale testing using sensitive pcr - based assays , and the fact that infections are often
resolved by the time patients seek medical care ( if they seek such care at all ) . instead
,
indirect modelling approaches have been used for many years to estimate the burden of
influenza [ 12 , 23 ] .
we used a previously described linear regression model , which adopted a recent refinement of regression techniques guided
by virus surveillance data as described in studies by pitman et al . and
other models used to estimate the burden of
respiratory infections , such as poisson regression with a natural logarithm for the link
function [ 11 , 26 ] , imply multiplicative effects of respiratory viruses ( i.e. the effect of
multiple simultaneously circulating respiratory viruses is different from the sum of their
individual effects ) .
the
linear regression model has the advantage over other methods of being an additive model ,
which corresponds to the view that the total burden in the population is due to the sum of
outcomes due to different causes , including viral infections [ 27 , 29 ] .
the strengths of our model include the use of large nationally representative databases ,
which limited the potential for sampling error ; inclusion of data over a long period that
covered 14 seasons ; and the use of control outcomes with no inherent seasonality and no
association to influenza . by defining a new outcome category ( respiratory disease broadly
defined ) , we aimed to improve the sensitivity in capturing the full burden of
influenza - attributable disease , while retaining sufficient specificity ; for example , the
additional codes in the respiratory broad outcome increased the average seasonal all - age
estimate by 14% over the stricter respiratory outcome , but left the confidence intervals
and the model fit virtually unchanged ( data not shown ) .
age stratification allowed detailed
estimation of the age - specific burden , controlled for the observed age effect on
consultation rates and overcame possible confounding due to higher frequency of viral
testing in some age groups .
finally , weekly occurrences of outcomes were assumed to be
determined by the circulation of influenza , rsv , and other causes which follow a seasonality
that was estimated using a combination of sine and cosine terms .
the inclusion of both
cyclical terms ( sine and cosine functions ) controlled for confounders for which data are not
available .
tri - mean smoothing of pathogens and outcomes series mitigated short - term effects
such as national holidays and extreme weather conditions on viral testing and gp
consultations .
a potential limitation of the study is the exclusive analysis of influenza and rsv virus
time - series with no consideration of other respiratory pathogens ; if such pathogens
consistently circulate at the same time as influenza , the influenza burden could be
overestimated .
we did not generate time - series for influenza subtypes as we did not have
access to subtyped data .
we did not evaluate the effect of pneumococcal vaccination in
children or older age groups , or consider
changes in herd immunity during the period of study , nor did we consider the effects of
obesity [ 31 , 32 ] .
the study is also dependent on the quality and consistency of routine recording
in the practice network .
the burden of illness as encountered in primary care , which is the
primary objective of this study , includes all consulting persons regardless of their
propensity to consult .
we did not study the indirect burden of illness in individuals with
influenza who did not consult .
while our study provides up - to - date and detailed information
on health service utilization , the study does not provide information for evaluation of
indirect medical costs associated with influenza .
none of the potential limitations of the
study are likely to have influenced the model estimates in any major way .
in addition to influenza - attributable respiratory disease , we estimated the burden of
influenza - attributable otitis media , which has often been regarded as a common complication
of influenza .
our study found almost as many
children ( aged < 5 years ) were diagnosed at presentation with influenza - attributable
otitis media as with influenza - like illness , suggesting that otitis media is part of the
primary symptomatology of influenza .
the diverse range of presenting illnesses identified as
attributable to influenza in our study questions the relevance of diagnostic criteria by
which influenza is defined clinically .
like other studies that have tried unsuccessfully to
correlate specific symptoms with a diagnosis of influenza [ 34 , 35 ] , our study suggests that
collections of symptoms with / without fever are largely inadequate for measuring the burden
of illness .
finally , the model estimated that more than 600 000 antibiotic courses were prescribed for
influenza - associated respiratory disease per season .
the potential misuse of antibiotics is
a well - recognized cause for concern , particularly in relation to common respiratory
infections .
a simple test that is able to
discriminate between bacterial and viral infection would be a great step forward , as would
timely local influenza surveillance data that would allow physicians to assess the
probability that an acute respiratory infection is caused by influenza .
health economic models can inform policy choices , but they depend on accurate and
age - specific parameter estimates of the disease burden . furthermore , it is critical to use
estimates based on recent assessments of the burden : earlier publications could overestimate
the current burden . because the effectiveness of influenza vaccination in preventing otitis
media appears similar to that of pneumococcal conjugate vaccines ,
it is important that estimates of the otitis media burden and the
potentially preventable fraction be included in cost - benefit estimates of influenza
interventions in this age group .
our study provides a source for understanding the national
influenza burden and undertaking cost evaluations . | summaryinfluenza is rarely laboratory - confirmed and the outpatient influenza burden is rarely
studied due to a lack of suitable data .
we used the clinical practice research datalink
( cprd ) and surveillance data from public health england in a linear regression model to
assess the number of persons consulting uk general practitioners ( gp episodes ) for
respiratory illness , otitis media and antibiotic prescriptions attributable to influenza
during 14 seasons , 19952009 .
in cprd we ascertained influenza vaccination status in each
season and risk status ( conditions associated with severe influenza outcomes ) .
seasonal
mean estimates of influenza - attributable gp episodes in the uk were 857 996 for
respiratory disease including 68 777 for otitis media , with wide inter - seasonal
variability . in an average season ,
24%/05% of children aged < 5 years and
13%/01% of seniors aged 75 years had a gp episode for respiratory illness attributed to
influenza a / b .
two - thirds of influenza - attributable gp episodes were estimated to result
in prescription of antibiotics .
these estimates are substantially greater than those
derived from clinically reported influenza - like illness in surveillance programmes .
because health service costs of influenza are largely borne in general practice , these are
important findings for cost - benefit assessment of influenza vaccination programmes . | INTRODUCTION
METHODS
RESULTS
DISCUSSION
Supplementary material |
PMC3483861 | we failed to find a suitable temporal window to record a doppler signal in one individual .
the mean age of the final 27 participants was 26.56 ( sd = 10.7 , min : 20 , max : 66 ) ; there were 9 males , and 4 reported writing with their left hand , 23 with their right hand .
a doppler ultrasonography device ( dwl multidop t2 : manufacturer , dwl elektronische systeme , singen , germany ) was used to examine the blood - flow velocity through the left and right mcas .
participants were fitted with a flexible head - set that held in place a 2-mhz transducer probe over each temporal skull window , and were seated at a viewing distance of approximately 120 cm from the screen .
experimental tasks were presented using a personal computer with a single , centrally aligned speaker and 21-inch digital vrc21-w3 monitor .
the procedures were programmed using matlab r2009a ( mathworks , natick , ma , usa ) , which sent pulses to the multidop system to denote trial onsets . the pulses in all tasks were sent using cogent 2000 developed by the cogent 2000 team at the fil and the icn and cogent graphics developed by john romaya at the lon at the wellcome department of imaging neuroscience .
ftcd tasks the running order of the three ftcd tasks was counterbalanced and the entire procedure lasted approximately 1 hour and 20 minutes .
schematic diagram of the word generation ( upper panel ) , auditory naming ( middle panel ) and picture story ( lower panel ) trial sequence .
bold font represents in - task presentation of instructions ; italicised represents events or responses .
word generation the word generation ( wg ) paradigm was based on knecht et al .
( knecht , deppe , & ebner , et al . 1998 ; knecht et al . 1996 ) .
there were 23 trials , one for each letter of the alphabet excluding q , x , and z. each trial included a 5-second written clear mind
instruction , 12.5 seconds of letter presentation with silent word generation , 5 seconds where the participant was asked to say aloud the generated words , followed by 25 seconds of relaxation .
a 500-ms event marker was sent to the multi - dop system at the onset of the letter presentation .
the auditory naming ( an ) task was based on procedures used by bookheimer et al .
( 1998 ) . on each trial the participant heard a short definition and was asked to say the defined word as quickly as possible .
high- and low - frequency words were selected from the whole english corpus ( http://ucrel.lancs.ac.uk/bncfreq/lists/5_1_all_rank_noun.txt ) and definitions were devised using the oxford english dictionary .
the average frequency of the high - frequency words was 767.5 ( sd = 287.75 ) words per million and all low - frequency words occurred at 10 words per million .
definition length ranged from 2 to 6 words with an average length of 4.15 ( sd = 1.19 ) ; there was no difference in length between the high- and low- frequency words , t(19 ) < 0.1 , p = 1 .
the average definition duration was 2.0 seconds ( sd = 0.43 ) and there was no difference in duration between the high- and low - frequency definitions , t(19 ) = 1.4 , p = 0.15 .
each trial included a 10-second visual clear mind instruction , an average 2-second auditory definition , a
5-second ( maximum ) interval for a verbal response , 3-second visual feedback of the correct word , and a 10-second relax instruction . immediately following
the definition participants were asked to make a speeded , single - word , verbal response .
visual feedback was presented immediately after the key - press or after 5 seconds in the absence of a response .
a 500-ms event marker was sent to the multi - dop system at the onset of the definition .
the picture story ( ps ) paradigm was based on that used by wilke et al .
the audio tracks of four stories were provided by the imaging research centre at cincinnati children 's hospital medical centre ( vannest et al . , 2009 ) .
the final word in each track was replaced by a 200-hz sine wave using audacity 1.2.6 ( http://audacity.sourceforge.net ) .
these samples were presented with accompanying high - definition photographs representing the replaced words , set on black backgrounds .
the stories were presented in a random order across 20 trials , each lasting approximately 27 seconds .
each trial included an auditory story ( mean 5.6 seconds ) , a 2-second picture presentation , an 8-second rest interval , a 500-ms visual fixation , and an 11.5-second blank interval .
the computer presented a black background throughout the presentation , with the exception of the picture and fixation presentations .
the fixation consisted of 10 white concentric circles . in turn , each outer circle disappeared to maintain attention across the blank interval .
a 500-ms event marker was sent to the multi - dop system at the onset of the story presentations . to assess attention to the task , 20 four - option multiple - choice questions , 5 for each story ,
participants were told that it was a passive task and they would hear a story accompanied by pictures .
the data were analysed using a custom matlab program based on average ( deppe , knecht , henningsen , & ringelstein , 1997 ) .
this down - sampled the data from 100 to 25 hz , adjusted mean left and right channel values to 100 on an epoch - by - epoch basis , performed heart cycle integration , and artefact rejection .
for each task , baseline - corrected , left minus right difference values were used to calculate laterality indices ( lis ) .
individual lis were estimated by calculating the average left - right difference across a 2-second window centred on the maximum peak difference within a task - specific period of interest ( poi ) for all suitable epochs .
laterality categorisation ( left , right , or bilateral ) was tested using one - sample t - tests to determine whether individual li values were significantly different from zero .
task - specific baseline and poi values ( in seconds ) were used relative the initial stimulus event markers ; wg task : baseline = 15 to 5 , poi = 3 to 13 ; an task : baseline = 10 to 0 , poi = 3 to 13 ; ps task : baseline = 8 to 0 , poi = 5 to 17 .
the internal reliability of each task was estimated using cronbach 's a ( cronbach , 1951 ) , which should be interpreted as a correlation coefficient ranging from 0 to 1 , with higher values indicating higher reliability .
the mean activation was calculated as the average left and right blood flow velocity relative to baseline levels at the peak left minus right difference and li latency reflects the time in seconds of the peak left minus right difference , relative to the onset of the stimulus . task difficulty analysis .
additional summaries were conducted for the wg and an tasks to assess the effect of task difficulty on lateralisation indices . in the wg task , letters were grouped into three difficulty levels based on the average number of reported words for the whole group of participants . in the an task , trials corresponding to the high- and low - frequency word definitions were taken to correspond to low and high difficulty levels respectively .
we failed to find a suitable temporal window to record a doppler signal in one individual .
the mean age of the final 27 participants was 26.56 ( sd = 10.7 , min : 20 , max : 66 ) ; there were 9 males , and 4 reported writing with their left hand , 23 with their right hand .
a doppler ultrasonography device ( dwl multidop t2 : manufacturer , dwl elektronische systeme , singen , germany ) was used to examine the blood - flow velocity through the left and right mcas .
participants were fitted with a flexible head - set that held in place a 2-mhz transducer probe over each temporal skull window , and were seated at a viewing distance of approximately 120 cm from the screen .
experimental tasks were presented using a personal computer with a single , centrally aligned speaker and 21-inch digital vrc21-w3 monitor .
the procedures were programmed using matlab r2009a ( mathworks , natick , ma , usa ) , which sent pulses to the multidop system to denote trial onsets .
the pulses in all tasks were sent using cogent 2000 developed by the cogent 2000 team at the fil and the icn and cogent graphics developed by john romaya at the lon at the wellcome department of imaging neuroscience .
ftcd tasks the running order of the three ftcd tasks was counterbalanced and the entire procedure lasted approximately 1 hour and 20 minutes .
schematic diagram of the word generation ( upper panel ) , auditory naming ( middle panel ) and picture story ( lower panel ) trial sequence .
bold font represents in - task presentation of instructions ; italicised represents events or responses .
word generation the word generation ( wg ) paradigm was based on knecht et al .
there were 23 trials , one for each letter of the alphabet excluding q , x , and z. each trial included a 5-second written clear mind
instruction , 12.5 seconds of letter presentation with silent word generation , 5 seconds where the participant was asked to say aloud the generated words , followed by 25 seconds of relaxation .
a 500-ms event marker was sent to the multi - dop system at the onset of the letter presentation .
the auditory naming ( an ) task was based on procedures used by bookheimer et al .
( 1998 ) . on each trial the participant heard a short definition and was asked to say the defined word as quickly as possible .
high- and low - frequency words were selected from the whole english corpus ( http://ucrel.lancs.ac.uk/bncfreq/lists/5_1_all_rank_noun.txt ) and definitions were devised using the oxford english dictionary .
the average frequency of the high - frequency words was 767.5 ( sd = 287.75 ) words per million and all low - frequency words occurred at 10 words per million .
definition length ranged from 2 to 6 words with an average length of 4.15 ( sd = 1.19 ) ; there was no difference in length between the high- and low- frequency words , t(19 ) < 0.1 , p = 1 . the average definition duration was 2.0 seconds ( sd = 0.43 ) and there was no difference in duration between the high- and low - frequency definitions , t(19 ) = 1.4 , p = 0.15 .
each trial included a 10-second visual clear mind instruction , an average 2-second auditory definition , a 5-second ( maximum ) interval for a verbal response , 3-second visual feedback of the correct word , and a 10-second relax instruction . immediately following
the definition participants were asked to make a speeded , single - word , verbal response .
visual feedback was presented immediately after the key - press or after 5 seconds in the absence of a response .
a 500-ms event marker was sent to the multi - dop system at the onset of the definition .
all instructions were presented centrally in white arial font on a black background . picture story . the picture story ( ps ) paradigm
the audio tracks of four stories were provided by the imaging research centre at cincinnati children 's hospital medical centre ( vannest et al . , 2009 ) .
the final word in each track was replaced by a 200-hz sine wave using audacity 1.2.6 ( http://audacity.sourceforge.net ) .
these samples were presented with accompanying high - definition photographs representing the replaced words , set on black backgrounds .
the stories were presented in a random order across 20 trials , each lasting approximately 27 seconds .
each trial included an auditory story ( mean 5.6 seconds ) , a 2-second picture presentation , an 8-second rest interval , a 500-ms visual fixation , and an 11.5-second blank interval .
the computer presented a black background throughout the presentation , with the exception of the picture and fixation presentations .
the fixation consisted of 10 white concentric circles . in turn , each outer circle disappeared to maintain attention across the blank interval .
a 500-ms event marker was sent to the multi - dop system at the onset of the story presentations . to assess attention to the task , 20 four - option multiple - choice questions , 5 for each story ,
participants were told that it was a passive task and they would hear a story accompanied by pictures .
the data were analysed using a custom matlab program based on average ( deppe , knecht , henningsen , & ringelstein , 1997 ) .
this down - sampled the data from 100 to 25 hz , adjusted mean left and right channel values to 100 on an epoch - by - epoch basis , performed heart cycle integration , and artefact rejection .
for each task , baseline - corrected , left minus right difference values were used to calculate laterality indices ( lis ) .
individual lis were estimated by calculating the average left - right difference across a 2-second window centred on the maximum peak difference within a task - specific period of interest ( poi ) for all suitable epochs .
laterality categorisation ( left , right , or bilateral ) was tested using one - sample t - tests to determine whether individual li values were significantly different from zero .
task - specific baseline and poi values ( in seconds ) were used relative the initial stimulus event markers ; wg task : baseline = 15 to 5 , poi = 3 to 13 ; an task : baseline = 10 to 0 , poi = 3 to 13 ; ps task : baseline = 8 to 0 , poi = 5 to 17 .
the internal reliability of each task was estimated using cronbach 's a ( cronbach , 1951 ) , which should be interpreted as a correlation coefficient ranging from 0 to 1 , with higher values indicating higher reliability .
the mean activation was calculated as the average left and right blood flow velocity relative to baseline levels at the peak left minus right difference and li latency reflects the time in seconds of the peak left minus right difference , relative to the onset of the stimulus .
additional summaries were conducted for the wg and an tasks to assess the effect of task difficulty on lateralisation indices . in the wg task , letters were grouped into three difficulty levels based on the average number of reported words for the whole group of participants . in the an task , trials corresponding to the high- and low - frequency word definitions were taken to correspond to low and high difficulty levels respectively .
there were a total of 22 individuals data for wg , 25 for an , and 27 for ps . within task analyses of variance
the wg task participants reported an average of 4.18 ( sd = 0.38 , min : 1 , max : 7 ) words per letter . in the an task
for the ps task post - test accuracy was 90% correct ( median = 95 , sd = 10 , min 60 , max 100 ) .
baseline corrected doppler velocities for the left and right hemisphere activation , as well as the left minus right difference are presented for each task in figure 2 .
the descriptive statistics for each task are presented in table 1 . at the group level ,
it is also possible , using the one - sample t - tests for each individual , to categorise their lateralisation as left , right , or bilateral .
both the overall task lis and the individually significant left lis , indicated the wg ( 77% of participants ) and an ( 72% ) tasks to be more strongly lateralised than the ps task ( 56% ) .
word generation , auditory naming , and picture story left and right hemisphere activation ( left half ) and left - right difference activation ( right half ) at a function of epoch time in seconds .
mean laterality indices ( li ) are displayed in the difference graphs with standard deviation in brackets .
all values are accompanied by * to indicate statistical difference from 0 at p < .05 .
task statistics including n accepted trials , internal consistency , laterality indices ( li ) and lateralisation counts ( left , right , and bilateral ) , as well as the relationship between lis and handedness .
categorisation of laterality status is based on one - sample t - tests for each individual testing whether l
the lis for each task were not significantly different ( paired - sample t - tests ) , although there was a trend for higher lis in wg and an compared with ps : wg : an , t(20 ) = 0.25 , p = 0.81 , cohen 's d = 0.07 ; wg : ps , t(20 ) = 1.98 , p = 0.06 , cohen 's d = 0.61 ; anps , t(24 ) = 2.04 , p = 0.05 , cohen 's d = 0.39 .
wg was not significantly related to either an or ps and there was a positive relationship between an and ps ; wg and an r = .32 ( ns ) , wg and ps , r = .13 ( ns ) , and an and ps , r = .55 ( p < .01 ) .
one multivariate outlier was removed from the wg correlations ; however , this did not influence the relationships .
although there was only one correlation , between an and ps , that reached statistical significance , it should be noted that these correlations need to be corrected for attenuation ; i.e. , the imperfect reliability of the measures ( spearman , 1904 ) . the internal reliability estimated using cronbach 's alpha were 0.52 for wg , 0.77 for an , and 0.68 for ps .
when correlation for attenuation was made using these values , the correlation between wg and an rises to .56 ( p < .05 ) , and that between an and ps rises to .76 ( p < .001 ) .
individual laterality index scatter plots between task ; top : word generation ( wg ) versus auditory naming ( an ) , middle : wg versus picture story ( ps ) , and lower : an versus ps .
the data points for left - handed individuals are filled in black . disattenuated pearson product moment correlations coefficients are presented for each relationship excluding the outlier ( grey fill ) for the wg comparisons ( * p < .05 , * * p < .001 ) .
n o j v i , m = 3.77 ( sd = 0.26 ) ; medium , a e c w f p r g : m = 4.25 ( sd = 0.12 ) ; and high , l t h m s b d : m = 4.56 ( sd = 0.14 ) .
lis , mean left right activations , and peak left minus right difference latencies are displayed by word production grouping in the top row of figure 4 .
none of the differences was statistically significant : repeated measures anovas ; li , f(2 , 42 ) = 0.25 p = 0.78 , p = 0.01 ; mean activation , f(2 , 42 ) = 1.53 p = 0.23 , p = 0.07 ; and latency , f(2 , 42 ) = 0.79 p = 0.46 , p = 0.04 .
thus word production is not related to the physiological li , mean activation , or peak latency of silent word generation in the wg task .
laterality indices ( left column ) , mean left right activation ( middle column ) , and peak left minus right difference latencies ( right column ) for word generation behavioural word production ( top row ) and auditory naming word frequency conditions ( bottom row ) .
the x - axis of the laterality index graphs ( left column ) have been reversed to clearly depict left ( positive ) and right ( negative ) lateralisation .
were more accurate and faster to the high - frequency words in comparison to the low - frequency words ; accuracy as proportion correct : high , m = 0.69 , sd = 0.09 ; low , m = 0.60 , sd = 0.10 ; and reaction time in seconds : high , m = 1.81 , sd = 0.36 ; low , m = 2.37 , sd = 0.53 .
both of these effects were statistically significant ; accuracy , t(24 ) = 4.16 , p < .001 , cohen 's d = 0.99 ; reaction time , t(24 ) = 7.99 , p < .001 , cohen 's d = 1.26 .
therefore the word frequency manipulation was successful , behavioural responses indicating that descriptions of high - frequency words were more easily and rapidly named in comparison to low - frequency words .
lis , mean left right activations , and peak left minus right difference latencies are displayed by word frequency in the bottom row of figure 4 .
none of the differences was statistically significant : paired sample t - tests ; li , t(24 ) = 0.29 , p = 0.77 , cohen 's d = 0.05 ; mean activation , t(24 ) = 0.77 , p = 0.45 , cohen 's d = 0.14 ; and latency , t(24 ) = 1.65 , p = 0.11 , cohen 's d = 0.4 .
this indicates that word frequency is not related to the physiological li , mean activation , or peak latency of word finding in the an task . picture story .
although no behavioural manipulation was conducted for the ps task , we were able to consider whether individual differences in post - test accuracy affected li .
accuracy was unrelated to li ( spearman r = 0.36 ) , mean activation ( spearman r = 0.17 ) , or li latency ( spearman r = 0.11 ) .
letter groupings based on average group report were low : k u y n o j v i , m = 3.77 ( sd = 0.26 ) ; medium , a e c w f p r g : m = 4.25 ( sd = 0.12 ) ; and high , l t h m s b d : m = 4.56 ( sd = 0.14 ) .
lis , mean left right activations , and peak left minus right difference latencies are displayed by word production grouping in the top row of figure 4 .
none of the differences was statistically significant : repeated measures anovas ; li , f(2 , 42 ) = 0.25 p = 0.78 , p = 0.01 ; mean activation , f(2 , 42 ) = 1.53 p = 0.23 , p = 0.07 ; and latency , f(2 , 42 ) = 0.79 p = 0.46 , p = 0.04 .
thus word production is not related to the physiological li , mean activation , or peak latency of silent word generation in the wg task .
laterality indices ( left column ) , mean left right activation ( middle column ) , and peak left minus right difference latencies ( right column ) for word generation behavioural word production ( top row ) and auditory naming word frequency conditions ( bottom row ) .
the x - axis of the laterality index graphs ( left column ) have been reversed to clearly depict left ( positive ) and right ( negative ) lateralisation .
were more accurate and faster to the high - frequency words in comparison to the low - frequency words ; accuracy as proportion correct : high , m = 0.69 , sd = 0.09 ; low , m = 0.60 , sd = 0.10 ; and reaction time in seconds : high , m = 1.81 , sd = 0.36 ; low , m = 2.37 , sd = 0.53 .
both of these effects were statistically significant ; accuracy , t(24 ) = 4.16 , p < .001 , cohen 's d = 0.99 ; reaction time , t(24 ) = 7.99 , p < .001 , cohen 's d = 1.26 .
therefore the word frequency manipulation was successful , behavioural responses indicating that descriptions of high - frequency words were more easily and rapidly named in comparison to low - frequency words .
lis , mean left right activations , and peak left minus right difference latencies are displayed by word frequency in the bottom row of figure 4 .
none of the differences was statistically significant : paired sample t - tests ; li , t(24 ) = 0.29 , p = 0.77 , cohen 's d = 0.05 ; mean activation , t(24 ) = 0.77 , p = 0.45 , cohen 's d = 0.14 ; and latency ,
t(24 ) = 1.65 , p = 0.11 , cohen 's d = 0.4 .
this indicates that word frequency is not related to the physiological li , mean activation , or peak latency of word finding in the an task . picture story .
although no behavioural manipulation was conducted for the ps task , we were able to consider whether individual differences in post - test accuracy affected li .
accuracy was unrelated to li ( spearman r = 0.36 ) , mean activation ( spearman r = 0.17 ) , or li latency ( spearman r = 0.11 ) .
participants completed three language tasks in an ftcd paradigm : word generation ( wg ) , auditory naming ( an ) , and picture story ( ps ) .
wg required participants to silently generate words to a visually presented letter , an required participants to verbally report a single word from an auditory definition , and ps required participants to passively listen to stories accompanied by pictures depicting acoustically masked words from the stories .
all tasks were left lateralised , wg and an more clearly with respect to the number of left - lateralised individuals , 77 and 72% respectively , than ps ( 56% ) .
furthermore , behavioural responses were unrelated to physiological responses , despite significant behavioural differences due to task difficulty .
wg is commonly used for assessing language lateralisation and has been validated in ftcd with respect to the wada technique ( knecht , deppe , ebner , et al . , 1998 ) and fmri ( knecht et al . , 1999 ) .
each trial is relatively long and participants report many different approaches to the task : some rehearsing a small set of words , others chunking sentence - like combinations of words , and some reporting new words that were not silently generated .
therefore , while the component of interest is the word finding , the activity due to the additional components ( visual recognition , covert productive , short - term memory rehearsal ) is likely to vary between individuals and potentially within individuals .
furthermore the long periods during which participants are required to clear their minds varies in reported ease between participants .
this variability may be reflected in the mean left - right activation at peak difference ( see figure 4 ) .
there is large variability in this task , mean activity varying from much lower than baseline to much higher than baseline .
decreases may reflect concerted efforts to think of nothing during periods of baseline and normalisation , whereas others may find this simple .
this is not the case with the an and ps paradigms , mean activation predominantly ( all but one individual ) being positive .
one of the clear advantages of the an task is the duration of the procedure .
approximately double the number of an trials were run relative to the wg task in the same time period .
potentially , the larger number of trials will reduce noise in the data and could underpin the significant relationship between an and ps .
previous research has identified lateralisation differences as well as varying relationships between language tasks using ftcd ( buchinger et al . , 2000 ;
, 2010 ; stroobant et al . , 2009 ; stroobant et al . , 2010 ) .
these studies have examined word fluency ( similar to wg ) , sentence construction , reading , semantic decision making , picture description , and story listening .
all studies have found variability between tasks and varying relationships between them , consistent with our current results . in a story - listening task with children consisting of 30 seconds of listening followed by questions , stroobant et al .
( 2010 ) found equivalent left lateralisation as with our own ps task . therefore our findings are consistent with prior research , which has shown that expressive tasks are more strongly lateralised than receptive tasks .
however , different expressive tasks are not interchangeable ; it seems that direction and degree of lateralisation can vary from task to task within individuals , and that this is not totally explicable in terms of poor task reliability or variations in task difficulty .
previous work suggests that receptive language is more bilaterally distributed ( boatman et al . , 1999 , 1998 ;
hertz - pannier et al . , 2002 ) ; we suggest that the relatively low li scores in the ps task may reflect weaker or inconsistent activation of implicit production in this task compared to the an or wg tasks .
the major limitation of this research is restricted range for the laterality indices , and the low internal reliability of the tasks .
the sample included few left - handed individuals and only two individuals demonstrated significant right lateralisation on any of the tasks , neither individual showing consistently right activity between tasks .
greater within sample variability may have shown a stronger relationship between tasks ; however , it is unlikely to affect the findings of the difficulty manipulation .
relative to previously reported internal reliability of ftcd language tasks ( e.g. , around .9 in bishop et al .
, 2009 ) , the reliability of our tasks was low ( .52 to .77 ) .
this will attenuate the observed correlation between tasks , although we were able to apply a standard attenuation correction to take this into account .
future investigations could adopt a targeted approach to subject recruitment , actively seeking left - handed individuals to increase the likelihood of atypical lateralisation ( see knecht et al . , 2000 ) . in conclusion ,
language lateralisation derived from performing expressive and receptive procedures varies between tasks , expressive tasks demonstrating the highest degree of lateralisation .
differences were not explained by task difficulty but may be due the balance of expressive and receptive language demands .
it may be best to use tasks that minimise the possibility of participants adopting idiosyncratic task - specific strategies . | language is lateralised to the left hemisphere in most people , but it is unclear whether the same degree and direction of lateralisation is found for all verbal tasks and whether laterality is affected by task difficulty .
we used functional transcranial doppler ultrasonography ( ftcd ) to assess the lateralisation of language processing in 27 young adults using three tasks : word generation ( wg ) , auditory naming ( an ) , and picture story ( ps ) .
wg and an are active tasks requiring behavioural responses whereas ps is a passive task that involves listening to an auditory story accompanied by pictures .
we also examined the effect of task difficulty by a post hoc behavioural categorisation of trials in the wg task and a word frequency manipulation in the an task .
ftcd was used to measure task - dependent blood flow velocity changes in the left and right middle cerebral arteries .
all of these tasks were significantly left lateralised : wg , 77% of individuals left , 5% right ; an , 72% left : 4% right ; ps , 56% left : 0% right .
there were significant positive relationships between wg and an ( r = 0.56 ) as well as an and ps ( r = .76 ) but not wg and ps ( r = 0.22 ) .
the task difficulty manipulation affected accuracy in both wg and an tasks , as well as reaction time in the an task , but did not significantly influence laterality indices in either task .
it is concluded that verbal tasks are not interchangeable when assessing cerebral lateralisation , but that differences between tasks are not a consequence of task difficulty . | METHOD
Participants
Materials and procedure
RESULTS
Task difficulty
DISCUSSION |
PMC4251271 | perceived risk is defined as one s belief about the likelihood of personal harm ( weinstein and klein,1995 ) .
personal health risk perceptions may be an important determinant of specific health - related behaviour and it is the pivot in a number of theoretical models on health behaviour , the health belief model being generally well - known ( janz and becker , 1984 ) . it is therefore clarifying to explore the elements underlying risk perception and to study the relationship between perceived risk and consequent health - related behaviour in order to communicate health risks to the general public in a way that health - related behaviour is changed for the better ( lipkus et al . , 2000 ) .
this review summarizes present knowledge on breast cancer risk perception and risk communication , two topics that are inherently related .
the subject of breast cancer risk assessment and risk communication is well researched but lacks implementation in daily clinical practice as physicians are hardly familiar with it .
how often does one hear women say : it seems to me breast cancer is all over the place : in my family , in my neighbourhood , in the newspapers and on tv .
indeed women worry a lot about getting breast cancer more than about any other disease ( wang et al . , 2009 ) .
age - adjusted incidence of breast cancer indeed has increased over time ( horner et al . , 2008 ) .
there is a high frequency of thinking about breast cancer probably triggered by events in our immediate environment , the so - called social construction of the breast cancer epidemic
most common triggers reported are media reports , daily social contacts , and meeting someone who suffered breast cancer ( mc caul et al . , 1998 ,
societies in western europe characterised by an ageing population with a higher prevalence of breast cancer , a recent introduction of breast cancer screening programmes associated with an increase of breast cancer diagnoses , and a widening of the public debate about breast cancer as a result of improved treatment outcome and an increasing number of breast cancer survivors are ideal grounds to nourish this availability heuristic .
when people worry about breast cancer they most often think about their personal risk , about family or friends affected , and the high prevalence of the disease .
lerman et al . reported that 30% of women with first - degree relatives who had breast cancer said that they worried about breast cancer so much that it interfered with their daily functioning and quality of life ( lerman et al .
women commonly misjudge their breast cancer risk , overestimating their lifetime risk and cancer - related death risk of a significant degree ( mc caul et al . , 1998 ;
in one study women between 40 and 50 years of age overestimated their lifetime risk by 12-fold and their short - term risk of dying from breast cancer by 22-fold ( black et al . , 1995 ) .
only 7 - 37% of women , depending on the demographics and the education level of the women studied , had a realistic notion of the population lifetime breast cancer risk .
the proportion of women that overestimate their risk of breast cancer varies widely between 50 - 90% depending on the population studied and the way information was obtained ( lerman et al . , 1994 ;
, 1996 ; alexander et al . , 1996 ; bowen et al . , 1997 ; herbert - croteau et al . , 1997 ; dolan et al . , 1997 ;
the mean perceived lifetime risk of developing breast cancer in studies varies between 30 and 46% , much higher than the real lifetime risk of 12% .
women in particular those who are young and those with a family history of breast cancer have unreal risk perceptions ( evans et al . , 1993 ; daly et al . , 1996 ; zakowski et al . , 1997
breast cancer risk perception is influenced by both medical ( subjective and objective ) ( lipkus et al . , 2000 ) and psychological factors ( gerend et al . , 2004
there is body of evidence that links a family history of breast cancer to perceived risk . notwithstanding
women s risk of developing breast cancer increases with age , older women perceive themselves as less susceptible to breast cancer than do younger women ( wilcox and stefanick , 1999 ; katapodi et al . , 2004 ) .
there is a well documented discrepancy between subjective and objective medical risk ( katapodi et al . , 2004 ) .
objective medical risk is measured in terms of absolute risk ( e.g. how likely is it that one will develop breast cancer in a lifetime ? ) and direct comparative risk ( e.g. compared with other women of the same age , how likely is it one will develop breast cancer ? ) .
objective medical risk can be calculated by various mathematical tools based on the gail model ( see risk calculation and communication , gail et al . , 1989 ) .
psychological factors that influence breast cancer risk perception include the perceived nature and the heuristic processing of breast cancer as a health threat . regarding the nature of a threat it is shown that the more prevalent , severe , devastating , unexpected , difficult to control the nature of a threat is , the more it is associated with higher risks of perception ( slovic , 1987 )
. heuristic processing of a threat involves two cognitive functions : i ) the availability heuristic judging the probability of an event by the ease with which examples of that event come to mind ( for example , experience with a threat be it personal , via family / friends or via the media increases the availability of the threat and , as a result , its perceived likelihood ( weinstein , 1987 ) ; ii ) the representativeness heuristic judging the probability of an event by its similarity to events with comparable features ( for example , the belief that one is similar to the type of person who develops a specific disease is associated with a higher risk perception to that disease ) ( lek and bishop , 1995 ) . this specific factor perceived similarity to the person who develops
breast cancer- is the single strongest correlate of perceived breast cancer risk followed by the perceived prevalence of breast cancer and the vicinity of a relative / friend with breast cancer ( gerend et al . , 2004 ) .
the first question that comes up in one s mind once diagnosed with breast cancer is : how long have i to live ?
health care professionals should at some stage in the follow - up of breast cancer treatment picture the risk of dying of breast cancer within a broader context taking into account other real risks such as death from cardiovascular disease or car accident .
non - oncological diseases as these which pose a much greater risk to a woman s life with a worse prognosis are underestimated and their threat is perceived to be low ( phillips et al . , 1999 ) . again
this disproportionate fear of breast cancer relative to other non - cancer diseases is irrational .
it is a well accepted statement that the media has a substantial effect on a women s health perceptions ( frost et al . , 1997 ) .
we referred to availability heuristic as one of the mechanisms that influences one s perception of risk .
availability heuristic probably mediates the heightened personal and general vulnerability perceptions induced by media coverage of health topics .
women who had been exposed occasionally to programs on breast cancer on tv were twice ( 95% ci 1.1 - 3.4 ) more likely to be very afraid of breast cancer .
depending on the degree of media exposure the odds ratio of expressing high levels of fear of breast cancer increased to 7.5 ( 95% ci 2.4 - 23.8 ) ( lemal and van den bulck , 2009 ) .
frequently involves personal stories with accent on emotional experiences ( atkin et al . , 2008 ) to give it a dimension of newsworthiness
the same has been found in the popular written press were stories on breast cancer misrepresent the age distribution of the disease , focusing on atypical cases of early - onset breast cancer and their often dramatic social impact .
these magazines are ( were ) known to be an important source of health information ( meissner et al .
, 1992 ; okeefe et al . , 1998 ) certainly before the era of the internet .
the emphasis on young age found in popular magazines follows women s misperceptions about risk and may nourish them .
the effect of risk factors related to breast cancer is another item that is miscommunicated by the popular press . in 1996
, many women were concerned after publication of a review of the association of breast cancer and hormonal contraceptives which stated that the risk of breast cancer was increased by 20% in users ( collaborative group on hormonal factors in breast cancer , 1996 ) .
if the risk was communicated as an absolute risk increase from 16 to 18.7 per 10 000 women or one additional breast cancer diagnosis per year among 3700 women taking oral contraceptive , risk perception would have been totally different .
similarly , the women s health initiative study results of hormone replacement therapy as a primary prevention trial of chronic diseases such as cardiovascular disease were summarized in the media as a 26% increase of breast cancer in users being the main message ( rossouw et al . , 2002 ) .
again if the risk was communicated in terms of absolute risk ( 19 additional breast cancer for combined therapy and 5 for estrogen - only therapy per 10 000 user - years ) the message would have been more shaded .
pleas made by the medical community ( woloshin and schwartz , 2002 ; schwartz and woloshin , 2004 ) and guidelines proposed by journalists ( russell , 1999 ; brody , 1999 ) for media to present health issues in a scientifically justified and balanced way have not filtered through yet as they are inferior to commercial interests .
with respect to the autonomy of women seeking medical care , providers should inform their patients to that extent that they feel comfortable in making decisions about their care . with regard to breast cancer risk perception and how to respond appropriately in terms of preventive strategies ,
is assumed to be a part of routine care / education provided by health care professionals ( sabatino et al . , 2007 ) .
in fact receiving information on heath - related issues from health care professionals in particular information about cancer risk is very much appreciated by patients ( schwartz et al . , 1999 ; buchanan et al . , 2005 ) .
very little is known about how breast cancer risk is assessed in daily practice . a study on risk communication defined as a discussion on health behaviour , participation in early detection or
other preventive health strategies revealed that risk was discussed in only 26% of primary care visits and that risk was presented numerically in only 3% of visits ( kalet et al . , 1994 ) .
lack of knowledge and a busy consultation time schedule are obvious reasons why physicians fail to deliver effective health risk information .
breast cancer risk information is important information to be communicated with patients for a number of reasons . in the first place it is an attempt to correct inappropriate perceived risk .
secondly , evaluation of risk status may influence health - related decisions about screening ( pichert et al . , 2003 ) , chemoprevention ( howell a , 2008 ) , referral for genetic counselling and testing ( nelson et al . , 2005 ) , and even prophylactic surgery ( evans et al . , 2009 ) .
there are a number of tools available that may be worthwhile in facilitating communication about breast cancer risk ( euhus , 2001 ; jacobi et al . , 2009 ) .
providing women with a personalized numerical breast cancer risk estimates on the one hand could bring the message much more clearly than general messages on breast cancer risk ( skinner et al . , 1995 ) .
however , focusing on a single disease - specific risk could overemphasize that risk disproportionately to other even more important health issues ( schwartz et al . , 1999 ) .
the risk of developing breast cancer of 1 in 10 is a statistic that is often used in the popular press as it is appealing and frightening at the same time .
however i do not see initiatives to ease the public opinion most probably out of fear that rectification of this sort of disinformation wo nt help breast cancer screening programmes . from an ethical point of view
it is not easy task and it requires a sense of numeracy ( quantitative literacy ) on the patient s part .
low numeracy is pervasive and constrains patient choice , limits access to treatments , reduces compliance with therapy , and impairs risk communication as it is associated with difficulty in understanding and assessing probabilities of risk and risk - related information , especially when that information was presented to them in a quantitative , numerical format .
( schwartz et al . , 1997 ; lipkus et al . , 2001 ; davids sl et al . , 2004 ; nelson et al . , 2008 ) .
intervention trials designed to improve communication on quantitative data about health risk or treatment benefit showed variable results with regard to risk perception ( woloshin and schwartz , 1999 ; vernon , 1999 ) .
graphic displays on understanding health risks are of general benefit and may be especially helpful to those who are least numerate ( ancker et al . , 2006 ) .
who , running the models for two healthy women from brca - negative / unknown status families , found that the tyrer - cuzick and the boadicea model were most appropriate in predicting breast cancer risk ( jacobi et al . , 2009 )
the most basic method of communicating breast cancer risk involves the use of the seer data ( surveillance , epidemiology , and end - results ) , the most exhaustive cancer database presently available on the internet ( http://seer.cancer.gov/statistics/ ) .
a lifetime table analysis of the cumulative incidence of breast cancer in the population will help to explain the 1 in 8 ( 12% ) lifetime risk for breast cancer ( table i ) . from figure 1
it is clear that at the age of 39 a women has a risk of 1 in 1000 to contract breast cancer in that calender year . for a women at the age of 69
table 1 shows us that a women at the age of 35 have a 1 in 115 risk of getting breast cancer in the next 10 years .
this risk increases with age becoming 1 in 40 by age 50 , 1 in 25 by age 65 .
this type of age - specific data should take care of any disproportionate fear of breast cancer amongst women .
these life table estimates are population - based data and represent averages that do not take into account patient - specific risk variables such as age at menarche , age at first pregnancy , a family history of breast cancer , etc .
ideally an individual should be counselled about her risk of developing breast cancer taking in consideration not only her age but also her risk factors .
the most important risk factor for the development of breast cancer apart from age is a positive family history for breast cancer ( dumitrescu and cotarla , 2005 ) .
ref : http://seer.cancer.gov/faststats/selections.php#output the calculation of a more personalized objective medical risk is based on the gail model ( gail et al . , 1989 ) .
the original gail model is a statistical model based on relative risk conferred by specific risk factor populations defined in the breast cancer detection demonstration project , a large american mammography screening trial between 1973 and 1980 . to date
a modified version linked to updated seer data is known as the breast cancer risk assessment tool and can be found on the website of the national cancer institute ( http://www.cancer.gov/bcrisktool/ ) .
risk is expressed as a 5-year and a life - time absolute and comparative average risk .
risk factors that are taken into account are age , age at menarche , age at first live birth , number of first - degree relatives with breast cancer , and number of previous breast biopsies .
other risk factors , such as age at menopause , dense breast tissue on mammography , use of hormonal contraceptives or replacement therapy , high - fat diet , alcohol intake , low physical activity , obesity , or environmental exposures , are not included in risk estimates as they do not increase the accuracy of gail model .
the modified gail model has been validated to provide accurate estimates of breast cancer risk of women without prior history of breast cancer ( costantino et al . , 1999 ) .
in contrast to the gail model which incorporates basic information on family history limited to the number of first - degree relatives only , the claus model provides age - specific breast cancer risk estimates based on a more extensive family history including first- and second - degree relatives as well as the age at the time of diagnosis of breast cancer ( claus et al . , 1994 ) .
risk is presented as a probability of developing cancer at a certain age with five - year increments up to the age of 79 .
brcapro , also known as the john hopkins bayesian model , is another statistical tool which analyzes family history information on breast and ovarian cancer even more extensively in order to determine the probability of a brca1/brca2 gene mutation in the proband ( berry et al .
, 2002 ) . to do so the model uses the observed incidences of breast and ovarian cancer among brca1/brca2 gene mutation carriers and noncarriers
. given this brca1/brca2 gene mutation probability the risk of getting breast cancer is estimated .
the accuracy of this estimation is based on the knowledge of the exact mutation frequency of the brca1/brca2 gene in the population this individual belongs to .
the brcapro model was further refined by incorporating non - genetic risk factors such as age at menarche , age at first born , age at menopause , atypical hyperplasia , lobular carcinoma in situ , height and body mass index , known as the tyrer - cuzick model ( tyrer et al . , 2004 ) . finally , cancergene is a software computer program ( http://www8.utsouthwestern.edu/utsw/cda/dept47829/files/65844.htlm ) that provides an interface for collecting risk factor information needed to run the gail , claus , and bayesian model based risk predictions .
older models such as the gail and the claus model tend to underestimate the baseline life - time risk for breast cancer as they were developed in the eighties and nineties at a time when the incidence of breast cancer was significantly lower ( jacobi et al .
. therefore newer models with updated epidemiological data ( nci breast cancer risk assessment tool ) and those incorporating also personal risk factors , such as a family history of ovarian cancer , age at menarche , and age at first born ( boadicea model ( antoniou et al . , 2004 ) , jonker model ( jonker et al . ,
2003 ) , claus - extended formula ( van asperen et al . , 2004 ) , tyrer - cuzick model ( tyrer et al . , 2004 ) ) , yield higher life - time risk estimates . at present breast cancer risk assessment is an active research topic where in an attempt to improve risk prediction , new models using novel risk factors are developed ( gail , 2009 ) . adding more information or incorporating new risk factors to the proband
this implies that current nice guidelines regarding threshold risk for increased mammographic surveillance of
17% life - time risk based on family history risks only , need to be reconsidered ( http://www.nice.org.uk/cg041 ) .
some researchers believe that excessive worry inhibits whilst others argue that worry facilitates screening behaviour .
to date general opinion ( evidence level ii-3 ) is that thinking and worrying about breast cancer in women with a realistic perceived breast cancer risk may motivate self - protective behaviour ( leventhal et al . , 1999 ;
however high levels of breast cancer worry in general are said to be uncommon ( hay et al . , 2006 ) .
a flemish study though reported high levels of breast cancer fear in about 30% of a random sample of 500 women ( lemal and van den bulck , 2009 ) .
researchers distinguish between content - based worry ( i.e. worry about a particular worry domain such as breast cancer ) and general worry which is more related to general anxiety .
the former is a constructive form of worrying and predicts problem solving , the latter is more predictive of avoidant coping ( davey , 1993 ) .
this is in accordance with the findings that worry about breast cancer promotes mammography screening uptake ( mccaul et al . ,
1996 ) whereas anxiety appears to inhibit mammography screening ( kash et al . , 1992 ) .
studies examining the association between perceived risk and worry have noted that these variables operate independently in predicting cancer screening behaviours ( mcqueen et al .
to date evidence is conflicting as to whether educational interventions as part of breast cancer early detection programs that aim to correct inappropriate perceived breast cancer risk can improve subsequent cancer screening ( vernon , 1999 ) .
this is a crucial deficit in the evidence supporting risk assessment and communication , a gap in our understanding of the mechanisms between perceived risk and impact on health behaviour that needs to be bridged before recommendations can be made .
an important caveat associated with mammography breast cancer screening is the detection of an increasing number of precursor lesions with uncertain clinical significance . in those cases
the ultimate diagnosis remains uncertain and in a climate of defensive medicine these women are often overtreated to anticipate progression into an invasive lesion .
indeed overdiagnosis and overtreatment are inherent to cancer screening ( zackrisson et al . , 2006 ) .
clinicians who take care of an increasing number of women with screen - detected ductal carcinoma in situ should be aware that many of these women have inaccurate perceptions of the breast cancer risk .
in fact women with ductal carcinoma in situ , in spite of a better prognosis , have comparable perceptions of risk of recurrence and risk of dying of breast cancer as women with invasive disease and this causes persistent anxiety ( van gestel et al .
this review summarizes the factors that play in the construction of breast cancer worry and perceived risk . although physicians recognize the daily scenarios where women state that they do not wish to perform a breast self - examination out of fear of detecting a nodule as frequently as there are women who insist on having a yearly mammography , they have not taken up the challenge to rectify inappropriate health behaviour .
although physicians spent much time in counselling patients within the curative setting , counselling about preventive health care strategies including health risk communication and lifestyle management should be stepped up as an integral part of patient education .
as the evidence of benefit is lacking at the moment these actions should be taken within a setting of clinical research . | breast cancer risk assessment and communication are much neglected aspects of women s health care .
breast cancer is the most prevalent cancer - related disease that touches the deepest of a women s feelings and the subject thus attracts much of the attention of the media .
disease prevalence and media coverage are the roots of inappropriate breast cancer risk perception .
many women overestimate their personal breast cancer risk .
inappropriate risk perception precedes inappropriate health behaviour and it is pivotal to understand the underlying mechanisms in order to plan intervention .
whether interventions such as patient education through counselling and objective risk assessment are effective in restoring inappropriate breast cancer risk perception remains a question unanswered , but the tools to measure breast cancer risk are available and were validated . | Introduction
Breast cancer worry
Breast cancer risk perception
Factors underlying breast cancer risk perception
Media coverage and worry about breast cancer
Breast cancer risk calculation and communication
Worry and self-protective behaviour
Conclusion |
PMC4959407 | pneumocephalus is characterized by the presence of air within the cranial vault and can be subdural , subarachnoid , epidural , intraventricular , or intraparenchymal depending on the location of the air .
, pneumocephalus is asymptomatic ; however , rapid expansion of a trapped air cavity within the cranial vault can create a mass effect on the brain and result in intracranial hypertension .
this condition , termed tension pneumocephalus , is a neurosurgical emergency and if left untreated may cause rapid neurological deterioration , herniation , and death .
nitrous oxide ( n2o ) , often administered as a component of general anesthesia , has also been implicated in pneumocephalus . since the blood :
gas coefficient of n2o is almost 34 times more than nitrogen , it would diffuse from blood into a closed space ( such as cranial vault ) at significantly faster rate than the nitrogen / air would exit from the closed space into the blood .
this would result in increased volume of gas in the closed space . in a noncompliant space such as a cranial vault , the increase in volume is restricted .
hence , with increased amount of intracranial gas , there is a severe and rapid increase in intracranial pressure ( icp ) , especially with a preexisting pneumocephalus .
we present a case of intra - operative tension pneumocephalus secondary to n2o in a patient with preexisting pneumocephalus and a history of ventriculoperitoneal ( vp ) shunt and two right parietal craniotomies for resection of glioblastoma multiforme ( gbm ) .
a review of studies discussing the role of n2o in tension pneumocephalus has also been included .
the article heightens awareness among neurosurgeons and anesthesiologists of the potential risk of tension pneumocephalus , while using n2o especially in the context of preexisting pneumocephalus .
the patient was a 61-year - old male who was diagnosed with right temporoparietal gbm and treated with right temporoparietal resection and adjuvant chemo - radiation .
unfortunately , he developed recurrence of his tumor and was re - operated for recurrent tumor and ultimately started on avastin chemotherapy . during
a routine follow - up magnetic resonance imaging , he was found to have a right middle cerebral artery pseudoaneurysm for which he required another craniotomy for definitive obliteration of the pseudoaneurysm .
he then developed wound dehiscence with cerebrospinal fluid ( csf ) leak , which was likely from a combination of steroid use , prior irradiation , multiple operations through the same incision , and avastin use the latter , through its anti - angiogenic properties , is particularly harmful to healing incisions .
he underwent wound debridement with the removal of his bone flap and previously placed titanium hardware and a titanium cranioplasty was done along with multiple layered scalp closure .
he continued to have multiple episodes of csf leak over next few months with elevated spinal fluid pressure on multiple lumbar punctures .
given the diminished wound leakage with lumbar drainage , a vp shunt was placed for definitive csf diversion .
he was discharged after uneventful recovery , but returned 5 days later with acute onset left sided weakness on his upper and lower extremity along with decreased sensation on the entire left side .
a head computed tomography ( ct ) scan was obtained which revealed significant right - sided tension pneumocephalus beneath his craniotomy site .
he was emergently operated on for right parietal craniotomy for the relief of his tension pneumocephalus .
intra - operatively , he was found to have frank wound dehiscence and his previous craniotomy wound was debrided and replaced with a new titanium implant measuring 6 cm 6 cm .
he continued to have a nonhealing portion in the central portion of his incision with a small opening which was being managed conservatively .
unfortunately , he presented again to the emergency room with left sided numbness and weakness with gait instability .
a ct scan was done which demonstrated concern for acute pneumocephalus [ figure 1 ] .
he was taken to the operating room for exploration and large scalp rotational flap based on the superficial temporal artery for definitive closure of another episode of wound dehiscence .
however , while the patient was undergoing debridement and prior to entering his old resection cavity , he was receiving n2o as a part of his general anesthesia .
at this point , his brain was noted to be incredibly tense which was significantly different from the start of the operation . his blood pressure was significantly elevated and his heart rate dropped to 40 s raising the concern of tension pneumocephalus and cushing phenomenon .
the n2o was immediately discontinued and emergently the old tumor cavity was entered with a curved snap followed by a forceful large whooshing sound , indicating escape of a large amount of trapped intracranial air ( cerebral flatulence ) . following this maneuver ,
his heart rate returned to the normal range and the vital signs were reversed from the cushing phenomenon .
the remainder portion of the surgery was uneventful and the wound was covered with superficial temporalis artery - based flap .
the patient had an unremarkable postoperative recovery and discharged 5 days postoperatively . however , his tumor continued to progress resulting in worsening neurological dysfunction and he died 24 months later from progressive illness .
sagittal ( a ) and axial ( b ) sections of computed tomography scan demonstrating pneumocephalus in the index patient
nitrous oxide is used in combination with other general anesthetic agents since it helps in rapid onset and termination of anesthetic effects and has minimal effect on the blood pressure .
the rapid onset of anesthesia with n2o can be attributed to smaller blood - gas partition coefficients ( 0.47 ) compared to other inhalational agents .
blood - gas partition coefficient is defined as the ratio of the concentration in blood to the concentration in gas that is in contact with that blood , when the partial pressure in both compartments is equal . during the anesthetic administration
these spaces mainly contain nitrogen ( from air ) , whose low blood / gas partition coefficient ( 0.013 ) limits its removal by blood .
thus , the rate of entrance of n2o into the closed gas spaces is significantly more than the exit of nitrogen from the closed gas spaced to the blood stream , resulting in a rapid increase in volume . in a nondistensible cavity like cranial vault ,
such rapid increase of volume could result in tension pneumocephalus especially with preexisting pneumocephalus .
based on this argument , it is recommended to avoid n2o altogether or discontinue its use prior to the dural closure .
the rationale behind discontinuing the usage of n2o until dural closure is that a pneumocephalus formed intraoperatively would mainly contain n2o , which would then be reabsorbed rapidly into the blood on discontinuation of n2o due to the concentration gradient .
the most significant study refuting the role of n2o in tension pneumocephalus was published by domino et al . who demonstrated that there was no difference in the icp when the patients undergoing elective craniotomy were randomized to continuing n2o following dural closure versus replacing the n2o with nitrogen before the dural closure .
in another study published by friedman , discontinuation of n2o 30 min before the dural closure did not seem to have any preventive effect on tension pneumocephalus . however , artru reported more than two - fold increase in the icp on continuation of n2o on dural closure which got back to normal reference range on discontinuing n2o .
few other studies also demonstrated a temporal increase in icp with n2o intra - operatively and reversal of increased icp on discontinuation of n2o .
since then , n2o has been implicated as a cause of tension pneumocephalus in other case reports . a history of multiple craniotomies , radiation to the brain , steroid use , and avastin administration contributed to wound dehiscence in our patient , which resulted in a communication between the intra - cranial resection cavity and the external environment .
negative pressure created by the vp shunt placed to divert csf away from the incision and prevent meningitis further conspired to the ingress of atmospheric air into intracranial cavity resulting in two episodes of tension pneumocephalus in our patient .
however , during the second episode , there was a definite acute increase in icp after inadvertent n2o inhalation as evidenced by extremely tense brain and a clear cushing response .
return of vital signs to normal range after releasing the pressure and discontinuation of n2o further confirms the role of n2o in tension pneumocephalus .
our study supports the association of n2o with tension pneumocephalus especially in the setting of preexisting pneumocephalus .
while it might be reasonable to use n2o in elective craniotomy cases , it should be discontinued prior to the dural closure and it should be avoided in the setting of known pneumocephalus . | rapid buildup of gas within the cranial vault can result in a life - threatening condition known as tension pneumocephalus , necessitating immediate surgical intervention .
nitrous oxide ( n2o ) , a commonly used inhaled anesthetic , is associated with the development of tension pneumocephalus and its role in neurosurgical procedures has been debated in the literature .
we present a case of tension pneumocephalus with preexisting pneumocephalus secondary to the usage of n2o as an inhaled anesthetic .
included is a literature review of studies discussing the role of n2o in the development of tension pneumocephalus .
n2o is associated with tension pneumocephalus especially in the setting of preexisting pneumocephalus .
tension pneumocephalus can manifest as cushing response and immediate decompression is life - saving .
nitrous oxide should be used cautiously in neurosurgical procedures , especially with preexisting pneumocephalus . | INTRODUCTION
CASE REPORT
DISCUSSION
Financial support and sponsorship
Conflicts of interest |
PMC4428008 | over the past decades
our atomistic understanding of protein structure
and function has been revolutionized by x - ray crystallography and
nmr spectroscopy , and magnetic resonance techniques have provided
information on the time scales of protein conformational dynamics .
however , until recently , no direct experimental techniques existed
that could combine atomistic structural information with time resolution
sufficient to observe the intrinsic functional time scales of many
proteins . among the new approaches to studying protein conformational
dynamics ,
infrared ( ir ) spectroscopy of amide i vibrations ( the amide carbonyl
stretch between 1600 and 1700 cm ) has seen a surge
of interest as a result of advances in 2d ir spectroscopy and structure - based
modeling , which provides an atomistic interpretation of protein ir
spectra .
amide i vibrations are delocalized , involving the concerted vibration
of numerous coupled carbonyls of peptide units of the protein backbone .
the sensitivity of the coupling between carbonyl groups to their relative
position and orientation in space means that the amide i band is influenced
by the underlying symmetry , size , and backbone structure of the protein .
though interpretation of protein ir spectra is often complicated by
the presence of many broad peaks in a small frequency window , a new
generation of atomistic spectral modeling that draws from molecular
dynamics ( md ) simulations of the protein provides new avenues by which
to interpret these experiments . in this context , the molecular origin of the observed high ion
throughput ( > 10 ions s ) and highly
selective transport of k ions of na ( > 1000:1 ) in the bacterial potassium channel kcsa is still
hotly debated .
traditional models of the selectivity
and transport assume the selectivity filter is inflexible and that
differences in the ion binding can be explained by a geometry optimized
to bind k ions ( the snug - fit model ) .
this is in contrast to other proposed explanations , where significant
fluctuations in the selectivity filter are invoked .
more recently , simulations have suggested the assumption
of alternating k ions and water molecules transporting
through the selectivity filter may be incorrect , and the k ions are more densely packed than previously thought .
what is clear , however , is that understanding
the behavior of the selectivity filter is essential to understanding
k transport in kcsa , which requires experimental characterization
of the dynamical interaction between the ions and the selectivity
filter .
( a ) structure of the transmembrane region of kcsa highlighting
the extended filter region ( red ) and k ions in the ( s1 ,
s3 ) configuration ( purple ) .
( b ) schematic illustration of the ion
occupancy within the selectivity filter for the three configurations
investigated .
the tvgyg structural
motif constituting the selectivity filter
is a highly conserved sequence among potassium channels .
each subunit of the kcsa tetramer consists of
an intracellular gating domain and a 15 narrow pore
domain in which the selectivity filter resides ( figure 1a ) .
the carbonyl groups in this 20-residue filter define a
set of binding sites , labeled s1s4 , in which k binds in an 8-fold coordinated geometry ( figure 1b ) .
x - ray crystallography indicates that under physiological
concentration k can bind in one of two configurations ,
either at sites s1 and s3 or at sites s2 and s4 .
water is assumed to occupy the respective other binding
sites for each configuration , though concerns have been raised over
whether this is the case during transport .
na has been observed to bind primarily in an in - plane
position ( figure 1b ) , though there is still
some debate over which residues coordinate na .
here we demonstrate on three well - characterized
examples of ion - binding
in kcsa that the ion - induced conformational changes of kcsa can be
characterized using amide i ir spectroscopy in combination with computational
modeling . using attenuated total reflection ( atr ) ir spectroscopy
,
it was recently shown that significant differences in the amide i
spectrum can be observed when the buffer cation composition is varied
between k and na , but the origin of these
differences remained elusive . here
we
present new difference ir spectra between k and na buffers designed to prepare the binding configurations pictured
in figure 1b and use structure - based spectroscopic
modeling that draws on md simulations to interpret the data .
our analysis
reveals the sensitivity of amide i vibrations of the selectivity filter
and pore region to the conformational changes which occur on binding
k or na .
the full - length kcsa
channel ( 167 amino acid length monomer ) , containing a 6xhis - tag inserted
directly after met1 and cloned into prsf1 ( merck ) , was expressed in escherichia coli bl21 ( de3 ) cells on induction with 1 mm
iptg ( isopropyl--d - thiogalactopyranoside ) .
bacteria
were collected and resuspended in buffer a ( 100 mm nacl , 50 mm kcl ,
50 mm sodium phosphate , ph 8.0 ) solution and lysed by sonication in
the presence of a protease inhibitor cocktail ( complete edta - free ,
roche ) .
the protein was solubilized by 1 h incubation with 20 mm n - dodecyl--d - maltopyranoside ( ddm , avanti )
which is known to not interfere with the protein s spectral
features in the amide i band , followed by centrifugation at 30000 g for 30 min .
the supernatant was incubated with co affinity resin ( thermo ) in the presence of 20 mm imidazole
for 1 h at 4 c , loaded onto a column , and washed with 40 mm
imidazole and 1 mm ddm in buffer a. the protein was eluted with 400
mm imidazole and 1 mm ddm in buffer a and extensively dialyzed versus
nacl buffer b ( 150 mm nacl , 1 mm ddm , 10 mm hepes , ph 7.5 ) to remove
k ions . to assist with sample transparency in the
infrared , all exchangeable protons of kcsa were deuterated by multiple
concentration dilution cycles with d2o - based buffer
b in a 10 kda mwco spin concentrator ( amicon , millipore ) over the
course of several days until the h2o content was below
1% as determined by absorption at 3400 cm on a
ftir spectrometer ( thermo ) .
temporary heating of the sample to 37
c and subsequent incubation at 4 c ensured proper deuteration
of the buried parts of the protein .
the desalted and deuterated kcsa
was concentrated to 78 mg / ml , frozen in liquid n2 , and stored at 80 c until further use .
correct secondary
structure of kcsa in k - free buffer b was confirmed via
cd spectroscopy .
the fraction of correctly folded tetrameric protein
was estimated by loading unexchanged samples of kcsa on a normal sds - gel ,
and from the densiometry of the monomeric and tetrameric band at approximately
18 and 75 kda , it was estimated that > 95% of the sample was composed
of correctly folded tetrameric protein . to introduce k ions into the sample
in a controlled fashion , the kcsa was
subjected to further concentration dilution cycles with a modified
buffer b ( 150 mm kcl , 1 mm ddm , 10 mm hepes pd 7.9 in d2o ) . for the ftir absorbance spectra ,
approximately 2.5 l of kcsa sample in different salt conditions
was sandwiched between two 1 mm thick caf2 windows ( cening
optics ) , separated by a 50 m ptfe spacer . for each sample ,
2048 averages were collected on a nicolet 380 ftir spectrometer ( thermo
scientific ) at 1 cm resolution against a background
of dry air at 20 c .
ftir spectra of each buffer , without protein ,
were also collected in order to subtract the background d2o absorption from the kcsa spectra .
difference spectra are calculated
as ftir spectra of ( kcsa in kcl , buffer subtracted )
( kcsa
in nacl , buffer subtracted ) . in
order to obtain a molecular interpretation of the spectral features
above
, we used a model of the amide i vibration that maps structure
vibrational frequencies and couplings between oscillators .
this procedure draws on md simulations of kcsa for each ion - binding
configuration of interest and uses it to parametrize a quantum - mechanical
local mode hamiltonian for the coupled peptide units of the protein
backbone , which govern the vibrational spectroscopy of the amide i
band .
md simulations were
performed using the gromacs software package and the charmm36 force
field .
we started with the full - length tetrameric kcsa crystal structure
by uysal et al .
( pdb accession code 3eff ) and embedded this
in a dmpc membrane using the g_membed function in
gromacs . the protein
this system was equilibrated
for a total of 2 ns ( 1 ns nvt equilibration , 1 ns npt equilibration ) ,
before running a further 7 ns trajectory to use as a basis for the
spectral calculations .
all stages of the simulation were run with 2 fs time steps , nos hoover
thermostat , semi - isotropic parrinello
rahman pressure coupling
( where appropriate ) , and the lincs constraint algorithm . to compare
the effects of different salts on the spectra , we replaced the contents
of the selectivity filter prior to equilibration with either 2 k ions and 2 water molecules ( with k in either
s1 , s3 or s2 , s4 , water occupying with remaining sites ) or 2 na and 2 water molecules ( using the positions from thompson
et al . ) . during the md production run ,
the k ( na ) ions were held in place with harmonic
position restraints .
individual peptide groups and their associated amide i
transition dipoles are identified in structures sampled from md simulations .
a local mode hamiltonian is parametrized from the structure using
the molecular electric field to set the diagonal site frequencies
and off - diagonal couplings between sites .
the delocalized eigenstates
and their corresponding transition dipole moments are used to calculate
the ir spectrum and use doorway modes to visualize the vibrations .
figure 2 illustrates the simulation protocol
used to link the experimentally
observed ir spectra with md simulations , as reviewed in detail in
ref ( 10 ) .
the local
mode hamiltonian is constructed in the basis set of the 668 backbone
peptide units and was parametrized using frozen configurations of
the protein and water .
the structure determines the position and orientation
of each peptide unit , and the corresponding amide i transition dipole
moment for that site is positioned in the plane of the peptide unit
between the carbon and oxygen .
the diagonal elements of the hamiltonian
are the vibrational transition energies ( site frequencies ) between
the = 0 and = 1 states of the amide i vibration .
hydrogen - bonding
interactions with the carbonyl oxygen downshift these frequencies
by an amount that depends on the local electrostatic environment .
for this calculation
, we used the empirical spectral map in which
the frequency of each oscillator is correlated with the electric field
acting on the oxygen of the peptide unit along the carbonyl bond developed
by reppert et al .
the hamiltonian s
off - diagonal elements , or couplings , are determined by the spatial
configuration of the peptide units . in the case of through - space
interaction ,
we used transition dipole coupling , which depends on the distance
and angle between the two oscillators . for bonded peptide units ,
the through - bond coupling was obtained
from the nearest - neighbor coupling model developed by jansen et al . , which maps the and angles of
the bonded peptide units onto a coupling .
diagonalizing this hamiltonian
led to eigenstates and eigenvalues which were used to calculate an
ftir spectrum of individual configurations of protein and water .
since
parameters used depend intimately on the sub - angstrom configuration
of water and protein , we generate hamiltonians for structural snapshots
at 1 ps intervals of the trajectory . in the case of the k calculations , an average of the s1 , s3 and
this approach is known
to qualitatively reproduce spectral features . while it does not quantitatively
reproduce exact peak frequencies , line widths , or absolute intensities ,
the relative frequency shifts and intensities of resonances are reliable
metrics for comparison .
our calculations also neglect side chain vibrations
that may absorb in the 16001700 cm region ,
such as the amide vibrations from glutamine and asparagine and the
cn vibrations of arginine . while side chains certainly contribute
,
it is at a significantly lower level than amide i , and their frequencies
are well - known for the interpretation of experimental spectra .
although
the spectral map we use here neglects side chains , it is important
to note that other maps have been developed which are developed with
the aim of capturing the vibrations of side chain amide groups ( asp ,
glu ) , though the application of this
maps to side chains in larger proteins has not been characterized .
the diagonalization of the local
mode hamiltonian provides not only the energies of the vibrational
modes but also the corresponding eigenstates .
interpretation of these
eigenstates , however , is hampered by the sheer number of them ( > 30 000
in this case ) , even for a small subset of the protein structure at
a reduced sampling rate .
analogously to how principal component analysis
can be used to describe which input variables are principally responsible
for observed trends , doorway mode analysis shows which amide units
contribute most significantly to the ir spectrum in a specific frequency
window . to obtain a set of representative
strongly ir - active states from a subset of eigenvectors ,
we apply
the doorway mode analysis in ref ( 34 ) , using a 3 cm window
about the stated center frequency .
though outlined in detail
in ref ( 34 ) , we briefly
review doorway mode analysis .
the aim is to find a transformation
matrix i , which will transform
vectors in the site basis to the doorway basis .
the doorway mode basis
is one where most of the intensity of the transition is contained
within the first three modes .
first , we define the matrix ti , a subset of the transformation
matrix obtained from the eigenvalue decomposition of the hamiltonian .
the subset is defined to only include eigenstates with eigenvalues
within the frequency range ri .
a singular value decomposition ( svd ) of the eigenstates of
the hamiltonian transforms them to a basis where each subsequent component
contributes less to the transition intensity . combining the svd transformation
matrix ui with our eigenstate
transformation matrix ti yields the overall transformation matrix i = uiti . with this matrix ,
as shown in figure 3 , the ftir spectra of
kcsa in both kcl and nacl
show a broad peak centered at 1655 cm , consistent
with the high -helix content of the protein .
the asymmetric
line shape features a broad shoulder at lower frequencies , which includes
contributions from the side chain absorptions of asp , arg , gln , tyr ,
trp , and gln residues .
to specifically
capture the spectral changes which arise solely from the binding of
ions by the selectivity filter , we focus on the analysis of the difference
spectra , i.e. , the spectrum of kcsa in kcl buffer minus the spectrum
of kcsa in nacl buffer .
the ftir difference spectrum , in figure 3 , is highly structured , with several distinct features .
of these features , we select three for analysis ( labeled 1 , 2 , and
3 ) because they are the largest amplitude differences within the amide
i window , are not overlapping other features , and are well - reproduced
by our calculations .
peak 1 is a strong and broad negative peak at
1625 cm , peak 2 is a strong positive band at 1660
cm , and peak 3 is a less intense narrow positive
band at 1680 cm . given that the amide i vibrations
of 668 peptide units contribute to the full ftir spectra , and that
only 16 carbonyls are in direct contact with the bound ions at any
one time , it is not surprising that the largest spectral changes between
kcl and nacl buffers are on the order of 4% .
nevertheless , this change
is large enough to suggest that a significant number of amide carbonyls
are sensitive to the identity of the bound ion .
experimental spectra
of kcsa with kcl or nacl buffers and the difference
spectrum ( kcl nacl ) illustrating the correspondence of key
features .
our experimental ftir spectra
and difference spectrum are consistent
with the difference atr infrared spectra reported recently by futurani
et al .
our peak positions differ by < 5
cm , but this readily explained by our use of deuterated
sample as opposed to experiments in h2o , and the differing
chemical environment of the sample ( ddm micelles in this work versus
asolectin bilayer ) .
simulated
ftir and ftir difference spectra , shown in figures 4a and 4b , respectively , are able to
qualitatively reproduce the key experimental features .
the full ftir
center frequency and line width are in good qualitative agreement ,
though as noted earlier , quantitative agreement should not be expected
from these calculations .
the experimental line shape is not fully
reproduced by calculation , notably in the 16201650 cm region , in which there is a shoulder in the experimental
spectra which is not captured in the calculations .
we attribute this
primarily to side chain absorptions in this region ( namely arg at
1605 cm , tyr at 1615 cm , trp
at 1618 cm , and gln at 1640 cm ) , which are not included in our calculation
since vibrational frequency and coupling models do not presently exist . comparison
of the experimental and calculated spectra for kcsa
in kcl buffer ( a ) and the difference spectra ( b ) .
peaks 1 , 2 , and
3 ( see main text ) are highlighted in red , green , and blue respectively ,
and the side chain region is highlighted in orange . in the calculated ftir difference spectrum ,
we
find the three features
highlighted in the experimental data are reproduced well both in peak
frequency ( differing by only a few wavenumbers ) and in relative intensity .
the reproduction of both the frequency and intensity in our calculations
suggests the nature of the delocalized vibrational modes is well captured .
the most notable region of mismatch between the experimental and
calculated difference spectra is again the 16201650 cm region .
while this could reflect a limitation of
the vibrational coupling model , is it more likely to reflect the neglect
of side chain vibrations in our calculations .
previous work has shown
that the y78f mutant , which shows similar functional behavior to the
wt protein , gives rise to a difference
spectra distinct from the wild - type , with the most notable differences
being in the 16201650 cm region .
however , we note that , while the side chain
absorptions may indeed contribute significantly to the experimental
difference spectrum , our analysis focuses on peaks which are clearly
reproduced by the carbonyl - only modeling , indicating that a discussion
of these peaks in terms of delocalized amide i vibrations only is
appropriate . comparison of calculated difference spectra for different
subsections
of kcsa .
the filter - only calculation is unable to reproduce the features
observed in the extended filter region , the full transmembrane region ,
and the experimental spectra . to determine the participation of different regions of the
protein
to the spectral features observed , we calculated difference ftir spectra
using various subdomains of the full tetrameric protein complex .
these
sections are ( a ) the four tvgy chains comprising the selectivity filter ,
( b ) the extended filter
region comprising the selectivity
filter and the four pore helices , and ( c ) the entire transmembrane
region of the protein .
we found that the calculated ftir spectra for
the transmembrane region and the extended filter differed only in
the intensity of some features but were otherwise consistent with
each other .
the selectivity filter alone could not reproduce the difference
spectra , notably the 1660 cm peak , suggesting
the larger protein complex and coupling between the monomer units
are actively participating in generating the observed experimental
spectra .
calculated spectra for the extended filter region for the various
ion - binding configurations .
the calculated difference spectrum is
shown in gray . in order to elucidate
the origins of the features in the difference
spectra
, we show the calculated ftir spectra for the individual ion - binding
configuration of the extended filter region in figure 6 . from this
, we see that peaks 1 and 3 come from distinct
peaks in the na- and k - bound calculations ,
respectively , while peak 2 comes from a change in intensity and line
shape of a peak present in the kand na calculations . taken in conjunction with the calculations
on truncated regions of the protein
, this suggest the 1660 cm peak in the difference spectra ( peak 2 ) arises from
changes in the -helices surrounding the pores .
the nature of
these structural changes is discussed below . to visualize the molecular
origins of the vibrations which contribute
to the difference spectrum
, we apply a doorway mode analysis to our
calculated eigenvectors for the extended filter region .
the first at 1630 cm corresponding to peak 1 , 1660 cm ( peak 2 ) , 1674
cm ( peak 3 for s1 , s3 binding ) , and 1680 cm ( peak 3 for s2 ,
on the basis of this calculation , the four frequencies can be assigned
to four modes with distinct vibrational motions involving amide units
within the extended filter region ( see figure 7 ) .
the amide units contributing most significantly to the 1630 cm mode , designated the b - site mode ,
are t75 carbonyls with some contribution from the carbonyls of the
pore -helices .
these carbonyls and the direction of their transition
dipoles are identified with outlined arrows in figure 7a .
carbonyls on opposite monomers of the filter mode vibrate
out - of - phase , contracting on one side and extending on the other ;
however , the net transition dipole moment of all oscillators constructively
adds so that the total transition dipole moment ( green arrows ) is
orthogonal to the filters pore .
the 1660 cm mode
( labeled helix ) is present in all cases and is constituted
almost exclusively by vibrations from the pore -helices .
carbonyls
in all monomers are in - phase and contribute to an overall dipole moment
that is oriented along the pore ( figure 7b ) .
the 1674 cm mode of the ( s1 , s3 ) configuration
is termed s3 , due to the strong involvement of the
t75 and v76 residues in the filter which define the s3 binding site
( figure 7c ) .
similarly the 1680 cm mode of the ( s2 , s4 ) configuration is termed
s2
due to the large amplitude in the v76 and g77 residues which define
the s2 binding site ( figure 7d ) .
visualizations
of calculated doorway modes of the extended filter
region presented as side view ( left ) and top view looking down the
pore ( right ) .
color indicates the relative sign of the co vibrational
motion ( contraction / extension ) , while the color intensity indicates
its amplitude .
outlined arrows indicate the direction of the transition
dipole moment of individual carbonyls contributing most to the mode .
the direction of the net transition dipole moment for the whole extended
monomer ( ) is shown with dotted green arrows .
the colors in
circles on the right indicate the overall displacement of the selectivity
filter carbonyls in that strand . na and k ions
are blue and purple , respectively .
the extent of delocalization
determines how specific an ir resonance will be to interactions at
a particular site of the protein .
the extent of delocalization of
vibrations is determined by two competing factors : the strength of
couplings between oscillators ( j ) and the variation
in individual site frequencies ( ) .
increasing j and decreasing leads to increasing delocalization .
in the case of the helix mode ( peak 2 )
; the near - uniform
electrostatic environment of the -helix minimizes
while also locking the oscillators into a geometry which gives rise
to significant coupling between oscillators , resulting in a vibrational
mode which is spread over the length of the pore helix .
we believe
the delocalized nature of the helix mode is the key
to why peak 2 is observed in the difference spectrum .
slight changes
in the structure of -helices , such as a slight bending of the
helix from the helical axis , have been predicted to change the intensity
and line widths of ir peaks .
the increase in intensity
at 1660 cm in the k bound data suggests
that the -helices deviate further from an ideal -helix
geometry than the na bound structure .
the remaining
three modes we identify , however , are more local
in character , because the electrostatic environments that arise from
binding k and na strongly and selectively
influences the frequency of the coordinating carbonyls .
for the b mode , the proximity of na lowers the frequency of the four t75 carbonyls by > 25
cm relative to the v76 carbonyls .
considering
the weaker
coupling between t75 and v76 carbonyls ( j < 8
cm ) , binding nain plane with the t75 residues largely decouples these vibrations from the
rest of the extended filter , creating a site - specific infrared transition .
for the s3 mode , the similar frequencies of the k - binding
v76 and g77 carbonyls ( 10 cm ) and their strong coupling ( j > 8 cm ) lead to a vibrational transition that is largely localized on the
eight carbonyls defining the s3 site .
similar factors seem to be responsible
for the s2 mode which strongly involves the t75 and v76 residues . in reality , all of the vibrational motions we observe are neither
localized to a single oscillator nor delocalized over the entire protein
but have varying degrees of delocalization .
furthermore , the doorway
modes do not reflect how these characteristic vibrational motions
influence one another or act in concert as would be appropriate for
developing a dynamical picture of the protein ion interaction .
our results show that ir spectroscopy
coupled with structure - based
spectral modeling can be used as a sensitive tool to investigate functionally
relevant conformational changes in large membrane proteins and , furthermore ,
detect subtle local changes .
we have demonstrated this capability
in this study by resolving the different environments of the binding
sites in the selectivity filter of kcsa and identified unique ir signatures
of ion binding states .
structure - based spectroscopic modeling of the
extended filter region allowed us to perform the doorway mode analysis
which established the link between our experimental data and the ion - induced
structural rearrangements in the protein .
we found different vibrational
couplings for the k- and na - bound states between
the tvgy core of the channel and the pore helices , which suggest that
the helices are not simply a structural support but may indeed play
a central role in determining the different transport properties of
kcsa for these ions .
it has previously been suggested that the pore
helices may act as a mediator between selectivity filter and activation
gate , with particular emphasis on the mechanism of c - type inactivation .
our work suggests the role of the pore helices may extend beyond
this and even have a role in determining the energetics and dynamics
of ion binding .
to what extent these vibrational couplings map onto
functionally relevant interactions could be further studied by mutations
that alter the interface between the pore helix and selectivity filter .
though our work was not designed to directly
study kcsa under transport
conditions , some of our conclusions have important implications in
this area .
we find experimental evidence for strong interactions between
the t75 carbonyls and na . in their recent paper ,
this suggests
perhaps that na ions are not conducted , in part , because
of the influence of the na on the coordinating carbonyl
dynamics ( rigidifying them ) . on the technical
side , one possible extension of our work here
this will
capture the dynamics of the protein in real time , shedding light on
the underlying molecular mechanisms of the early time events in binding
in transport , thus far inaccessible to standard techniques .
this provides
the tantalizing possibility that mechanisms suggested by md , such
as those for c - type inactivation , could be directly
tested . | the effect of ion binding in the
selectivity filter of the potassium
channel kcsa is investigated by combining amide i fourier - transform
infrared spectroscopy with structure - based spectral modeling .
experimental
difference ir spectra between k+-bound kcsa and na+-bound kcsa are in good qualitative agreement with spectra
modeled from structural ensembles generated from molecular dynamics
simulations .
the molecular origins of the vibrational modes contributing
to differences in these spectra are determined not only from structural
differences in the selectivity filter but also from the pore helices
surrounding this region . furthermore , the coordination of k+ or na+ to carbonyls in the selectivity filter effectively
decouples the vibrations of those carbonyls from the rest of the protein ,
creating local probes of the electrostatic environment .
the results
suggest that it is necessary to include the influence of the surrounding
helices in discussing selectivity and transport in kcsa and , on a
more general level , that ir spectroscopy offers a nonperturbative
route to studying the structure and dynamics of ion channels . | Introduction
Methods
Results and Discussion
Conclusions |
PMC3810434 | airborne microorganisms are typically present in the air we breathe and are potential causes of infectious diseases .
influenza viruses are among the respiratory pathogens that can be transmitted by airborne routes [ 14 ] .
breathing , coughing , sneezing , and talking during the course of influenza generate a cloud of airborne particles containing influenza virus .
the airborne particles have diameters that range from a few millimeters ( large droplets formed during coughing and sneezing ) to submicron ( < 1 m ) ( formed during breathing ) . small particles ( 5 m ) including droplet nuclei from evaporated larger particles can remain airborne for hours ( these concepts and their relevance to influenza viruses are discussed in [ 1 , 314 ] ) . presently , the us centers for disease control and prevention ( cdc ) and the world health organization ( who ) state that influenza virus transmission occurs mostly by large - particle respiratory droplets that travel only a short distance from the source ( 1.829 m ( 6 feet ) according to the cdc and 1 m ( 3.3 feet ) according to the who ) [ 1517 ] .
noteworthy , deposition of influenza virus into the lungs ( as small particles ) versus the upper respiratory tract ( as large droplets ) may increase infection risk and illness severity [ 3 , 9 , 1820 ] . assessments of microbiological air quality are useful for studies of the airborne transmission of pathogens and of other biological particles in the air that may cause noninfectious diseases ( such as allergy to pollen ) .
the two principle methods of assessing microbiological air quality are passive monitoring and active sampling .
passive monitoring is typically performed using settle plates , which are opened and exposed to the air for a given time , then incubated and resulting bacterial and fungal colonies are analyzed .
active sampling is performed with a microbiological air sampler that mechanically draws a known volume of air over , or through , a particle collection device .
air samplers are also used to collect airborne viruses , but it is difficult to do so primarily due to their small size and relatively low concentration in ambient air .
also , most of the available bioaerosol samplers are not suitable for the collection of viruses for reasons that include ( a ) they typically run for short periods of time ( min ) , making it difficult to capture large volumes , ( b ) most do not separate particles by size , and ( c ) , they are inefficient at collecting submicron particles [ 2325 ] . the pcis separates airborne particles in a cascading fashion and simultaneously collects the size - fractionated particles by impaction on polytetrafluoroethylene ( ptfe ) filters .
it has collection filters on four impaction stages ( a d ) , and optionally , an after - filter can be added onto a 5th stage .
the pcis is designed to separate and collect airborne particulate matter above the cutpoint of five size ranges : > 2.5 m ( stage a ) , 1.0 to 2.5 m ( stage b ) , 0.50 to 1.0 m ( stage c ) , 0.25 to 0.50 m ( stage d ) , and < 0.25 m ( collected on an after - filter ) ( figure 1 ) .
ptfe filters , better known as teflon filters , can collect particles at high efficiency above the cutpoints without the need for coatings .
lastly , for the efficient collection of airborne influenza virus , the filters are not prewetted with methanol prior to use .
made out of glass , the skc biosampler is purpose - designed to collect particles of a size range that would go through human nasal passages .
the skc biosampler has three 0.630 mm tangential sonic nozzles ; air passing through the nozzles creates a swirling flow of collection medium during sampling .
compared to other impingers , particle skip and damage to the collected microbial agent are potentially minimized by the collection swirling liquid . whereas the skc biosampler has worked well for the collection of airborne bacteria and fungi in the field ,
it is nevertheless inefficient at recovering submicrometer and ultrafine virus aerosols , with collection efficiencies of < 10% for particles in the 30 to 100 nm size range . nevertheless ,
mostly because significantly better alternative devices are not readily available , the skc biosampler has been used for the collection of airborne influenza virus [ 23 , 2931 ] .
an adult and a child living in an apartment in gainesville , florida , were diagnosed with influenza caused by type a influenza virus . as both occupants had been vaccinated against influenza a and b viruses in november of 2012 ,
there was a possibility that they were infected with a new influenza virus strain significantly different than from those in the vaccines .
we ( opportunistically ) evaluated airborne material from the apartment that had been collected on pcis filters and in skc biosampler liquid collection media for the presence of viable influenza a virus and/or viral genomic rna .
the dwelling that was evaluated in this report is a single - family four - bedroom apartment in gainesville , florida , usa , that has its own heating , ventilation , and air conditioning ( hvac ) system .
passive air exchange with the outside was minimal , as windows and an entry door were kept closed due to cold weather .
relative humidity ( rh ) and temperature ( t ) were measured using an electronic nist traceable rh and t meter ( thermofisher , catalog number 11 - 661 - 19 ) .
virology work was performed in a biosafety level 2-enhanced laboratory at the university of florida .
the skc ( skc , inc . , eighty four , pa , usa ) biosampler ( skc catalog number 225 - 9595 ) is a widely - used device for microbiological air quality assessments .
skc biosamplers were operated with 115 volt vac - u - go sampling pumps ( skc , inc . , catalog number 228 - 9605 ) .
the pumps were warmed up by running them for 5 min prior to use . to protect the pumps against moisture , in - line vapor traps ( skc inc . , catalog number 225 - 22 - 01 )
skc biosamplers were sterilized by autoclaving prior to use then filled with 15 ml of sterile phosphate buffered saline with calcium and magnesium ( pbs ) , 0.5% w / v purified bsa fraction v ( life technologies , grand island , ny , usa ) , and 0.25% ( v / v ) molecular - grade antifoam agent b ( sigma - aldrich , inc . , st .
the operating pressure was measured using the pump 's built - in manometer ( pressure gauge ) ; the sampling flow rate was 12.5 l / min , determined using a flow meter attached to the inlet of the skc biosampler prior to use .
this suggested that the skc biosampler was operating normally , because at normal atmospheric conditions , the flow rate should always be around 12.5 l / min when there is a pressure drop of at least 0.5 atm . for each unit , a collection time of 30
min was used to sample approximately 375 liters ( 0.375 m ) of air .
, catalog number 225 - 370 ) units were used with leland legacy pumps ( skc , inc . ,
cat number 100 - 3002 ) and operated at a flow rate of 9 l / min .
polytetrafluoroethylene ( ptfe ) filters ( 25 mm , 0.5 m pore , skc , inc .
ptfe after - filters ( 37 mm , 2.0 m pore , skc inc .
the pump 's operating flow rate was calibrated by measuring volume displacement using a defender primary standard calibrator ( skc , inc . ,
a collection time of 41 min 36 sec was used to sample approximately 0.375 m of air .
pcis and skc biosampler units were tandemly situated beneath ceiling vents so that air samplings would be made in areas with active air - flow ( figure 2 ) .
the intake openings of the air samplers were at a height of 1 m and perpendicular to the ground , facing the patients . for comparisons of indoor to outdoor counts , outside air
was evaluated using a pcis and an skc biosampler in a courtyard outside the apartment .
the patients , an adult female ( age 50 ) and young female ( age 10 ) , were diagnosed with influenza at a local clinic in early february 2012 .
nasal discharges from both were positive for influenza a virus using a binax now flu a and flu b test ( binax inc .
both individuals exhibited classic signs of influenza , starting with a sudden onset fever ( 40c ) , quickly followed by body ache , chills , dry cough , headache , malaise , muscle ache , pain on motion of the eyes , photophobia , red ( and warm ) flushed skin , sneezing , sore throat , watery eyes , and a feeling of severe weakness .
less characteristically , the adult but not the child suffered from diarrhea during the first day of illness . the adult had been vaccinated against influenza by injection , and the child by inhaled vaccine , the previous november ( 2012 ) .
neither individuals had underlying health conditions and were otherwise healthy and physically fit . within 30
min of collection , virus collection media in the skc biosamplers was concentrated to a volume of about 400 l by ultrafiltration using amicon ultra 15 filter units with a molecular - weight cut off of 100 kd ( millipore , bedford , ma , usa ) .
the concentrated skc biosampler fluids were adjusted to a volume of 500 l by addition of pbs with 0.5% w / v bsa fraction v , and were stored at 80c .
the pcis filters for virus tests were individually scraped with flocked swabs wetted with pbs with 0.5% w / v bsa fraction v then each swab eluted into separate 10 ml aliquots of pbs with 0.5% w / v bsa fraction v. the eluates were individually concentrated to a volume of about 400 l by ultrafiltration using amicon ultra 15 filter units , the volume adjusted to 500 l by the addition of sterile pbs with 0.5% w / v bsa fraction v and the concentrated pcis fluids stored at 80c .
mdck ( ccl-34 ) and mv1 lu ( ccl-64 ) were obtained from the american type culture collection ( manassas , va , usa ) and were propagated as monolayers at 37c and 5% co2 in advanced dulbecco 's modified eagle 's medium ( admem ) ( invitrogen corp . ,
carlsbad , ca , usa ) supplemented with 2 mm l - alanyl - l - glutamine ( glutamax , invitrogen corp . ) , antibiotics ( psn ; 50 g / ml penicillin , 50 g / ml streptomycin , 100 g / ml neomycin ( invitrogen corp . ) ) , and 10% ( v / v ) low igg , heat - inactivated gamma - irradiated fetal bovine serum ( hyclone , logan , utah ) .
mdck - siat2,6-uf and mv1 lu - siat2,6-uf are cell lines prepared in this laboratory from the atcc mdck and mv1 lu cells and were propagated as described for atcc cells .
mdck - siat2,6-uf and mv1 lu - siat2,6-uf overexpress influenza virus 2,6-linked sialic acid receptors .
prior to use , all cell lines were treated for 3 weeks with plasmocin and verified free of mycoplasma dna by pcr .
aliquots ( 50 l ) of concentrated fluids from the skc biosampler and pcis filters were inoculated onto mdck , mv1 lu , mdck - siat2,6-uf , and mv1 lu - siat2,6uf in serum - free dmem otherwise supplemented as previously described plus l-1-tosylamido-2-phenylethyl chloromethyl ketone ( tpck)-treated mycoplasma- and extraneous virus - free trypsin ( worthington biochemical company , lakewood , nj ) in 5% co2 at 33c .
the tpck - trypsin was used at a final concentration 2 g / ml for mdck and mdck - siat2,6-uf cells and at 0.2 g / ml for mv1 lu - siat2,6-uf cells .
a commercial solid phase elisa test ( quickvue influenza a and b kit , quidel corp . , san diego , ca , usa ) was used to quickly detect influenza virus in cell cultures .
viral rna was extracted from mv1 lu cells using a qiaamp viral rna mini kit , and rt - pcr was performed using primers described in a who manual for influenza virus diagnostics .
briefly , vrna was denatured for 5 min at 67c in the presence of superase - in rnase inhibitor ( invitrogen corp . ) , cooled rapidly , and in separate reactions , first strand synthesis was performed with omniscript reverse transcriptase ( rt ) ( qiagen , inc . ) for 1 hr at 37c with primers h1f1 ( 5-agcaaaagcaggggaaaataaaagc-3 ) for influenza a(h1n1 ) 2009 virus , thaf2 ( 5-gcaggggaaaataaaaacaacc-3 ) for former seasonal influenza a(h1n1 ) , and h3a1f6 ( 5-aagcaggggataattctattaacc-3 ) for influenza a(h3n2 ) .
pcr was performed using one taq dna polymerase ( new england biolabs , ipswich , ma .
primers h1f1 and h1r1264 ( 5-cctactgctgtgaactgtgtattc-3 ) were used for influenza a(h1n1 ) 2009 virus , with an expected product size of 1,264 bp , thaf2 , and sphar11 ( 5-tattttgggcactctcctattg-3 ) for former seasonal influenza a(h1n1 ) , with an expected product size of 990 bp , and h3a1f6 and h3a1r1 ( 5-gtctatcattccctcccaaccat-3 ) for influenza a(h3n2 ) , with an expected product size of 1,127 bp .
pcr was performed as initial denaturation step ; 94c ( 30 sec ) ; 30 cycles of 94c ( 30 sec ) , 46c ( 60 sec ) , and 68c ( 1 min 25 sec ) ; terminal extension step at 68c ( 5 min ) ; 4c . gel electrophoresis ( 1.5% ethidium bromide - stained agarose gel ) was used to visualize the pcr product .
rna was extracted from aliquots of concentrated skc biosampler and pcis fluids using a qiaamp viral rna mini kit then subjected to rt - pcr analyses .
reverse transcription was performed with omniscript rt using primer h3a1f6 , and pcr was performed as described above with primers h3a1f6 and h3a1r1 to generate a 1127 bp amplification product .
the consensus sequences of the hemagglutinin and neuraminidase genes of the virus isolated in this work ( designated a / gainesville/07/2013 ( h3n2 ) ) were determined from pcr amplicons .
the vrna was reverse transcribed using accuscript high fidelity reverse transcriptase ( agilent technologies , inc . , santa clara , ca ) in the presence of superase - in rnase inhibitor .
pcr was performed using phusion polymerase ( new england biolabs ) with denaturation steps performed at 98c .
a combination of primers mentioned in the who manual for influenza virus diagnostics were used ( information to be provided upon request ) .
amplicon sequences were analyzed using an applied biosystem 3130 dna analyzer by using bigdye terminator ( v. 3.1 ) chemistry and the same used for amplifications .
the hemagglutinin and neuraminidase gene sequences of influenza virus a / gainesville/07/2013 ( h3n2 ) were deposited in genbank ( accession numbers kf061021 and kf061022 ) .
plaque assays were performed as in , though mdck ( and not mv1 lu ) cells were used so that our results would be easier to compare and contrast with those of others ( since most laboratories use mdck cells for influenza virus plaque assays ) .
the rh of indoor air at the residence ranged from 44 to 45% , and indoor t 's ranged from 21 to 23c during the testing period of this work .
viable virus was successfully isolated from samplers located up to 3.7 m away from one of the sick occupants ( table 1 ) .
only samplers located at sites 1 , 2 , and 3 , which were close to and in a direct path with sick individuals ( figure 2 ) , collected rt - pcr detectable or viable virus .
the pcis collected a range of particle sizes containing influenza virus when it was 1.2 m away from the ill person resting on a couch ( at site 1 ) but presumably only fine to ultrafine particles ( 2.5 m ) at distances of 2.1 and 3.7 m away from the sick persons ( table 3 ) . for each sample
set , influenza virus - specific cpe were apparent earlier in mdck - siat2,6-uf and mv1 lu - siat2,6-uf cells than in conventional mdck and mv1 lu cells .
an example is shown in figure 3 for cpe ( granulation of the cytoplasm , enlargement of nuclei , followed by rounding of the cells and detachment from the growing surface ) three days postinfection of mdck and mdck - siat2,6-uf cells that had been inoculated with eluate from the after - filter of a pcis positioned at site 3 . however , for each sample wherein virus was isolated , cpe developed in all the cell types that were used by 6 days postinfection . a quidel quickvue influenza a and b kit was used to quickly confirm the presence of virus in the cells .
as observed by others [ 13 , 14 ] , virus was detected by rt - pcr in some samples wherein viable virus was not isolated ( table 3 ) .
rt - pcr analyses indicated that influenza a(h3n2 ) had been isolated in cell culture , as primers for the detection of pandemic h1n1 and seasonal h1n1 virus did not amplify a specific product , whereas primers for h3n2 amplified the expected 1,127 bp amplicon corresponding to the anterior half of the hemagglutinin gene sequence ( figure 4 ) .
the virus that had been isolated was different from strains present in the laboratory , ruling out laboratory contamination .
a simple blast homology search against sequences in genbank as of 27 may 2013 indicated that the ha gene of influenza virus a / gainesville/07/2013 ( h3n2 ) was most similar to that of influenza virus ( a / washington/3262/2012(h3n2 ) ) .
the rh within the apartment ( 44 - 45% ) was within the optimal comfort level for humans ( 4060% ) , and the interior t was also comfortable .
various studies indicate that aerosol transmission of influenza virus is decreased at an rh greater than about 50% , and that transmission of airborne influenza virus is best under cold and dry conditions ( and references therein ) .
since the rh of the apartment was 44 - 45% , and the t was between 22 and 23c , we predicted that viable airborne virus would be detected , and this turned out to be the case .
viable live virus was present in the liquid collection media of the skc biosampler , and some but not all of the filters of the pcis ( table 3 ) . prior to drawing conclusions ,
modeling is needed with in vitro generated influenza virus aerosols to determine if the results of table 3 truly reflect a difference in collection efficiency between the skc biosampler and the pcis .
it is possible that a large proportion of the collected airborne virus was damaged during impaction with the ptfe filters , yet the actual airborne virus collection efficiency of the pcis was higher than that of the skc biosampler .
alternatively , it is also possible that under a constantly flowing airstream during active sampling with a pcis , that influenza virus collected on the ptfe filters gets desiccated ( and thus inactivated ) on the filters . whereas it is already acknowledged that influenza patients expel large droplets as they cough and sneeze , our findings suggest that influenza patients also produce small aerosol particles ( 5 m ) that contain viable virus .
viable virus was detected in areas close to the two patients but not in adjoining rooms separated by curved paths . because fluid dynamics modeling was not used to study air - circulation in this work , it was not clear whether positioning of the samplers in a direct path to sick individuals was important ; perhaps the overriding factor was closeness to the persons .
the hemagglutinin gene of influenza virus a / gainesville/07/2013(h3n2 ) is essentially identical to that of a / washington/3262/2012(h3n2 ) ( genbank accession number cy141277 ) .
it has less sequence homology to the corresponding gene of a / gainesville/01/2012(h3n2 ) ( genbank accession number kf142471 ) , which circulated in gainesville in 2012 . since florida and washington
are situated on opposite coasts of the usa , it is plausible that a / gainesville/07/2013(h3n2 ) stemmed from someone who had traveled to florida from the west coast .
sequence analyses of the other genes of a / gainesville/07/2013(h3n2 ) indicated that the virus was similar to other nonvariant h3n2 strains in circulation in the usa , suggesting influenza vaccines were not protective for the two patients of this report .
these findings of this work are consistent with recent reports of influenza viruses in fine particle aerosols in the exhaled breath of influenza patients [ 13 , 14 ] , and collectively , these reports challenge the notion that influenza virus is only transmitted through mucous contact with large droplets . | the air we breathe contains microorganisms that can cause infectious respiratory diseases . after two occupants of an apartment
were diagnosed with influenza in february of 2013 , efforts were made to detect and isolate airborne influenza virus using two different types of active air samplers : a sioutas personal cascade impactor sampler ( pcis ) and an skc biosampler .
the pcis collects size - fractionated particles by impaction on polytetrafluoroethylene filters , whereas the skc biosampler collects airborne particles in liquid media .
influenza h3n2 virus was collected by both types of air samplers .
the pcis collected a range of particle sizes containing influenza virus near one of the sick individuals but only ultrafine particles when the samplers were positioned farther away .
viable virus was present in the liquid collection media of the skc biosampler and some pcis filters .
these findings suggest that influenza patients produce ultrafine aerosol particles that contain viable virus . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC4621524 | chylous ascites is an accumulation of milky lipid - rich lymph in the peritoneal cavity caused by a disruption in the lymphatic system .
it was first identified in 1694 by richard morton in a 2-year - old male who died with tuberculosis .
chylous ascites is fairly rare in the clinical setting , with a reported incidence of approximately 1 in 20 000 presenting to the hospital [ 13 ] .
left untreated , it can lead to nutritional deficiency and in some cases immunosuppression . in rare cases ,
patients presenting emergently with volvulus or appendicitis have incidentally been found to have chylous ascites upon surgical exploration [ 24 ] .
paraesophageal hernia can present acutely with gastric obstruction or strangulation at an estimated incidence of 1.2% per patient per year , the treatment for which involves laparoscopic or open hernia reduction , resection of the hernia sac , repair of the hiatal defect and fundoplication .
we report the unique observation of spontaneous chylous ascites , chylothoraces and chylomediastinum occurring in the setting of an incarcerated paraesophageal hernia .
a 60-year - old female with a long - standing gastroesophageal reflux disease and known type iii paraesophageal hernia presented to the emergency department with acute epigastric pain , dysphagia and vomiting .
she noted a 1-month history of progressive dysphagia as well . computed tomography ( ct ) revealed a large paraesophageal hernia with significant surrounding fluid concerning for incarceration ( figs 1 and 2 ) .
owing to these findings and her discomfort , emergent laparoscopic repair of her paraesophageal hernia was recommended .
figure 1:axial ct view of the paraesophageal hernia and mediastinal fluid .
figure 2:coronal ct view of the paraesophageal hernia and mediastinal fluid .
access to the peritoneal cavity began with a 2-cm incision at the umbilicus using the hasson technique .
after insufflation , four 5-mm trocars were introduced to the left and right upper quadrants .
chylous fluid was immediately visualized in the vicinity of the greater curvature of the stomach with the preponderance of the fluid at the splenic hilum and splenic flexure . upon entering the lesser sac
, chyle was found covering the pancreas as well . at this point , 20 ml of this chylous fluid was aspirated and sent for fluid analysis .
the report later returned with triglyceride levels of 3362 mg / dl , consistent with chyle . during the hernia sac dissection ,
two 19-fr blake drains were placed : one in the lesser sac extending to the splenic flexure and one in the mediastinum .
chest tubes drained about 100 ml of chylous fluid each , after which the output transitioned to serous .
an abdominal and pelvic ct scan revealed no signs of malignancy that may have caused the chyle leak .
the etiologies for chylous ascites can be categorized as traumatic or atraumatic , with malignancy and cirrhosis comprising the majority of atraumatic causes in the developed world via disruption of lymphatic flow .
spontaneous chylothoraces due to compression of the thoracic duct by a large substernal goiter and thoracic aorta aneurysm have been reported in the literature as well [ 6 , 7 ] .
because the chyle leak resolved rapidly after reduction of the hiatal hernia in this case , it could be postulated that acute incarceration of the hernia within the chest led to significant inflammation and subsequent compression of the thoracic duct above the hiatus .
the chyle found within the abdomen could be explained as a transudate from the chest , where the initial disruption to the lymphatic system likely occurred .
the most common presenting symptom for isolated chylous ascites is painless abdominal distention , but a subset of patients may present with peritoneal irritation or unspecified abdominal pain . whether chylous ascites is suspected in a clinical setting or incidentally in the operative room , analyzing fluid to confirm diagnosis is critical . for this reason , abdominal paracentesis may be indicated when suspicion for ascites exists at the time of presentation .
chyle is classically defined as turbid and milky in character with triglyceride levels above 200 mg / dl [ 1 , 8 ] . in our patient ,
analysis of the fluid encountered during laparoscopy showed triglyceride levels of 3362 mg / dl , well above the diagnostic marker .
imaging studies , such as ct , lymphoscintigraphy and lymphangiography , also have roles in diagnosing chylous ascites preoperatively .
the most important initial step is treatment of the underlying cause of the chyle leak [ 3 , 9 ] . in this case ,
reduction of the paraesophageal hernia was imperative to relieve potential compression of the thoracic duct as well as to treat the incarcerated hernia .
medical management is typically aimed at improving nutritional status and decreasing chyle flow , thereby allowing spontaneous closure of the chyle leak .
a high - protein and a low - fat diet with middle - chain fatty acids has proved successful .
numerous case reports have endorsed pharmacotherapy such as octreotide and somatostatin either alone or in combination with total parenteral nutrition in managing a refractory chyle leak [ 1 , 2 , 9 ] . in cases of chylothorax , pleuro - peritoneal shunts or chest tubes are found to be useful , especially if severe dyspnea is present . although there currently lacks a universal approach to treating chylous leakage , surgical reduction of the hernia and dietary restriction were successful in our atypical case . | chylous ascites is an accumulation of milky lipid - rich lymph in the peritoneal cavity .
spontaneous chyle leak is a rare occurrence , often associated with malignancy or cirrhosis .
this is a report of spontaneous chylous ascites , chylomediastinum and chylothoraces encountered in the setting of an incarcerated paraesophageal hernia .
a 60-year - old female presented to the emergency department with epigastric pain , dysphagia and vomiting . clinical presentation and imaging
were concerning for an incarcerated paraesophageal hernia , and the patient was taken to the operating room . during laparoscopic reduction of the hernia and repair ,
chyle was encountered in the peritoneal cavity , mediastinum and pleural spaces .
postoperatively , the chyle leak resolved with a nonfat diet .
the unusual occurrence of a chyle leak in this case may have been due to compression of the thoracic duct within the chest by the incarcerated paraesophageal hernia . | INTRODUCTION
CASE REPORT
DISCUSSION
CONFLICT OF INTEREST STATEMENT |
PMC5067398 | a 19-year - old man presented to the orthopedic surgery department with pain in the left buttock after military training which including running with hyper - flexion of hip for 3 hours , twice a day for 5 days . when he hyper - flexed his left hip joint , he felt and heard a crack .
initially about one week later , he referred to the local hospital because of persisting hip pain . on radiographic evaluation
however , he refused to get more examination due to the alleviation of the hip pain .
although he got rest without any military training for the next six weeks , there was no improvement and tingling sense developed over the posterior aspect of left thigh . during the first consultation with the patient , he complained difficulty on sustained standing and sitting on a locked position .
his body weight , height , hip and lumbar t - score for dual - energy x - ray absorptiometry ( dxa ) scan were 76 kg , 183 cm , 0.7 and 0.1 respectively . on physical examinations , there was no limitation on range of motion of the left hip joint .
also he could walk without any pain except mild tenderness on the area distal from the ischial tuberosity . on the radiographs of the pelvis anteroposterior and frog leg view ( fig .
1 ) , there was a 354020 mm sized bony fragment , 15 mm distance from the left ischial tuberosity .
after the patient admitted to our hospital , he had an examination of computed tomography ( fig .
2 ) and magnetic resonance imaging for rule out pathologic conditions . there was a bony fragment at left ischial tuberosity , which was mainly from conjoined origin of biceps femoris and semitendinosus tendons without any bone marrow edema ( fig .
he determined to get conservative treatment on condition of a gradual alleviation of the symptom . during the admitted 2 months
thanks to the improvement except discomfort on running , he discharged and returned to his military unit .
stress fracture can divided into two types , depending on the relations between bone and muscle .
one is fatigue fracture , which generated by excessive muscular stress applied to the bone of normal strength .
the other is insufficiency fracture that occurred when normal muscular stress is applied to weakened bone .
it is important to distinguish one from another for understanding the patient 's medical status .
it is hard to distinguish avulsion fracture of ischial tuberosity from hamstring muscle strain since its symptoms and injury mechanism are similar .
many authors suggest displacement of 2 to 3 cm , painful nonunion , exostosis formation , neurologic symptom as an indication for surgery . while others report that some patients who had displacement of more than 5 cm eventually regained muscle strength nearly equal to contralateral leg4 ) . also , there is no definitive classification system or operative indication exists for avulsion fractures of the hip and pelvis yet . therefore , more studies are needed to provide the reasonable treatment to patients .
the hamstring muscle complex consists of three biarticular structures ( semitendinosus , semimembranosus , long head of biceps femoris ) and one uniarticular structure ( short head of biceps femoris ) .
though , there are several studies and different results that estimating the power of the hamstring muscle , commonly all the reports represented that the biomechanical load on biarticular hamstring muscles are the greatest during terminal swing phase of sprinting and the semimembranosus muscle generates the largest peak musculotendon force5 ) . for instance , in a study , the peak musculotendon forces which stress to the ischial tuberosity as tensile force during stance and late swing was 13.09 and 19.22 nkg in average respectively6 ) .
then the ischial tuberosity avulsion fracture can occur when suddenly flex the hip forcefully . however , the pelvis stress fracture accounts for less than 1% of all stress fractures1 ) .
despite , the prevalence of hip pain , it can be difficult to find the exact underlying causes .
initial pelvis radiographs will be normal in up to 70% of patients with pelvis stress fracture1 ) .
so , the careful history taking , including medical history , strength and duration of activities and others should be done to prevent the misdiagnosis .
repetitive stress to the bone followed by crack and the accumulation of which results in the stress fracture7 ) .
though various biologic , mechanical and many other predisposing factors can increase the patient 's risk for stress fracture , crucial modifiable risk factors are participation condition of the bone , frequency , duration and intensity of the causative activity . according to literature
it can also occurs in experienced runner if the training period or intensity rapidly rises . in this case , the patient got pain , 3 months after the date when he join to the military service .
furthermore , the strength of the bone estimated to have no problem regarding the patient had no specific underlying disease or fracture history .
this case has its singularity that avulsion fracture has occurred during the 5 days of hip hyper - flexion running motion .
notable point of this case is , unlike others normally do , this patient transmitted stress to hip using excessive hamstring stretching during the hyper - flexion , which is the extension of terminal swing phase , while in extension he gave avulsion stress to ischial tuberosity by hamstring muscle contracture .
this proves running that include hyper - flexion running motion exerts excessive stress to ischial tuberosity which can be accompanied by fracture in short term . | fatigue fracture of the pelvis is the form of fracture due to repetitive micro - stress accumulation , can be affected by a number of factors such as patient 's nutritional status , biomechanics , social status and so on .
still there is no study about precise standard degree of external force that lead to stress fracture , but it may caused by compression force , traction force or complex force and others .
avulsion stress to ischial tuberosity or anterior superior iliac spine by attached muscle is known as the main factor for the avulsion fracture .
this report will deal with 19 years old conscripted policeman who occurred ischial tuberosity avulsion fracture after training of 6-hour running for 5 days accompanying hip hyper - flexion motion .
this reports aims to provide case study of stress fracture occurred after 5 days of exercise which is relatively short period who had no specific trauma history or pain . | CASE REPORT
DISCUSSION |
PMC3819047 | in the united states , foodborne pathogens cause 76 million illnesses , 325,000 hospitalizations , and 5,000 deaths annually . some of the main pathogens behind these sicknesses are gram - negative bacteria such as salmonella , escherichia , and campylobacter . infections with gram - negative bacteria may lead to endotoxemia caused by lipopolysaccharides ( lps ) which is a complex of glycolipids made up of two distinct regions .
these are hydrophilic polysaccharide region ( composed of o antigen and core oligosaccharide ) and hydrophobic regions known as lipid a .
lipid a is responsible for most of the lps induced biological effects . since lipid a
is the innermost component of lps , it is closely adherent to the inner cell wall of bacteria and is generally not released ( and therefore not toxic ) until the death of the bacterial cell .
lps is heat stable and not strongly immunogenic , so it can not be converted to a toxoid .
histone modification is the central point for the control of cardiac growth and gene expression in response to acute and chronic stress stimuli . in the prevention and treatment of cardiac disease , histone modification and the signaling pathways manipulation is a major therapeutic step .
histone acetyltransferases ( hats ) mediate acetylation of histone tails , loosening the interaction between dna and histones .
acetylation of histones reduces their overall positive charge , thus decreasing their tight interactions with negatively charged dna .
histone deacetylases ( hdacs ) can remove the acetyl groups on amino - terminus of histones , restore tight interactions between dna and histones , and deactivate the transcriptional pathway .
therefore , hats ( histone acetyltransferases ) and hdacs ( histone deacetylases ) activity determines the transcriptional activation or repression .
hat activity in cardiac muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression .
studies done by yanazume et al . , 2003 , showed that agonist - induced cardiac hypertrophy accentuates p300 transcriptional activity and that this agonist - mediated cardiac hypertrophy is reversed by the blocking of p300-hat activity .
curcumin is an inhibitor of p300-hat but very little is known about whether this regulatory effect is related to a protective role in cardiac dysfunction .
curcumin which is derived from turmeric ( curcuma longa plant ) is a tropical plant that is native to southern and southeastern tropical asia .
it is a perennial herb within the ginger family and is known for its yellow - orange color and for numerous therapeutic applications .
curcumin is a polyphenolic compound and has been used in the treatment of many conditions , including cardiovascular diseases .
this study was designed to determine the potential for curcumin to attenuate lps induced cardiac hypertrophy in rodents .
curcumin c3 complex ( r ) was a kind gift from sabinsa corporation , hyderabad , india .
the patented c3 complex ( r ) contains curcumin and its derivates demethoxycurcumin and bisdemethoxycurcumin , also known as curcuminoids .
lps sc-3535 was obtained from santa cruz biotechnology as a white to yellow lyophilized powder . for the live and dead experiment ,
lps was used as 1.5 mg / mice ( dissolved in distilled water ) , and in curcumin attenuation of lps induced cardiac hypertrophy , lps was used as 60 mg / kg body weight dose ( dissolved in distilled water ) .
histone h3 ( n-20 ) , was produced against a peptide mapping at the n - terminus of histone h3 of human origin .
histone h4 ( n-18 ) , purified goat polyclonal antibody , was produced against a peptide mapping at the n - terminus of histone h4 of human origin .
p300 ( c-20 ) was obtained from santa cruz biotechnology , santa cruz , ca .
p300 ( c-20 ) , affinity purified rabbit polyclonal antibody , was raised against a peptide mapping at the c - terminus of p300 of human origin .
acetyl - histone h3 ( lys9 ) antibody was obtained from cell signaling technology , danvers , ma .
endogenous levels of histone h3 were detected by acetyl - histone h3 ( lys9 ) antibody only when acetylated at lysine 9 .
acetyl - histone h4 ( lys12 ) antibody was obtained from cell signaling technology , danvers , ma .
endogenous levels of histone h4 were detected by acetyl - histone h4 ( lys12 ) antibody only when acetylated at lys12 .
acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody was obtained from cell signaling technology , danvers , ma .
acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody detects endogenous levels of cbp or p300 only when acetylated at lysine 1535 or lysine 1499 , respectively .
the animal care and use committee of tuskegee university approved the experimental protocol ( protocol number r0804 - 12 - 1 ) .
eight breeding pairs of mice ( mus musculus ) strain 129s6/sveev , il-10 , were received from the gnotobiotic unit of the mutant mouse resource center , university of north carolina , chapel hill , nc ( iacuc approval number r076 - 10 - 2 ) .
the mice were housed under specific pathogen - free ( spf ) conditions in the comparative medicine resource center ( cmrc ) , tuskegee university , and bred via brother : sister mating to produce the mice used in this study .
subsequently , 48 of the resulting mice were randomly assigned to one of four treatment groups .
the adult 129svev mice ( 68 weeks old ) used in the current study were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100 g / kg of body weight , ( c ) lps : 60 mg / kg , and ( d ) lps ( 60 mg / kg ) + curcumin ( 100 g / kg of body weight ) .
then , following a 24 acclimation period , curcumin and curcumin + lps treated groups received intraperitoneal ( i.p . ) injections of curcumin ( 100 g / kg of body weight ) .
one day after the pretreatment with 100 g / kg curcumin , the lps and curcumin + lps groups received lps : 60 mg / kg i.p .
all mice in the 12 hrs subgroups were sacrificed with co2 12 hrs after lps injection .
all mice in the 24 hrs subgroups were sacrificed with co2 , 24 hrs after lps injection .
hearts and lungs of the sacrificed mice were dissected and weighed to compare heart weight / brain weight ( mg / g ) and lung weight / brain weight ( mg / g ) ratios in lps and curcumin treated mice .
the collected organs were then preserved in 10% regular formalin in 50 ml tubes .
immunohistochemistry is the process of detecting antigens in cells of a tissue section by exploiting the principle of antigen - antibody binding specificity .
the sections of murine tissue were placed on the heated water bath and picked up , placed on the slides , and placed in a rack . after all slides had been cut and placed in a rack they were put into a 60 degrees celsius oven over night .
the slides were deparaffinized by going through 3 changes of xylene 2 minutes each , followed by 3 changes of 100% alcohol at 1 minute and one change of 95% alcohol at 1 minute .
slides were washed in running tap water for 1 minute and stored in distilled water slides .
each group was normally distributed , and the variances were equal in the two groups . the test statistic , ts , was calculated using a formula that has the difference between the means in the numerator .
the denominator was the standard error of the difference in the means , which got smaller as the sample variances decrease or the sample sizes increase .
thus ts got larger as the means got farther apart , the variances got smaller , or the sample sizes increased .
mice in the lps treated group showed significant cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio .
figure 1 illustrates that the lps treated group showed significant cardiomegaly . in 12 hrs subgroup , the p = .04 denotes a significant difference between the control and lps , and in the 24 hrs subgroup , the p = .0026 denotes a significant difference between the control and lps treated groups .
mice , in the lps + curcumin treated group , showed attenuation of cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio ( figure 1 ) . in 12 hrs subgroup , the p = .005 denotes a significant difference between the lps and lps + curcumin , and in 24 hrs subgroup , the p = .04 denotes a significant difference between the lps and lps + curcumin treated groups .
no significant changes were observed in the mice treated with either curcumin or control group ( figure 1 ) . in figure 2 , the heart collected from lps treated mice group showed significant cardiomegaly .
dna wound around eight core histone proteins ( two each of h2a , h2b , h3 , and h4 ) form the nucleosome which is the primary building block of chromatin .
the amino - terminal tails of core histones pass through various posttranslational modifications , including acetylation , phosphorylation , methylation , and ubiquitination .
these modifications happen in response to various stimuli and serve to open the transcriptional machinery , increase the accessibility of chromatin to transcription factors , and ultimately result gene expression .
acetylation of h3 has a central role in histone deposition and chromatin assembly in some organisms .
hats acetylate histone proteins , relax chromatin , and expose prohypertrophic genes for activation by cardiogenic transcription factors .
inhibition of histone acetylation has a central role for the antihypertrophic activity of curcumin . in order to evaluate
the mechanism of action of curcumin against lps induced cardiac hypertrophy , we determined the condition of histone h3 acetylation by using acetylation specific antibodies . in this study ,
curcumin repressed the lps induced acetylation of histone-3 in mice ( figure 4 ) . as a control , the paraffin blocks were exposed to histone h3 antibody , and endogenous levels of histone h3 were detected by h3 antibody ( figure 3 ) .
as expected , lps induced a significant increase in histone acetylation that was blocked and sustained by curcumin ( figure 4 ) .
histone proteins were detected by histone h3 antibody in control ( figure 3(a ) ) , curcumin ( figure 3(b ) ) , lps ( figure 3(c ) ) , and curcumin + lps ( figure 3(d ) ) groups .
but the cardiac cells in the lps treated group showed more staining in the cytoplasm indicating hypertrophy .
figure 4 illustrates the degree of histone acetylation in cardiac tissue occurring in each treatment group . as can be seen in figure 4(c ) , lps induced a significant increase in histone acetylation as indicated by the intense staining .
the histone proteins were mainly acetylated in the n - terminal end of the histone protein on lysine residues . in the curcumin + lps treatment group ( figure 4(d ) ) , there was a much lesser degree of nuclear staining with staining being similar to the control group , indicating that curcumin inhibited the histone acetylation activity of lps .
these findings indicate that acetylation of histone tails by histone specific antibodies , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium .
conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) .
modulation of chromatin structure has a central role in the regulation of transcription in eukaryotes . in eukaryotes ,
histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a reversible posttranslational chromatin modification .
the incorporation of this modified histone into the nucleosome inhibits the formation of compact 30 fibers and inhibits the ability of chromatin to form cross - fiber interactions .
acetylation of histone tails , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium .
conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) .
histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a posttranslational chromatin modification .
in addition to histone h3 we also measured histone h4 acetylation by measuring the [ h ] acetate incorporation into histones using h4 acetylation specific antibody .
figure 5 illustrates the level of histone proteins as detected by histone h4 antibody in cardiac tissue from control ( figure 5(a ) ) , curcumin ( figure 5(b ) ) , lps ( figure 5(c ) ) , and curcumin + lps ( figure 5(d ) ) treatment groups . as can be seen in the figure , there was little variation in histone h4 basal expression levels among any of the treatment groups .
as can be seen in figure 6(c ) , the lps treated group showed a significant increase in histone [ h ] acetylation .
control and curcumin alone treatment groups , figures 6(a ) and 6(b ) , respectively , showed little difference in the staining of the acetylated histone h4 .
similar to histone h3 acetylation , lps induced h4 acetylation was also blocked by curcumin treatment ( figure 6(d ) ) .
this data strongly indicates that curcumin inhibits the acetylation of histones induced by lps .
hats ( histone acetyl - transferase ) acetylate histone proteins , relax chromatin , and expose genes for activation by cardiogenic transcription factors .
two highly related and widely expressed molecules , camp regulated enhancer binding protein ( cbp ) and p300 , have emerged as important cofactors for a broad number of transcription factors .
cbp was originally discovered based on its ability and to interact with the camp response element binding protein , whereas p300 was isolated as a cellular target of the adenoviral oncoprotein e1a .
sequence analysis of cbp and p300 has shown substantial homology that these proteins are transcriptional coactivators .
p300 , high molecular weight transcriptional factor , has three cysteine and histidine - rich regions .
p300 knockout mice showed the defects of cardiac muscle differentiation and trabeculation , indicating the importance of p300 for early cardiac morphogenesis and heart development .
curcumin ( diferuloylmethane ) , a major curcumanoid in the spice turmeric , is a specific inhibitor of the p300/creb - binding protein ( cbp ) .
to further check the mechanism of action of curcumin , we analyzed p300 protein acetylation levels .
curcumin inhibited the lipopolysaccharide - induced acetylation resulting in an increase in the binding of p300 to the gene promoter ( figure 7(c ) ) .
as was seen with histone acetylation , p300 acetylation was also inhibited by curcumin ( figure 7(d ) ) .
the results of the present study indicate that inhibition of histone acetylation is a key mechanism for the anti - cardiac hypertrophy activity of curcumin and that p300-hat serves as its molecular target .
p300 may maintain basal function in the normal heart but it also promotes cardiac hypertrophy under lps infusion .
we can conclude from our study that curcumin attenuated lps induced cardiac hypertrophy in vivo .
curcumin plays its anti - cardiac hypertrophy role by the involvement of chromatin remodeling , especially histone acetylation .
acetylation of lysine residues upon a single histone tail ( hyperacetylation ) would produce a stronger effect .
histone acetyltransferases have a critical role to acetylate histone proteins , relax chromatin , and expose cardiac hypertrophy genes for activation by cardiogenic transcription factors .
hat activity in muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression .
lps induced cardiac hypertrophy accentuates p300 transcriptional activity , and lps mediated cardiac hypertrophy is reversed by blocking of p300-hat activity . therefore ,
blocking p300-hat activity may be a viable method for the treatment or prevention of myocardial hypertrophy .
the results showed that curcumin attenuated lps induced cardiac hypertrophy in rodents and that the most probable mechanism of action involves inhibiting the p300-hat activity . | to study the ameliorating effects of curcumin in lipopolysaccharide ( lps ) induced cardiac hypertrophy , mice were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100 g / kg of body weight by intraperitoneal route ( ip ) , ( c ) lps : 60 mg / kg ( ip ) , and ( d ) lps + curcumin : both at previously stated concentrations by ip route .
all mice were sacrificed as 12 hr and 24 hrs groups accordingly after lps injection .
the hearts were collected , photographed for cardiomegaly , and weighed to compare heart weight / brain weight ( hw / bw ) in mg / mg . for immunohistochemistry , the tissue sections were exposed to histone h3 , h4 and acetylated histone h3 , h4 antibody .
lps induced a significant increase in histone acetylation as shown by intense staining . in curcumin
+ lps treated mice nuclear staining was similar to the control group indicating that curcumin traversed the histone acetylation activity of the lps . to further check the mechanism of action of curcumin , p300 protein acetylation levels were analyzed .
this study suggests that the probable mechanism of action of curcumin is via the reduction of p300 hat activity . | 1. Introduction
2. Methods
3. Results and Discussion
4. Conclusion |
PMC4958433 | protein - based therapeutic drugs have an inherent potential to elicit undesired immune response in human subjects .
the impact of treatment - induced anti - drug antibody ( ada ) responses may range from inconsequential to potentially life - threatening .
regulatory agencies mandate testing for the presence of ada in all phases of clinical development and require assessments of potential impact on safety , drug exposure , and efficacy [ 15 ] .
it is therefore crucial to ensure that ada testing results are accurate and consistent throughout the drug development cycle by implementing long - term maintenance and monitoring of the functional integrity of critical reagents [ 68 ] .
one of the common assay formats for ada evaluation is the solution phase bridging electrochemiluminescent ( ecl ) assay , which typically provides high levels of sensitivity and drug tolerance combined with ability to detect most ada isotypes . in this format ,
the ecl signal is generated by ada simultaneously binding two different conjugated forms of the drug : one biotinylated and one conjugated with a ruthenium complex .
conjugation chemistry requires the protein being labeled to be in a buffer free of primary and secondary amines . to achieve this ,
proteins are typically buffer - exchanged into phosphate - buffered saline ( pbs ) prior to the chemical reaction . for convenience ,
conjugated proteins are frequently maintained in the pbs buffer after labeling since pbs is compatible with a large variety of analytical methods , including those used to determine protein concentration .
the use of pbs for long - term storage of proteins is fairly common as evidenced by the many commercially available antibodies formulated in pbs and stored at 20c or below . while pbs is convenient and widely used ,
numerous literature reports demonstrate that this buffer is far from ideal for cryostorage of proteins .
freezing of sodium phosphate buffers leads to precipitation of dibasic sodium salts which in turn causes a significant drop of ph .
for example , ph of a 50 mm sodium phosphate solution may drop from 7.00 when measured at 25c down to 3.36 when measured at 30c . in addition , formation of the na2hpo412h2o crystals leads to a local increase of protein concentration due to sequestration of water from the solution . localized high protein concentration combined with low ph and the presence of the liquid - solid interface on the surface of the dibasic sodium phosphate crystals may stimulate protein unfolding and aggregation .
problems related to precipitation of dibasic sodium phosphate crystals may be eliminated by the use of 50 mm potassium phosphate containing 6.5% sucrose ( a cryoprotectant ) , which was proposed by staack et al . as an adequate buffer for long - term cryostorage of most antibodies . as an alternative to cryostorage ,
long - term refrigeration of proteins at 5c can eliminate problems caused by aggregation ; however it presents a separate set of challenges such as potential for microbial contamination , protein hydrolysis reactions , and possibility of assay interference caused by the use of protein stabilizers ( e.g. , bovine serum albumin ) .
it should be remembered that even well - developed formulation buffers may not be able to completely eliminate protein degradation and formation of protein aggregates which are driven by a complex interplay between the storage temperature , protein concentration , and formulation buffer components as well as by the rate of cooling / thawing and the storage container material and size [ 12 , 13 ] .
selection of formulation buffers suitable for long - term storage of critical reagents used in ada assays may be of crucial importance due to emerging evidence that aggregation of conjugated reagents can play a critical role in generation of reliable immunogenicity data .
an ecl solution phase bridging assay on meso scale diagnostics ( msd ) platform for detection , confirmation , and titration of anti - drug antibodies against a therapeutic human monoclonal antibody was developed and validated by medimmune .
the method was subsequently transferred to a second laboratory site to support clinical studies for two disease indications ( diseases a and b ) .
the initial assay transfer was deemed successful based on the performance of the provided quality control samples : negative controls and low and high level ada positive controls gave responses within the ranges established during the original method validation when tested at the second site .
unexpectedly , however , screening and confirmatory cut points obtained at the second laboratory site were significantly higher than those obtained during the original method validation in the same disease populations .
for both disease a and disease b , the responses of serum samples from drug - nave subjects were more variable and many were much higher than those observed during original validation .
high responses were detected only in certain individuals but not in the negative control pool which suggested that the high sample - to - sample variability could be caused by aggregation of conjugated reagents similar to that reported by tatarewicz et al . .
tatarewicz and colleagues demonstrated that the presence of very small amounts ( 0.55% ) of high molecular aggregates in the preparations of ruthenylated monoclonal antibody used in a bridging electrochemiluminescent immunogenicity assay caused a significant increase of baseline ada responses in subjects with rheumatoid arthritis . while both biotinylated and ruthenylated reagents showed a similar level of aggregation , it was the ruthenylated conjugate that was found to be chiefly responsible for elevated signal . based on this report and our own accumulated experience with the ecl immunogenicity assays , we evaluated reagent aggregation and its impact on ada assays focusing on the ruthenylated conjugate as being more critical for assay performance than its biotinylated counterpart .
we performed an investigation into the effects of different formulation buffers on aggregation of ruthenylated antibody and the resulting ada responses of drug - nave subjects .
two storage buffers were compared : phosphate - buffered saline ( pbs : 3 mm dibasic sodium phosphate , 1 mm potassium phosphate , and 155 mm nacl , ph 7.4 ) and histidine - sucrose buffer ( hsb : 25 mm histidine , 250 mm sucrose , ph 6.0 ) as examples of formulations that may , respectively , promote and prevent protein aggregation .
our selection of the histidine - sucrose buffer was driven by the relative ease of its preparation and its general similarity to most formulation buffers used for long - term cryostorage of monoclonal antibodies .
we found that relatively small amounts of aggregate present in the ruthenylated reagent stored in pbs caused a disproportionately high increase of nonspecific signal in some individual serum samples . replacing pbs with histidine - sucrose buffer ( hsb ) effectively eliminated protein aggregation and significantly reduced the amount of sample - to - sample variability in the immunogenicity assay .
we also developed a reagent monitoring control comprised of pooled individual sera that are sensitive to the aggregation level of conjugated reagents and implemented it for monitoring of reagent quality during long - term clinical studies .
positive ada assay controls were generated by medimmune and consisted of affinity - purified goat polyclonal antibody directed against the idiotype of the human monoclonal antibody drug .
individual human serum samples and pooled human serum were obtained from bioreclamation ( hicksville , ny ) .
multiarray high binding streptavidin - coated plates ( ma6000 ) , blocker a , read buffer 4xt , and ruthenium ( ii ) sulfo - trisbipyridine n - hydroxysuccinimide ester ( sulfo - tag ) were obtained from meso scale diagnostics ( gaithersburg , md ) .
ez - link biotin sulfo - n - hydroxysuccinimide ester and 40 kda zeba spin desalting columns were purchased from life technologies .
all antibody and antibody conjugates concentrations were measured using pierce bca protein assay kit with bovine gamma globulin standards ( life technologies ) .
histidine was purchased from jt baker , and sucrose was purchased from emd millipore . prior to conjugation ,
the human therapeutic monoclonal antibody was buffer - exchanged into pbs ( 3 mm dibasic sodium phosphate , 1 mm potassium phosphate , and 155 mm nacl , ph 7.4 ) , using a 40 kda zeba spin desalting column .
conjugations were performed by treating a 2 mg / ml solution of the antibody for 1 hour at room temperature either with nhs - sulfo - tag at a 15 : 1 challenge ratio or with sulfo - nhs - biotin at a 10 : 1 challenge ratio .
ruthenylated antibody was split into two portions ; the first portion was purified using a 70 ml bed volume of g75 sephadex media equilibrated with pbs , ph 7.4 .
the pbs eluate was diluted to 1 mg / ml with pbs and stored as single use aliquots ( 250 l / vial ) at 80 10. the second portion was purified using a 70 ml bed volume of g75 sephadex media equilibrated with 25 mm histidinehcl , ph 6.0 .
the eluate in the 25 mm histidine buffer was diluted to 1 mg / ml using the same buffer and concentrated sucrose solution so that the final buffer formulation was 25 mm histidine and 250 mm sucrose , ph 6.0 ( henceforth referred to as histidine - sucrose buffer or hsb ) . biotinylated antibody was purified using a 70 ml bed volume of g75 sephadex media equilibrated with 25 mm histidinehcl , ph 6.0 .
the eluate was diluted to 1 mg / ml with hsb and stored as single use aliquots ( 250 l / vial ) at 80 10. for normal use , individual aliquots of both ruthenylated and biotinylated reagents were thawed and stored at 5 3c for up to one month before being discarded .
ruthenylated antibody solutions prepared in pbs buffer and hsb were placed in 500 l polypropylene cryovials equipped with o - ring screw caps at 250 l / vial .
each cycle consisted of freezing the aliquots for approximately 16 hours ( overnight ) at 80 10c ( nominal ) and then thawing them at 5 3c ( nominal ) for approximately 8 hours .
control aliquots and aliquots subjected to 1 , 3 , and 5 freeze / thaw cycles for both buffer formulations were analyzed by high performance size - exclusion chromatography ( hpsec ) and used in the immunogenicity assay .
levels of aggregation were assessed by hpsec on an agilent hplc-1200 system ( agilent technologies , santa clara , ca ) operated by agilent openlab software .
high resolution tskgel super sw mab hr column ( 4 m , 250 , 7.8 mm i d 300 mm ) from tosoh bioscience ( tosoh bioscience , montgomeryville , pa ) was isocratically eluted by 0.1 m sodium sulfate and 0.1 m sodium phosphate buffer , ph 6.8 , and protein elution was monitored by uv absorbance at 280 nm .
wyatt technologies dawn eos mals detector coupled with uv detection was used in size exclusion chromatography multiangle light scattering ( sec - mals ) analyses .
structure designations were made based on comparisons with stressed antibody reference standard analyzed by sec - mals .
column performance was verified by running a gel filtration standard protein mixture ( bio - rad , hercules , ca ) .
serum samples were diluted 50-fold with assay buffer ( phosphate - buffered saline/10% msd blocker a/0.1% tween 20 , ph 7.2 ) containing a mixture of the ruthenylated and biotinylated forms of the drug at 0.8 g / ml and 0.4 g / ml , respectively .
samples were incubated for 1722 hours with gentle agitation at room temperature ( rt ) in the dark .
high binding streptavidin - coated ( hb - sa ) 96-well msd plates were blocked with msd blocker a ( 150 l / well ) for approximately 2 hours at rt and washed four times with wash buffer ( pbs/0.1% tween 20 , ph 7.2 ) .
the reaction mixture was transferred to the blocked hb - sa plate ( 50 l / well ) and incubated for 1 hour at rt with gentle agitation in the dark .
plates were washed four times with the wash buffer prior to addition of 2x msd read buffer t ( 150 l / well ) and read immediately on msd sector 6000 imager . screening and confirmatory evaluations were performed on the same assay plate , each in duplicate wells .
confirmatory wells were treated the same as the screening assay wells except for the addition of excess unconjugated drug in the overnight incubation step ( final concentration 10 g / ml ) .
screening and confirmatory data were obtained by testing 48 drug - nave human serum samples each from disease a and disease b patient populations using conjugated reagents stored in pbs or in hsb each exposed to 1 f / t cycle .
different sets of a and b disease samples were used for each reagent storage buffer .
each sample was tested in duplicate wells in four separate experiments performed by two different operators on two different occasions , generating a total of 192 screening and confirmatory data points for each disease indication .
each assay plate contained a set of system suitability controls consisting of a negative control measured in four duplicate wells ( nqc ) and low and high ada positive controls , each measured in two duplicate wells ( lqc and hqc : 15 ng / ml and 1000 ng / ml , resp . , of goat anti - idiotypic antibody in nqc ) .
the nqc consisted of a commercial serum pool collected from multiple healthy human male and female donors .
the screening assay responses were expressed as s : b ( signal to baseline ) values by dividing the mean signal of an individual sample by the mean response of nqc measured on the same assay plate .
confirmatory assay percent inhibition values were calculated as % inhibition = 100% [ 1 ( response in the presence of drug)/(response in the absence of drug ) ] .
the screening and confirmatory cut points were obtained with the respective false positive rates set to 5% and 1% using statistical methods described in detail elsewhere [ 15 , 16 ] .
individual serum units from patients diagnosed with disease b ( 160 in total ) were analyzed in the screening assay using ruthenylated and biotinylated reagents stored in pbs and subjected to one freeze / thaw cycle .
sera with s : b signals at or above the original validation screening cut point ( 31 in total ) were retested using both conjugated reagents stored in pbs and in hsb .
samples that changed classification from negative when tested using reagents in hsb to positive when tested with reagents stored in pbs and also showed increase of the s : b signal by > 30% ( 10 in total ) were pooled together to create a reagent monitoring control ( rmc ) .
chromatograms and results of the hpsec analysis of the ruthenylated antibody stored in hsb or in pbs and subjected to 0 ( stored at 5 3c ) , 1 , 3 and 5 freeze / thaw ( f / t ) cycles are shown in figure 1 and table 1 .
ruthenylated antibody stored in hsb was 99.6% monomeric and contained about 0.2% of dimers / trimers with no unordered multimers .
it was very stable and showed virtually no changes of aggregate and monomer content upon repeated freeze / thaw cycles .
on the other hand , the preparation stored in pbs contained unordered multimers ( peak at 11.45 minutes with molecular weight of approximately 1900 kda ) and had > 10 times more dimers / trimers ( peak at 12.45 minutes with molecular weight of approximately 360 kda ) than the hsb preparation , even prior to freezing . the area of the dimers / trimers peak remained relatively unaffected by the number of freeze / thaw cycles and ranged between 2.6% and 3.6% of the total protein .
the unordered multimer peak , however , increased with the number of freeze / thaw cycles starting with 0.09% prior to freezing and growing to 0.44% after three freeze / thaw cycles .
the rise of unordered multimer content upon repeated freeze / thaw cycles correlated well with the increased responses in the immunogenicity assay ( see figure 2 ) suggesting that these larger aggregates may be directly responsible for the elevation of ecl signal intensity .
serum samples from drug - nave individuals ( 8 from disease a and 8 from disease b ) were tested in the screening assay alongside a set of system suitability controls : nqc , lqc , and hqc .
all assays utilized biotinylated antibody reagent prepared in hsb and stored at 80 10c until time of use . the samples and controls were analyzed using ruthenylated antibody stored in hsb or in pbs that was subjected to 0 , 1 , 3 and 5 freeze / thaw cycles , and all four freeze / thaw cycles were analyzed on the same plate .
the s : b value for each sample was calculated by dividing its mean response by the mean response of nqc analyzed using ruthenylated reagent subjected to the same number of freeze / thaw cycles ( figure 2 ) .
the signal to baseline ( s : b ) responses of samples tested with ruthenylated antibody prepared in hsb were very consistent and were not affected by the number of freeze / thaw cycles applied to the ruthenylated reagent . for all samples in both disease groups
the absolute percent difference of response across 5 f / t cycles of ruthenylated reagent was 14.7% .
of the eight disease a samples , one was consistently classified as positive and one measured slightly above the screening cut point only with the 0 freeze / thaw cycles ' reagent preparation .
a dramatically different outcome was observed when the immunogenicity assay was performed with the ruthenylated drug stored in pbs . while only two of eight disease a samples were classified as screen positive for ada using 0 f / t cycles ' reagent ,
six measured positive after the first f / t cycle . for disease b samples ,
this number changed from one positive classification at 0 f / t cycles to six after the first f / t cycle . for the majority of samples ,
the change in classification status was accompanied by a severalfold increase of the s : b signals , most of which occurred upon the initial freezing of the ruthenylated antibody stored in pbs . the increased s : b ratios were due to increased sample responses and not to reduction of the nqc response ( see figure 3 ) . in both disease
a and disease b sets there were two samples whose responses were only slightly affected by the reagent storage in pbs .
overall , when the 0 f / t cycles ' ruthenylated reagent was used to test the 16 samples , one measured ada positive using the hsb preparation ( 6.3% ) compared to 12 using the pbs preparation ( 75.0% ) .
storage of the ruthenylated reagent in pbs buffer or in hsb did not have an equally dramatic effect on the % inhibition values ( data not shown ) .
however , it should be noted that testing of samples with the reagent stored in pbs exposed to 0 and 5 freeze / thaw cycles resulted in changes of the positive / negative classifications of individual samples as well as the overall number of positive classifications .
three disease a samples ( numbers 1 , 5 , and 8) and two disease b samples ( numbers 4 and 6 ) were above the confirmatory cut point ( 29.9% and 21.2% for diseases a and b , resp . ) when tested with the ruthenylated reagent stored in pbs at 0 f / t cycles .
after exposure of the ruthenylated reagent to 5 f / t cycles , three disease a samples ( numbers 3 , 6 , and 8) and no disease b samples were confirmed as positive .
on the other hand , confirmatory responses for samples tested with the ruthenylated reagent stored in hsb remained constant after 5 freeze / thaw cycles with only two disease a samples ( numbers 1 and 8) consistently classified as confirmed positive . unlike individual sera , control pool responses were not significantly impacted by the use of different storage buffers for the ruthenylated reagent .
application of multiple freeze / thaw cycles to the ruthenylated reagent did not produce a strong effect on responses of the assay quality controls ( figure 3 ) .
after 5 freeze / thaw cycles , the ecl signal of the low and high positive controls remained within 12.7% of the values obtained with fresh ( i.e. , never frozen ) reagents stored in either pbs buffer or in hsb .
a more pronounced effect was observed for the negative control where 5 freeze / thaw cycles applied to the ruthenylated reagent stored in pbs led to a 30% increase of the signal generated with fresh reagents .
this increase of the nqc signal is relatively small and can not account for the observed severalfold upsurge of the s : b responses for some individual samples .
it should be noted that , despite a positive trend in signal intensity of the assay controls related to the number of f / t cycles applied to the ruthenylated reagent , all responses remained within acceptable ranges established during the original method validation .
negative and positive control pools did not respond to changes in the conjugated reagent integrity and , therefore , are insufficient to monitor increases in sample responses associated with reagent aggregation . as shown in figure 3 , a regular set of system suitability controls may be inadequate to detect degradation of conjugated reagents and the resulting increase of positive classifications . in order to prepare a control sensitive to aggregation status of the conjugated reagents , we focused on disease b population where the sample - to - sample differences caused by the presence of aggregates were more common and more striking ( figure 4 ) .
we screened 160 individual serum units from individuals with disease b using conjugated reagents in pbs ( 1 f / t cycle ) and selected 31 samples with responses above the screening cut point .
these samples were retested following the same procedure and on the same assay plate but using ruthenylated reagents stored in either pbs or hsb in order to identify sera that changed ada classification status ( negative to positive ) and showed increase of the s : b signal by > 30% .
ten samples met these criteria and were pooled to create a reagent monitoring control ( rmc ) . when tested with aggregated reagents ( in pbs ) ,
rmc responses increased approximately 3-fold in comparison to the result obtained with nonaggregated reagents ( in hsb ) thus demonstrating that rmc could be used to monitor reagent aggregation status during clinical studies .
the large differences in sample responses observed across different reagent storage buffers led us to suspect that even slight aggregation of the conjugated reagents may have a tremendous impact on the screening and confirmatory cut point values and , consequently , on the reported baseline reactivity in drug - nave populations .
screening and confirmatory data were generated for disease a and disease b populations using conjugated reagents stored in pbs or hsb , each exposed to 1 f / t cycle .
screening and confirmatory data for the drug - nave disease a and disease b populations and the resulting cut point values are shown in figure 5 . as expected , screening cut points established using reagents stored in pbs ( and containing aggregates ) were much higher than those obtained with reagents stored in hsb ( little or no aggregation ) with the difference being approximately 4-fold for disease a population and almost 15-fold for disease b population .
it should be noted that all cut point values were obtained after removal of statistical outliers following the customary procedures detailed in [ 15 , 16 ] .
the elevated screening cut point values obtained with the pbs reagent preparations were caused by broadening of signal distributions in both disease a and disease b populations .
confirmatory cut points obtained using reagents stored in pbs were also higher than those generated with reagents stored in hsb but the differences were less pronounced than those observed for the screening cut points .
increase of the confirmatory cut point for disease a samples was caused by a combination of broader distribution and increased median , while for disease b population increased median accounted for most of the change in the confirmatory cut point value .
we conducted a stability evaluation of the ruthenylated monoclonal antibody and in lieu of storing the reagent for several years at < 70c , which would be expected for its typical use during testing of clinical samples , we chose to subject the reagent to multiple freeze / thaw cycles which may simulate the stress of long - term cryostorage [ 13 , 17 ] .
we evaluated two storage buffers : phosphate - buffered saline ( pbs : 3 mm dibasic sodium phosphate , 1 mm potassium phosphate , and 155 mm nacl , ph 7.4 ) and histidine - sucrose buffer ( hsb : 25 mm histidine , 250 mm sucrose , ph 6.0 ) in order to , respectively , enhance and reduce protein aggregation .
changes of protein purity caused by storage in these two buffers were monitored by high performance size - exclusion chromatography .
concentration of aggregates in the preparation of ruthenylated antibody stored in hsb remained constant at about 0.2% through five freeze / thaw cycles which provides assurance that conjugates stored in this buffer should be able to remain in cryostorage for years without any appreciable degradation .
an elevated amount of dimers and trimers ( 2.6% ) appeared prior to freezing of the preparation in pbs which implies that certain degree of aggregation may be due to phosphate buffer alone and is not related to protein degradation caused by freezing .
this is underscored by the observation that the amount of dimers / trimers was not appreciably affected by the number of freeze / thaw cycles .
species that formed in response to the freeze / thaw induced stress were identified as unordered multimers with molecular weight of about 1900 kda . increased concentrations of unordered multimers in the ruthenylated antibody preparation were associated with the increased responses of selected individual samples in both disease a and disease b , suggesting that this species is directly responsible for high ecl responses . despite formation of aggregates in pbs , it should be noted that the monomer content remained 95.9% throughout all five freeze / thaw cycles , which for most antibody applications would be considered acceptable and would not trigger a follow - up investigation .
our observation that the presence of 0.44% of high molecular aggregates can have a profound impact on the individual responses in the immunogenicity assay suggests that quality requirements for conjugated reagents used in immunogenicity testing should be more stringent than those typically applied to monoclonal antibodies . to the best of our knowledge , the publication by tatarewicz et al . is the only report linking the presence of aggregates in the conjugated reagents used for detection of ada with high apparent baseline reactivity in a drug - nave population .
this report demonstrates that high signals in the ecl immunogenicity assay could be reduced by modifying the conjugated antibody formulation buffer with sucrose , a cryoprotectant , which was able to prevent formation of aggregates .
their study was performed using samples from patients with rheumatoid arthritis known to contain significant amounts of rheumatoid factor ( rf ) which may bind to aggregated immunoglobulins ; however no correlation between the rf levels and the intensity of response in the ada assay was detected .
we also tested sixteen individual serum samples with rf ranging from 12.2 u / ml to 650.0 u / ml and we did not observe any correlation between the rf level and response in the screening assay using either of the two different formulations of the ruthenylated antibody ( data not shown ) . using ruthenylated reagent stored in pbs , we observed high baseline reactivity in drug - nave samples from patients diagnosed with disease a and disease b , two populations typically not associated with high levels of rf .
similar to the previous report , storage of conjugated reagents in pbs ( containing no cryoprotectant ) instead of hsb ( containing 250 mm sucrose ) caused formation of aggregates , which in turn resulted in a dramatic increase of the screening responses in both populations .
inexplicably , this increase was far from uniform : it reached as much as 6-fold for some individual disease a and disease b samples while other samples appeared to be unaffected .
it is far from obvious how the aggregates present in the ruthenylated reagent can cause a significant signal increase for certain samples .
one could hypothesize that high molecular weight aggregates ( composed of 12 - 13 molecules based on the observed average molecular weight of 1900 kda ) are capable of amplifying ecl signal to a higher degree than a monomeric molecule , thus strongly enhancing otherwise insignificant sample - to - sample differences .
dramatic differences in behavior of individual serum samples in response to aggregated reagents can have a significant impact on screening and confirmatory cut point values as well as the number of positive classifications generated by the immunogenicity assay .
thus , integrity of conjugated reagents becomes of utmost importance for ensuring quality of immunogenicity data as discussed below .
one can envision a scenario where , due to gradual degradation of conjugated reagents , screening and confirmatory responses of some individual samples show a marked increase similar to that illustrated in figures 2 and 5 .
such increases may be misinterpreted as developing immune responses , especially if ada positive / negative classifications are made using cut points obtained with intact conjugated reagents .
for example , screening and confirmatory cut point values generated in disease a and disease b populations using nonaggregated reagents ( i.e. , stored in hsb ) are 1.37 and 29.9% and 1.14 and 21.2% , respectively .
as shown in figure 5 , application of these cut points to data generated with aggregated reagents ( i.e. , stored in pbs ) would result in a very high number of confirmed positive classifications .
a common practice in clinical studies is to establish in - study cut points using either nontreated commercial samples or pretreatment patient samples prior to immunogenicity testing .
however , cut points obtained with aggregated reagents can be very high as shown in figure 5 .
cut point values for disease a and disease b populations obtained with reagents stored in pbs were 6.07 ( screening ) and 38.2% ( confirmatory ) and 16.7 ( screening ) and 27.4% ( confirmatory ) , respectively .
such high screening cut point values would significantly reduce assay sensitivity and drug tolerance and may be invalid for sample testing .
it is expected that loss of reagent integrity should be detected by unacceptable performance of the system suitability controls resulting in assay failure that should preclude reporting of inaccurate immunogenicity results . as shown in figure 3 ,
however , aggregation of ruthenylated reagents had negligible effect on performance of both positive and negative assay controls , thus raising the likelihood of unwittingly reporting immunogenicity results that are heavily contaminated with false positives . in order to eliminate such a possibility
, we generated a reagent monitoring control ( rmc ) by pooling together individual serum samples selected for their ability to strongly increase their ecl signal in response to the presence of aggregates in conjugated reagents .
as shown in figure 4 , rmc is classified as negative when nonaggregated reagents ( stored in hsb ) are used and positive when tested with aggregated reagents ( stored in pbs ) .
the observed 3-fold increase of s : b response upon testing with reagents stored in pbs makes rmc more sensitive to reagent aggregation than the traditional set of positive and negative controls and can be used as a real - time functional test of reagent quality to be performed together with testing of unknown samples .
the rmc is included in the confirmatory assay tier ( 4 replicates tested in the absence of added drug ) for the purpose of monitoring the performance of reagents during sample analysis .
rmc is expected to be classified as negative and repeated positive classifications of rmc should trigger a halt to sample testing until completion of an investigation into quality of conjugated reagents .
due to difficulties in its preparation , rmc can not easily replace the negative control pool but it can be used in addition to regular system suitability controls .
rmc or related controls could also be included in functional testing of stress - induced stability of critical reagents and selection of appropriate formulation buffers similar to the approach described by staack et al . .
while we expect that use of optimal formulation buffer and proper storage of conjugated reagents should prevent their degradation and subsequent changes of assay performance , rmc can be used as an additional precaution ensuring high quality of the immunogenicity data .
integrity of conjugated reagents used for measurement of ada in clinical samples is critical for generation of reliable immunogenicity data .
small amounts of aggregates present in preparations of conjugated reagents may lead to a spurious increase of ada positive classifications creating an appearance of immune response developing in certain individuals .
system suitability controls prepared in normal serum or plasma pools may be unaffected by the presence of aggregates and perform within specification thus failing to alert the investigators about potential problems with the quality of immunogenicity results .
presence of aggregates in conjugated reagents is best detected using individual samples known to be sensitive to reagent aggregation analyzed either individually or being pooled together to create controls for monitoring of reagent quality .
phosphate - buffered saline may be especially ill suited for cryostorage of proteins and its use should be discouraged .
presence of cryoprotectants such as sucrose in formulation buffers may eliminate or at least reduce aggregation of conjugated reagents during long - term storage at < 70c . | consistent performance of anti - drug antibody ( ada ) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of pk , pd , safety , and efficacy .
the electrochemiluminescent assays commonly employed for ada measurement use drug conjugated with ruthenium and biotin to bind ada in samples . here
we report an association between high nonspecific ada responses in certain drug - nave individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ada assay . ruthenylated reagents stored in phosphate - buffered saline ( pbs ) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects .
reagents stored in a histidine - sucrose buffer ( hsb ) had lower aggregate levels and produced low sample responses . in contrast to pbs , conjugated reagents formulated in hsb remained low in aggregate content and in sample response variability after 5 freeze / thaw cycles . a reagent monitoring control ( rmc ) serum was prepared for the real - time evaluation of conjugated reagent quality .
using appropriate buffers for storage of conjugated reagents together with rmcs capable of monitoring of reagent aggregation status can help ensure consistent , long - term performance of ada methods . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Summary |
PMC4525466 | the vertebrate immune system consists of a conserved innate system complemented by a highly specialized ( adaptive ) immune system .
both branches of immunity communicate and collaborate in a bipartisan way to ensure the effective destruction of potentially harmful microbes .
pattern recognition receptors ( prrs ) are crucial germ - line encoded components of the innate branch , as they recognize directly and immediately conserved microbial structures and molecular motifs ( mamps , microbe - associated molecular patterns , previously known as pamps ) as well as immunogenic endogenous molecules released from the infected host ( damps , damage - associated molecular patterns ) [ 24 ] .
toll - like receptors ( tlrs ) are the best characterized innate immune receptors . more than 20 tlrs clustered in six subfamilies have been identified in more than a dozen of fish species [ 57 ] ( figure 1 ) .
binding , tlrs dimerize and undergo conformational changes to recruit the myddosome to the activated toll / interleukin-1 receptor domain ( tir ) . in mammals ,
this helical structure consists of six myd88 ( myeloid differentiation primary response protein 88 ) adaptor molecules , onto which a layer of four irak4 ( interleukin-1 receptor - associated kinase 4 ) serine / threonine kinases and another layer of four irak2 or irak1 factors are assembled .
the composition of teleost myddosome is unknown so far , although functional interaction of the complex myd88-irak4a with the tir domain has been reported .
however , no irak2 factor has yet been found in any teleostean fish species [ 5 , 6 ] .
the activated receptor complex promotes the dissociation of irak1 from its functional repressor tollip ( toll - interacting protein ) allowing its association with traf6 ( tnf receptor - associated factor 6 ) and further downstream factors to activate either nf-b or interferon regulatory transcription factors or mitogen - activated protein kinases .
this tlr - myd88-irak - traf6 signaling pathway is well conserved , not only in vertebrates but also in drosophila [ 11 , 12 ] .
the activated cascade results in enhanced expression of immune factors such as cytokines provoking inflammation and allowing the communication with the adaptive branch of immunity .
salmonicida is the causative agent of furunculosis , a serious disease of salmonid fish inducing high mortality even after a low - dose intraperitoneal injection .
pathogenic challenges induce not only the massive activation of proinflammatory mediators [ 1417 ] , but also enhanced transcription of tlr - encoding genes in fish [ 1820 ] as reported previously for mammals [ 2123 ] .
the dominant mamp from gram - negative bacteria is lipopolysaccharide ( lps ) , known in mammals to be specifically and solely recognized by tlr4 .
no tlr4 ortholog has been identified so far in salmonid fish , and lps recognition in bony fish is unclear to date [ 6 , 25 , 26 ] .
moreover , modulation of the expression pattern of the entire tlr panel in response to a. salmonicida has not been reported from rainbow trout .
we therefore profiled the expression of 13 tlrs belonging to five subfamilies of these receptors ( figure 1 ) in immune tissues ( spleen , head kidney , liver , and thymus ) from healthy and a. salmonicida - infected rainbow trout .
moreover , we also included other genes encoding downstream factors of tlr signaling into analysis , that is , the tlr adapter myd88 ; the key kinases irak4a and irak1 ; the nf-b - activating factor traf6 ; and the inhibitor of tlr signaling , tollip .
this panel of candidate factors should provide a comprehensive overview of the transcriptional regulation of tlr signaling in trout during a. salmonicida infection .
rainbow trout ( steelhead ; trout lodge , tacoma , usa ) were kept in 300-l tanks at 15c in partially recirculating water systems .
salmonicida ( wild type strain jf 2267 ) was used for experimental infection of trout .
the bacteria were cultivated from cryoconserved batches ( microbank , pro - lab diagnostics , cheshire , uk ) in lb broth ( sifin ) at 15c for 72 h. the initial cultures were checked for purity by gram - staining and observation of cell morphology .
the bacterial suspension was concentrated by centrifugation ( 4300 rpm , 10 min , 4c ) .
the bacterial pellet was washed once in sterile 0.9% sodium chloride solution and diluted to 1 10 bacteria / ml . we injected lethal doses of a. salmonicida to conceivably induce uniform physiological reactions in all individual fish .
one group of fish ( n = 30 ) was infected by peritoneal injection with 200 l pbs ( phosphate - buffered saline ) containing 1 10 a. salmonicida while a control group ( n = 5 ) received 200 l pbs only . five fish per group were sampled at 0- , 6- , 12- , 24- , 48- , and 72-hour postinfection ( hpi ) .
peritoneal injection and anaesthetization of rainbow trout with phenoxyethanol prior to sampling were conducted in compliance with terms of the german animal welfare act ( 4(3 ) tierschg ) .
the experimental protocol was approved by the animal care committee of the state mecklenburg western pomerania ( landesamt fr landwirtschaft , lebensmittelsicherheit und fischerei , mecklenburg - vorpommern , germany ; lallf m - v / tsd/7221.3 - 2.5 - 008/10 ) .
tissue samples of spleen , head kidney , liver , and thymus were immediately snap - frozen and stored in liquid nitrogen .
tissue samples were powdered in a mortar under liquid nitrogen and total rna was subsequently extracted using qiazol lysis reagent followed by purification with rneasy mini spin columns , as provided in the rneasy plus universal kit ( qiagen , hilden , germany ) .
the rna concentrations were determined with the nanodrop 2000 photometer ( thermo scientific , waltham , ma , usa ) . 1.5 g total rna from each sample was transcribed into cdna using the super script ii kit ( invitrogen / life technologies , karlsruhe , germany ) and gene - specific antisense oligonucleotides ( table 1 ) .
complementary dna aliquots equivalent to an input of 75 ng total rna were used in subsequent rt - qpcr reactions .
we derived rainbow trout specific oligonucleotide primer pairs to amplify cdna sequence fragments of 13 tlr genes ( tlr1 , tlr2 , tlr3 , tlr5 , tlr7 , tlr8a1 , tlr8a2 , tlr9 , tlr19 , tlr20 , tlr21 , tlr22a1 , and tlr22a2 ) ; of five genes coding for downstream signaling factors of the tlr pathway ( myd88 , irak4a , irak1 , tollip , and traf6 ) ; of the nod1 gene ( nucleotide - binding oligomerization domain - containing 1 ) representing an alternative prr .
primers for five cytokine - encoding genes ( il1b , tnf , il8 , il10 , and tgfb ) and two immune cell marker genes ( ighm , trb ) were also derived ( table 1 ) .
the oligonucleotide primers were designed using the pyrosequencing assay design software v.1.0.6 ( biotage , uppsala , sweden ) .
the resulting pcr products were cloned ( pgem - t easy ; promega , mannheim , germany ) and sequenced ( applied biosystems 3130 genetic analyzer ; life technologies ) to ensure the authenticity of the respective gene fragments .
cdna copy numbers were quantified on the lightcycler 96 system ( roche , basel , switzerland ) using the sensifast sybr no - rox kit ( bioline , luckenwalde , germany ) .
after each rt - qpcr run , pcr products were visualized on 3-% agarose gels to validate product size and quality .
melting curve analyses evaluated the amplification of single products per sample ( specific tm values are listed in table 1 ) .
standard curves were generated based on 10-fold dilutions ( 10 to 10 copies ) of the respective cloned fragments serving as external standards in the analytical runs .
copy numbers were calculated on the basis of linear regression of the standard curve ( r > 0.99 in each case ) .
the cells in the peritoneal cavity , the site of experimental infection , were retrieved through lavage with 5 ml ice - cold pbs containing 5 mm edta ( ethylenediaminetetraacetic acid ) . for the analysis of cell number and distribution of lymphoid and myeloid cell populations , 100 l cell suspension
cells counts were acquired by facscalibur ( becton dickinson , germany ) in high - throughput mode for 20 seconds .
the cell composition was analyzed using a set of monoclonal antibodies as previously described . in brief , the total number of leukocytes was incubated with diluted antibodies for 30 minutes .
antibodies were either directly labeled with fluorochrome or cells were first incubated with an unlabeled specific antibody and subsequently for another 30 minutes with the corresponding mouse isotype - specific antibody , labeled either with fluorescein isothiocyanate ( fitc ; rockland , limerick , pa , usa ) or r - phycoerythrin ( rpe ; jackson immunoresearch laboratories , west grove , pa , usa ) .
the data are presented as the mean standard error of the mean sem . to assess statistical significances , we performed one - way analysis of variance ( anova ) followed by tukey 's post hoc test as provided by sigmaplot ( systat software inc . , san jose , ca , usa ) .
we performed a quantitative real - time pcr ( rt - qpcr ) profiling across 19 factors constituting the recognition of mamps in rainbow trout . regarding the tissue - specific expression
, we found that all 13 tlr genes were significantly expressed in spleen , head kidney , liver , and thymus ( figure 2(a ) ) .
spleen tissue featured the highest mrna concentrations of almost all the tlr - encoding genes , as frequently found in other fish species .
the copy numbers ranged from a minimum of 0.12 0.02 10 ( tlr5 ) to a maximum of 1.3 0.18 10 copies ( tlr21 ) per g rna .
the concentrations in head kidney amounted to approximately one - third of the values found in spleen with tlr5 featuring the lowest abundance of only 7% of the level in spleen .
levels in liver were generally much lower than in spleen , amounting for eleven of the considered tlr genes on average to less than 10% of the levels as recorded in spleen . yet , tlr5 and tlr3 were found to be exceptional since their mrna concentrations reached 163% and 58% of those values measured in spleen .
we could not find distinctive tissue - specific expression profiles distinguishing the expression levels of the known transmembrane tlr genes ( e.g. , tlr1 , tlr2 , and tlr5 ) from those of the known endosomal tlrs ( tlr3 , tlr7 , tlr8a1 , tlr8a2 , and tlr9 ) or of the fish - specific tlrs ( tlr19 , tlr20 , tlr21 , tlr22a1 , and tlr22a2 ) , the latter belonging all to the tlr11 family ( figure 1 ) . comparing the relative expression intensities between the various tlr genes , we recorded the highest mrna abundances ( > 220,000 transcripts/g rna ) for tlr9 , tlr20 , and tlr21 in spleen , head kidney , and thymus , while tlr5 and tlr19 were found to be expressed on comparatively low levels in those tissues ( < 160,000 transcripts/g rna ) .
nod1 copy numbers were on similar levels as tlr2 , tlr9 , and tlr20 in spleen and liver and exceeded in head kidney by more than twofold the level of the quite strongly transcribed tlr9 or tlr21 .
transcripts encoding downstream factors of the tlr pathway were also abundant ( figure 2(b ) ) .
copy numbers of irak4a , irak1 , traf6 , and tollip genes were in a similar range as the tlr genes .
myd88 , in contrast , exceeded by 3- to 15-fold the level of those other factors .
rainbow trout were intraperitoneally infected with a high dosage ( 1 10 cfu ) of a. salmonicida ensuring establishment of a uniform clinical infection in all individuals . at the end of the trail , infected trout displayed classical apathetic behavior ; hemorrhages in liver ; enlarged spleen and liver ; and swollen intestine as typical symptoms of acute infection .
to characterize the course of the up - running immune defense after infection , we profiled the expression of several cytokine - encoding genes in our four target tissues spleen , head kidney , liver , and thymus . il1b
[ 31 , 32 ] , tnf , and il8 [ 34 , 35 ] play key roles during inflammation of rainbow trout .
as early as 6 hpi , we found significantly elevated il8 transcript levels ( p 0.03 ) in liver ( 60-fold ) , spleen ( 28-fold ) , and head kidney ( 27-fold ) compared to nave trout ( figure 3 ; table 2 ) . at 12 hpi , we recorded strongly increased il1b mrna abundances ( with p < 0.01 ) in head kidney ( 393-fold ) , liver ( 152-fold ) , spleen ( 48-fold ) , and thymus ( 10-fold ) accompanied by moderately increased tnf mrna levels ( with p < 0.03 ) in head kidney ( 12-fold ) , liver ( 12-fold ) , and spleen ( 8-fold ) . at 72 hpi
, we found a second , less pronounced upregulation of il8 gene expression ( with p < 0.02 ) in liver ( 39-fold ) , head kidney ( 24-fold ) , and spleen ( 18-fold ) .
il10 expression was highly upregulated in head kidney at 12 hpi ( 54-fold ) indicating the onset of immune - dampening mechanisms restricting inflammation .
a key aspect of our study was to profile the expression of all our candidate tlr factors during a. salmonicida infection identifying prominently regulated members of this receptor family .
surprisingly , from the 13 tlr genes studied , only the expression of tlr22a2 was clearly and quickly induced in head kidney ( 6 hpi : 6-fold ; 12 hpi : 4-fold over controls ) and liver ( 12 and 48 hpi : 4-fold over controls ) in the infected trout .
the genes encoding tlr9 , -19 , and -20 were induced in liver to a low extent ( 2- to 3-fold ; p < 0.05 ) .
transcript levels of tlr1 and tlr22a1 remained stable over time and those encoding tlr2 , -3 , -5 , -7 , -8a1 , -8a2 , -9 , -21 , and nod1 were even downregulated ( < 4-fold ) in spleen or head kidney .
no downregulation was found for any of our candidate downstream factors of tlr signaling in any of the four tissues ( figure 4 ; table 3(b ) ) .
levels of all these factors remained virtually stable in thymus during the entire infection period .
in contrast , all these factors were significantly upregulated in liver , at least at some time point during infection . the mrna concentration of myd88 was most prominently upregulated ( > 6-fold , 12 hpi and 72 hpi ) and remained on elevated levels throughout .
the traf6 mrna concentration rose at 12 hpi to a subsequently sustained 2-fold increased level .
the mrna concentration of tollip was raised at 24 hpi by ~3-fold and increased further until 72 hpi .
the induction profile of irak1 was remarkable in so far as it significantly increased > 2-fold in three organs , spleen , head kidney , and liver already 6 hpi .
this elevated level was sustained in liver throughout the infection period ( p > 0.05 at 24 and 48 hpi ) , while it clearly dropped down later compared to 12 hpi in spleen and head kidney .
activation of the early cellular immune response in the peritoneal cavity , the site of infection , was indicated by a very strong increase in the total number of cells ( figure 5(a ) ) .
the number of peritoneal leukocytes increased during the first 12 hpi by > 40-fold ( p < 0.05 ) .
their number remained on this high level for another 60 h in all infected fish .
only slight and statistically insignificant changes were observed in the control group injected with pbs ( < 2-fold , p = 0.6 ) .
flow cytometry was used to differentiate myeloid from lymphoid cells in peritoneal lavages ( figures 5(b ) and 5(c ) ) .
myeloid cells ( mainly monocytes / macrophages and granulocytes ) were the first to become massively recruited .
their number increased already during the first 6 hpi by 43-fold ( p < 0.05 ) and reached a plateau level of a 162-fold ( p < 0.01 ) increase at 12 hpi ( figure 5(b ) ) .
significant amounts of lymphoid cells ( most likely b cells ; maybe t cells ; natural killer - like cells ) were also recruited into the peritoneum , but with a slower , yet steady rate .
their number was increased by 41-fold ( p < 0.02 ) at 72 hpi .
the different rates for recruiting both cell types eventually resulted in a grossly altered composition of the peritoneal cell population .
the relative proportion of myeloid ( 44% ) and lymphoid cells ( 55% ) was quite balanced in control trout ( figure 5(c ) ) .
myeloid cells constituted more than 80% of the peritoneal cells already at 6 hpi , whereas lymphoid cells accounted for only 18% .
the data show that infection activated very swiftly the cellular branch of innate immune defense by recruiting well - known effector cells and this occurred concomitantly and was conceivably triggered by the induced expression of key cytokines .
we profiled the dynamics of lymphoid cell migration in the four immune organs in order to validate the onset of adaptive immune activities involving t and b cells .
t cell receptor ( trb ) and immunoglobulin m , heavy chain ( ighm ) are broadly used gene markers indicating the presence of cells of the t- or b - type lineage .
we found only in the head kidney clear and significant changes of these markers ( p = 0.03 ) .
the trb level remained stable for 12 hpi and subsequently rose to reach its maximum 12-fold increase at 72 hpi .
concomitantly , the ighm level dropped steeply after 12 hpi and reached a lower level plateau ( 11-fold reduction ) at 24 hpi ( figure 6 ) .
this observation indicates either that b cells emigrated from the head kidney or that t or myeloid cells immigrated into this organ or that lymphocytes were strongly induced to proliferate here , thereby affecting the proportion of immune cells .
changes of trb and ighm levels were all less than 3-fold and mostly statistically insignificant in the other three organs ( thymus , spleen , and liver ; table 4 ) .
toll - like receptors are key components of the innate immune system promoting a proinflammatory state after invasion of pathogens . analyzing the tissue - specific and immune - modulated expression of a comprehensive set of factors contributing to tlr signal transduction
may therefore inform about their role to overcome microbial threats ( see for a review ) .
rt - qpcr is often the method of choice for the analysis due to the lack of fish - specific antibodies and its superior sensitivity .
moreover , a significant and positive correlation between the concentration of mrna and its encoded protein has been found for a vast number of genes and also for tlr genes in fish .
the prime interest of our study was to analyze in trout the role of tlr signaling to combat infection with the gram - negative pathogen a. salmonicida , since no tlr is known in teleost fish to recognize lps , the major component of the outer cell wall of this pathogen .
prominent feature of our comprehensive survey of tlr expression during infection was that their expression was only moderately regulated , if at all .
we conclude from this observation that a. salmonicida infection strongly induced proinflammatory mechanisms but failed to induce those pathways controlling the expression of genes encoding tlrs and associated factors .
tlr expression is known to be regulated by the jak / stat signaling cascade and hence our data hint , by inference , that also the latter signaling cascade was not largely activated by the infection . beyond that , such comparatively small modulations in the levels of tlr transcripts can not unequivocally be attributed to altered gene expression since they might as well reflect altered cell composition in the respective organs due to cell migration during the up - running immune defense .
for example , large differences have recently been reported regarding the organ - specific content of tlr - expressing igm cells in trout .
this general pattern of only a moderate regulation of tlr expression during infection in o. mykiss contrasts reports from other organisms .
pronounced upregulation of tlr expression after infection with relevant pathogens has been documented for mammals [ 21 , 23 ] , invertebrates [ 45 , 46 ] , and also bony fish .
distinct sets of tlr genes showed enhanced expression in channel catfish ictalurus punctatus , atlantic salmon salmo salar , and antarctic bullhead notothen notothenia coriiceps after parasitic , bacterial , and viral infection , respectively . after infection , some of those tlr genes revealed distinctively high mrna abundances with changes of more than 10-fold above controls .
the unique and quick sixfold upregulation of tlr22a2 already 6 hpi in head kidney would highlight this tlr as a candidate for specifically contributing to a. salmonicida recognition . yet , attributing that specific role to this factor is highly unlikely since the expression of the fish - specific tlr22 is known to be modulated by a variety of apparently unrelated signals .
it was significantly upregulated in several tissues and immune cells after infection and/or stimulation ( i ) with gram - negative bacteria , that is , aeromonas sp . in trout , in goldfish carassius auratus , and in rohu labeo rohita , as well as with vibrio anguillarum in sea bream sparus aurata ; ( ii ) with gram - positive mycobacterium chelonae in c. auratus ; ( iii ) with reovirus in grass carp ctenopharyngodon idella [ 51 , 52 ] ; and ( vi ) with the ectoparasite argulus siamensis in common carp cyprinus carpio .
the broad spectrum of apparent ligands for this factor suggests that its interaction with mamps can structurally not be similar to the sophisticated key - lock principal as known from mammalian tlrs interacting with their specific ligands [ 54 , 55 ] .
perhaps trout tlr22a2 senses some endogenous damps resulting from aeromonas - induced traumata rather than exogenous mamps as previously discussed by ingerslev et al . .
however , assuming ligand - independent activation of tlr22a2 expression is even more puzzling since we noted in our original description of the twin receptors tlr22a1 and tlr22a2 that both factors share a high degree of identical amino acid residues ( 94% ) , with most of the few exchanged residues being located in the n - terminal , distal leucine - rich repeat region ( lrr ) .
we also note that the expression of both tlr22a twin factors is not coregulated since only one of them was upregulated .
our candidate factors contributing downstream to tlr signaling were eventually upregulated in the livers of infected fish and none of them
upregulated mrna concentrations of such factors in liver were reported from a variety of challenge studies and infection experiments in different fish species [ 5659 ] .
irak1 was distinct from all those other factors in so far as its mrna abundance was increased already at 6 hpi , not only in liver , but also in spleen and head kidney .
early stimulation of the expression of this factor in several immune organs connects its activation to the very early events of mounting an immune defense .
a prominent function of irak1 in mammals is its integration into the myddosome , which is built up around the activated tir domain of tlrs .
viral infection of the grass carp was demonstrated to recruit irak1 to the cell membrane possibly indicating that it is recruited to transmembrane tlrs . relating
this information with our data could suggest that rate - limiting low levels of irak1 factors might constrain tlr signaling in healthy trout and its organs .
given the absence of strong transcriptional tlr regulation in trout , this would conceptually allow shifting the regulatory level of the tlr signaling cascade away from the transcriptional regulation of some key receptors ( such as tlr2 and tlr4 in mammals ) towards the expression level of rate - limiting downstream factors .
moreover , irak1 is also the factor being functionally inactivated by tollip [ 58 , 60 ] .
intriguingly enough , we found a consistent and significant upregulation of tollip expression in liver from 24 hpi onwards .
hence , the data altogether allow the conclusion that irak1 plays a prominent role in controlling the activity of tlr signaling in trout .
fast and strong recruitment of myeloid cells into the peritoneum indicates that the infected fish were prepared to combat the infectious pathogens .
this almost instantaneous reaction against the invaders was in stark contrast to the sluggish modulation of the expression of factors contributing to the tlr signaling cascade . recognizing the presence of pathogens by the host activated vibrantly the immune defense . the first step consisted of mounting a massive cellular defense to fight off the bacteria rather than inducing resilience or tolerance .
those myeloid cells ( conceivably monocytes / macrophages and granulocytes ) , having been recruited into the peritoneum , belong to the cellular arm of innate immune defense .
the second step of harnessing cellular immune mechanisms consisted of activating lymphoid cells , which belong to the adaptive branch of immune defense .
the activation of adaptive immunity involves time - consuming processes such as induction of cell proliferation and cellular redifferentiation . only then
, significant amounts of cells might start migrating out from their site of proliferation and maturation to the site of infection .
our rt - qpcr data suggest belated onset of adaptive immunity , which was not surprising .
it took more than 12 hpi for a considerable amount of the cells of the b - type lineage to probably move out from their reservoir in the head kidney into the peritoneum . at the same time , the t lymphocytes presumably started moving into this organ , conceivably to be functionally primed by relevant antigen - presenting cells .
the presence of infectious a. salmonicida is quickly recognized by trout since the expression of key cytokine - encoding genes is instantaneously induced . however , even the severe infection with high doses of this gram - negative pathogen only modestly regulated the expression of most tlr genes in immune organs indicating a mainly constitutive expression of these factors .
no specific tlr could be singled out as a dominant receptor for perceiving the presence of a. salmonicida , albeit that tlr22a2 was quickly and strongly induced after infection .
rather , this factor is known to be regulated by several nonrelated stimuli and might serve as unspecific alarm switch .
the massive recruitment of granulocytes and monocytes / macrophages into the peritoneum highlights their pivotal importance .
hence , recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is of key importance for mounting a first immune defense against invading a. salmonicida . | toll - like receptors ( tlrs ) are known to detect a defined spectrum of microbial structures .
however , the knowledge about the specificity of teleost tlr factors for distinct pathogens is limited so far .
we measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune - relevant tissues of rainbow trout oncorhynchus mykiss .
intraperitoneal injection of a lethal dose of aeromonas salmonicida subsp .
salmonicida induced highly increased levels of cytokine mrnas during a 72-hour postinfection ( hpi ) period .
in contrast , only the fish - specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels , while the mrna concentrations of many other tlr genes decreased .
flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells .
t and b lymphocytes were differentiated with rt - qpcr revealing that b lymphocytes emigrated from and t lymphocytes immigrated into head kidney . in conclusion , no specific tlr can be singled out as a dominant receptor for a. salmonicida .
the recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading a. salmonicida . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
PMC2686497 | protein protein interactions ( ppis ) regulate many fundamental cellular processes . as a consequence ,
a key step in understanding the function of a protein in its cellular context is to identify potential interacting partners .
ppis are typically identified on a small scale by pull - down experiments or similar techniques , but this approach is too slow and expensive to meet the goal of identifying all the ppis necessary to provide a rich picture of the functional and dynamic properties of the cell ( 1 ) .
high - throughput methods , such as yeast two - hybrid seek to overcome the time constraints of traditional protein - by - protein methods and have been applied to the study of ppis in many organisms , including saccharomyces cerevisiae ( 2,3 ) caenorhabditis elegans ( 4 ) , drosophila melanogaster ( 5,6 ) , escherichia coli ( 7 ) and more recently human ( 8,9 ) .
although high - throughput methods provide data for large numbers of potential interacting pairs , they unfortunately often have much higher error rates than traditional approaches ( 10 ) . computational methods to predict ppis complement experimental methods .
they can efficiently integrate data from numerous sources in order to make predictions of the likelihood of interaction between two proteins ( 11 ) .
databases , such as the hprd ( 12,13 ) , dip ( 14 ) , intact ( 15 ) , biogrid ( 16 ) and mint ( 17 ) all provide lists of experimentally determined interactions .
many of these resources contain only interactions that have been observed experimentally , but these data are not yet representative of a complete interactome .
it has been suggested that the human proteome includes around 300 000 ppis ( 18 ) out of a potential > 300 000 000 .
this estimate does not account for the numerous variations in interacting pairs due to post - translational modifications and alternative splicing .
however , the number of human ppis that have been experimentally determined is an order of magnitude less as shown in table 1 .
the importance of prediction in filling this gap has been recognized by a number of groups and led to the development of databases , such as ophid ( 19 ) and point ( 20 ) which predict ppis as well as string , a database of predicted protein protein associations ( direct and indirect ppis ) ( 21 ) .
all three services computationally predict likely ppis ( whether direct or indirect ) based on orthology , annotations and/or experimental information and have substantially increased the size of the human interactome . however , neither ophid nor point ranks the predictions in order of likelihood .
furthermore , the breakdown of the evidence for interaction is limited to a summary of correlation scores or a binary indication of co - occurrence .
string provides an aesthetically pleasing , informative and user - friendly method of accessing its predictions and the primary data , but does not distinguish between direct physical interactions and indirect relationships , which include transcriptional relationships as well as co - pathway membership ( 21 ) .
table 1.number of human ppis that have been determined experimentally and the results made available via publically accessible databasesdatabaseno . of interactionsno . of proteinswebsitereferencedip19231298http://dip.doe - mbi.ucla.edu/(14)hprd38 16725 661http://www.hprd.org/(12,13)intact*24 2748766http://www.ebi.ac.uk / intact/(15)mint20
8326106http://mint.bio.uniroma2.it / mint / welcome.do(17)mips355423http://mips.gsf.de / cgi - bin / proj / ppi / prot2ppi.cgi(31)all values were extracted from the respective databases statistics pages except where identified ( * ) .
values obtained 8 august 2008.*the number of unique proteins and interactions was calculated by searching for all human binary interactions within intact then analysing the downloaded psi - mi data file .
number of human ppis that have been determined experimentally and the results made available via publically accessible databases all values were extracted from the respective databases statistics pages except where identified ( * ) .
values obtained 8 august 2008 . * the number of unique proteins and interactions was calculated by searching for all human binary interactions within intact then analysing the downloaded psi - mi data file . in this article , a new database pips of
the predictions stored in pips are derived by a bayesian prediction method that combines information on the likelihood of interaction from a variety of sources ( 11 ) .
transitive module that gathers evidence for interaction from examination of predicted common interactors to a pair of proteins . the unique combination of features examined allowed the generation of a set of predictions that are mostly orthogonal to other ppi databases ( 11 ) .
the database and its interface allow the user to see the full evidence trail for each predicted interaction . in this way , pips is a resource not only for large - scale modelling of protein interaction networks , but also as an exploratory tool for the cell / molecular biologist who wishes to understand more about the predicted interaction network for the protein they are studying .
the pips database is a resource of ppis in human predicted by a nave bayesian model as described in scott and barton ( 11 ) .
briefly , the method ( 11 ) combines information from gene co - expression , orthology , co - occurrence of domains , post - translational modifications , co - localization of the proteins within the cell and analysis of the local topology of the predicted ppi network .
the different evidence types are programmed as separate modules with each module giving a score of interaction .
the individual module scores are combined to give a prediction for the overall likelihood of interaction given the available data .
the full database of predicted interactions includes details about 69 965 human proteins imported from the ipi ( 22 ) together with interaction scores for 17 643 506 protein pairs , of which 37 606 are predicted to interact . for each protein pair
, the overall score is stored along with a breakdown of the scores provided by each of the modules .
further information is stored that details the evidence that was used in calculating the final score .
the evidence includes 5872 s. cerevisiae , 23 195 c. elegans and 27629 d. melanogaster proteins that were analysed by inparanoid ( 23 ) to identify orthologous protein pairs , where each protein was known to be involved in an interaction .
details of the interpro ( 24 ) motifs and domains , the sites of post - translational modifications , and each protein 's sub - cellular localization are also stored , as well as the pearson 's correlation coefficients from analysis of expression data . in order to simplify exploration of the predicted interactions ,
links are stored to external data sources including , refseq ( 25 ) , uniprot ( 26 ) and entrez ( 27 ) . comparisons to other publicly available databases of interactions are simplified by the inclusion of links to hprd ( 12,13 ) , dip ( 14 ) , bind ( 28 ) and ophid ( 19 ) for protein pairs that are represented in those databases .
the pips database was constructed on a linux server running the mysql database software and apache / tomcat for the web server .
the front - end utilizes java server pages ( jsp ) to provide a dynamic and easy to navigate web interface .
the front page of the pips interface allows for simple searches with the ipi , uniprot or refseq identifier for a protein , or a text search with keywords .
the advanced search allows the query protein sequence to be compared with the protein sequences stored in the pips database by magicmatch ( 29 ) which returns exact matches to the query sequence .
if no match is found , a blast ( 30 ) search may optionally be run to find sequences that are similar to the query .
a batch mode is available to allow larger numbers of protein ipi identifiers to be run against the database as a single set .
figure 1 illustrates the result of searching with ipi00016572 ( snrpg small nuclear ribonucleoprotein g ) via the quick search from the front page and selecting to view the scores from each module .
the interaction summary page for snrpg shows interacting pairs of proteins ranked in descending order by the final interaction score .
the output includes the name of the protein and scores obtained by each of the different modules .
for example , the interaction between snrpg and lsm8 seen in figure 1 shows that a low contribution was made by the orthology and combined modules , but the expression and transitive modules provide the major contribution to the final score .
in contrast , the interaction between snrpg and snrpd3 , the modules expression , orthology , combined and transitive are all predictive of this interaction .
the evidence column provides a link to view the evidence that was used by each of the modules in calculating the final interaction score , while the database column lets the user know if the pair of proteins has been reported as interacting in other databases [ currently bind ( 28 ) , dip ( 14 ) , hprd ( 12,13 ) and ophid ( 19 ) ] .
figure 1.interaction summary for the protein ipi00016572 ( snrpg ) : this page shows the predicted interactors , ordered by the score in descending order from the most probable interactor .
the name of the predicted interactor and a breakdown by predictive feature is also shown with links to retrieve the evidence for the predicted interaction .
interaction summary for the protein ipi00016572 ( snrpg ) : this page shows the predicted interactors , ordered by the score in descending order from the most probable interactor . the name of the predicted interactor and a breakdown by predictive feature is also shown with links to retrieve the evidence for the predicted interaction .
figure 2a c show the evidence of interaction page for the interaction predicted between snrpg and snrpd3 that was identified in figure 1 .
the page is organized into six sections which provide a break - down of the information on expression , orthology , domains , post - translational modifications , localization and topology ( transitive ) score .
figure 2.(a ) evidence of interaction summary page for the interaction between snrpg and snrpd3 : sections gene expression and orthology provide details about the predictions based on expression and orthology for the interaction pair .
( b ) sections domains , post - translational modification and localization provide the information that was used by the combined module describing the co - occurrence of domains within the protein pair , post - translational modifications and localization of the proteins within the cell .
( c ) section transitive score provides a list of the common interactors with an integrated interaction score > 0.025 for the expression , orthology and combined modules .
these common interactors are considered by the transitive module for calculating the likelihood of interaction between snrpg and snrpd3 . in total , there are 236 predicted common interactors ; the figure shows only the top six common interactors .
( a ) evidence of interaction summary page for the interaction between snrpg and snrpd3 : sections gene expression and orthology provide details about the predictions based on expression and orthology for the interaction pair .
( b ) sections domains , post - translational modification and localization provide the information that was used by the combined module describing the co - occurrence of domains within the protein pair , post - translational modifications and localization of the proteins within the cell .
( c ) section transitive score provides a list of the common interactors with an integrated interaction score > 0.025 for the expression , orthology and combined modules .
these common interactors are considered by the transitive module for calculating the likelihood of interaction between snrpg and snrpd3 . in total , there are 236 predicted common interactors ; the figure shows only the top six common interactors . for each protein
analysed in the prediction , a protein summary page is available as a link from the main prediction result page .
for example , figure 3 shows the protein summary page for the snrpg protein . the summary shows the number of predicted interactions above a given threshold ( 57 predicted interactors with a score 1.0 of which four have a score 2500 ) .
the table also provides links to external protein databases including refseq ( 25 ) , hprd ( 12,13 ) , uniprot ( 26 ) and entrez ( 27 ) .
figure 3.protein summary for the protein snrpg : information about the selected protein including a breakdown of the number of predicted interactions and the number of interactions within external databases .
links are also provided to obtain further details about the protein from the hprd , refseq , entrez and uniprot .
protein summary for the protein snrpg : information about the selected protein including a breakdown of the number of predicted interactions and the number of interactions within external databases .
links are also provided to obtain further details about the protein from the hprd , refseq , entrez and uniprot .
figure 4 illustrates the display of interactions through a new java applet that can be accessed from the protein summary page .
users are able to view the network of the predicted protein interactions out to a path length of two from the query protein . within the applet
the user is able to view the network with and without proteins that have only a single connection .
the user can also grow the graph by selecting a protein and clicking on the grow network option . once the network has been created it is possible to save the network as an image or save an adjacency list of the proteins so that they can be represented in an external application , such as cytoscape ( http://cytoscape.org/ ) or graphviz ( http://www.graphviz.org/ ) .
figure 4.network view of the predicted interactors of snrpg : java application to view the local topology of the predicted ppi network . left : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) .
right : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) , with all interactors that have only a single predicted interaction removed from the image .
network view of the predicted interactors of snrpg : java application to view the local topology of the predicted ppi network . left : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) .
right : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) , with all interactors that have only a single predicted interaction removed from the image .
the pips database is a resource of ppis in human predicted by a nave bayesian model as described in scott and barton ( 11 ) .
briefly , the method ( 11 ) combines information from gene co - expression , orthology , co - occurrence of domains , post - translational modifications , co - localization of the proteins within the cell and analysis of the local topology of the predicted ppi network .
the different evidence types are programmed as separate modules with each module giving a score of interaction .
the individual module scores are combined to give a prediction for the overall likelihood of interaction given the available data .
the full database of predicted interactions includes details about 69 965 human proteins imported from the ipi ( 22 ) together with interaction scores for 17 643 506 protein pairs , of which 37 606 are predicted to interact . for each protein pair
, the overall score is stored along with a breakdown of the scores provided by each of the modules .
further information is stored that details the evidence that was used in calculating the final score .
the evidence includes 5872 s. cerevisiae , 23 195 c. elegans and 27629 d. melanogaster proteins that were analysed by inparanoid ( 23 ) to identify orthologous protein pairs , where each protein was known to be involved in an interaction .
details of the interpro ( 24 ) motifs and domains , the sites of post - translational modifications , and each protein 's sub - cellular localization are also stored , as well as the pearson 's correlation coefficients from analysis of expression data . in order to simplify exploration of the predicted interactions ,
links are stored to external data sources including , refseq ( 25 ) , uniprot ( 26 ) and entrez ( 27 ) . comparisons to other publicly available databases of interactions are simplified by the inclusion of links to hprd ( 12,13 ) , dip ( 14 ) , bind ( 28 ) and ophid ( 19 ) for protein pairs that are represented in those databases .
the pips database was constructed on a linux server running the mysql database software and apache / tomcat for the web server .
the front - end utilizes java server pages ( jsp ) to provide a dynamic and easy to navigate web interface .
the front page of the pips interface allows for simple searches with the ipi , uniprot or refseq identifier for a protein , or a text search with keywords .
the advanced search allows the query protein sequence to be compared with the protein sequences stored in the pips database by magicmatch ( 29 ) which returns exact matches to the query sequence .
if no match is found , a blast ( 30 ) search may optionally be run to find sequences that are similar to the query .
a batch mode is available to allow larger numbers of protein ipi identifiers to be run against the database as a single set .
figure 1 illustrates the result of searching with ipi00016572 ( snrpg small nuclear ribonucleoprotein g ) via the quick search from the front page and selecting to view the scores from each module .
the interaction summary page for snrpg shows interacting pairs of proteins ranked in descending order by the final interaction score .
the output includes the name of the protein and scores obtained by each of the different modules .
for example , the interaction between snrpg and lsm8 seen in figure 1 shows that a low contribution was made by the orthology and combined modules , but the expression and transitive modules provide the major contribution to the final score .
in contrast , the interaction between snrpg and snrpd3 , the modules expression , orthology , combined and transitive are all predictive of this interaction .
the evidence column provides a link to view the evidence that was used by each of the modules in calculating the final interaction score , while the database column lets the user know if the pair of proteins has been reported as interacting in other databases [ currently bind ( 28 ) , dip ( 14 ) , hprd ( 12,13 ) and ophid ( 19 ) ] .
figure 1.interaction summary for the protein ipi00016572 ( snrpg ) : this page shows the predicted interactors , ordered by the score in descending order from the most probable interactor .
the name of the predicted interactor and a breakdown by predictive feature is also shown with links to retrieve the evidence for the predicted interaction .
interaction summary for the protein ipi00016572 ( snrpg ) : this page shows the predicted interactors , ordered by the score in descending order from the most probable interactor . the name of the predicted interactor and a breakdown by predictive feature is also shown with links to retrieve the evidence for the predicted interaction .
figure 2a c show the evidence of interaction page for the interaction predicted between snrpg and snrpd3 that was identified in figure 1 .
the page is organized into six sections which provide a break - down of the information on expression , orthology , domains , post - translational modifications , localization and topology ( transitive ) score .
figure 2.(a ) evidence of interaction summary page for the interaction between snrpg and snrpd3 : sections gene expression and orthology provide details about the predictions based on expression and orthology for the interaction pair .
( b ) sections domains , post - translational modification and localization provide the information that was used by the combined module describing the co - occurrence of domains within the protein pair , post - translational modifications and localization of the proteins within the cell .
( c ) section transitive score provides a list of the common interactors with an integrated interaction score > 0.025 for the expression , orthology and combined modules .
these common interactors are considered by the transitive module for calculating the likelihood of interaction between snrpg and snrpd3 . in total , there are 236 predicted common interactors ; the figure shows only the top six common interactors .
( a ) evidence of interaction summary page for the interaction between snrpg and snrpd3 : sections gene expression and orthology provide details about the predictions based on expression and orthology for the interaction pair .
( b ) sections domains , post - translational modification and localization provide the information that was used by the combined module describing the co - occurrence of domains within the protein pair , post - translational modifications and localization of the proteins within the cell .
( c ) section transitive score provides a list of the common interactors with an integrated interaction score > 0.025 for the expression , orthology and combined modules .
these common interactors are considered by the transitive module for calculating the likelihood of interaction between snrpg and snrpd3 . in total , there are 236 predicted common interactors ; the figure shows only the top six common interactors .
for each protein analysed in the prediction , a protein summary page is available as a link from the main prediction result page .
for example , figure 3 shows the protein summary page for the snrpg protein . the summary shows the number of predicted interactions above a given threshold ( 57 predicted interactors with a score 1.0 of which four have a score 2500 ) .
the table also provides links to external protein databases including refseq ( 25 ) , hprd ( 12,13 ) , uniprot ( 26 ) and entrez ( 27 ) .
figure 3.protein summary for the protein snrpg : information about the selected protein including a breakdown of the number of predicted interactions and the number of interactions within external databases .
links are also provided to obtain further details about the protein from the hprd , refseq , entrez and uniprot .
protein summary for the protein snrpg : information about the selected protein including a breakdown of the number of predicted interactions and the number of interactions within external databases .
links are also provided to obtain further details about the protein from the hprd , refseq , entrez and uniprot .
figure 4 illustrates the display of interactions through a new java applet that can be accessed from the protein summary page .
users are able to view the network of the predicted protein interactions out to a path length of two from the query protein . within the applet
the user is able to view the network with and without proteins that have only a single connection .
the user can also grow the graph by selecting a protein and clicking on the grow network option .
once the network has been created it is possible to save the network as an image or save an adjacency list of the proteins so that they can be represented in an external application , such as cytoscape ( http://cytoscape.org/ ) or graphviz ( http://www.graphviz.org/ ) .
figure 4.network view of the predicted interactors of snrpg : java application to view the local topology of the predicted ppi network . left : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) .
right : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) , with all interactors that have only a single predicted interaction removed from the image
. network view of the predicted interactors of snrpg : java application to view the local topology of the predicted ppi network . left : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) .
right : network image of the predicted primary and secondary interactors of the protein snrpg ( blue ) , with all interactors that have only a single predicted interaction removed from the image .
it has been estimated that only 10% of the human interactome has been identified ( 18 ) .
the pips database allows the user to browse and easily access many additional high probability predicted human interactions and to see the evidence that led to each prediction .
it also provides a source of information to help improve the design of experiments to investigate further the function of proteins in the human proteome .
all predictions are ranked allowing the most probable interactions to be investigated first rather than being given a flat list of predicted interactions .
uk biotechnology and biological sciences research council ( bbsrc to m.d.m . ) ; canadian institutes of health research ( fellowship to m.s.s . ) . funding for open access charge : canadian institutes of health research . | the pips database ( http://www.compbio.dundee.ac.uk/www-pips ) is a resource for studying protein
protein interactions in human .
it contains predictions of > 37 000 high probability interactions of which > 34 000 are not reported in the interaction databases hprd , bind , dip or ophid .
the interactions in pips were calculated by a bayesian method that combines information from expression , orthology , domain co - occurrence , post - translational modifications and sub - cellular location . the predictions also take account of the topology of the predicted interaction network . the web interface to pips ranks predictions according to their likelihood of interaction broken down by the contribution from each information source and with easy access to the evidence that supports each prediction . where data exists in ophid , hprd , dip or bind for a protein pair this is also reported in the output tables returned by a search .
a network browser is included to allow convenient browsing of the interaction network for any protein in the database .
the pips database provides a new resource on protein protein interactions in human that is straightforward to browse , or can be exploited completely , for interaction network modelling . | INTRODUCTION
THE DATABASE
Overview
The PIPs web interface
SUMMARY
FUNDING |
PMC4286652 | in 1834 costa , a professor of zoology embarking on a big study of the rich fauna of the kingdom of naples , reclassified what , from a preserved cornish specimen , the german zoologist peter simon pallas had 60 years before described and depicted as a mollusc .
costa placed live animals from posillipo , just north of the city , as relatives of cyclostomes , the jawless hagfish and lampreys , though dissection soon showed that amphioxus deviated more from these than they did from salamanders ( willey 1894 , pp .
costa s interest in links between classes and europe - wide discussion as an intermediate form ( de ceglie 1999 , pp .
the jena zoologist ernst haeckel , with his anatomist colleague carl gegenbaur , evolutionized the science of form ( nyhart 1995 ; richards 2008 ; hopwood 2015 , pp .
haeckel filled the first darwinist system , his generelle morphologie ( general morphology ) , with cosmic ambition , new technical terms , ferocious polemic , bold pedigrees all the way up to human beings and taxonomic revisions which promoted amphioxus to a new subphylum , the acrania or skull - less ones .
probably the last mohican of a once richly developed and many - branched tree
the reference to james fenimore cooper s novel invoked imperialist elegies for the last representatives of lineages , tribes and species ( stafford 1994 , pp .
232260 ; brantlinger 2003 ) , but haeckel in fact picked winners , stressing the nobility of amphioxus as an ancestor of the dominant line and the respect owed to age .
the transition from the invertebrates to amphioxus remained mysterious , however ; he adopted a marine roundworm as a hypothetical link , but there was as yet no convincing bridge .
from 1868 haeckel s natrliche schpfungsgeschichte ( natural history of creation ) preached this darwinist gospel and , over several decades , a dozen german editions and as many translations took it to the general educated public .
lacking fossils , his conjectural pedigrees relied on embryos , via the theory that individual development ( ontogeny ) recapitulates that of the species ( phylogeny ) .
still cautious about vertebrate origins in 1866 , haeckel now used kowalevsky s work to argue that an invertebrate like an ascidian larva , itself derived from annelids , had developed in two directions : degenerating into the adult tunicates sitting on the sea floor and progressing to amphioxus and other vertebrates ( haeckel 1868a , pp .
, a popular scientific lecture proclaimed , as that venerable animal which of all still living animals is alone in a position to give us an approximate idea of our oldest silurian ancestors with backbones ( haeckel 1868b , p. 42 ) .
fine schematic plates of ascidians and amphioxus graced the second edition of the schpfungsgeschichte , bridging a deep gulf and allowing haeckel to trace humans back to worms ( fig . 1 ) .
speculative philosophers to stop building futile castles in the air and think about these facts instead ( haeckel 1870 , p. 673 ) .
liberals cared only how far a person had come and could go but the specificity and degree of consensus were new.fig .
1plates of the embryology and anatomy of ascidian ( a ) and amphioxus ( b ) in the second edition of haeckel s natrliche schpfungsgeschichte to show the
, plate x represents the ontogeny in five stages from eggs to larvae , and plate xi the adults with the internal organization visible through the transparent skin .
copper engravings with blue wash by wilhelm wagenschieber from haeckel ( 1870 , plates x
21 26 cm plates of the embryology and anatomy of ascidian ( a ) and amphioxus ( b ) in the second edition of haeckel s natrliche schpfungsgeschichte to show the blood relationship of vertebrates and invertebrates . simplified after kowalevsky
, plate x represents the ontogeny in five stages from eggs to larvae , and plate xi the adults with the internal organization visible through the transparent skin .
copper engravings with blue wash by wilhelm wagenschieber from haeckel ( 1870 , plates x
21 26 cm haeckel s pedigrees stirred up trouble from the start , but editors , journalists and general readers entrenched the schpfungsgeschichte in the optimistic , nationalist , anticlerical culture of progress of the new german empire and took these ancestors into middle - class homes.4 then , in 1874 , haeckel came out with the anthropogenie ( translated as the evolution of man ) , a major statement of the relations between ontogeny and phylogeny and a salvo in the kulturkampf , chancellor bismarck s anti - catholic campaign ( haeckel 1874 , pp . xi xvi ) . joining the nationalist monument - building ( belgum 1998 , pp .
5972 ) , the book proudly traced humanity back to amoeba , amphioxus , axolotl , australian lungfish and ape ( hopwood 2015 , pp .
ignorant theologians and anthropocentric philosophers objected that his attitude towards amphioxus had ridden roughshod over the dignity of humanity and most grievously insulted the divine human sense of reason .
but , he insisted , the thousand - year - old oak wood it seemed natural to respect was as nothing compared to this little fish .
, this flesh of our flesh and blood of our blood still deserved more admiration and reverence than all the useless rabble of so - called saints to whom our highly civilized nations build temples and dedicate processions ( haeckel 1874 , pp .
haeckel deployed fresh empirical research and his own new argument that all true animals descended from a swimming stomach , or gastraea , to strengthen and expand his coverage of the ascidian
he mobilized these animals as secular icons and in a struggle for embryology between phylogeny and physiology that politicized species choice ( hopwood 2011 , p. 5 ) .
one look at amphioxus development , he declared , and the chick - based theory of his rival , wilhelm his , collapses like a house of cards ( haeckel 1874 , p. 629 ; also 1875 , p. 23 ) .
graduates of the classical grammar schools ( haeckel 1874 , p. xi ) , the anthropogenie disappointed high expectations of a good read , but many learned the arguments at second hand ( hopwood 2015 , pp .
127134 ) . the liberal publicity machine announced the book with a review in the illustrirte zeitung ( illustrated news ) that told progressives to study comparative embryology .
large wood engravings presented amphioxus and ascidians as primordial ancestors of the tribe of backboned animals
2).5die gartenlaube ( the bower ) , the family magazine with the huge run of nearly 400,000 , also stressed their significance as a bridge . without the little lancet fish
we would barely know we came from worms , a kinship certainly more congenial to the christian feeling of humility than descent from the apes.6 but the anthropogenie did not just give journalists material ; it also goaded experts into warning the public ( hopwood 2015 , pp . 119126 , 132134).fig .
these wood engravings in otto zacharias s review of haeckel s anthropogenie are cruder than the original plates but reached more people .
the magazine omitted cross - sections and a lamprey larva that haeckel had added to present his more developed theory in the more specialized book .
these wood engravings in otto zacharias s review of haeckel s anthropogenie are cruder than the original plates but reached more people .
the magazine omitted cross - sections and a lamprey larva that haeckel had added to present his more developed theory in the more specialized book .
niederschsische staats- und universittsbibliothek gttingen haeckel basked in adulation , but his polarizing rhetoric also provoked howls of opposition from scientists as well as philosophers and theologians .
a disciplinary enemy , the physiologist emil du bois - reymond , had already attacked his overstepping of the bounds of natural knowledge ( bayertz et al .
even fellow zoologists , most of whom by now accepted the fact of evolution , worried about the dogmatic presentation of personal speculations , exaggeration of his own novelty and intolerance towards other researchers views .
few darwinists endorsed every detail of his pedigrees and many objected to his gastraea theory ( nyhart 1995 , pp . 168206 ) . yet
haeckel s advocacy and darwin s endorsement had established the lancelet as the primordial vertebrate and built momentum behind the more controversial ascidian theory , but from 1875 other darwinist theories began to compete ( beeson 1978).7 from naples , anton dohrn urged followers of haeckel and gegenbaur to pay living conditions more attention .
then they would see that ascidians and amphioxus merely continued the degeneration that the parasitic cyclostomes had begun . reduced to filtering food from sandy water , amphioxus represented a degenerate fish , and the ascidians had declined even more ( dohrn 1875 , p. 55 ; ghiselin 1994 , p. 63 ) .
for the wrzburg zoologist and authority on sea cucumbers carl semper , the other main advocate of the annelid theory , amphioxus was also no vertebrate ( semper 1875 , p. 66 ; 1876a ) .
this comment in semper s institute journal gained currency when the veteran materialist and early darwinist carl vogt deployed it in the liberal frankfurter zeitung to dismiss haeckel s mythical pedigrees , and anti - darwinists picked it up.8 few were in any position to adjudicate , but expert disagreement made the choices look arbitrary and helped those who rejected pedigrees out of hand .
haeckel defended himself , in a small section of a long polemic , by pretending that for consistency dohrn would have to make the whole animal kingdom degenerate from the sinless adam until the unfortunate amphioxus , which had burdened itself with the most serious guilt , finally lost even its head
as religious and political protests rained down on haeckel , and the press amplified colleagues censure of everything from pictures of embryos to speculations about cellular souls , objections to amphioxus as ancestor were still easily overlooked . for spokesmen of science
the bigger issue was the threat from his behaviour to their freedom in the new state . in early 1876 semper
publicly targeted haeckelism for falling into the same credulous dogmatism that its author repudiated in the churches , and so risking the status that darwinism had brought zoology , but he mentioned amphioxus only in a note ( semper 1876b , p. 35 ) .
haeckel s reputation would never recover fully from these battles , but most competent researchers still credited him for pushing the theory of evolution through . as tempers frayed , humour , which had
so far figured mostly in sarcasm between haeckel and his critics , appeared as mediator .
rhymes went some way towards defusing a tense situation by separating haeckel s excesses from commitments on which evolutionists just might agree .
nature researchers in the german lands had long sung together to foster ( male ) camaraderie and cultural nationalism , validate specialized knowledge ( jackson 2003 ) , and share the fun in songs such as joseph victor scheffel s mock - tragic lament by the last ichthyosaur .
a commission from the bern scientific society spurred the newspaper editor moritz reymond to write humorous verses on intellectual fashions such as the kulturkampf and medical advice , and haeckelism became his greatest theme.9 with a title referring to catholic books of daily prayers and the like for non - priests , and a focus on the old testament , das neue laienbrevier des haeckelismus ( the new lay breviary of haeckelism ) eventually ran to five books with cartoons by fritz steub of the fliegende bltter , the bavarian punch .
reymond prepared genesis , oder die entwickelung des menschengeschlechts ( genesis , or the development of the human race ) for christmas 1876 . shrinking the octavo lectures of the anthropogenie chapter by chapter to a sexto - decimo of songs
painlessly acquainted readers with the positive and negative sides of the original . engaging with its reception too
, the satire played on the symmetry between haeckel s charges of ignorance and the exclusivity of his own neologisms , on the one hand , and the irony of his condemning church dogma while setting himself up as the apostle of a new faith
reymond tackled the pedigrees at length , but left the hypothesis of amphioxus as ancestor unchallenged ; the consensus was too strong and dissent not yet prominent enough .
two songs reviewed the discoveries that had revealed respectively the structure of amphioxus and ascidians and their embryology and thematized the german tradition of finding origins in and around the mediterranean .
the first , to a folk tune previously used for a student song about diogenes the cynic and alexander the great , corresponded to chapter 13 of the anthropogenie and tells how zoologists lancets raised the status of the lancelet ( reymond 1877 , pp .
a series of encounters with costa , kowalevsky and haeckel , the extent of whose hands - on research is exaggerated , prove fatal for the individual animals .
invoking pallas fails to stop costa testing his hunch ( fig . 3 ) .
here is the complete song , der krperbau des amphioxus und der ascidie ( the structure of amphioxus and ascidian ) , with my translation but without the four endnotes that explained the history in quotations from the anthropogenie.fig .
the illustration by fritz steub , like the one in fig . 4 , was added in the second and kept in this third edition .
border 10.5 8.2 cmein schlpfriges lanzettenthiera lancet fish so slipperylag wohlgemuth i m sandelay cheerfully in the sandund dachte : ei wie schn ist s hierand thought ,
oh , is nt it lovelyan posilippos10 strande!here on posillipo s strand!da stieg ein grimmer zoologbut then a zoologist grimempor am horizonte , rose up on the horizon , der alles zu zergliedern pflog , who used to dissect with much vim , was er erwischen konnte.whatever he laid hands on.er sieht das thier und hebt es auf , he sees and snatches the creatureobwol es schneckenglatt ist , though smooth as a snail it be , und sagt : ich nehme gift darauf , and says : my own life i d wager , wenn das kein vertebrat ist!but vertebrate that must be!das thierchen seufzt : ach , gehn sie weg!the animal sighs : be off , you!das knnt ein jeder sagen!anybody could say that!ich bin ja nur ein nackter schneck a slug is all i am , it s true sie knnen pallas fragen!just go and ask pallas that!der zoologe aber sprachthe zoologist however spakezum groen schreck des findlings : to great terror in his find:der weise mann sieht selber nachthe wise man must his own look takeund glaubt nicht andern blindlings!and does nt trust others blind!drauf schob die pseudoschnecke erwith that the pseudo - snail he tuckedgefhllos in die tasche , callously in his pocket , zerschnitt und setzt sie hinterhercut it to bits and these then dunkedin eine weingeistflasche.in a bottle of spirit.dies kam , wie sehr auch paradoxhowever great a paradoxes scheint , dem thier zu statten;this seems , the beast uprated;denn seither zhlt der amphioxfor since that day ranks amphioxzur crme der vertebraten.as crme of the vertebrated.und wieder schwamm i m mittelmeeragain a zoologicalein zoolog spazieren , went for a swim in the med , wo die ascidien stationrwhere ascidians staticalam grunde vegetiren.vegetate on the seabed.mit einem schleppnetz und bedachtwith drag - net , care and attentionhat er an jenem ortehe in that same place did bringso manches stck zu tag gebrachtto light a splendid selectionvon dieser wrmersorte.of this type of wormy thing.es barg der cellulose - schlauchthe cellulose tunic it helddie wunderlichen altenthe wondrous adults matureund gleich die lieben kleinen auchand the dear babies also dwelledin der cloake falten.in cloacal folds secure.sie kamen unter s mikroskop , they came under the microscope , sie kamen unter s messer;they came under the cutter;sie starben sich zu tod darob
ein wunder hatte dafor see ! a miracle had thereenthllt mit einem mal sich : revealed itself once for all : die larve der ascidiathe larva of ascidiaerwies als vertebral sich!turned out to be vertebral!der hckel hrt s von ungefhrhaeckel heard , by chance twas really , und schnrte gleich den ranzenand packed his bags right awayund ging an s mittellndsche meerand went down to the middle seadrei monat in vakanzen.for three months of holiday.es forscht der groe zoologthat zoologist searched the groundvon frh bis abends spt hierfrom early till evening lateund fand , wie sehr doch analogand most analogous he foundascidie und lanzett-thier.ascidian and lance-let.was ihm gemangelt bis anhinwhat till then he had been lackingin seiner ahnenreihe , in his ancestral series , das bindeglied nach unten hin the downwards link a - connecting
das waren diese zweie!that was this pair of beasties!nun war die lcke ausgefllt , now the gap it was nicely healed , die reihe schn geschlossen , the series well closed henceforth , und das geheimni uns enthllt , and the secret to us revealed , wie wir dem wurm entsprossen!how from the worm we sprang forth!ascidie und amphiox , ascidian and amphiox , ein glas zu eurem ruhme!let s raise a glass to your fame!den wurzeln unsers ahnenstocksthe stem of our ancestral stocksbring jeder seine
the illustration by fritz steub , like the one in fig . 4 , was added in the second and kept in this third edition .
border 10.5 8.2 cm listeners were invited to drink to their newest - oldest ancestors . the next chapter , and so the next song , was about the embryology presented through a chance meeting over a beer between kowalevsky and brother straubinger , a travelling journeyman whose literary career had begun in the drinking song of a bavarian medical student some 50 years before ( reymond 1877 , pp .
there is space here only for a selection of verses in which straubinger recounts what happened after the st .
kowalevsky , whose report described amphioxus as starting to mate on warm may evenings between seven and eight , asks his acquaintance to appear on the beach behind vesuvius , sober and at half past seven precisely ( fig .
4alexander kowalevsky and brother straubinger watching amphioxus mating in naples . by fritz steub , from reymond ( 1878 , p. 113)der
professor wohlgeartetthe professor , good - humouredly , hat mich dort bereits erwartet , was there and waiting already , winkte mir und ruf : waved and gave a shout : passen sie man uf hallo , mate , look out allsogleich wird der schwindel losgehen!the show is going to start any minute now!als wir drauf zusammenrucktenwhen we moved closer , he and me , und in s meer hinunterguckten , and looked down , down into the sea , schwamm ein amphiuxan amphux swimmerum die ecke flugssped round the cornerund lie in gedanken etwas in s wasser fallen.and , in thought immersed , let something fall into the water.alldieweil traf unterwegs ihnwherepon an amphoxy ladydie madame amphixin;meeting him en route , silentlydachte sich : oho , thought to self : wahey , steht die sache so?are things now that way?und deponirte selbigenorts diverse amphioxeneier.and deposited various amphioxus eggs in the very same place.diese amphioxigen eierthese eggs so amphioxicalintressirten ungeheuerappeared of interest colossalkowalewsken , derto kowalevsky , alsbald hinterherwho now directlyin s meer sprang und dieselben mit nach hause nahm.jumped into the sea and took the same home with him .
alexander kowalevsky and brother straubinger watching amphioxus mating in naples . by fritz steub , from reymond ( 1878 , p. 113 )
straubinger goes back to the russian s lodgings and they watch the amphioxus develop.dorten konnten wir betrachten , there we could contemplate steadywie bereits um mitternachtenhow around midnight alreadyaus den eiern zartthose eggs so tendereine blase ward , became a bladderwelche kowalewsky das blastodermichen nannte.which kowalevsky called the blastodermlet .
the uneducated artisan , who symbolized conflict with the academic world , has difficulty with the damned latin names .
reymond thus both followed haeckel and his readers in thematizing the difficulty of the esoteric science , and borrowed straubinger s idiom to communicate some embryology : doch i m weiteren entwickelnyet as it develops quicklysprt die larve nun ein prickeln , the larva now feels all tinglyrcken auf und ab , up and down its bodund ein axenstaband an axis rodwchst ihr bratspieartig mitten durch s gedrm.grows right through its intestines like a skewer . working up an appetite
, straubinger would not have minded hearing more over some spare ribs , because you do nt never know what education will be good for , but kowalevsky sends him away hungry .
a catholic reviewer was pleased to see darwinism s true colors exposed , and for an editor of the leading anti - darwinist science magazine this food for the mind complemented the christmas sweetness as perfectly as herring salad with spanish pepper a stomach upset by marzipan.11 others saw reymond as helping haeckel , who was rumoured to have kept a copy in his jacket pocket ( may 1909 , p. 116 ) , even though many verses cut nearer the bone than those about amphioxus . in jena
zoology practical.12 english - speakers familiar with haeckel s books , and the debates over them , enjoyed reymond s verses too . in 1878 the british - born , gttingen - educated toronto palaeontologist and poet edward john chapman translated the first amphioxus song .
his biologist colleague robert ramsay wright had paid this animal much attention when reviewing the anthropogenie for the local scientific society , the canadian institute . like most professors
, he found the book admirably lucid while regretting the assertive tone ,
dogmatism and cavalier treatment of opponents ( wright 1876 ) . under the pseudonym dr .
agorastes ( marketeer ) , perhaps taken from lucian s sale of philosophies , chapman dedicated his translation to wright and the institute.13 but amphioxus and ascidian , our gelatinous ancestors imitated as much as it translated ; compare what follows with my more literal rendition above .
chapman added information , colour and drama , accommodated with an extra line in each of three fewer verses , as he turned a drinking song into a lyric poem , a pictureless piece of jocose rhyming in the style of samuel taylor coleridge , to be declaimed more than sung.14a slimy lancelet once lay half - hidden in the golden sandof naples blue and balmy bay and thought how pleasant , here , to - day is posilippo s strand.but on the horizon s shadowy brim a horrid vision doth arise a spectacled professor grim!all things that creep , crawl , fly , or swim , must he anatomize!a find his eager senses
he sees : with joy elatehe grasps the creature in a jiff and cries now , i ll be jiggered
if it is nt vertebrate!the victim groandcome , that s too pale do nt try on me that precious cram limpid and soft from top to tail , im nothing but a naked snail ask pallas what i am!the horrid zoo made answer , nay ! i mean , my dear , myself to fix it : no wise man trusts what others say , or heeds , in this far - searching day , the dead past s ipse dixit!so saying , without more ado , ( to tender feeling sadly callous)he slit poor slimy through and through , and bottled as he d bottle you this pseudo - snail of pallas!and thus , although the little nata- torial beast has no backbone , for reasons based on larval datait came among the vertebrata a place , at last , to own!pass thirty years and two ah me !
how quick time gallops!then there camea learned russ to that blue sea to fish for tunicates came he , also to fish for fame!and so before his zeal should flag , or fall below its high meridian15to work he went with dredge and drag , and fished up quite a thumping bag of things ascidian!and then came out the startling truth let all the world s four corners hear it!the ascidian in its frisky youthis half a vertebrate , in sooth , or something somewhere near it!swift sped the news oer land and sea , till reaching haeckel s ears that greatstem - framer in vacation free
packd up , and went post - haste to see this yea - nay vertebrate!and there , our great ontogenist whose word bewitches whilst it shocks us
beheld the links his system missd : yes , here they were or he d be hissd ascidian
amphioxus!all things are sure to one who waits : and here the links at last were seenmade manifest to meanest pates
invertebrated vertebrates fishes and worms between!thus , haeckelism s wondrous gleam makes clear , to all , how all arose backward and forward went the streamof shifting forms , like shapes of dream , and found in us its close!chapman thus replaced the toast with a romantic scene that perhaps foresaw endless revision to the pedigree .
a slimy lancelet once lay half - hidden in the golden sand of naples blue and balmy bay and thought how pleasant , here , to - day is posilippo s strand .
but on the horizon s shadowy brim a horrid vision doth arise a spectacled professor grim ! all things that creep , crawl , fly , or swim , a find his eager senses
he sees : with joy elate he grasps the creature in a jiff and cries now , i ll be jiggered
if it is nt vertebrate ! the victim groandcome , that s too pale do nt try on me that precious cram limpid and soft from top to tail , i m nothing but a naked snail ask pallas what i am ! the horrid zoo made answer ,
i mean , my dear , myself to fix it : no wise man trusts what others say , or heeds , in this far - searching day , the dead past s ipse dixit ! so saying , without more ado , ( to tender feeling sadly callous ) he slit poor slimy through and through , and bottled as he d bottle you this pseudo - snail of pallas ! and thus , although the little nata- torial beast has no backbone , for reasons based on larval data it came among the vertebrata a place , at last , to own !
how quick time gallops!then there came a learned russ to that blue sea to fish for tunicates came he , also to fish for fame !
and so before his zeal should flag , or fall below its high meridian15 to work he went with dredge and drag , and fished up quite a thumping bag of things ascidian ! and then came out the startling truth let all the world s four corners hear it !
the ascidian in its frisky youth is half a vertebrate , in sooth , or something somewhere near it !
swift sped the news oer land and sea , till reaching haeckel s ears that great stem - framer in vacation free packd up , and went post - haste to see this yea - nay vertebrate ! and there , our great ontogenist whose word bewitches whilst it shocks us beheld the links his system missd : yes , here they were or he d be hissd ascidian
all things are sure to one who waits : and here the links at last were seen made manifest to meanest pates invertebrated vertebrates fishes and worms between ! thus , haeckelism s wondrous gleam makes clear , to all , how all arose backward and forward went the stream of shifting forms , like shapes of dream , and found in us its close !
meanwhile , the german controversy over haeckel raged on . in the neue freie presse ( new free press ) of vienna vogt analysed haeckel s metamorphosis from apostle through prophet to oracle .
gravest sin that he had denied human kinship with ascidians and amphioxus and lampooned haeckel s enthusiastic exaggeration .
he now protested against even more extreme hyperbolicization by exclaiming with reference to the demand of the austrian tyrant defied by the swiss patriot wilhelm tell : how long will it be before we have an edict that we must doff our hats to a jar of amphioxus , like in front of geler s pole with a hat on top !
s verses , which developed this criticism , not long . following the second book of the breviary , the more critical exodus , a new instalment containing books three to five , leviticus
these still poked fun at the pedigrees , and now also satirized the latest debates by imagining the establishment of haeckelism as the state religion and haeckel s installation as high priest of an amphioxus cult .
leviticus , or the law for the prophets of the gospel of development ,
opened with steub s sketch of a kneeling haeckel ( fig . 5 ) and this verse ( reymond 1880a , pp . 34):fig .
5the cult of amphioxus in moritz reymond s leviticus : the high priest haeckel kneels as he gives instructions on bringing offerings .
border 10.5 8.2 cmwer staunte nicht das edle wesenwho would nt marvel with reverence , voll ehrfurcht und verwundrung an , full of admiration most bolddurch das die wissenschaft genesenfor the noble one which freed sciencevom alten blinden schpfungswahn?from blind creationism old?das unsers menschenstammbaums lckethat golden bridge of discoverymit seiner herrlichkeit erfllt , which with glory magnificentals goldene erkenntnisbrckefills a great gap in man s pedigree , uns den entwicklungsplan enthllt?!and reveals our development ? !
the cult of amphioxus in moritz reymond s leviticus : the high priest haeckel kneels as he gives instructions on bringing offerings .
border 10.5 8.2 cm the singer argued that if psalms must be sung , then to this little fish ; if offerings made , then to the first animal ever to develop vertebrae , a headless - unconscious great deed of development by the ancestor and creator of human beings .
haeckel was presented as tempted to do away with some senior professors he disliked
he maintained to the end , for example , the place of ascidians in the human stem - tree .
if , when ordaining the darwinist priests , reymond s haeckel detected any concession to creationism and teleology or scepticism towards recapitulation , he felled them with a lightning bolt and finished them off with
his pamphlet on freedom in science and learning slaughtered the traitors abihu , that is the left - liberal pathologist rudolf virchow , who had opposed the teaching of evolution in the schools , and nadab , alias reymond ( 1880a , pp .
peace came when , like yahweh quashing the rebellion of the tribes of israel by causing aaron s rod to bud , darwin ended his followers rivalry by keeping evergreen only haeckel s tree of
gastrae , amphiox and ape.16 reymond parodied haeckel s cult , but reflected the consensus on the ancestral status of amphioxus.fig .
du bois - reymond had declared ignorabimus ( we shall not know ) : scientists would never solve certain riddles .
( let us exercise restraint ) , as haeckel put it in order not to turn people against science .
by fritz steub , from reymond ( 1880a , p. 18 ) nadab - ignorabimus and abihu - restringamur killed by fire
du bois - reymond had declared ignorabimus ( we shall not know ) : scientists would never solve certain riddles .
( let us exercise restraint ) , as haeckel put it in order not to turn people against science .
having subsided in the 1880s , darwinist debate revived in the following decade . from 1899
haeckel s bestselling anticlerical synthesis die weltrthsel , translated as the riddle of the universe , expanded the audience for science , took him to dizzying heights of international notoriety and fame , and created a market for a fresh english translation of the evolution of man ( hopwood 2015 , p. 167 ) .
new readers also discovered the laienbrevier , including in the us ( kellogg 1908 ) . according to an anniversary edition
, haeckel recommended it to students too busy drinking and fencing for lectures ( reymond 1912 , p. 251 ) .
after man the most important and interesting of all vertebrate animals , a claim his translator exaggerated by omitting the vertebrate ( haeckel 1903 , ii , p. 441 ; haeckel 1905 , ii , p. 419 ; see also haeckel 1874 , p. 298 ; haeckel 1899 , p. 69 ) .
even as speculation about ancestors was going out of fashion , many biologists agreed on the merits of [ t]his exquisite form ( h. f. osborn in willey 1894 , p. ix ) .
encyclopaedias acknowledged that some scientists put the stem - father of all vertebrates in a degenerate line ( meyers groes konversations - lexikon1905 , pp . 454455 ) . yet as research on amphioxus produced a huge volume of articles , books and models ( e.g. , hatschek 1881 ; willey 1894 ; hopwood 2002 , pp .
3435 , 101 , 146 ) , it thrived as ( a relative of ) an ancestor and for other reasons too .
accessible on various coasts , if hard to culture , it featured prominently in surveys ( balfour 1881 , pp .
because all the fundamental structures of the body are laid down with schematic clearness , it provided an unrivalled introduction to embryology and a refuge to the perplexed embryologist
, produced in and for biological summer schools in the interwar us , rejoices in this classroom staple : a fish - like thing appeared among the annelids one day;it had nt any parapods or setae to display.it had nt any eyes or jaws or ventral nervous cord , but it had a lot of gill slits and it had a notochord.chorus:its a long way from amphioxus , it s a long way to us.it was a long way from the german tradition and reymond s haeckelism to these more technical , less historical verses .
but laughter over haeckel s cult still echoes in biologists amused pride in an animal which , for all his efforts , never quite became a household deity or even a household word.17 a fish - like thing appeared among the annelids one day ; it had nt any parapods or setae to display .
it had nt any eyes or jaws or ventral nervous cord , but it had a lot of gill slits and it had a notochord .
it s a long way from amphioxus , it s a long way to us .
this look at the public life of amphioxus in its early years of fame has sought , on the one hand , to do more justice to its dominance than accounts that have dwelled on controversy over the ascidian theory and then the diverse challenges to what long remained the dominant view .
it has contributed , on the other , to appreciation of the aesthetic dimensions of darwinism and of species choice . running through some of the fiercest battles in the history of science are smiles at haeckel s excesses easing acceptance that he spoke a core of truth , ancestor - worship that self - consciously imitated a religious cult , and affection for naples as the site of discoveries that revealed ancient history along the mediterranean shore . | biologists having rediscovered amphioxus , also known as the lancelet or branchiostoma , it is time to reassess its place in early darwinist debates over vertebrate origins . while the advent of the ascidian amphioxus theory and challenges from various competitors have been documented , this article offers a richer account of the public appeal of amphioxus as a primitive ancestor .
the focus is on how the
german darwin ernst haeckel persuaded general magazine and newspaper readers to revere this flesh of our flesh and blood of our blood , and especially on das neue laienbrevier des haeckelismus ( the new lay breviary of haeckelism ) by moritz reymond with cartoons by fritz steub . from the late 1870s these successful little books of verse introduced the neapolitan discoveries that made the animal s name and satirized haeckel s rise as high priest of its cult .
one song is reproduced and translated here , with a contemporary imitation by the canadian palaeontologist edward john chapman , and extracts from others .
predating the american it s a long way from amphioxus by decades , these rhymes dramatize neglected species politics of darwinism and highlight the roles of humour in negotiating evolution . | Flesh of our flesh
Haeckelism in rhymes
Hats off to amphioxus
This exquisite form |
PMC4456887 | reaction time ( rt ) is a measure of the quickness with which an organism responds to some sort of stimulus .
rt is defined as the interval of time between the presentation of the stimulus and appearance of appropriate voluntary response in the subject .
( 1 ) simple rt : here there is one stimulus and one response . ( 2 ) recognition rt : here there is some stimulus that should be responded to and other that should not get a response .
dutch physiologist franciscus cornelis donders ( 1865 ) was among the first to systematically measure human rt using a telegraph like device invented in 1840 by charles wheatstone .
prior to his studies , there is no significant traceable thread in the literature about human rts being measured .
human rt works by having a nervous system recognize the stimulus . the neurons then relay the message to the brain .
the message then travels from the brain to the spinal cord , which then reaches person 's hands and fingers .
the accepted figures for mean simple rts for college - age individuals have been about 190 ms for light stimuli and about 160 ms for sound stimuli .
rt in response to a situation can significantly influence our lives due its practical implications .
fast rts can produce rewards ( e.g. in sports ) whereas slow rt can produce grave consequences ( e.g. driving and road safety matters ) .
factors that can affect the average human rt include age , sex , left or right hand , central versus peripheral vision , practice , fatigue , fasting , breathing cycle , personality types , exercise , and intelligence of the subject . in the literature
the present study seeks to determine ( i ) whether rt varies with the receptor system involved , ( ii ) the difference if any to rts between the two sexes ( iii ) difference in rt in medical students with sedentary lifestyle and regularly exercising students .
a total of 130 medical students were taken out of which 10 were excluded by various exclusion criteria comprising of any history of hearing or visual disorder , smoking , alcoholism , cardiovascular and respiratory disease , on any medication affecting cognitive performance .
thus , this cross - sectional study was conducted on a total of 120 ( 60 male and 60 female ) healthy medical students who were physically normal in the age group 1820 years .
participation in the test was voluntary and informed written consent was taken from every participant .
the tests were done using inquisit 4.0 computer software released in 2013 by millisecond software in seattle , washington . during the visual rt ( vrt ) task , in the center of the white screen background ,
the participant gets presented a black fixation cross that is followed after variable time intervals by a target stimulus that is , red circle .
the students were asked to concentrate on the fixation cross and press the space bar key , as soon as possible once the red circle ( target stimulus ) appears on the screen . in a simple auditory rt ( art )
task after variable time intervals , the sound is played for 30 s to the participant through the speakers .
all the subjects were thoroughly acquainted with the procedure and practice trial was given every student before taking the test . by default ,
the time intervals are randomly chosen from 2000 ms , 3000 ms , 4000 ms , 5000 ms , 6000 ms , 7000 ms , 8000 ms .
five readings of each stimuli were taken , and their respective fastest rt 's for each stimuli were recorded .
the readings were taken between 10 a.m. and 12 p.m. in a quiet secluded room .
a comparison was made between a. vrt and art ; b. vrt and art between males and females ; and c. vrt and art between sedentary and regularly exercising healthy students .
both male and female medical students who participated in at least 30 min of moderate physical activity which made them sweat or breathe hard , such as fast walking , slow bicycling , skating , pushing a lawn mower , etc .
, on 5 or more days / week or in at least 20 min of vigorous physical activity that made them sweat or breathe hard , such as basketball , soccer , running , swimming , fast bicycling , fast dancing etc . ,
on 3 days or more / week for a minimum duration of 6 weeks were considered to be regularly exercising healthy adult students , whereas those students who did not participate in these activities were considered to have a sedentary lifestyle .
the statistical analysis was carried out with statistical package for social sciences version 17.0 manufactured by spss inc .
the statistical analysis of the results show that there is highly significant difference between the two and the art is faster than the vrt in medical students .
comparison between vrt and art of medical students unpaired t - test was used for overall comparison between male ( n = 60 ) against female medical students ( n = 60 ) for art and vrt separately as shown in table 2a .
p < 0.001 was obtained , which is highly significant indicating that males have faster rts when compared to females for both auditory , as well as visual stimuli .
table 2b shows comparison between sedentary male medical ( n = 44 ) and female medical students ( n = 56 ) .
p < 0.001 was obtained indicating sedentary male students have faster rts ( vrt and art ) as compared to sedentary female students .
however when regularly exercising male ( n = 16 ) and female students ( n = 4 ) were compared as shown in table 2c , nonsignificant differences were obtained .
overall comparison between male and female medical students according to our statistical analysis of the overall comparison between sedentary and regularly exercising healthy medical students in table 3 , a significant difference was found indicating that sedentary medical students have slower rts .
comparison between sedentary male and female medical students comparison between regularly exercising male and female medical students overall comparison between sedentary and regularly exercising healthy medical students
the study was conducted on 120 healthy medical students to study the vrts and arts and how do they vary on the basis of gender , and sedentary and regularly exercising healthy students .
our study shows that the art is faster than the vrt [ table 1 ] .
research done by pain and hibbs , shows that simple art has the fastest rt for any given stimulus .
has documented that the mean rt to detect visual stimuli is approximately 180200 ms , whereas for sound it is around 140160 ms
. researches by kemp show that an auditory stimulus takes only 810 ms to reach the brain , but on the other hand , a visual stimulus takes 20 - 40 ms .
therefore , since the auditory stimulus reaches the cortex faster than the visual stimulus , the art is faster than the vrt . shelton and kumar also concluded that simple rt is faster for auditory stimuli compared with visual stimuli and auditory stimuli has the fastest conduction time to the motor cortex along with fast processing time in the auditory cortex . on the other hand
, there is a research done by yagi et al . , which shows that rt to visual stimuli is faster than to auditory stimuli .
hence , our study further supports the evidence that art is faster than the vrt even in medical college students when confounding factors are removed . a review of the literature on the influence of gender on rt shows that in almost every age group , males have faster rts as compared to females , and female disadvantage is not reduced by practice .
researches done by misra et al . also showed that males responded faster than females .
study done by shelton and kumar , nikam and gadkari also reported similar findings to support females have longer rts than males .
the results of our study [ table 2a ] agree with these studies and indicate that male medical students have faster rts when compared to female medical students for both auditory , as well as visual stimuli . in our study
when sedentary male and female medical students [ table 2b ] were compared removing any confounding factors , rts of male students were faster ; thereby further supporting the evidence that males have faster rts than females .
the male - female difference is due to the lag between the presentation of the stimulus and the beginning of muscle contraction .
it is documented in the literature that the muscle contraction time is the same for males and females and motor responses in males are comparatively stronger than females , this explains why males have faster simple rts for both auditory as well as visual stimuli .
nowadays the male advantage is getting smaller , possibly because more women are participating in driving and fast - action sports .
this is evident from table 2c in which nonsignificant differences were obtained when regularly exercising male and female medical students were compared .
the findings of our study [ table 3 ] indicate that regularly exercising healthy medical students have faster rt than medical students with sedentary lifestyles and the difference between the two groups was statistically significant .
these findings confirm the effect of physical activity and doing sports on improving rt which is supported by literature review done in this regard .
nakamoto and mori found that college students who played basketball and baseball had faster rts than sedentary students .
spirduso proposed that less rt of athletes as compared to nonathletes was attributed to faster central nervous system processing times producing faster muscular movements in athletes . according to gavkare et al .
shorter rt in athletes could be due to improved concentration and alertness , better muscular coordination , improved performance in the speed and accuracy task . also , motor response execution is a physical task , so it is logical that people trained in physically reactive sports may have superior motor response ability .
it is also thought that individuals who exercise at moderate to intense levels have higher rates of cerebral blood flow .
this increased amount of blood flow in the brain results in improvements in cognitive functioning due to increased supply of necessary nutrients , such as oxygen and glucose .
our study shows that the art is faster than the vrt [ table 1 ] .
research done by pain and hibbs , shows that simple art has the fastest rt for any given stimulus .
has documented that the mean rt to detect visual stimuli is approximately 180200 ms , whereas for sound it is around 140160 ms
. researches by kemp show that an auditory stimulus takes only 810 ms to reach the brain , but on the other hand , a visual stimulus takes 20 - 40 ms .
therefore , since the auditory stimulus reaches the cortex faster than the visual stimulus , the art is faster than the vrt . shelton and kumar also concluded that simple rt is faster for auditory stimuli compared with visual stimuli and auditory stimuli has the fastest conduction time to the motor cortex along with fast processing time in the auditory cortex . on the other hand
, there is a research done by yagi et al . , which shows that rt to visual stimuli is faster than to auditory stimuli .
hence , our study further supports the evidence that art is faster than the vrt even in medical college students when confounding factors are removed .
a review of the literature on the influence of gender on rt shows that in almost every age group , males have faster rts as compared to females , and female disadvantage is not reduced by practice .
study done by shelton and kumar , nikam and gadkari also reported similar findings to support females have longer rts than males .
the results of our study [ table 2a ] agree with these studies and indicate that male medical students have faster rts when compared to female medical students for both auditory , as well as visual stimuli . in our study
when sedentary male and female medical students [ table 2b ] were compared removing any confounding factors , rts of male students were faster ; thereby further supporting the evidence that males have faster rts than females .
the male - female difference is due to the lag between the presentation of the stimulus and the beginning of muscle contraction .
it is documented in the literature that the muscle contraction time is the same for males and females and motor responses in males are comparatively stronger than females , this explains why males have faster simple rts for both auditory as well as visual stimuli .
nowadays the male advantage is getting smaller , possibly because more women are participating in driving and fast - action sports .
this is evident from table 2c in which nonsignificant differences were obtained when regularly exercising male and female medical students were compared .
the findings of our study [ table 3 ] indicate that regularly exercising healthy medical students have faster rt than medical students with sedentary lifestyles and the difference between the two groups was statistically significant .
these findings confirm the effect of physical activity and doing sports on improving rt which is supported by literature review done in this regard .
nakamoto and mori found that college students who played basketball and baseball had faster rts than sedentary students .
spirduso proposed that less rt of athletes as compared to nonathletes was attributed to faster central nervous system processing times producing faster muscular movements in athletes . according to gavkare et al .
shorter rt in athletes could be due to improved concentration and alertness , better muscular coordination , improved performance in the speed and accuracy task . also , motor response execution is a physical task , so it is logical that people trained in physically reactive sports may have superior motor response ability .
it is also thought that individuals who exercise at moderate to intense levels have higher rates of cerebral blood flow .
this increased amount of blood flow in the brain results in improvements in cognitive functioning due to increased supply of necessary nutrients , such as oxygen and glucose .
male medical students have faster rts as compared to female medical students for both auditory , as well as visual stimuli . regularly exercising medical students have faster rts as compared to medical students with sedentary life styles .
thus , it is strongly suggested that regular exercising must be encouraged in both male and female medical students to improve their efficiency levels . | background : reaction time ( rt ) is a measure of the response to a stimulus .
rt plays a very important role in our lives as its practical implications may be of great consequences .
factors that can affect the average human rt include age , sex , left or right hand , central versus peripheral vision , practice , fatigue , fasting , breathing cycle , personality types , exercise , and intelligence of the subject.aim:the aim was to compare visual rts ( vrts ) and auditory rts ( arts ) on the basis of gender and physical activity levels of medical 1st year students.materials and methods : the present cross - sectional study was conducted on 120 healthy medical students in age group of 1820 years .
rt for target stimulus that is , for the beep tone for measuring art , and red circle for measuring vrt was determined using inquisit 4.0 ( computer software ) in the laptop .
the task was to press the spacebar as soon as the stimulus is presented .
five readings of each stimulus were taken , and their respective fastest rt 's for each stimuli were recorded .
statistical analysis was done.results:in both the sexes rt to the auditory stimulus was significantly less ( p < 0.001 ) as compared to the visual stimulus .
significant difference was found between rt of male and female medical students ( p < 0.001 ) as well as between sedentary and regularly exercising healthy medical 1st year students.conclusion:the art is faster than the vrt in medical students .
furthermore , male medical students have faster rts as compared to female medical students for both auditory as well as visual stimuli . regularly exercising medical students have faster rts when compared with medical students with sedentary lifestyles . | I
M
R
D
Auditory reaction time versus visual reaction time
Gender
Sedentary and regularly exercising healthy students
C
Limitation of our study |
PMC4733387 | despite meticulous attention to surgical technique , an incidental durotomy ( idt ) is one of the most common complications of spinal surgery.1
2
3
4
5
6
7
8 this complication has led to innovative developments in materials and methods to assist surgeons in repairing these complications to avoid cerebrospinal fluid ( csf ) leaks .
primary closure with polytetrafluoroethylene ( ptfe ) such as 7.0 gore - tex ( l. gore & associates , newark , delaware , united states ) has historically been the method of choice.9 other primary methods used to repair these defects are microdural staples , muscle and fat grafts , xenograft dural patches ( e.g. , bovine pericardium ) , and collagen matrix grafts.9 to improve the repair , secondary augmentations are often utilized.9 two main categories are dural sealants and fibrin glues.9 numerous comparisons demonstrate that ptfe sutures along with secondary fibrin glue is significantly more effective than just sutures at reducing csf leaks.10
11 success rates as high as 97.7% have been reported for preventing csf leak using these methods.8 similarly , success rates of 93 to 95% have been reported with tight dural suture and fibrin glue.12
13 a recent study looking at idt repair , without the use of sutures , using duragen ( integra , plainsboro , new jersey , united states ) , a collagen matrix graft , combined with duraseal ( covidien , mansfield , massachusetts , united states ) , a dural sealant , reported a success rate of 95.7%.14 although this figure falls within the same range , this method is much more efficient than suturing.14 moreover , during dural suturing , pinholes are made , which may cause persistent csf leakage .
suturing the dura whenever possible , with or without augmentation , remains the gold standard for idt repairs .
food and drug administration ( fda ) to include the following statement : duraseal can swell after application , it should not be used in areas where neural structures could be compressed , and no difference in the prevention of long - term csf leakage has been demonstrated and standard diligence in wound closure should be performed when using this device.15 based on fda approval documents , when tested in phosphate - buffered solution , duraseal swells no more than 200%.16 based on instructions provided by the fda , in vivo , hydrogel may swell up to 50% of its size in any dimension.15 through a search of the literature , we have found that the expansion properties of duraseal are not always predictable.17
18
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20
21
22 one study reports a 333% expansion after the use of a dural sealant.21
the aim of this case series is to report a rare complication ( hydrogel cutaneous fistula ) of the use of a dural sealant .
this is a case series report ( n = 2 ) of a previously unreported complication of the use of a dural sealant . in both cases ,
duraseal was used for the correction of idt and led to transcutaneous drainage of clear gel - like substance .
duraseal is fda - approved for its use as an adjunct to sutured dural repair.15 it is a resorbable synthetic hydrogel sealant that is composed of two solutions : a polyethylene glycol ester solution and a trilysine amine solution.15 duraseal is provided in a syringe applicator that mixes both solutions as pressure is applied.15 this application causes a crosslink between the two compounds forming the hydrogel sealant.15
both patients were born in mexico and were of spanish descent .
these cases were performed by one surgeon ( f.t . ) in a single institution .
a 19-year - old man presented to our clinic with intractable back and leg pain that failed to improve with standard conservative treatment .
three weeks postoperatively , a transcutaneous csf fistula was diagnosed due to an idt not recognized intraoperatively .
the patient was taken back to the operating room and the durotomy was closed with dural suturing ( 7.0 gore - tex ) , followed by a collagen matrix graft ( 1 cm by 1 cm ) and dural sealant ( 5 ml ) .
a four - layer closure was done to repair the incision ( interrupted 0 vicryl [ ethicon , somerville , new jersey , united states ] ; running 0 vicryl , running 2.0 vicryl , and running 3.0 nylon ) .
the patient presented to the clinic on postoperative day 14 with mild wound dehiscence and transcutaneous drainage of a clear , gel - like material .
approximately 3 ml of a clear gel - like substance was expressed from a 3-mm fistula on the superior aspect of the incision .
the sample was negative for 2-transferrin , bacterial culture growth , gram staining , and it demonstrated a low white blood count .
18 months ' postoperative follow - up , the patient was free of any symptoms and the incisions were well healed .
a 42-year - old man presented to our clinic with intractable low back and leg pain that failed to improve with standard conservative treatment .
imaging showed a left - sided disk herniation at the l5s1 level . a single - level microdiskectomy was conducted .
the operation was complicated by an idt , which was repaired by dural suturing ( 7.0 gore - tex ) , followed by a collagen matrix graft ( 1 cm by 1 cm ) and dural sealant ( 5 ml ) .
a four - layer closure was done to repair the incision ( interrupted 0 vicryl ; running 0 vicryl , running 2.0 vicryl , and running 3.0 nylon ) .
the patient presented to the clinic on postoperative day 16 with mild wound dehiscence and transcutaneous drainage of a clear gel - like material .
the sample was negative for 2-transferrin , bacterial culture growth , gram staining , and it demonstrated a low white blood count .
the patient was taken back to the operating room for a washout . during the operation
two weeks after the washout operation , the patient presented with recurrence of a similar clear gel - like drainage and small wound dehiscence . at this point ,
18 months ' postoperative follow - up , the patient was free of any symptoms and the incisions were well healed .
because the second irrigation and debridement surgery was done in mexico , there is no information on any analysis of surgical tissue or fluids that may have been performed .
a 19-year - old man presented to our clinic with intractable back and leg pain that failed to improve with standard conservative treatment .
three weeks postoperatively , a transcutaneous csf fistula was diagnosed due to an idt not recognized intraoperatively .
the patient was taken back to the operating room and the durotomy was closed with dural suturing ( 7.0 gore - tex ) , followed by a collagen matrix graft ( 1 cm by 1 cm ) and dural sealant ( 5 ml ) .
a four - layer closure was done to repair the incision ( interrupted 0 vicryl [ ethicon , somerville , new jersey , united states ] ; running 0 vicryl , running 2.0 vicryl , and running 3.0 nylon ) .
the patient presented to the clinic on postoperative day 14 with mild wound dehiscence and transcutaneous drainage of a clear , gel - like material .
approximately 3 ml of a clear gel - like substance was expressed from a 3-mm fistula on the superior aspect of the incision .
the sample was negative for 2-transferrin , bacterial culture growth , gram staining , and it demonstrated a low white blood count .
18 months ' postoperative follow - up , the patient was free of any symptoms and the incisions were well healed .
a 42-year - old man presented to our clinic with intractable low back and leg pain that failed to improve with standard conservative treatment .
the operation was complicated by an idt , which was repaired by dural suturing ( 7.0 gore - tex ) , followed by a collagen matrix graft ( 1 cm by 1 cm ) and dural sealant ( 5 ml ) .
a four - layer closure was done to repair the incision ( interrupted 0 vicryl ; running 0 vicryl , running 2.0 vicryl , and running 3.0 nylon ) . a drain was placed and removed on postoperative day 3 .
the patient presented to the clinic on postoperative day 16 with mild wound dehiscence and transcutaneous drainage of a clear gel - like material .
the sample was negative for 2-transferrin , bacterial culture growth , gram staining , and it demonstrated a low white blood count .
the patient was taken back to the operating room for a washout . during the operation
two weeks after the washout operation , the patient presented with recurrence of a similar clear gel - like drainage and small wound dehiscence . at this point ,
the drainage stopped after this operation . at 18 months ' postoperative follow - up , the patient was free of any symptoms and the incisions were well healed .
because the second irrigation and debridement surgery was done in mexico , there is no information on any analysis of surgical tissue or fluids that may have been performed .
the case reports we have presented are unique due to the transcutaneous discharge after the use of a dural sealant .
although the amount of gel expelled from the wound site was not measured , we estimate it to be 3 ml in both cases . in both patients ,
a 2-transferrin electrophoresis test was done on the gel - like secretion , and the results were negative , concluding that the fluid was not csf . due to the similarity of this gel to the dural sealant used and the negative results for csf , immune response , and infection , it is our impression that the discharge was due to the cutaneous fistula formation and drainage of the in vivo expansion of a dural sealant . because the drainage in both cases was through the operative incision and not through the drain orifice
although the fda - approved product information for duraseal reports an expansion up to 50%,15 there have been multiple reports of inappropriate expansion of this hydrogel sealant . through our search of the literature , we discovered six similar reported cases of duraseal expansion .
our study is the first one reporting a cutaneous fistula complication after the use of a collagen matrix graft along with dural sealant . unlike other reported cases
kacher et al used computed tomography and magnetic resonance imaging to investigate the expansion properties of duraseal in two canines .
they found that the hydrogel sealant had its maximal expansion between 3 days and 2 weeks.23 they also report a complete absorption of the hydrogel and closure of the remaining void by 8 weeks after implantation.23 the expansion properties of duraseal have also been reported to have protective effects when recombinant human bone morphogenetic protein type 2 is used in transforaminal lumbar interbody fusion ( tlif ) surgeries.24 its use to seal off the annulotomy site and as a protective covering to the nerve root reduces the incidence of postoperative radiculitis from 20.4 to 5.4% ( p = 0.047).24
abbreviation : n / a , not available .
we used 5 ml of a dural sealant for each spinal level , which seems to be higher than the other cases that we have cited in this report .
neuman et al use a maximum of 2 ml for each spinal level in their case of cauda equina after tlif.20 duraseal 's fda - approved instructions do not suggest an amount ; instead , it recommends making a 1- to 2-mm coat and it permits immediate irrigation after application.15
duraseal 's expansion is properties are noted in its manufacturer 's documentation , numerous case reports , and in vivo and in vitro studies . the amount of expansion , however , is not uniform among these sources .
complications such as neurologic compression and the now reported cutaneous fistula may arise from the material expansion .
duraseal is effective in reducing postoperative csf leakage and in most cases reduces surgical complications and operative time .
we suggest applying duraseal conservatively and using caution with respect to volume and location to reduce possibility of symptomatic expansion . the hydrogel fistula was easily controlled in the two cases .
simple expression of the gel from the wound in the clinic resolved the first case , and a small irrigation and debridement , removing the hydrogel , concluded the second one . |
study design case report .
objective incidental durotomy ( idt ) is a common complication of spinal surgery .
the use of collagen matrix graft along with hydrogel dural sealant is a common method of idt repair . with this method ,
there have been several reported cases of detrimental dural sealant expansion in the literature .
one case study reported an expansion rate greater than 300% ; many report neurologic damage .
this article reports the clinical course of two patients who developed postoperative transcutaneous drainage of a gel - like substance after the use of a dural sealant , which is a previously unreported complication .
methods the clinical course and treatment outcome of two patients is presented .
results both patients experienced postoperative transcutaneous drainage of a gel - like substance at the surgical site .
case one began draining this substance on postoperative day 14 .
this patient required no further intervention , and the drainage ended after 3 ml of a gel - like substance was expressed from his incision while in the clinic .
case two began draining the gel on postoperative day 16 .
this patient underwent two washout procedures and resolution of the drainage .
no infection was ever detected .
conclusions to our knowledge , our patients are the first reported cases of transcutaneous drainage of expanded dural sealant .
it is important to take into consideration the unexpected expansion of a dural sealant when using it for the repair of idt . | Introduction
Materials and Methods
Case Reports
Case One
Case Two
Discussion
Conclusion |
PMC4875992 | radiation , as a monotherapy or as a component of a polytherapeutic approach , is used to treat malignant cancers due to its high degree of efficacy .
unfortunately , radiation does not distinguish among the types of cells it destroys , and so healthy tissues in and around the cancerous ones are also compromised .
thus , survival carries with it a risk of deleterious side effects as a result of radiation - induced normal brain tissue injury .
this presents a unique challenge with regard to the pediatric population , wherein cranial radiotherapy ( crt ) is part of treatment for acute lymphoblastic leukemia ( all ) and solid tumors of the brain , which comprise a significant proportion of malignant cancers .
the nonspecific radiation - induced perturbations to the developing brain can have adverse consequences , both acute and long - term , for the brain and body [ 39 ] .
clinical imaging studies provide evidence at the functional network level that crt has detectable effects on cerebral integrity [ 1012 ] , connectivity [ 1315 ] , and volume [ 11 , 1621 ] .
numerous studies have found changes in metabolites and/or diffusion tensor parameters in the brain after irradiation in rodents . using magnetic resonance spectroscopy changes in n - acetylaspartate ( naa ) ,
glutamate , choline , lactate , taurine , and myo - inositol have been measured in different brain regions after radiation exposure [ 2224 ] .
in addition , diffusion tensor imaging ( dti ) parameters have been measured in a variety of brain regions , such as the fimbria , external capsule , and corpus callosum , after brain irradiation [ 2326 ] . in this study , we employ these imaging measurements to investigate the effect of crt on adolescent rats .
crt also disrupts endocrine function by suppressing growth hormone expression [ 2729 ] resulting in growth retardation [ 9 , 30 ] and significant alterations in body composition and weight are often observed in survivors of radiation - treated childhood cancers [ 7 , 31 , 32 ] .
although there is a great deal of individual variability within survivors in terms of diagnosis , sex , age at treatment , and type , dose , and regimen of treatment , childhood crt is recognized as being the biggest risk factor for negative neurocognitive and psychosocial outcomes in adulthood , leading to a poorer quality of life overall [ 33 , 34 ] . as such , it is imperative to characterize the scope and precise mechanisms of crt - induced damage in order to determine how best to minimize the cost - benefit ratio of crt and establish effective interventions to mitigate late - onset neurocognitive deficits that are generally irreversible
. it is widely accepted that the adverse response of the normal tissue in the brain to single dose irradiation occurs early , while its response to fractionated irradiation is late .
differences in the expression levels of inflammatory molecules , between single and fractionated doses , have been reported .
however , most of these studies have focused on the early phase of radiation damage and little has been done to elucidate the long - term difference between these two regimens .
animal models of pediatric radiotherapy provide the opportunity to examine radiation effects at the cellular , structural , and circuit levels in the developing brain .
animal studies of single dose radiation effects on neurogenesis show a dramatic decrease [ 3638 ] , but the effects of fractionated irradiation of the adolescent brain on hippocampal cell genesis have been less studied . in this work ,
we investigated the effects of single dose and fractionated crt in adolescent rats on body weight , brain weight , ki67 ( a marker of cell proliferation ) , and fractional anisotropy ( a measure of functional integrity of fiber tracts ) in the fimbria fornix as well as metabolite changes in the hippocampus .
the hippocampus and its primary efferent projection ( the fimbria fornix ) are critical for learning and memory [ 39 , 40 ] .
indeed , it is thought that the cognitive impairments associated with cancer treatment are due in large part to effects on this structure [ 4143 ] . in this paper
, we will present data on proliferation , microstructural , and metabolite changes measured in these two structures .
animal care was in accordance with guidelines set by the national institutes of health guide for the care and use of laboratory animals ( 2011 ) and all procedures were approved by the institutional animal care and use committee of baylor college of medicine ( houston , tx ) .
rats were 24 days old upon arrival and were housed in pairs in a temperature - controlled vivarium under a 12-hour dark / light cycle ( lights off 7:00 am7:00 pm ) with unrestricted access to food and water .
body weights were recorded daily during the course of radiation and then weekly and prior to euthanasia for brain harvest .
their teeth were trimmed under isoflurane anesthesia once every week until termination of the experiment .
the overgrowth of the incisors prevented irradiated animals from being able to consume the standard , hard , food pellets .
therefore , all rats , including shams , were given fresh , water - softened food pellets in their home cages , every day , throughout the course of the experiment .
this was initiated immediately after the first session of crt to ensure that the drop in body weight during the course of radiation was not due to a crt - induced inability to chew the standard pellets or fatigue / illness that would prevent only the irradiated animals from accessing the pellets on top of their home cages . at 28 days of age
, animals were anesthetized using isoflurane and randomly assigned to either the radiotherapy ( crt ) or sham group .
animals in the crt group were individually irradiated at a dose rate of 128 cgy / min using a rs 2000 biological x - ray irradiator 150 kvp , 25 ma ( rad source technologies , inc . , suwanee , ga ) .
each rat was placed prone and lead shielding was used to ensure that only the region of the head beginning behind the eyes and extending to approximately 5 mm behind the ears received radiation , so that the whole brain ( cerebrum and cerebellum ) , but not the eyes , would be within the field of radiation .
approximately half of the crt animals received a single dose of 20 gy ( n = 19 ) and the other half received the same dose divided into 4 gy fractions across 5 consecutive days ( n = 18 ) .
animals in the sham group ( n = 18 ) were anesthetized for the same length of time as those in the crt group but did not receive radiation .
animals from each group were sacrificed 1 d ( n = 6 per group ) , 5 d ( n = 6 per group ) , and 3.5 mo ( n = 6/7 per group ) following start of crt ( figure 1 ) .
animals euthanized at the 3.5 mo after crt interval were first imaged approximately between 3 and 3.5 mo after crt ( figure 1 ) . a 9.4 t biospec mri scanner ( bruker , ma ) with a 20 cm bore and a quadrature rat brain array ( bruker , ma )
a respiratory pillow was placed under the abdomen of the animals and a rectal probe was used to monitor respiration and temperature , respectively .
a t2-rapid acquisition with relaxation enhancement ( t2-rare ) scan with the following parameters , te = 20 ms , tr = 2600 ms , # of averages = 1 , 3 cm field of view , 12 slices , 1 mm slice thickness , 1.1 mm interslice distance , and 256 256 matrix size , was then performed and used to localize the placement of the spectroscopy voxel .
voxel placement was in the hippocampus ( 2 mm height , 4 mm width , 3 mm anterior to posterior ) .
prior to mr spectroscopy a fastmap sequence was performed to correct for local field inhomogeneity by adjusting first- and second - order shim coil currents .
all mr spectra acquired using a stimulated echo ( steam ) sequence with the following parameters , tr = 2 s , te = 2.22 ms , # of averages = 512 , and voxel size = 24 mm , were executed .
water suppression was achieved using variable pulse power and optimized relaxation delay ( vapor ) . for quantification purposes ,
metabolites were excluded when they did not pass the criterion of n 4/group and cramr - rao lower bounds < 20% .
following spectroscopy , dti was performed ( n = 6 - 7/group ) using a spin echo planar imaging ( epi ) sequence with a tr = 500 ms , te = 33.2 ms , 2 repetitions , slice thickness = 12.8 mm , b value = 800 s / mm , # of directions 30 , and matrix size 128 256 16 giving a spatial resolution of 150 m 150 m 800 m .
total imaging time , for spectroscopy and dti , was approximately 1 hour per animal .
fractional anisotropy ( fa ) maps were processed through medinria and analyzed using inveon research workstation ( siemens , washington , dc ) .
briefly , in slices in which the fimbria fornix was visible , a region of interest ( roi ) was drawn around the fimbria fornix ( left and right sides ) and a fa threshold value of 0.6 and 0.75 applied and mean fa calculated .
mean fa values obtained were averaged between left and fight fimbria and between slices for each animal .
first , total fimbria volume ( tfv ) was calculated using a 0.5 fa threshold , to isolate the entire fimbria ( figure 4(a ) ) , in each imaging slice in which the fimbria was visible .
second , a fa threshold value of 0.6 and 0.75 was applied to the tfv and a resulting thresholded volume ( tv0.6 , tv0.75 ) was calculated ( figure 4(b ) ) .
third , the tvx was calculated and averaged between left and fight fimbria and between slices for each animal to arrive at tvx .
finally , a ratio of tvx / tfv was calculated ( figure 4(c ) ) .
animals were given an overdose of ketamine / xylazine ( i.p . ) and transcardially perfused with 4% paraformaldehyde at 1 d , 5 d , or 3.5 mo after the start of crt ( figure 1 ) .
brains were removed , postfixed for 24 h , and stored in 30% sucrose until sectioned .
brains ( forebrain and cerebellum only ) were weighed and then cut into 50 m serial , coronal sections on a freezing - stage sliding microtome ( leica microsystems , sm2000r , nussloch , germany ) and stored in cryoprotectant in 96-well microtiter plates at 20c . every 6th section throughout the rostrocaudal extent of the hippocampus was processed by standard immunohistochemical procedure as reported previously to label proliferating cells .
briefly , sections were rinsed in 0.1 m tris - buffered saline ( tbs ) , quenched for 30 min at room temperature in 0.6% hydrogen peroxide , and rinsed in tbs again .
next , sections were pretreated in 10 mm sodium citrate buffer ( ph 8.5 ) for 30 min in a 80c water bath , allowed to cool down to room temperature , and rinsed in tbs before incubation in 3% normal donkey serum ( sigma - aldrich , st .
louis , mo ) for 1 h. this was followed by incubation at 4c for 72 h in primary antibody ( rabbit , polyclonal anti - ki67 , 1 : 1800 ; vector laboratories , burlingame , ca ) . sections were then rinsed in tbs , blocked in 3% normal donkey serum for 15 min , and incubated overnight at room temperature in secondary antibody ( biotinylated donkey , anti - rabbit , 1 : 250 ; jackson immunoresearch laboratories , west grove , pa ) .
next , sections were rinsed in tbs , treated for 90 min in avidin - biotin complex ( abc , vector laboratories ) , and rinsed again .
sections were reacted and visualized with diaminobenzidine , rinsed before being mounted onto gelatin - coated slides , and allowed to dry overnight .
sections were then counterstained with methyl green , cleared in xylene , and cover - slipped using permount mounting medium ( fisher scientific , pittsburgh , pa ) .
ki67 + cells were quantified in the subgranular zone ( sgz ) of the dentate gyrus ( dg ) using a nikon eclipse 80i upright microscope in conjunction with stereoinvestigator ( microbrightfield , williston , vt ) .
approximately , 10 - 11 sections per animal were analyzed for ki67 + cells at 40x magnification throughout the septotemporal axis of the hippocampus by an experimenter blind to the conditions .
since ki67 staining was sparse , cells were counted nonstereologically , and the number of cells was summed across sections for each animal .
ki67 + cell counts in the sgz ( 1 d , 5 d , and 3.5 mo after crt ) , body weights ( recorded just before euthanasia at each interval ) , and normalized brain weights ( 24 h and 3.5 mo after the end of crt ) were analyzed by two - way anovas with crt and post - crt interval as factors .
one - sample t - tests were used to compare differences between mean brain weight per group ( at each interval ) and shams ( i.e. , a value of 100 ) .
mean fa values , fimbria volume - ratios per applied threshold , and hippocampal metabolite levels were analyzed by one - way anovas .
data on body weight , body length , and body weight / length collected from a small , separate cohort of animals ( see figure 6 ) were analyzed by one - way repeated measures anovas .
there was a significant main effect of crt [ f(2,46 ) = 59.72 , p < 0.0001 ] , post - crt interval [ f(2,46 ) = 20.13 , p < 0.0001 ] , and interaction [ f(4,46 ) = 17.81 , p < 0.0001 ] on the mean number of ki67 + cells in the sgz of the hippocampus ( figure 2 ) .
post hoc analyses revealed that single dose crt as well as fractionated crt significantly reduced the number of ki67 + cells relative to shams at the 1 d and 5 d post - crt intervals . however , due to the substantial drop in ki67 + cells in shams due to age alone [ 1 d versus 5 d : p < 0.0001 , 5 d versus 3.5 mo : p = 0.001 ] , this difference did not reach statistical significance in adulthood . in total ,
13 metabolites were successfully measured ( n 4/group and 20% cramr - rao lower bound ) in all experimental groups . however , only two metabolites , myo - inositol and n - acetylaspartate + n - acetylaspartylglutamate ( naa + naag ) , had measureable significant change .
there was a significant effect of crt on myo - inositol [ f(2,16 ) = 8.3 , p < 0.01 ] and naa + naag levels [ f(2,15 ) = 8.75 , p = 0.003 ] at 3 mo after crt ( figure 3 ) .
post hoc analyses showed that myo - inositol was significantly lower in single dose crt animals compared to shams [ p < 0.01 ] .
naa + naag levels were significantly higher in single dose crt animals relative to shams [ p < 0.01 ] and fractionated crt animals [ p < 0.05 ] . at 3 mo after crt , no changes in mean fa were found in the fractionated crt animals .
however , single dose crt animals demonstrated a significant reduction compared to shams [ p < 0.05 ] in mean fa values within voxels that were equal to or greater than the set threshold of 0.6 fa ( figure 4(b ) ) .
further , post hoc analyses revealed that , relative to shams , only single dose crt animals had a significant reduction in the ratio of the fimbria volume above fa thresholds of 0.6 [ p < 0.01 ] and 0.75 [ p < 0.05 ] ( figure 4(c ) ) .
furthermore , the ratio of the volume of the fimbria above fa thresholds of 0.6 and 0.75 in fractionated crt was also reduced , albeit not significantly .
there was a significant main effect of crt [ f(2,46 ) = 56.55 , p < 0.001 ] and post - crt interval [ f(2,46 ) = 556.9 , p < 0.001 ] on body weight as well as a significant interaction between crt and post - crt interval [ f(4,46 ) = 39.14 , p < 0.001 ] .
post hoc tests revealed that , at 3.5 mo after crt , both fractionated and single dose - treated animals had significantly reduced body weights relative to age - matched shams [ p < 0.0001 ] ( figure 5 ) .
in addition , single dose crt suppressed body weight even relative to fractionated crt [ p < 0.001 ] . at 5 d after crt , that is , 24 h after the last fractionated dose of radiation , only the single dose crt group had significantly reduced body weight relative to shams [ p < 0.05 ] .
we also measured differences in body length and body weight / length ratios ( figure 6 ) .
brain weights of crt groups were normalized to that of age - matched shams and compared at 24 h and 3.5 mo following end of treatment ( figure 7(a ) ) .
there was a significant effect of interval on brain weight [ f(1,21 ) = 141.1 , p < 0.0001 ] and a significant interaction between crt and interval [ f(1,21 ) = 108.6 , p < 0.0001 ] .
post hoc analyses showed that , the day after end of treatment , single dose crt significantly increased brain weight relative to shams ( 9.2% increase , [ p < 0.01 ] ) and fractionated crt ( 15.7% increase , [ p < 0.0001 ] )
. however , in the fractionated crt group , mean brain weight was significantly lower than sham ( 6.5% reduction , [ p = 0.0001 ] ) .
3.5 mo after end of treatment , single dose crt significantly reduced brain weight relative to shams ( 18.9% reduction , [ p < 0.0001 ] ) and fractionated crt ( 10.6% reduction , [ p < 0.0001 ] ) .
the mean brain weight of the fractionated crt group was also significantly reduced relative to sham ( 8.4% reduction , [ p = 0.0001 ] ) .
mean brain weight of the single dose crt group was reduced 28.1% at 3.5 mo relative to 24 h [ p < 0.0001 ] .
crt - induced suppression of cell proliferation in the sgz was evident the day after the start of radiation and persisted into adulthood ( figure 2 ) .
there were no significant differences between single dose and fractionated regimens at any interval , although there were more ki67 + cells at the 1 d post - crt interval in the animals that received a single 4 gy fraction relative to those that received a single 20 gy dose while this pattern was reversed at 5 d. it may be that there was some recovery in the single dose animals 5 days after their last exposure to radiation or that this is simply an artifact of within - group variance .
if assessed independently at each interval , the differences between single dose and fractionated crt reach statistical significance only at the 1 d interval which is not surprising as at this point the comparison is between doses of 20 gy and 4 gy .
note that while proliferation in neither irradiated group differed significantly across intervals , the number of ki67 + cells in shams dropped significantly as a function of age .
our observations concur with those of others reporting a comparable number of ki67 + cells in the sgz of nave rodents of similar age [ 38 , 46 ] and a significant drop in neurogenesis between adolescence and adulthood [ 4749 ] .
the decrease in proliferation in shams from 1 d to 5 d may in part be a function of error variance and/or due to repeated isoflurane exposure which is reported to suppress hippocampal neurogenesis in juvenile but not in adult rodents [ 50 , 51 ] .
however , studies on isoflurane - induced neurotoxicity typically use long durations of anesthesia , whereas , in our experiment , animals received , on average , 1015 min ( 13% , 0.51
furthermore , as sham animals were matched to irradiated animals for isoflurane exposure , the differences in proliferation between the irradiated groups and age - matched sham groups can not be attributed to isoflurane .
our results agree with others [ 38 , 52 , 53 ] , as independent anovas for each post - crt interval reveal a significant main effect of crt even at 3.5 mo after crt .
however , when the data are analyzed as a whole , the significance is lost .
we chose a factorial analysis to control for familywise error rate , but the fact remains that , in the absence of data at earlier time points , we would have detected a statistically significant difference at this time point . in summary ,
our results indicate that 20 gy of radiation , delivered either as a single dose or in 5 daily fractions , suppresses hippocampal cell proliferation acutely relative to controls , and there is no change in proliferation from the acute to early delayed phase after radiation in both crt groups .
the sgz , one of the primary neurogenic niches of the brain , contains progenitor cells which proliferate and subsequently migrate to the adjacent granule cell layer whereupon they differentiate and mature .
the survival of these neurons is typically determined 1116 days after mitosis ; therefore , any damage sustained during this period could disrupt normal brain development resulting in adverse long - term effects .
our data show that a dose of radiation as low as 4 gy is sufficient to suppress cell proliferation to approximately 6% of control levels , 24 hours after exposure .
this is consistent with evidence that the majority of apoptosis among progenitor cells in the sgz occurs within 24 hours after radiation and other reports of a long - lasting decrease in proliferation or neurogenesis in the rodent hippocampus following cranial radiation in early development [ 5558 ] .
disruption of hippocampal neurogenesis during development , especially if persistent , may well contribute to the decrease in brain size that we have observed [ 59 , 60 ] ( figure 7(b ) ) as well as late cognitive sequelae of childhood crt [ 43 , 61 , 62 ] . to assess radiation - induced damage ,
we used dti , a noninvasive mri technique commonly used to measure the diffusion of water within the brain , which can be altered by organizational or microstructural changes in tissue matter . a decrease in fa value (
an increase in unrestricted water movement ) in the brain has been correlated with edema , inflammation , and a decrease in neuronal myelination . in our model , a significant decrease in mean fa was measured in the fimbria of animals treated with single but not fractionated dose crt , implying increased radiation - induced damage compared to fractionated crt .
our findings are consistent with those of others who have reported that fa decreases after single dose radiation exposure in the fimbria [ 23 , 65 ] and other fiber tracts [ 24 , 26 , 65 , 66 ] but is not detectable following fractionated treatment .
histological studies also report white matter damage following a single exposure to radiation [ 23 , 6769 ] .
we also found that the ratio of fimbria volume was decreased in the single dose - treated animals at both thresholds applied , whereas the fractionated crt animals showed no reduction at either threshold .
another study using adolescent mice also reported that volumetric differences in the fimbria emerge as early as 1 week after single dose crt and show limited subsequent recovery .
given that the fimbria fornix comprises a bundle of afferent and efferent myelinated projections to and from the hippocampus , radiation - induced damage to this white matter tract may contribute to late cognitive effects . using magnetic resonance spectroscopy , we found that , in the single dose , but not in the fractionated , crt group , myo - inositol decreased significantly , while naa + naag increased significantly relative to shams .
these two metabolites are involved in astrocyte metabolism ; myo - inositol , which is an osmolyte and is also involved in membrane turnover [ 71 , 72 ] , is more abundant in astrocytes than in neurons , and the production and metabolism of naa + naag are through a cycle that involves neurons , astrocytes , and oligodendrocytes [ 56 , 57 ] .
coupling these metabolites findings with our observed reduction in cell proliferation ( figure 2 ) seems to indicate that in the case of severe radiation - induced injury , as demonstrated by the single dose crt , the onset of gliosis and demyelination could offer an explanation for these changes .
we have previously reported an increase in gliosis following single dose cranial irradiation that persists for months after treatment [ 74 , 75 ] .
further , we and others have observed demyelination onset at around three months after irradiation [ 76 , 77 ] . further experimentation and maybe the use of nmr spectroscopy of brain tissue samples would be needed to confirm our observation . within the first few days after the start of radiation , body weights of irradiated animals were lower than those of age - matched shams ( figure 5(a ) ) . while both regimens suppressed growth , only the difference between single dose and sham reached statistical significance at the 5 d post - crt interval , but , by 3.5 mo , all three groups differed significantly from one another .
we have observed the retarding effect of crt on body weight in other strains of rats as well ( long - evans hooded and fisher 344 , data not shown ) .
we also replicated our findings of crt - induced overall reduction in body weight in a small , separate cohort of animals ( figure 6(a ) ) .
note that this cohort was followed over time and when data for 5 d after crt ( i.e. , day 4 on x - axis , figure 6(a ) ) are analyzed by one - way anova , shams weigh significantly more than both single dose [ p < 0.001 ] and fractionated crt [ p < 0.01 ] animals . additionally , we documented significant effects of crt , post - crt interval , and crt x post - crt interval on body length ( figure 6(b ) ) and the ratio of body weight to body length ( figure 6(c ) ) .
all groups differed significantly from each other in terms of body length and body weight / length ratio and single dose crt had the most severe impact . furthermore , while growth eventually plateaued with age for all groups , irradiated animals never recovered to sham levels .
however , note that while crt decreased body weight and length , the rate of weight gain and increase in length was similar across groups .
post - irradiation weight loss in rodents has been reported in mice and rats irradiated with different doses and at different ages [ 25 , 7881 ] . in order to mitigate the effects of possible damage to the salivary gland , we provided softened food to the animals inside their home cages on a daily basis , starting immediately after the first session of radiation .
our brain weight data at 3.5 mo after radiation show that both single dose and fractionated crt stunted brain growth ( figure 7(a ) ) . once again , a single dose of crt suppressed growth to a significantly larger extent ( 18.9% decrease ) than fractionated did ( 8.4% decrease ) .
brains of single dose crt animals weighed , on average , 10.6% less than those of fractionated crt animals , which is visible to the naked eye ( figure 7(b ) ) .
other studies have also reported radiation - induced suppression of brain growth in rodents [ 25 , 49 , 79 , 82 , 83 ] .
in contrast , 24 h after the end of crt , that is , when both groups had received a total dose of 20 gy , brains of single dose - treated animals were , on average , heavier compared to shams and fractionated crt animals , whereas brains of fractionated crt animals weighed less compared to shams .
the initial increase in brain weight among single dose - treated animals may be a result of acute inflammation - induced edema which is absent in the fractionated crt group , because they receive radiation over 5 days which allows for bouts of recovery between exposures that are 1/5th the dose .
collectively , our findings show that whole brain radiation has long - lasting consequences for growth , paralleling the clinical literature [ 8486 ] , and that despite some differences , primarily related to inflammation , crt delivered in childhood / adolescence causes growth stunting of the brain and body and severe suppression of proliferation .
indeed , hypopituitarism is frequently observed in both adults [ 8791 ] and children [ 84 , 88 , 9294 ] treated with cranial radiation even for nonpituitary cancers .
animal studies also show that cranial radiation disrupts endocrine function in a dose - dependent manner [ 78 , 95 ] .
we are currently investigating to what extent our findings can be explained by the interaction of radiation and the endocrine system .
to our knowledge , this is the first report of the acute and long - term effects of crt delivered during childhood / adolescence in an animal model which measures proliferation , microstructural metabolites ( using translational imaging tools ) , and phenotype changes .
importantly , the observed early radiation - induced alterations were present 3.5 mo after exposure coinciding with the early delayed phase of radiation injury .
ongoing investigations in our lab are aimed at evaluating the effects of fractionated crt on cognition and the inhibition of the radiation - induced inflammatory response as a possible mechanism for mitigating the long - term sequelae . | cranial radiotherapy ( crt ) increases survival in pediatric brain - tumor patients but can cause deleterious effects .
this study evaluates the acute and long - term impact of crt delivered during childhood / adolescence on the brain and body using a rodent model .
rats received crt , either 4 gy fractions 5 d ( fractionated ) or a cumulative dose of 20 gy ( single dose ) at 28 d of age .
animals were euthanized 1 d , 5 d , or 3.5 mo after crt .
the 3.5 mo group was imaged prior to euthanasia . at 3.5 mo , we observed significant growth retardation in irradiated animals , versus controls , and the effects of single dose on brain and body weights were more severe than fractionated . acutely single dose
significantly reduced body weight but increased brain weight , whereas fractionation significantly reduced brain but not body weights , versus controls .
crt suppressed cell proliferation in the hippocampal subgranular zone acutely .
fractional anisotropy ( fa ) in the fimbria was significantly lower in the single dose versus controls .
hippocampal metabolite levels were significantly altered in the single dose animals , reflecting a heightened state of inflammation that was absent in the fractionated .
our findings indicate that despite the differences in severity between the doses they both demonstrated an effect on cell proliferation and growth retardation , important factors in pediatric crt . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions |
PMC4054052 | mineral trioxide aggregate ( mta ) was first introduced in 1990s and it has been proven to be a superior material regarding with its excellent sealing ability , biocompatibility and hard - tissue forming capacity .
it also possesses ideal properties such as its antimicrobial effect , dimensional stability , radiopacity and tolerance to moisture .
clinically , mta is currently being used for a variety of endodontic and restorative dental applications such as vital pulp therapy , apexification , repair of root perforations , and root - end filling .
although mta is considered to have ideal properties , its usage remained limited due to its high - cost , difficult handling characteristics , long setting time and potential of discoloration .
these shortcomings of mta led to the continuous efforts in developing the modified versions of mta .
first modification was performed on the original gray formula ( proroot mta ) by the manufacturer ( dentsply tulsa dental , tulsa , ok , usa ) producing a tooth - colored formulation ( white mta ) in order to overcome the potential discoloration problem .
white mta mainly differs from the gray mta in the absence of iron . in 2001 , mta angelus ( mta - a ) was introduced as an alternative to proroot mta and used in certain regions with its lower price .
the chemical difference between proroot mta and mta - a is that mta - a lacks calcium sulfate dehydrate as one of its main compounds , resulting in that mta - a has shorter setting time than proroot mta ( 165 minutes for proroot mta , while 10 minutes for mta - a ) .
mta - a is less radiopaque than proroot mta due to the lower content of bismuth oxide in its composition .
bioaggregate ( ba ) was released in 2007 as a bioceramic material , in which most of the constituents are similar to those in white mta .
it differs from mta by being aluminum -free and containing calcium phosphate monobasic and tantalum pentoxide .
aluminum - free content is one of the most significant features of the ba since it is known that aluminum has a toxic effect on the human body . a novel mta material called mta - plus ( mta - p ) , which is claimed to have a finer particle size than the currently available mta products that has been introduced on the market .
because mta - p has been recently available in the market , there are not many studies reported on this material .
x - ray diffraction ( xrd ) is a powerful method used for the identification and characterization of the crystalline phase composition of the materials . although xrd analysis of the relatively new material , mta - p , has recently been carried out and studies comparing the major constituents of this material with other mta derivatives are still lacking .
the purpose of this study was to investigate and compare the crystalline structures of mta - p , white mta - a and diaroot ba by xrd analysis .
materials used in this study included mta - p ( compounded by prevestdenpro , jammu , india for avalon biomed inc . , bradenton , fl , usa ) , white mta - a ( angelus , londrina , pr , brazil ) and diaroot ba ( innovative bioceramix , vancouver , bc , canada ) .
the mta - p is provided with either water or a gel for mixing . in the current study ,
phase analysis was carried out on both the powder and the set forms of tested materials .
for the powder form , each specimen was placed into the sample holder and packed with a glass slide to provide a uniform surface . for the set form
, each mixture was prepared according to their manufacturer 's instructions was placed into a mold and the upper surface of the specimen was swept with a spatula .
the set samples were set for 3 days at 37c and 100% humidity in the incubator .
an x - ray diffractometer ( xpert pro mpd , panalytical , almelo , the netherlands ) with ni filter and cuka radiation running at 45 kv voltage and 40 ma current was used in the present study .
the scan range was set at 2702 and with a scan speed of 22 per minute .
each phase ( crystalline substance ) of a compound has a characteristic diffraction pattern consisting of several x - ray peaks .
the peaks at the specified intensity representing the diffraction patterns of the tested materials were matched with the standard data documented in the powder diffraction files ( pdf ) found in the international centre for diffraction data ( icdd ) database .
x - ray diffraction patterns of mta - plus in the set ( a ) and powder ( b ) forms .
( bo , bismuth oxide ; c3s , tricalcium silicate , c2s , dicalcium silicate ; ch , calcium hydroxide ; a , anhydrite ) x - ray diffraction patterns of mta - angelus in the set ( a ) and powder ( b ) forms .
( bo , bismuth oxide ; c3s , tricalcium silicate , c2s , dicalcium silicate ; c3a , tricalcium aluminate ) x - ray diffraction patterns of bioaggregate in the set ( a ) and powder ( b ) forms .
( to , tantalum oxide ; c3s , tricalcium silicate , c2s , dicalcium silicate ; ch , calcium hydroxide ) both mta - p and mta - a showed strong peaks of bismuth oxide at 27.37 , 33.03 and 46.30 2. there were no peaks for bismuth oxide in the ba sample .
all samples comprised tricalcium silicate indicated by the main peaks at 32.14 , 32.49 and 34.31 and dicalcium silicate indicated by the main peaks at 32.05 , 32.13 and 32.59 2. mta - a showed peaks at 33.16 , 47.62 and 59.27 2 representing tricalcium aluminate .
portlandite ( calcium hydroxide ) was identified in mta - p groups and powder form of ba .
anhydrite was observed in powder mta - p and ettringite ( hexacalcium aluminate trisulphate hydrate ) was observed in the set mta - p .
the identification of the major constituents or compounds present in a material is important as it will contribute to understanding of the material 's physical , chemical and mechanical properties .
the use of xrd permits the identification of the major constituents or compounds present in a material .
the key principle of the technique is based on identifying the diffraction pattern of each crystalline phase characterized by a unique set of peaks ( known as bragg 's peaks ) , with a specific diffracted intensity ( y - axis ) and diffracted angle at a specified position .
phase identification is accomplished by comparing the data of the tested specimens by using peaks and relative intensities with a very large set of standard data provided by the icdd .
mta contains largely crystalline phases , with the calcium silicate hydrate being the only amorphous phase .
all the tested materials in this study had two common constituents , tricalcium silicate and dicalcium silicate .
tricalcium silicate is the major component in the formation of calcium silicate hydrate which gives early strength to the cement .
dicalcium silicate hydrates more slowly than tricalcium silicate and is responsible for the latter strength .
mta - a has also included the tricalcium aluminate phase as one of its main phases .
tricalcium aluminate is the most reactive constituent and reacts rapidly with water , however , its contribution to the strength is very little .
the crystal structure of ba has been recently investigated by xrd analysis and all the crystalline phases were identified .
the xrd results obtained from ba samples in this study were largely similar to those of park et al . tantalum oxide , tricalcium silicate
ba also includes hydroxyapatite and amorphous silicon oxide together with the main cementitious and radiopacifier phases .
the hydroxyapatite and amorphous silicon oxide are added by the manufacturer to reduce the levels of the calcium hydroxide , which is a weak hydration product of calcium silicates .
portlandite was identified in the powder form of ba and this is in contrast to the study by park et al . , which demonstrated portlandite only in set samples .
hydroxyapatite peaks were not observed in this study differing from the study by park et al .
the absence of aluminum specified by the manufacturer was also verified in the present study .
mta - p is a new version of mineral trioxide aggregate , which is claimed to be similar in composition to the other mta products .
it has been released by the manufacturer highlighting the features of finer particle size and lower cost .
mta - p is sold with either distilled water or anti - washout gel intending to improve its anti - washout resistance .
determined that the crystalline particles in mta - p were smaller than those present in proroot mta although the chemical compositions were found to be similar .
smaller particle size is important for the physical properties as it will increase the surface available for hydration and cause greater early strength as well as ease of handling .
there are not many studies published comparing the new mta - p and the other mta products , which have been extensively investigated .
formosa et al . were first to investigate the chemical and physical properties of the novel mta - p .
they used the xrd method to identify the crystalline phases of mta - p and made a comparison between its two set forms , one mixed with the anti - washout gel and the other mixed with the distilled water .
the phases identified in their study were similar for each mixture and included tricalcium silicate , dicalcium silicate , bismuth oxide and portlandite , which also corroborated with the findings of this study .
they also observed weak peaks of ettringite , which were also identified in the set sample of mta - p of the present study .
it is known that the ettringite peaks are formed as a result of reaction between tricalcium aluminate and water in the presence of calcium sulfate .
our results showed that mta - p powder consisted of anhydrite as the calcium sulfate phase and it is likely that small amounts of tricalcium aluminate are present considering the production of ettringite in set samples .
the aluminate phase results from the addition of alumina in the mixture and alumina helps reducing the burning temperatures required to make the cement .
the quantity of anhydrite is important for controlling the flash set phenomenon attributed to the high reactivity of the aluminate phase with water .
it can be controlled by adding a sulfate - involving agent such as gypsum or anhydrite , which delays the setting time .
although mta - a had an aluminate phase , it did not contain calcium sulfate and this results in a shorter setting time ( ~10 min ) of the material .
mta - p and mta - a contained the same constituent , bismuth oxide , as a radiopacifier while ba included tantalum oxide instead .
considering that tantalum oxide is the major difference between mta and ba , it may be important to examine the differences between bismuth oxide and tantalum oxide in terms of toxicity and biocompatibility .
bismuth oxide , which is one of the main compounds of mta , may not contribute to its excellent biocompatibility .
steinemann reported a strong inhibition zone when osteoblasts were grown , whereas fibroblasts proliferated well on the tantalum disc .
however , they could not infer whether those results were due to the surface roughness or the chemical nature of the material .
the set mta - p exhibited strong peak of calcium hydroxide , which is in accordance with the results of a previous study by camilleri et al .
the calcium hydroxide peak in this study was found to be much more pronounced in mta - p than in the other mta products tested .
the high portlandite peak , which is indicative of a greater rate of reaction , is reported to be related to the fine grinding of mta - p cement .
all the tested materials were similarly composed of tricalcium silicate and dicalcium silicate crystalline structures .
mta - p showed anhydrite in a powder sample and ettringite in a set sample differing from mta - a and ba .
in addition , set mta - p exhibited a strong peak of portlandite , which is attributable to its fine grinding property .
mta - a differed from mta - p and ba by the presence of tricalcium aluminate phase .
ba mainly differed from mta - a and mta - p by the radiopacifier added .
mta - p and mta - a contained the same constituent , bismuth oxide , as a radiopacifier while ba included tantalum oxide instead . | objective : the purpose of this study was to investigate and compare the crystalline structures of recently released mta plus ( mta - p ) , mta angelus ( mta - a ) , diaroot bioaggregate ( ba ) by x - ray diffraction ( xrd ) analysis.materials and methods : phase analysis was carried out on powder and set forms of tested materials .
the powder specimens placed into sample holders that were packed with a glass slide and the set samples prepared according to the manufacturer 's instructions were placed into molds .
the samples after being set for three days at 37c and 100% humidity in an incubator were mounted onto the xrd machine and phase identification was accomplished using a search - match software program.results:xrd findings indicated that major constituents of mta - p were bismuth oxide , portlandite , dicalcium silicate and tricalcium silicate . the crystal structure of mta - a were similar to those of mta - p except for the absence of portlandite .
additionally , mta - a had tricalcium aluminate differing from mta - p .
ba mainly differed from mta - p and mta - a by the radiopacifier ( tantalum oxide - to ) in its composition.conclusions:the majority of constituents of the tested materials have shown similarity except for the presence of tricalcium aluminate in mta - a and the inclusion of to in ba . in addition , set mta - p showed a strong peak of portlandite . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC5300865 | loss - of - function mutations in scn5a , that encodes the alpha - subunit of the cardiac na channel , lead to variable phenotypes , viz .
brugada syndrome ( brs ) , progressive cardiac conduction disease , atrial fibrillation , and sick sinus syndrome ( sss ) . in young children carrying scn5a mutations ,
reportedly , cardiac conduction disturbance appears as the most common manifestation of disease , with both atrial and ventricular arrhythmia .
the phenotype of the patients with the same scn5a mutations is variable because various modifiers might modulate it .
we describe a proband , and his family members with a novel nonsense scn5a mutation , who displayed variable phenotypes , and searched for the genetic modifiers that might affect the phenotype .
gene analysis was performed for the proband , his parents , and two siblings as summarized in the family tree ( fig .
genomic dna was isolated from blood lymphocytes , and screened for the open reading frame of kcnq1 , kcnh2 , kcne1 - 3 , kcne5 , kcnj2 , and scn5a .
genetic screening was performed using denaturing high - performance liquid chromatography ( dhplc wave system , transgenomic , omaha , ne , usa ) . for abnormal screening patterns ,
direct sequencing was performed using an automated sequencer ( abi prism , 3100x , applied biosystems , foster city , ca , usa ) . for detection of the genetic modifiers
, we performed targeted panel sequencing for coding region of 46 genes ( supplementary information ) that were related to primary arrhythmia syndrome for proband and 4 family members , using a bench - top next generation sequencer , the miseq ( illumina , san diego , ca , usa ) .
we then evaluated the detected variants by frequently referring to the ncbi dbsnp database ( http://www.ncbi.nlm.nih.gov/snp/ ) , human genetic variation browser that is a japanese snp database http://www.genome.med.kyoto-u.ac.jp/snpdb/ ) , and the 1000 genomes database ( http://www.1000genomes.org/home ) .
the pathogenicity of the variants was estimated by three different types of prediction software : ( 1 ) combined annotation dependent depletion ( cadd ) ( http://cadd.gs.washington.edu/ ) , ( 2 ) polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) , and ( 3 ) sift ( http://sift.jcvi.org/ ) .
index proband was a 13-year - old ( yo ) boy who was suspected to have a sino - atrial block because of an auscultation abnormality at the age of three ( family tree in fig .
his pr interval was 179 ms , qrs was 107 ms , and qt interval was 394 ms ( qtcf 451 ms ) .
the sinus node recovery time was significantly prolonged to 7,268 ms , and hv interval to 70 ms .
the atrio - ventricular nodal effective refractory period was 360 ms . against a diagnosis of sss
the electrocardiogram ( ecg ) monitored by an autonomic external defibrillator ( aed ) showed ventricular fibrillation ( vf ) ( fig .
his resting ecg in v2 showed st elevation of a saddleback type at the 4th , and coved type at the 3 intercostal leads sites ( fig .
2b ) . during atrial pacing at 60 bpm , his pr interval prolonged to 262 ms , qrs interval was 125 ms , and qtc was 436 ms ( table 1 ) .
2c depicts temporal changes in the ecg ( v1-v3 ) recorded from the age 4 to 13 . at age 4
, we failed to find any brugada pattern , while the j point gradually elevated from the age of 9 .
the signal averaged electrocardiogram ( saecg ) revealed positive late potentials , and microvolt - t wave alternans ( v - twa ) was negative .
the proband had asymptomatic parents and two siblings as shown in the family tree in fig .
his mother ( 46-yo ) had a coved type st elevation in the 3rd precordial leads ( fig .
had sss , and a coved type brugada ecg during a pilsicainide ( class ic antiarrhythmia drug ) challenge test ( fig .
3b ) , and his elder brother ( 21-yo ) had a saddle back brugada ecg during pilsicainide administration ( fig .
the prolongation of the pr interval was detected in his mother at rest , and in his sister and brother on pilsicainide administration ( table 1 ) .
his maternal grandfather , who died of cancer , had received a pacemaker for sss .
we obtained his ecg recording that showed sinus rhythm at 70 bpm , a pr interval of 190 ms , qrs duration of 106 ms , and no brugada ecg pattern ( data not shown ) .
we identified a scn5a mutation in the proband , and the three family members except for his father .
the mutation , a single - base substitution at nucleotide 2335 ( c.2335c > t ) , is located in the cytoplasmic loop of the na channel alpha - subunit and changed the codon cag ( glutamine : q ) to an in - frame stop codon , tag , at position 779 , q779x ( fig .
a premature stop codon in this position results in nonsense mediated decay ( nmd ) , leading to decay of mrna .
therefore , the expression level of mrna and protein of cardiac na channel is presumably low in scn5a - q779x carriers . to clarify the phenotypic variation among the mutant scn5a carriers in the index family
in the analysis , we further detected four non - synonymous variants of arrhythmia - related genes in the proband , his mother and his sister , but not in his brother exhibiting a milder phenotype .
the first one was desmoglein 2 ( dsg2)-r773k ( rs2278792 ) and the others were scn1b l210p ( rs55742440 , ) , s248r ( rs67701503 ) , and r250 t ( rs67486287 ) .
scn1b encodes an ancillary beta - subunit of the cardiac na channels and affects the cardiac conduction , and was reported as a causative gene for brs .
table 2 summarizes minor allele frequencies ( maf ) and prediction scores for the pathogenicity of each variant .
none of them seemed to be the main cause of the disease because their mafs of them were greater than 0.1 , and were predicted to be of benign or mild pathogenicity .
index proband was a 13-year - old ( yo ) boy who was suspected to have a sino - atrial block because of an auscultation abnormality at the age of three ( family tree in fig .
his pr interval was 179 ms , qrs was 107 ms , and qt interval was 394 ms ( qtcf 451 ms ) .
the sinus node recovery time was significantly prolonged to 7,268 ms , and hv interval to 70 ms .
the atrio - ventricular nodal effective refractory period was 360 ms . against a diagnosis of sss
the electrocardiogram ( ecg ) monitored by an autonomic external defibrillator ( aed ) showed ventricular fibrillation ( vf ) ( fig .
his resting ecg in v2 showed st elevation of a saddleback type at the 4th , and coved type at the 3 intercostal leads sites ( fig .
2b ) . during atrial pacing at 60 bpm , his pr interval prolonged to 262 ms , qrs interval was 125 ms , and qtc was 436 ms ( table 1 ) .
2c depicts temporal changes in the ecg ( v1-v3 ) recorded from the age 4 to 13 . at age 4
, we failed to find any brugada pattern , while the j point gradually elevated from the age of 9 .
the signal averaged electrocardiogram ( saecg ) revealed positive late potentials , and microvolt - t wave alternans ( v - twa ) was negative .
the proband had asymptomatic parents and two siblings as shown in the family tree in fig .
1 . all except for his father had abnormal ecg . his mother ( 46-yo ) had a coved type st elevation in the 3rd precordial leads ( fig .
had sss , and a coved type brugada ecg during a pilsicainide ( class ic antiarrhythmia drug ) challenge test ( fig .
3b ) , and his elder brother ( 21-yo ) had a saddle back brugada ecg during pilsicainide administration ( fig .
the prolongation of the pr interval was detected in his mother at rest , and in his sister and brother on pilsicainide administration ( table 1 ) .
his maternal grandfather , who died of cancer , had received a pacemaker for sss .
we obtained his ecg recording that showed sinus rhythm at 70 bpm , a pr interval of 190 ms , qrs duration of 106 ms , and no brugada ecg pattern ( data not shown ) .
we identified a scn5a mutation in the proband , and the three family members except for his father .
the mutation , a single - base substitution at nucleotide 2335 ( c.2335c > t ) , is located in the cytoplasmic loop of the na channel alpha - subunit and changed the codon cag ( glutamine : q ) to an in - frame stop codon , tag , at position 779 , q779x ( fig .
a premature stop codon in this position results in nonsense mediated decay ( nmd ) , leading to decay of mrna .
therefore , the expression level of mrna and protein of cardiac na channel is presumably low in scn5a - q779x carriers . to clarify the phenotypic variation among the mutant scn5a carriers in the index family
, we performed a targeted panel sequence using miseq , for the mutation carriers . in the analysis
, we further detected four non - synonymous variants of arrhythmia - related genes in the proband , his mother and his sister , but not in his brother exhibiting a milder phenotype .
the first one was desmoglein 2 ( dsg2)-r773k ( rs2278792 ) and the others were scn1b l210p ( rs55742440 , ) , s248r ( rs67701503 ) , and r250 t ( rs67486287 ) .
scn1b encodes an ancillary beta - subunit of the cardiac na channels and affects the cardiac conduction , and was reported as a causative gene for brs .
table 2 summarizes minor allele frequencies ( maf ) and prediction scores for the pathogenicity of each variant .
none of them seemed to be the main cause of the disease because their mafs of them were greater than 0.1 , and were predicted to be of benign or mild pathogenicity .
we identified a novel , heterozygous nonsense scn5a mutation , q779x , in a family with overlapped phenotype with progressive conduction defects and brs .
the proband suffered from sss at the age of three , and subsequent ventricular fibrillation with a coved type brugada ecg pattern at age of 13 was detected .
mild prolongation of the hv intervals had been observed at the age of four , indicating the presence of an extensive conduction delay . at 9 years of age ,
sss is complicated in children with a left isomerism heart , or on surgical intervention for congenital heart disease .
it is also seen in channelopathies such as long qt syndrome , brs , or as a part of a progressive cardiac conduction disease .
loss - of - function scn5a mutants have been shown to be related to brs as well as profound bradyarrhythmias . in children with these mutations ,
prolonged cardiac conduction intervals have been reported to emerge as the most common manifestation of the disease , and are accompanied with both atrial and ventricular arrhythmias .
the intravenous challenge test with a na channel blocker was conducted in two of our genotype - positive members , and the drug produced pr prolongation ( table 1 and fig .
the pr prolongation , which results from common loss - of - function scn5a mutation , was enhanced by a na channel blocker in our family members .
the heterozygous nonsense scn5a mutation of the family was at amino acid 779 that lies in the middle of the cytoplasmic loop of domain 2 ( fig .
a missense scn5a mutation at the same position , q779k , was previously reported for a japanese pedigree of long qt syndrome , which harbored a compound heterozygous mutation , e284k , in kcnq1
.
a change in the residual charge from neutral ( q : glutamine ) to basic ( k : lysine ) may cause a gain - of - function outcome .
in contrast , the nonsense mutation at the same codon leads to nmd , thereby exerting severe loss - of - function effects .
the index boy presented progressive conduction defects and vf during earlier stage of his life .
his mother and two siblings had similar ecg abnormalities at rest and when challenged with na - channel blockers , but remained asymptomatic in spite of the same nonsense scn5a mutation .
this variation in phenotypic severity could be partially explained by the gender difference , because brs is predominantly noted in males as the disease has an autosomal - dominant mode of transmission .
the reason for the variation in phenotypic severity between the index boy and his elder brother is unknown .
both had the same scn5a mutation , however , the proband had additional dsg2 and scn1b variants .
dsg2 is located on chromosome 18 , and encodes a calcium - binding transmembrane glycoprotein component of desmosomes , desmoglein 2 .
few mutations in dsg2 have been shown to decrease the na current , leading to conduction disturbances , and are associated with arrhythmogenic right ventricular dysplasia .
the scn1b gene encodes a beta-1 subunit of the sodium channel that is known to modulate the sodium channel through a non - covalent interaction with the alpha - subunit encoded by scn5a .
maf for each variant was not low , indicating that they were not pathogenic mutations ; although , the pathogenicity estimation scores of scn1b - s248r and r250 t were relatively high ( table 2 ) . therefore , the phenotype variation among the scn5a - q779x carriers could be partially explained by modifier effects of these variants , in addition to the gender difference .
we could only once record the ecgs of family members ; therefore , we might have failed to record coved type brugada pattern ecg , which might have been recorded during follow - up .
we did not perform functional analysis of genetic modifiers to confirm the effect on the phenotype .
additionally , we only screened 46 genes related with cardiac arrhythmias , and did not screen introns that might affect the transcription or expression .
thus , we might have overlooked mutation in other genes or in the intronic regions that could be related to the phenotype .
scn5a - positive proband and the female members of the family with overlap syndrome phenotype carried additional variants in scn1b and dsg2 .
in contrast , these variants were absent in their scn5a - positive male family member who showed a milder phenotype .
therefore , in addition to the gender difference , scn1b and dsg2 variants could be modifiers of brs and sss phenotypic expression , and our results may help explain the enigma of variable phenotypes for those with scn5a mutations . | backgroundphenotypes often differ even within family members carrying the same scn5a mutation .
we aimed to evaluate the genetic modifiers in a family with brugada syndrome ( brs ) and sick sinus syndrome ( sss ) with an scn5a mutation that causes the truncated alpha - subunit of cardiac na channel protein.methodsto detect the genetic modifiers , we performed targeted panel sequencing of the coding region of 46 genes that are related to primary arrhythmia syndrome , by using a bench - top , next generation sequencer .
phenotype
genotype relationships were evaluated among the family members.resultsindex proband was a 13-year old ( yo ) boy with cardiac conduction defect as well as brs .
genetic analysis revealed that he and his three asymptomatic family members carried a novel nonsense mutation : scn5a - q779x .
both genotype - positive mother and sister exhibited coved type st elevation and his sister had sss , whereas his elder brother exhibited saddleback type st elevation induced by pilsicainide administration .
we detected four non - synonymous variants ( dsg2-r773k , scn1b - l210p , -s248r , and -r250 t ) in the proband , his mother and his sister , but not in his brother.conclusionphenotypic differences between the proband and his brother carrying the same nonsense scn5a mutation could be explained by modifiers such as scn1b , and dsg2 gene variants . | Introduction
Material and methods
Results
Case presentation
Phenotypes of the family members
Genetic analyses
Discussion
Conclusion
Conflict of interest
Supplementary material |
PMC3824355 | induced pluripotent stem ( ips ) cells can be directly generated from fibroblast cultures by expression of four factors ( 4f ) , including octamer - binding transcription factor 4 , sex - determining region y - box 2 , kruppel - like factor 4 , and myelocytomatosis viral oncogene homolog .
although these factors present attractive sources for stem cell therapy , the mechanisms by which they are generated are not fully understood .
the inefficiency of ips cell generation has prompted the development of two contending models , namely , the stochastic and elite models .
a recent multilineage - differentiating stress - enduring cell study suggested the greater utility of the elite model over the stochastic model . in either model , stem cells or progenitor cells isolated for experimental or therapeutic research are usually heterogeneous populations ; therefore , a more favorable subfraction for use in stem cell therapy may exist .
indeed , several reports have suggested that the differentiation stage of the starting cell line has a critical influence on the efficiency of reprogramming into ips cells in hematopoiesis .
immature cell populations , such as linc - kitsca1cd48cd150cd34 ( lack of lineage / hardy - zuckerman 4 feline sarcoma viral oncogene homolog / spinocerebellar ataxia / cluster of differentiation 48/150/34 ) hematopoietic stem cells , in general give rise to ips cells at higher efficiencies than terminally differentiated cell types .
furthermore , a recent study demonstrated that cd90 adipose tissue - derived mesenchymal stem cells ( adscs ) are more capable of forming bone both in vitro and in vivo than in cd105 cells .
cd90 ( thy-1 ) is a 2537-kda glycosylphosphatidylinositol - anchored glycoprotein expressed mainly in leukocytes that have been used as murine pan t - cell markers , like cd2 , cd5 , and cd28 .
multipotent mesenchymal stem cells ( mscs ) , which represent a nonhematopoietic cell population , can differentiate into mesenchymal tissues ( i.e. , bone , cartilage , or fat ) [ 5 , 6 ] ; they were first isolated from bone marrow and later from nonmarrow tissues , including umbilical cord blood and adipose tissue [ 5 , 6 ] .
immunologically , mcss are known to be less immunogenic because of , at least in part , the lack of surface - expressed human leukocyte antigens .
moreover , they have been shown to possess anti - inflammatory and immunosuppressive effects both in murine and human models of diabetes [ 7 , 8 ] .
thus , the immunoregulatory properties of mscs are appealing , because they inhibit t - cell proliferation and differentiation of monocytes to dendritic cells , modulate b - cell functions , and suppress natural killer cell cytotoxicity .
solid organ transplantation is an established and useful treatment option for end - stage organ failure . despite excellent short - term results , long - term survival of transplanted grafts
has not improved accordingly because of graft rejection , graft fibrosis , or side effects of immunosuppressants .
the aim of stem cell therapy for solid organ transplantation is to prevent or treat acute rejection and to involve autologous or allogeneic stem cell transplantation into patients , either through local delivery or systemic infusion . currently , there are over 100 ongoing clinical trials to evaluate the utility of mscs , especially in kidney transplantation [ 911 ] .
adscs have been shown to consist of heterogeneous subpopulations ; therefore , we hypothesized that cd90 adscs might exhibit increased reprogramming capacities and subsequently have better immunoregulatory effects .
hence , the aim of the present study was to compare the reprogramming capacity of sorted murine cd90 adscs .
our data demonstrated that cd90 selection improved the reprogramming capacity of murine adscs , suggesting that cd90 adscs could help prevent graft rejection .
murine adscs were isolated from b6 mice as previously described but with slight modifications . briefly , adipose tissue was obtained from the inguinal fat pads , washed with dulbecco 's phosphate - buffered saline ( d - pbs ; invitrogen , carlsbad , ca , usa ) containing 50 u / ml of penicillin and 50 g / ml of streptomycin ( psm ; invitrogen ) , and then cut into fine pieces , which were incubated with d - pbs containing 1.0 mg / ml of clostridium histolyticum collagenase ( sigma - aldrich , st .
louis , mo , usa ) in a 37c shaking incubator for 1 h. the digested tissue was filtered through sterile 70-m nylon mesh , centrifuged at 430 g for 5 min , and resuspended in dulbecco 's modified eagle 's medium ( dmem ) ( nacalai tesque , kyoto , japan ) supplemented with 10% fetal bovine serum ( fbs ; hyclone ; thermo scientific , waltham , ma , usa ) and psm .
murine adscs were washed with d - pbs and treated with accutase reagent ( emd millipore , billerica , ma , usa ) in a 37c incubator for 2 - 3 min to dissociate the cells , which were then resuspended in staining media [ d - pbs supplemented with fbs ( 1% ) and edta ( 2 mm ) ] .
next , the cells were stained with fluorescein isothiocyanate - conjugated anti - mouse cd34 ( ebioscience , san diego , ca , usa ) , phycoerythrin - conjugated anti - mouse cd31 , efluor450 antimouse cd45 , and allophycocyanin - conjugated anti - mouse cd90.2 for 60 min at 4c . the cells were then washed with staining media , stained with 7-aminoactinomycin d to exclude dead cells , and sorted using the facsaria cell sorter ( bio - rad , hercules , ca , usa ) .
the particular phenotypic subsets of cd90 and cd90 adscs , based on cd31 , cd45 , and cd90 expression , were evaluated by multicolor analysis and sorted by adequate gating . to prepare the lentiviruses , human embryonic kidney ( hek)-293ta cells were cultured in dmem supplemented with 10% fbs and psm .
then , the cells were seeded at 5.0 10 cells per 100 mm dish 1 day before viral transduction .
fugene 6 transfection reagent ( 22.5 l ; promega , madison , wi , usa ) was diluted with 500 l of dmem and incubated for 5 min at room temperature .
plasmid dna ( 2.5 g ) was added to the mixture , which was incubated for an additional 15 min at room temperature .
then , the culture media was replaced with fresh dmem supplemented 10% fbs and the dna / fugene 6 mixture was added dropwise onto the hek-293ta cells .
the medium was replaced after 24 h. after an additional 48 h , virus - containing supernatants , derived from the hek-293ta cultures , were filtered through a 0.22-l cellulose
sorted cd90 and cd90 cells were plated onto 6-well culture plates at a density of 1.0 10 cells / well , respectively .
after incubation for 24 h , the culture medium was retrieved and viral supernatants were added to each well .
mouse embryonic fibroblasts ( mefs ) were mitotically inactivated by the addition of 10 g / ml of mitomycin c ( mmc ; nacalai tesque inc . ,
kyoto , japan ) for 90 min and then washed with d - pbs three times .
the cells were then cultured with complete medium for at least 2 h , trypsinized , counted , and plated on gelatin - coated 60 mm dishes at a density of 5.0 10 cells / well .
on posttransduction day 5 , the cells were trypsinized ; suspended in dmem supplemented with 15% ( v / v ) fbs , psm , 1-mm sodium pyruvate ( invitrogen ) , 10 m 2-mercaptoethanol ( nacalai tesque , inc . ) , nonessential amino acid solution , and 1,000 u / ml of leukemia inhibitory factor ; after which they were counted . then
, 1000 cells were transferred to mmc - treated mef feeder cells . on post - transduction day 30 , alkaline phosphatase ( ap )
4f - transduced adscs were stained using an ap staining kit ( muto pure chemicals , tokyo , japan ) following the manufacturer 's recommended protocol .
briefly , cultured cells were rinsed twice with d - pbs and then fixed for 5 s in ice - cold methanol .
then , the cells were washed with tap water , stained with ap solution at 37 for 120 min , and washed again with running water .
as shown in figure 2 , parental adsc showed no colonies and ap staining was negative ( negative control ) .
next we used sorted adscs to assess the reprogramming capacities of cd90 and cd90 adscs .
as shown in figure 3(a ) , the cd90 marker was widely expressed on the surfaces of the cd31cd45cd34adscs .
after 24 h of incubation , sorted cd90 or cd90 adscs became attached to the culture dishes ( figure 3(b ) ) and both showed similar morphologies .
as shown in figure 4(a ) , 7-day culture resulted in the reexpression of cd90 even after sorted cd90 adscs .
these results suggest that sorted cells might be transduced as soon as possible . to show the successful induction of the undifferentiated state , we employed immunocytochemistry of oct4 and ssea1 .
as shown in figure 5 , colonies were positive for oct4 and ssea1 . sorted and 4f - transduced adscs also exhibited colony formation .
as shown in figures 6(a ) and 6(b ) , morphologically distinct colonies were visible in both cd90 and cd90 4f - transduced adscs on posttransduction day 14 .
there were ap+ colonies in both groups , although the cd90 adscs tended to form larger colonies than cd90 adscs ( figures 4(c ) and 4(d ) ) .
as shown in figure 7 , cd90 adscs exhibited more ap+ colonies than cd90 adscs .
the reprogramming efficiencies of unsorted , cd90-sorted , and cd90-sorted adscs were 100% , 116.5% , and 74.7% , respectively .
in the present study , we demonstrated that murine adscs can be reprogrammed by standard 4f transduction .
furthermore , ips cell formation was also observed in cd90-based sorted cells and the cd90 sorting resulted in enhanced reprogramming capacity of murine adscs compared with cd90 adscs with regard to colony number .
moreover , there was a trend in the association between cd90 expression level and individual colony size .
these results clearly demonstrated that adscs have heterogeneous subpopulations and that the cd90 adscs present favorable candidates for the application of clinical stem cell therapy .
the importance of cd90 in oncogenesis or properties of cancer stem cells has been reported .
cd90 expression reportedly has prognostic values in esophageal squamous cell carcinoma ( escc ) patients , because higher cd90 expression was significantly associated with a strong family history of escc and higher incidences of lymph node metastasis .
however , the importance of cd90 expression in mscs has not been fully elucidated ; therefore , our next project is to clarify the mechanisms of cd90-mediated immunomodulatory effects . of note , recent study reported that cd90-expressing niche stromal cells would support hematopoiesis .
stem cell therapy for autoimmune disease is also well described . in a murine inflammatory bowel disease model ,
thy-1 is also expressed by thymocytes , peripheral t cells , myoblasts , epidermal cells , and keratinocytes .
we and others previously reported that it is possible to reprogram murine and human cells to pluripotency by direct transfection of mature double - stranded micrornas without viral vectors [ 1618 ] .
currently , studies are underway to compare the immunoregulatory properties of viral - free strategies both in vitro and in vivo . |
background . mesenchymal stem cells ( mscs ) , including adipose tissue - derived mesenchymal stem cells ( adsc ) , are multipotent and can differentiate into various cell types possessing unique immunomodulatory features .
several clinical trials have demonstrated the safety and possible efficacy of mscs in organ transplantation .
thus , stem cell therapy is promising for tolerance induction . in this study , we assessed the reprogramming capacity of murine adscs and found that cd90 ( thy-1 ) , originally discovered as a thymocyte antigen , could be a useful marker for cell therapy .
method .
murine adscs were isolated from b6 mice , sorted using a facsaria cell sorter by selection of cd90hi or cd90lo , and then transduced with four standard factors ( 4f ; oct4 , sox2 , klf4 , and c - myc ) .
results .
unsorted , cd90hi - sorted , and cd90lo - sorted murine adscs were reprogrammed using standard 4f transduction .
cd90hi adscs showed increased numbers of alkaline phosphatase - positive colonies compared with cd90lo adscs .
the relative reprogramming efficiencies of unsorted , cd90hi - sorted , and cd90lo - sorted adscs were 100% , 116.5% , and 74.7% , respectively .
cd90hi cells were more responsive to reprogramming .
conclusion .
cd90hi adscs had greater reprogramming capacity than cd90lo adscs , suggesting that adscs have heterogeneous subpopulations .
thus , cd90hi selection presents an effective strategy to isolate a highly suppressive subpopulation for stem cell - based tolerance induction therapy . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC4823349 | the initial surgical treatment for osteochondral defect ( ocd ) of the talus is usually arthroscopic debridement via curettage
. however , many patients do not achieve adequate pain relief or restoration of mobility with debridement alone [ 34 , 36 ] .
in addition , even among those patients for whom debridement appears to be effective , symptoms often recur over time . for these patients ,
standard treatments include osteoarticular autograft ( or allograft ) transfer ( oats ) [ 18 , 19 ] , or autologous chondrocyte implantation ( aci ) [ 2 , 3 , 14 , 17 , 25 ] .
these open procedures are more invasive and often more expensive than arthroscopy and can result in considerable morbidity .
in addition , debridement does not repair the underlying bone defect and may thus allow continued progression of the lesion .
researchers have sought treatment alternatives for ocd that could stabilise the interior of the lesion and prevent further collapse [ 1 , 2 , 7 ] .
this report introduces a new arthroscopic procedure in which a volume - filling , osteoconductive material containing a diffusible growth factor is inserted into the ocd lesion to stimulate repair of the patient s own subchondral bone .
this growth factor , recombinant human platelet - derived growth factor ( rhpdgf ) , has been found to promote healing ; regeneration ; and repair of bone , ligament , periodontal cementum , and other tissues [ 9 , 2224 , 2633 , 35 ] . in this new procedure , arthroscopic debridement to stable , viable bone
is followed by arthroscopic placement of rhpdgf mixed with an osteoconductive matrix of beta - tricalcium phosphate ( -tcp ) .
the defect is then sealed with fibrin glue , and the patient is kept non - weight bearing for 6 weeks prior to being remobilised .
the primary objective of this case - series - based study was to evaluate the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus , over the course of an initial 6-month follow - up .
the hypothesis was that the procedure would result in improved pain and functional scores for patients with and ocd .
the clinical relevance of this study is that , if successful , the procedure could provide a less invasive option with improved healing compared to standard techniques .
this single - site , single - surgeon , prospective study , conducted with enrolment from february 2012 until november 2012 , was designed as a case - series - based clinical study .
patients diagnosed with a talar ocd were recruited for the study from the clinical practice of the authors .
inclusion criteria included signed reb - approved informed consent form ( icf ) prior to enrolment ; diagnosis of an isolated ocd larger than 5 mm long , 3 mm wide , and 5 mm deep , confirmed by mri ; independent , ambulatory and can comply with all post - operative evaluations and visits ; 21 years of age or older ; skeletally mature ; stable ankle joint on history and similar ligament stability with the opposite ankle ; < 15 of hindfoot valgus and 5 of hindfoot varus ; ocd is chronic and not secondary to acute trauma within the previous 6 months ; if history of fracture , no residual deformity of the tibia , fibula , or syndesmosis ; no prior fusions of the hindfoot ( subtalar and talonavicular joints ) ; body mass index ( bmi ) 35 ; american society of anesthesiologists ( asa ) physical status classification of 1 or 2 ; and has exhausted non - operative treatment .
exclusion criteria included > 15 of hindfoot valgus or 5 of hindfoot varus ; defect > 30 mm length , 25 mm width , or 20 mm depth in size on mri assessment ; allergy to yeast - derived products ; has metallic or electronically , magnetically , or mechanically activated implants that would contraindicate mri scans of the foot ; history of malignancy anywhere in the body ; physically or mentally compromised and unable or unlikely to remain compliant to follow - up ; history of drug / alcohol abuse within the 12 months prior to screening for study entry ; pregnant , or able to become pregnant but not practicing a medically accepted form of birth control ; current acute infection in area surrounding surgical site ; history of anaphylaxis ; condition is bilateral and surgery is to be scheduled over the course of the study ; requires concomitant osteotomy of tibia , fibula , or calcaneus for hindfoot deformity , or requires concomitant hindfoot fusion for hindfoot arthritis or deformity ; undergoing any concomitant surgery that may invalidate outcome scores for this study ; ocd of the tibia in isolation or in combination with the talar lesion ; nicotine addiction or using medication or substances containing nicotine ; cocaine abuse or use of cocaine derivatives ; undergoing revision debridement of an ocd . diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment .
location and size of defect ( width , depth , and length ) , were determined via ankle arthroscopy and recorded prior to treatment .
photographs of defect before and after debridement as well as any associated pathology ( osteophytes , synovitis ) were also obtained .
patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively .
clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays .
all adverse events affecting the ankles were also recorded at each study visit ( see appendix ) .
follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . in addition ,
ct scans at 2 and 24 weeks were compared in order to determine healing status at the base of the ocd and fill of the graft .
all scans were measured using inteleviewer software tools ( intelerad , calgary , ab , canada ) , which enable automated , quantitative segmentation and calculation of two - dimensional area and three - dimensional volume measurements ( with read - outs in 1-mm increments ) , as well as image registration for pre- and post - treatment comparisons of patient lesions .
the mri scans were evaluated with sagittal and coronal sequences cut with the greatest amount of oedema ( t2-weighted signal on fat suppressed images ) being measured for square surface area of involvement . the oedema signal was measured preoperatively and at each follow - up period .
the ct scan was evaluated for size of defect preoperatively and for fill with bone graft at both time periods .
the ct was measured on one 0.6-mm slice on the sagittal view and one 0.6-mm slice on the coronal view .
all procedures were performed by a single surgeon having advanced skills in arthroscopy and foot and ankle surgery .
patients were treated with arthroscopic debridement of the ocd to stable , viable bone via abrasive osteochondroplasty without microfracture or drilling .
( microfracture was not used because the subchondral bone was breached and softened in all cases ) .
debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) .
routine ankle arthroscopy was performed using anterior medial , anterior lateral , and medial portals to access and debride the joint . an augment bone graft preparation ( biomimetic inc . , memphis , tn ) containing -tcp and rhpdgf
was then prepared using standard techniques and placed into the joint via the arthroscopic portal . following mixing ,
the final consistency of the graft material is similar to that of granular wet sand . when placed near a viable host bone , it acts as scaffold for new bone growth ; it subsequently undergoes remodelling and is finally replaced by host bone .
all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects .
the foot was dorsiflexed while the fibrin glue set , then the ankle kept dorsiflexed until the wounds were closed and the limb placed in a below - the - knee slab in dorsiflexion for 2 weeks post - surgery .
if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit .
the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) .
the vas for pain is a unidimensional , self - reported measure of pain intensity [ 16 , 20 , 21 ] , which has been widely used in diverse adult populations , including those with rheumatic disease [ 12 , 13 ] .
the aos is a reliable , validated , patient - reported , ankle - specific functional outcome instrument .
( the score ranges from 0 to 100 , with a lower score indicating more normal function ) .
secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles .
the sf-36 is a generic , patient - reported measure of general health status [ 4 , 6 ] that has been shown to have acceptable criterion validity in patients with end - stage ankle arthritis .
this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments .
all patients provided informed consent for study enrolment and for the surgical procedure prior to questionnaire administration .
treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test .
ninety - five per cent confidence intervals were used to assess variability using aos scores and mean area of oedema ( mm ) via mri .
diagnosis of ocd was confirmed via baseline magnetic resonance imaging ( mri ) scans of the lesion site prior to enrolment .
location and size of defect ( width , depth , and length ) , were determined via ankle arthroscopy and recorded prior to treatment .
photographs of defect before and after debridement as well as any associated pathology ( osteophytes , synovitis ) were also obtained .
patient assessments were completed preoperatively and at 2 , 6 , 12 , and 24 weeks post - operatively .
clinical outcomes were recorded preoperatively and at each follow - up visit using the visual analogue scale ( vas ) for pain , the ankle osteoarthritis scale ( aos ) , the short - form-36 ( sf-36 ) health survey , and ap and lateral x - rays .
all adverse events affecting the ankles were also recorded at each study visit ( see appendix ) .
follow - up mri was conducted at 12 and 24 weeks , in order to evaluate bone marrow oedema . in addition , ct scans at 2 and 24 weeks were compared in order to determine healing status at the base of the ocd and fill of the graft .
all scans were measured using inteleviewer software tools ( intelerad , calgary , ab , canada ) , which enable automated , quantitative segmentation and calculation of two - dimensional area and three - dimensional volume measurements ( with read - outs in 1-mm increments ) , as well as image registration for pre- and post - treatment comparisons of patient lesions .
the mri scans were evaluated with sagittal and coronal sequences cut with the greatest amount of oedema ( t2-weighted signal on fat suppressed images ) being measured for square surface area of involvement .
the ct scan was evaluated for size of defect preoperatively and for fill with bone graft at both time periods .
the ct was measured on one 0.6-mm slice on the sagittal view and one 0.6-mm slice on the coronal view .
all procedures were performed by a single surgeon having advanced skills in arthroscopy and foot and ankle surgery .
patients were treated with arthroscopic debridement of the ocd to stable , viable bone via abrasive osteochondroplasty without microfracture or drilling .
( microfracture was not used because the subchondral bone was breached and softened in all cases ) .
debridement was followed by arthroscopic placement of a preparation containing rhpdgf and -tcp ( see below ) .
routine ankle arthroscopy was performed using anterior medial , anterior lateral , and medial portals to access and debride the joint .
an augment bone graft preparation ( biomimetic inc . , memphis , tn ) containing -tcp and rhpdgf was then prepared using standard techniques and placed into the joint via the arthroscopic portal .
following mixing , the final consistency of the graft material is similar to that of granular wet sand . when placed near a viable host bone , it acts as scaffold for new bone growth ; it subsequently undergoes remodelling and is finally replaced by host bone .
all materials used in the components have been previously approved by health canada for the treatment of foot and ankle fusion procedures and by the fda for the treatment of periodontal bone defects .
the foot was dorsiflexed while the fibrin glue set , then the ankle kept dorsiflexed until the wounds were closed and the limb placed in a below - the - knee slab in dorsiflexion for 2 weeks post - surgery . if all appeared stable at 2 weeks , subjects began joint range - of - motion exercises in a walker boot and remained non - weight bearing until the 6-week follow - up visit .
the primary outcome measures were the vas for pain and the ankle osteoarthritis scale ( aos ) .
the vas for pain is a unidimensional , self - reported measure of pain intensity [ 16 , 20 , 21 ] , which has been widely used in diverse adult populations , including those with rheumatic disease [ 12 , 13 ] .
the aos is a reliable , validated , patient - reported , ankle - specific functional outcome instrument .
( the score ranges from 0 to 100 , with a lower score indicating more normal function ) .
secondary outcome measures included the physical component summary ( pcs ) and mental component summary ( mcs ) subscales of the sf-36 health survey questionnaire , and mri and ct scans of the affected ankles .
the sf-36 is a generic , patient - reported measure of general health status [ 4 , 6 ] that has been shown to have acceptable criterion validity in patients with end - stage ankle arthritis .
this study was approved by the institutional review board of the providence health care research institute at the university of british columbia ( reb # h03 - 50062 ) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments .
all patients provided informed consent for study enrolment and for the surgical procedure prior to questionnaire administration .
treatment safety and efficacy were assessed by comparing the preoperative baseline scores on the primary and secondary outcome measures with the scores recorded during follow - up evaluations at 6 , 12 , and 24 weeks , using student s t test .
ninety - five per cent confidence intervals were used to assess variability using aos scores and mean area of oedema ( mm ) via mri .
thirty patients with verified ocd were initially assessed for inclusion in this pilot study . of these , 25 patients were excluded due to ankle instability or other associated pathology , or were from the practice of another surgeon .
five patients with six ocd lesions of the talus met all inclusion and exclusion criteria , and underwent arthroscopic debridement followed by arthroscopic filling of the defect with rhpdgf - bb/-tcp matrix .
none of these patients withdrew from the study before the final follow - up visit at 24 weeks ; all patients were seen for follow - up at the correct intervals and had complete data .
mean patient age was 52 8.5 years , and mean bmi was 26.3 5.0 .
three of the patients were female and two were male . in one patient , the ocd occurred in the left ankle ; in the remainder , it occurred in the right ankle .
one patient had two lesions on the same ankle ( one on the medial and one on the lateral side of the talus ) .
four patients had undergone a prior debridement , and three had undergone more than one prior debridement .
four lesions were shoulder lesions and two were not.table 1dimensions of ocd lesions from arthroscopic assessmentaveragestandard deviationlength ( mm)143width ( mm)92depth ( mm)112surface area ( mm)10337volume ( mm)285139
surface area was calculated as an ellipse
volume was calculated as a hemi - ellipsoid dimensions of ocd lesions from arthroscopic assessment
surface area was calculated as an ellipse
volume was calculated as a hemi - ellipsoid mean scores for pain and disability at baseline and at 24 weeks post - operative follow - up ( as measured by the vas , sf-36 , and aos scales ) are listed in table 2 .
changes in these scores are illustrated in figs . 1 , 2 and 3.table 2mean outcome scores , pain , and disabilityvas pain scaleaossf-36non - wbwbpaindisabilitytotalpcsmcspreoperative baseline3.44.137.238.237.735.153.2week 241.12.125.927.626.742.953.5
vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summaryfig .
1average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
2average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
3average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
b
post - operative sagittal view showing reduction in the oedema signal . c
preoperative coronal view showing bone marrow oedema .
d
post - operative coronal view showing reduced marrow signal mean outcome scores , pain , and disability
vas visual analogue scale , aos ankle osteoarthritis scale , sf-36 short - form ( 36 ) health survey , wb weight bearing , pcs physical component summary , mcs mental component summary average scores on the vas pain questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
a lower score indicates decreased pain average scores on the mental and physical components of the sf-36 health survey questionnaire , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
increased scores indicate improvement average scores on the total ankle osteoarthritis scale ( aos ) and on the pain and disability components , from preoperative baseline ( preop ) to final follow - up ( week 24 ) .
decreased scores indicate improvement magnetic resonance imaging ( mri ) scans . a
preoperative sagittal view showing bone marrow oedema .
b
post - operative sagittal view showing reduction in the oedema signal . c
preoperative coronal view showing bone marrow oedema . d
post - operative coronal view showing reduced marrow signal no major adverse events related to the graft were observed . over the course of the study
, there were no wound infections , wound breakdowns , reoperations , loss of motion , symptoms of loose body / impingement , or synovitis .
( any additional adverse events affecting the ankle are listed by patient in the appendix ) .
evaluation of bone marrow oedema as visualised by mri , preoperatively and at 24 weeks post - operatively , is shown in table 3 and fig .
4 ( note that retrograde drilling was not possible in this joint , as only minimal cartilage remained).table 3mean area of oedema ( mm ) via mriparameterpreop24 weekscoronal oedema95 ( ci 20171)71 ( ci 0208)sagittal oedema140 ( ci 50229)37 ( ci 093)average118 ( ci 45190)54 ( ci 0150)table 4mean area of ocd lesions and percentage filled by graft , as visualised via ct scanparameter2 weeks24 weekscoronal view ( mm)6958sagittal view ( mm)8976amount of defect filled by graft ( coronal view ) ( % ) 85.254amount of defect filled by graft ( sagittal view ) ( % ) 84.661 mean area of oedema ( mm ) via mri mean area of ocd lesions and percentage filled by graft , as visualised via ct scan incorporation of the graft as visualised by ct scan , at 2- and 24-week follow - ups , is shown in table 4 and fig .
5.fig . 5post - operative computed tomography ( ct ) scans . a
coronal view at 2 weeks showing fill of the osteochondral defect .
b
coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules post - operative computed tomography ( ct ) scans . a
coronal view at 2 weeks showing fill of the osteochondral defect .
b
coronal view at final scan ( 24 weeks ) showing healing of the bone in the base of the osteochondral defect and resorption of periarticular tricalcium phosphate granules
the most important finding of the present study was that a new arthroscopic procedure yielded improved pain and functional scores for patients with ocd , and enhanced healing of these lesions ( as confirmed by mri and ct scans ) .
no short - term complications were observed despite close monitoring of the patients . this prospective , single - centre , case - series - based , proof - of - concept study evaluated the initial safety , efficacy , and clinical utility of rhpdgf/-tcp as an arthroscopic treatment following debridement for ocd of the talus in five patients .
results at 6-month follow - up indicate that the method is both safe and effective , at least initially .
mean non - weight bearing vas pain scores decreased by 68 % , mean weight bearing vas pain scores decreased by 49 % ( vas ) , and mean scores on the aos disability component decreased by 28 % ( table 2 ; figs . 1 , 3 ) .
mri scans confirmed a reduction in subchondral oedema over time in all patients , with the mean area decreasing by 25 % on the coronal view and by 74 % on the sagittal view from preoperative baseline to final follow - up scans at 24 weeks ( table 3 ; fig .
no heterotopic ossification was evident on follow - up ct scans , and the graft was incorporated in all five cases ( table 4 ; fig . 5 ) .
in addition , no major adverse events related to the graft were observed , and none of the patients required reoperation for their ocd . with procedures such as oats , not only
is there increased risk of knee donor - site morbidity , but the force of impact during graft insertion can damage both the talus and the chondrocytes in the graft .
thus , gentler insertion of the non - cellular preparation used in our procedure confers the added advantage of reduced damage to ankle and graft , as well as the knee . in a large , multi - centre , prospective , randomised , controlled , clinical trial , digiovanni et al .
compared the safety and efficacy of pdgf/-tcp versus autogenous bone grafts ( autografts ) in patients requiring hindfoot or ankle fusion .
the study found that the pdgf treatment resulted in comparable fusion rates , less pain , and fewer side effects compared to treatment with autograft . due to its role
as a mitogenic and chemotactic factor for fibroblasts [ 30 , 32 ] , osteoblasts [ 2224 , 26 , 29 ] , chondrocytes [ 28 , 33 ] , and tenoblasts [ 9 , 27 , 35 ] , pdgf also holds considerable promise in restoring bone integrity in lesions such as ocds .
healing and restoration of the subchondral bone may thus make our method more effective in the long term than cartilage grafts , oats , or aci .
in addition , this new procedure offers an alternative that should prove to be less invasive and more economical than either oats or aci .
especially promising was the fact that four of the five patients had undergone previous debridements that required revision , but no revisions or additional procedures were required following the rhpdgf/-tcp treatment .
limitations included small sample size , a short follow - up period , lack of a concurrent control group , and potential selection bias due to patient recruitment from a single clinical centre . in addition , the improved area of marrow oedema observed at final follow - up could be due in part to a period of relatively limited mobility . a larger , multi - centre , randomised , controlled trial with a minimum 2-year follow - up period is needed and is planned in order to address these limitations
. the clinical relevance of this study is that it demonstrates that a new technique using a human growth factor for the treatment of ocd can be safe , well tolerated , and clinically useful .
if the results of the present study are confirmed in a larger trial , the potential benefits include lower cost , less post - operative pain , fewer wound complications , and shorter recovery time due to its less invasive nature ; no non - unions of osteotomies ; considerable reductions in patient - reported pain several months after surgery ; and better long - term outcomes , compared with more invasive procedures such as oats or aci , which are currently the standard of care .
in addition , there is no need for grafts ( and therefore no donor - site morbidity ) , and this mixture of growth factor and osteoconductive matrix may help to stabilise the lesion after debridement , preventing further progression .
in conclusion , the use of rhpdgf in an osteoconductive matrix shows promise as a clinically useful alternative for the treatment of ocd lesions of the talus following arthroscopic debridement . an expanded series or randomised ,
see tables 5 , 6 , and 7.table 5adverse events as reported by patientspatient idadverse event descriptiononset date001fell using crutches , stuck splint at study ankle12 march 2012slipped on stairs , muscles sore at ankle11 may 2012development of stress fracture at calcaneus of study foot ( osteoporosis - z - score-3.7 , severely osteoporotic)noted during may 2012small clot with superficial phlebitis right anklenoted during 17 may 2012 appointmentsevered acl right leg ( study side)noted during unscheduled visit 18 july 2012fell off bike , landed on right side ( knee area)early august 2012occasional catching and locking symptoms study anklenoted during 24 august 2012 visitmedial pain , study anklenoted during 24 august 2012 visitdropped 1-lb weight on study anklenoted during 24 august 2012 visit002right hip replacement6 march 2013continued pain left study ankle .
patient had injection for thisnoted during unscheduled visit 4 april 2013003cast causing pain removed and replacednoted during unscheduled visit 23 october 2012sharp , acute pain right study ankle5
november 2012shooting pains medial ankle , decreased sensation throughout entire dorsum of footnoted during 15 november 2012 visitpatient notes colour difference right study ankle , swelling in foot , lump on bottom of foot ( noticed during december 2012 ) , sore big toe with pain radiating to foot , numbnessnoted during unscheduled visit 21 december 2012severe oa left knee
referred to specialistreferral 27 december 2012after short walk , sharp pain in ankle6 january 2013corneal surgery for displaced lens10 january 2013004graft not contained as well as hopednoted during 7 june 2012 visitstiff anklenoted during 12 july 2012ankle becomes swollen after running any distancenoted during 15 november 2012 visit005slipped in elevator , caught himself24 june 2012patient notes occasional numb / tingling sensationnoted during 25 june 2012 visitresidual material back of jointnoted during 25 june 2012 visitdropped brick on ankle16 august 2012withdrawal symptoms from discontinuation of tramadol insomnia , loss of appetitenoted during 30 august 2012 visittable 6adverse event definitions : severity criteriadegreedescriptionmild ( grade 1)symptom(s ) barely noticeable to subject or does not make subject uncomfortable ; does not influence performance or functioning ; prescription drug not ordinarily needed for relief of symptom(s ) , but may be given because of the nature of subjectmoderate ( grade 2)symptom(s ) of a sufficient severity to make subject uncomfortable ; performance of daily activity is influenced ; subject is able to continue in study ; treatment of symptom(s ) may be neededsevere ( grade 3)cause severe discomfort ; symptoms cause incapacitation or significant impact on subject s daily life ; severity may cause cessation of treatment with study device ; treatment for symptom(s ) may be given and/or subject hospitalisedlife - threatening ( grade 4)extreme limitation in activity ; significant assistance required ; significant medical intervention / therapy required ; hospitalisation or hospice care requiredtable 7adverse event definitions : relationship to procedurerelationshipdescriptionnot relatedany reaction that does not follow a reasonable temporal sequence from administration of the study device and that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectunlikelyany reaction that does not follow a reasonable temporal sequence from administration of the study device or that is likely to have been produced by the subject s clinical state or other modes of therapy administered to the subjectlikelya reaction that follows a reasonable temporal sequence from administration of the study device or that follows a known response pattern to the suspended device and that could not be reasonably explained by the known characteristics of the subject s clinical state or other modes of therapy administered to the subjectdefinitea reaction that follows a reasonable temporal sequence from administration of the study device and that follows a known response pattern to the suspected device and that recurs with rechallenge , and/or is improved by removing the device adverse events as reported by patients adverse event definitions : severity criteria adverse event definitions : relationship to procedure
none of the authors have received financial support , including pharmaceutical company support , for authorship , or promotion of this study . | purpose
an arthroscopic procedure for the treatment of osteochondral defects using platelet - derived growth factor ( pdgf ) carried out in a matrix of tricalcium phosphate was developed .
this prospective , case - series - based study was designed to evaluate the safety and clinical utility of this procedure.methodspatients with an isolated osteochondral defect larger than 5 mm long , 3 mm wide , and 5 mm deep and smaller than 30 mm long , 25 mm wide , or 20 mm deep were considered for enrolment . only patients with chronic lesions
were enroled .
arthroscopic debridement was followed by the placement of recombinant human pdgf in a matrix of tricalcium phosphate .
the ankle osteoarthritis scale ( aos ) , visual analogue scale ( vas ) for pain , and sf-36 questionnaires were administered at 0 , 2 , 6 , 12 , and 24 weeks .
magnetic resonance imaging ( mri ) and computed tomography ( ct ) scans were taken before and after surgery.resultsfive patients were ultimately enroled in this proof - of - concept trial .
all outcome measures demonstrated marked improvement from baseline to final follow - up : the mean weight bearing vas pain score improved by 49 % , and the mean aos functional score improved by 28 % .
bone healing was seen on ct , and reduction in oedema signal was seen on mri.conclusionthis new procedure may offer a promising alternative for the treatment of osteochondral defects .
further high - quality studies are needed to confirm these results and to analyse the long - term effects of the procedure .
the clinical relevance of this study is that the procedure may provide a less invasive option with improved bone healing compared to standard techniques
.level of evidenceiv . | Level of evidence
Introduction
Materials and methods
Data collection
Surgical techniques
Outcome measures
Statistical analysis
Results
Discussion
Conclusion
Appendix: adverse events
Conflict of interest |
PMC3437157 | in the usa , where more than one quarter of all adults smoke cigarettes,1 tobacco smoking is considered the leading preventable cause of disease , disability and premature death.23 it has been estimated that direct and indirect economic losses attributed to tobacco use in usa approach 85 billion us dollars annually.2 the risk of premature death in smokers was about double that of non - smokers and was directly proportional to the frequency of smoking.24 recent data showed that 15% of all cancer cases in the world were estimated to be due to cigarette smoking.5 moreover , slightly less than 20% of all deaths in industrialized countries were attributable to tobacco.67 tobacco contains about 4000 substances , more than 40 of which are carcinogenic and many others that are hazardous to cardiopulmonary systems.2 many studies have demonstrated an association between cigarette smoking and cancers of the oral cavity , pharynx , larynx , lungs , esophagus , stomach , colon , rectum , pancreas , kidney and urinary bladder,35,811 and a less strong association with leukemia and cervical cancer.8 a causal association was found to exist between smoking and coronary artery disease , peptic ulcer , chronic obstructive pulmonary disease and stroke.3101213 cigarette smoking is not only harmful to those using it deliberately , but also to involuntary smokers ( the so - called passive smokers ) .
passive smoking is associated with coronary artery disease , chronic obstructive pulmonary disease , asthma , leukemia and cancers of the lung , sinuses , cervix , breast and brain.121416 in children , passive smoking is associated with higher incidence of asthma attacks , pneumonia , ear disease , sudden infant death syndrome and upper and lower respiratory tract infections.121417 fetuses are not exempted from hazards of smoking .
it was found that pregnant mothers exposed to passive smoking were more prone to have low birth weight infants.18 it is doubtful that an ex - smoker will fully resume his pre - smoking health state .
however , a substantial risk reduction with great improvement in life expectancy and quality is beyond doubt2111923 the benefit of quitting is documented even for old patients and those with an established tobacco - related disease.2325
more than half of americans are considered to have a sedentary life - style.3 this is considered the most preventable risk factor for coronary artery disease.32126 regular physical exercise decreases the risk for many health problems including non - insulin dependent diabetes mellitus , osteoporosis , osteoarthritis , cardiovascular disease , cancer , hypertension , obesity and hypercholestrolemia.32734 regular exer - cise was also found to be associated with better general well - being35 and life expectancy3637 in addition to lesser mortality rates .422 specific prescription of physical exercise should be given to almost all patients , bearing in mind the age and general condition of each individual .323 almost every patient including elderly people and those with severe coronary artery disease and patients with congestive heart failure , has the potential of benefiting from regular physical exercise.3242537 the physiologic effects of exercise depend on its type , intensity , duration and frequency.38 recreational physical exercise can be classified into the following two types:3839 this type is characterized by an increase in muscle tension without much change in fiber length , with substantial energy expenditure .
examples are weight lifting , hand grip exercises and pushing or pulling against a resistance .
anaerobic exercise increases total peripheral resistance and thus leads to considerable rise in blood pressure .
this is the type of exercise which is recommended to the general population on a regular basis .
the more frequent and prolonged the aerobic exercise , the better its effects , provided that the patient 's condition permits it.3340
this type is characterized by an increase in muscle tension without much change in fiber length , with substantial energy expenditure .
examples are weight lifting , hand grip exercises and pushing or pulling against a resistance .
anaerobic exercise increases total peripheral resistance and thus leads to considerable rise in blood pressure .
this is the type of exercise which is recommended to the general population on a regular basis .
the more frequent and prolonged the aerobic exercise , the better its effects , provided that the patient 's condition permits it.3340
the annual economic losses due to obesity are calculated in billions of dollars.41 the prevalence of obesity in uk ( 1986 ) approached 12% in males and 8% in females.42 the situation in usa is even worse , where about one quarter of all adult americans are overweight.4243 further- more , recent data indicated a linear increase in the average weight of american adults over the last few decades.41 obesity is defined by who as a body mass index ( bmi ) more than 30 kg per square metre.4244 the risk of overweight related problems , however , starts at body mass index of 25 kg per square metre.41 overweight adversely affects the general health of people.35 it is considered to be a risk factor for non insulin dependent diabetes mellitus , coronary artery disease , hypertension , hyperlipidemia , gallstones , low back pain , certain cancers ( colorectal and prostate in males , and cervical , breast and ovarian in females ) and premature death.4264348 distribution of regional , rather than total , body fat is increasingly gaining special importance .
recent research has demonstrated an association between the amount of visceral fat and the disturbances in glucose tolerance and lipid concentrations.46 visceral fat , which can be measured by computerized tomography,46 was found to correlate highly significantly with waist - to - hip ratio.49 cut off points for waist - to - hip ratio , above which high risk begins , were generally agreed to be 0.95 for men and 0.80 for women.50 high waist - to - hip ratio ( upper body obesity ) is associated with high risk for hypertension , diabetes mellitus , hypercholestrolemia , hypertriglyceridemia , low hdl , hyperinsulinemia and mortality from cardiovascular disease.44465054 causes of obesity include a variety of medical , genetic and lifestyle factors .
the easiest and most effective modality of prevention is to deal with the lifestyle factors , namely diet and physical activity.4255 prevention of obesity has been proved to be highly beneficial in preventing obesity - related health hazards , resulting in a better life expectancy and general health.112023325658
seat belts were introduced for the first time in the mid 1950s . by the late 1960s
the introduction of interlock devices that prevent ignition if the driver 's safety belt was not fastened , resulted in a significant improvement in the use of seat belts .
minimal improvement occurred with the introduction of warning lights and buzzers . in the usa ( 1984 ) , legislation for the use of seat belts took place and resulted in increased compliance from 15% to about 50% in a short time.59 studies from all over the world documented the benefits of seat belts and the hazards of non - use.1127356065 there was more than 60% reduction in the severity of injuries , hospital admissions and hospital charges for users of seat belts.66 in germany , morbidity and mortality due to road traffic accidents have shown substantial decrease when the rates of seat belts use increased.67 interestingly it was found that parents use of seat belts , influenced children to do the same68 and that non - use of seat belts was associated with other unhealthy behavior such as smoking , overweight and lack of exercise.6269 the airbag was recently introduced in many new motor vehicles as a supplement to seat belts .
this new device has proved quite effective but its effect is still limited due to the relatively small number of cars equipped with them.70
there is a belief that during the last few decades some unhealthy lifestyles have been adopted by saudis . however , published national studies that quantify these important assumptions are few.71 most of the studies referred to below are apparently limited by being either retrospective , based on insufficient recording systems , clinic - based , hospital - based , or done on a small scale .
these limitations do not rule out the importance of such studies in providing us with rough estimates of the extent of the problem in our country .
available data point to cigarette smoking as an important health problem in saudi arabia.72 following the steady increase in the quantity of tobacco imported before 1987 , national statistical data7374 showed significant reduction in tobacco imports during the period from 1989 to 1994 ( table 1 ) .
tobacco , however , is still considered a burden on the health and economy of saudi population .
it was found that 33% of male medical students in riyadh smoke cigarettes.75 the prevalence among other university students was even higher ( 37%).76 in secondary schools , 22% of male students admitted being regular smokers.77 while smoking is considered socially unacceptable among females , up to 12% of university female students reported being current smokers.78 the weight ( in thousand tons ) and cost ( in million sr ) of tobacco imported during the period 1989 - 1994 in 1987 , a1-turki found that the smoking rate in the eastern province was 37% and 9% for adult males and females.79 in al - baha province , in 1989 , al - bedah reported a higher rate among males ; about 53% of adult males being smokers .
the overall rate , however , was 29% because cigarette smoking was absolutely unacceptable among females in such a rural community.80 over the past two decades , the per capita calory consumption of saudis has shown a steady increase ( table 2).8182 considering bmi of 25 as a cut - off point , binhemd et a183 found 52% of males and 65% of females visiting the primary care clinic of king fahd hospital of the university at al - khobar , to be obese . despite the limitation of such a study ,
the high figures it showed were supported by findings of al - shammari et al.47 al - abbad ( 1995 ) demonstrated a prevalence of obesity ( defined as a bmi of 85th percentile ) approaching 29% among female students aged 11 - 21 years.84 the per capita daily dietary consumption ( kcal / day ) of saudis over the period 1974 - 1989 data about recreational aerobic physical activity of saudis are not yet available . however
, if we consider the body weight as an indirect indicator , the physical activity of saudi population does not seem to be improving in relation to other healthy life styles .
the number of cars imported by gulf countries increased by 1031% between 1971 and 1984 , and deaths due to road traffic accidents increased by 307% in the same period . in saudi arabia , the number of cars increased by 2400% within the 10 years ending 1986 and this was associated with 469% increase in trauma cases and a 561% increase in related deaths.85 it is not surprising , therefore , that road traffic accidents constitute the leading cause of death in the age group 16 - 36 years . in 1984 , mortality due to road traffic accidents approached 15 deaths per day .
this number reflects only those who died at the scene of the accident . those who died en route to the hospital or following admission were almost double.86 in 1980 , khawashki estimated the economic loss in saudi arabia due to road traffic accidents as saudi rivals 4,776,836 per day.87 in asir region alone , which contains 2.5% of all cars in the country , about 7760 persons were involved in road traffic accidents in the period from 1989 - 1991 ; 62% of these were injured , and 12% died.88 fast driving , driving by children under 18 years of age and driving without a license were found to contribute enormously towards road traffic accidents.8890 the authorities have responded to this by taking different measures including the imposition of severe penalties to offenders .
surprisingly , the mandatory use of seat belts which is probably easier to enforce , is still receiving little attention . in a study done by marwa
et a1,91 none of the victims of road traffic accidents were found to have had the seat belts fastened .
a few small studies attempted to measure the rate of seat belts use in certain localities in the country and ended up with the rates ranging from 7 - -19%.909293 unfortunately , to the best of the author 's knowledge , there have been no nation - wide studies on the rate of seat - belt use .
indeed , there is a great need to enforce healthy behavior in the saudi community as means of promoting healthy lifestyle towards the eventual goal of positive health . | the negative effects on health by behavior such as cigarette smoking , lack of physical exercise , non - control of body weight and non - use of seat belts were empirically documented .
available findings of the various studies on lifestyle of the saudi arabian community were not encouraging .
if the general health status of the saudi population is to be improved , an enforcement of healthy lifestyles must be considered . | CIGARETTE SMOKING
LACK OF PHYSICAL EXERCISE
1. Isometric (static or anaerobic) exercise:
2. Isotonic (dynamic or aerobic) exercise:
NON-CONTROL OF BODY WEIGHT
NON-USE OF SEAT BELTS
THE SITUATION IN SAUDI ARABIA |
PMC5145912 | physical exercise is regarded as a promising treatment and complement to pharmacological treatments in several different neurological diseases ( svensson et al . , 2014 ) .
exercise can affect both the adrenergic and corticosteroid systems involved in stress response as well as the microglia function and other cells and signaling molecules involved in inflammatory processes ( svensson et al . , 2014 ) .
in the central nervous system ( cns ) , microglial cells are the major source of pro - inflammatory cytokines ( kim and de vellis , 2005 , carson et al . , 2006 ) .
in response to neuronal injury , such as stroke , resident microglia are immediately activated and start to proliferate and produce pro - inflammatory cytokines ( banati et al , 1993 , barone et al , 1997 ) .
it has been shown in both patients and animal models that cerebral ischemia leads to an inflammatory response , systemically as well as in the brain ( lambertsen et al . , 2012 ) .
neuroinflammatory response can further aggravate the neuronal damage and administration of the pro - inflammatory cytokine il-1 after induction of cerebral ischemia in animal models can exacerbate the brain injury ( yamasaki et al . , 1995 ) .
microglial cells or their inflammatory mediator molecules may therefore be suitable targets for treating or preventing deleterious neuroinflammation following brain ischemia ( yenari et al . , 2010 ) .
several studies have shown that the levels of pro - inflammatory cytokines decrease , and the levels of anti - inflammatory cytokines increase following physical exercise ( mota et al , 2012 , piao et al , 2013 ) . indeed , treadmill running in animals has been shown to reduce the microgliosis and inhibit the release of pro - inflammatory cytokines , as well as reducing lesion size and cell death in the hippocampus and prevent short - term memory disturbances following cerebral ischemia ( austin et al , 2014 , lovatel et al , 2014 , sim et al , 2004 , sim et al , 2005 ) .
out - of - hospital cardiac arrest is a severe complication that can lead to global cerebral ischemia with pronounced neuronal damage .
the survival rate is very low , below 10% , and patients that survive are often affected by severe neurological injury and life - long cognitive and motor disabilities ( sasson et al . , 2010 ) .
in an experimental setting , global cerebral ischemia can be modeled in mice by transient occlusion of the common carotid arteries ( olsson et al . , 2003 ) .
global cerebral ischemia results in neuronal cell death in vulnerable brain regions such as hippocampus ( kirino and sano , 1984 , back et al . ,
2004 ) evolving during the first week after the ischemic insult ( bottiger et al . ,
1998 ) . by altering detrimental neuroinflammatory reactions , physical exercises could make the brain more resistant to ischemic injuries . on the negative side
, exercise can also induce a chronic stress response , which may result in detrimental effects in the event of a brain injury .
for example , forced running exercise in rodents can lead to anxiety and increase the levels of the stress hormone corticosterone in serum ( leasure and jones , 2008 , brown et al , 2007 ) .
it has been shown that stress can evoke a pro - inflammatory response in the brain , with increased expression of nlrp3 inflammasome involved in the cleavage and secretion of pro - inflammatory il-1 ( gadek - michalska et al , 2013 , frank et al , 2014 ) .
therefore , the overall positive effect of physical exercise in experimental models could potentially be masked by the stress response .
to the best of our knowledge , the effects of treadmill exercise pre - conditioning on stress and neuroinflammatory responses following global cerebral ischemia have not previously been studied in mice .
therefore , the aim of this study was to investigate the effect of pre - conditioning forced treadmill running on stress response , neuroinflammation , neuronal damage and behavioral alterations following global cerebral ischemia .
an additional aim was to compare the stress response after forced running with the response after voluntary running .
all proceedings and animal treatment were in accordance with the guidelines and requirements of the government committee on animal experimentation at lund university .
we used 63 male c57bl/6 mice , aged 810 weeks , weighing 2227 g that were obtained from charles river .
the mice were housed in standard laboratory cages ( 37 animals per cage ) , with sawdust bedding and free access to water and food .
the mice were weighed at the day of exercise introduction , and thereafter once every second week until the induction of global cerebral ischemia . thereafter the mice were weighed 34 days as well as 1012 days after ischemia .
when the experiment was initiated , the mice were assigned to different groups ensuring an even distribution in body weight and age .
briefly , some of the animals were subjected to treadmill running exercise for 4.5 weeks . during the third week of exercise
behavioral tests were then conducted 515 days after ischemia , after which , the animals were sacrificed and samples were collected ( 1516 days after ischemia ) . originally , the mice were divided into two different treatment groups , one subjected to exercise and one that was not subjected to any exercise ( sedentary ) .
exercise consisted of 30 min treadmill running at a speed of 25 cm / s with no inclination of the treadmill ( 5-lane treadmill , harvard apparatus , panlab ) .
the mice were exercised 3 days / week for 4.5 weeks of which the first week was an introduction week with a lower speed and duration of the exercise . on the first day of the introduction , the mice were subjected to 10 min of walking / running at a speed increasing from 5 to 18 cm / s .
the second day of the introduction consisted of 20 min of running with speeds up to 25 cm / s . during the third and last day of the introduction
, the duration was increased to 30 min , with a speed of 25 cm / s . to motivate the mice to run the researcher pushed them with a small stick if needed .
if the mouse refused to run it could be motivated by a transient and light electric stimulation from the grid at the beginning of the treadmill platform .
after this , if the mouse persisted in its refusal to run , it was removed from the treadmill and re - introduced to it at a later time point . in this study , all mice without exceptions had to be motivated at least some times by pushing them with the small stick at each day of exercise
. four of our best running mice even had to be motivated with a light electric stimulation at up to three different days . despite this , several mice refused to run after repeated trials .
exercise took place in a room separated from the housing room to which all mice were transferred and kept during the exercise procedures in order to minimize environmental confounders among the mice not subjected to the exercise protocol . to investigate the effect on stress response resulting from the enforcement to run compared to voluntary running exercise
, our main study was complemented with mice subjected to voluntary wheel running . for this , 10 male c57bl/6 mice at an age of 12 month were used .
these mice had the same housing conditions as the mice in the main study , except that they were single caged .
six of these mice had access to low - profile wireless running wheel for mouse ( med associates ) in their home cages during 6.5 weeks .
the running wheels had a wireless connection to a software measuring the distance each mouse ran every day , assuring that all mice with access to running wheels actually run .
the remaining four mice were sedentary controls with no access to running wheels in their homecage .
after 6.5 weeks of this running intervention , an open field test was performed ( as described in 2.5.1 ) and feces samples were collected ( as described in 2.6 ) in order to compare anxiety and corticosterone levels with the sedentary control group .
in order to model cardiac arrest patients with successful cardiopulmonary resuscitation , global ischemia was induced to the mice as previously described ( olsson et al .
, 2003 , burguillos et al , 2015 , lambertsen et al , 2011 , deierborg et al , 2008 ) .
the common carotid arteries were then isolated and encircled with a thin silk thread to allow bilateral occlusion using a micro aneurysm clip .
the wound was then sealed with absorbable stitches before the anesthesia was discontinued . during the surgery ,
the body temperature was monitored and controlled using a heating pad and an infrared lamp to keep the mice normothermic .
mice with body temperatures below 35.5 c a few hours after the intervention were put in an incubator at 34 c overnight in order to recover .
mice in the sham groups were subjected to the same surgical protocol , except that no occlusion to the common carotid arteries was made .
the person performing the surgery was blinded to the animals exercise regimen . in order to evaluate the locomotion and anxiety levels of the mice subjected to forced treadmill exercise ,
the test before the induction of ischemia was performed during the third week of exercise .
the mice were put in an open filed arena ( panlab , barcelona , spain , 45 cm by 45 cm ) and allowed to freely explore it for 10 min . an automated smart system ( panlab , barcelona , spain )
was used to measure the velocity of movements , distance moved and time spent in the center and periphery of the box . spending more time in the periphery
the box was cleaned first with ethanol and then with water before each mouse was introduced to the open field arena .
furthermore , anxious behavior in the open field test of mice subjected to voluntary wheel running with age - matched sedentary controls were also analyzed to test if voluntary running results in anxious behavior . in order to examine any defects in hippocampus - dependent spatial memory ,
anhedonic behavior of the mice was assessed using a sucrose preference test during the night between day1415 or 1516 .
fecal samples were collected from the open field box after conducting the open field test performed 1213 days following induction of ischemia in order to measure the stress levels of the mice . since anxiety in itself can increase the frequency of defecation , fecal samples from mice that did not defecated during the open field test were collected by putting those mice in individual cages until they had defecated .
this was done to prevent potential bias that could arise if only the corticosterone levels from those mice that indeed defecated during the test would have been analyzed .
corticosterone levels measured in feces is likely to reflect the level of corticosterone that was found in serum several hours earlier ( kalliokoski et al . , 2010 ) .
to compare the effect of forced running on the corticosterone levels without ischemic insult as a confounding factor , mice only subjected to sham surgery were used for this test .
corticosterone was then extracted and analyzed using a corticosterone elisa kit ( enzo life sciences ) as described by touma et al .
( touma et al . , 2003 ) , except that feces was homogenized in 1 ml of 80% methanol per 100 mg sample .
feces samples from mice subjected to voluntary wheel running with age - matched sedentary controls were also analyzed to investigate if running exercise per see results in corticosterone increase .
blood samples were kept in room temperature for 25 min and then stored on ice a few hours until the samples were centrifuged at 1300 g in 4 c for 10 min and the serum supernatants were collected and stored at 80 c until analysis .
the brains were dissected out and snap frozen in crushed dry ice and stored at 80 c until analysis .
the brains where sectioned with a leica cm3050 s cryostat ( leica microsystems nussloch gmbh , germany ) .
the hippocampus was extracted from 1.20 to 2.20 ( according to k. franklin , g. paxinos , the mouse brain : in stereotaxic coordinates , academic press , usa ) .
the brain sections were 30 m thick and collected into 6 series , with 3 series mounted onto microscope slides and 3 series stored in tubes for preparations of brain homogenates .
sections used for immunohistochemical stainings were fixated with 4% of pfa for 10 min prior to staining procedure .
viable neurons were stained on hippocampal sections using mouse - anti - neun antibody ( 1:200 , anti - neun , mab377 , emd millipore corporation , billerica , ma , usa ) .
4,6-diamidino-2-phenylindole ( dapi ) ( 1:1000 , sigma aldrich ) was used to stain cell nuclei .
the number of neun positive cells with viable cell morphology was counted in the hippocampal subregions ca1 , ca2 and ca3 of each hemisphere from two coronal subsequential sections ( bregma 1.2 and 1.4 ) from each mouse using an epifluorescence microscope ( nikon eclipse 80i microscope , europe ) .
inflammation and microglial activation was visualized using polyclonal rabbit - anti - iba1 ( 1:500 , wako , japan ) and monoclonal rat - anti - galectin-3 ( m3/38 , 1:300 made in - house , provided by professor hakon leffler ) antibodies .
to evaluate the overall number and activity of microglia in the hippocampus pictures were captured of the 3 subsequential sections of hippocampus ( bregma-1.2 mm , bregma-1.4 mm and bregma-1.6 mm ) from each mouse , using the epifluorescence microscope . the extent and intensity of the iba1 staining
the number of galectin-3 positive cells in the hippocampus was counted in the same sections using the epifluorescence microscope .
eight brain slices at the level of the hippocampus from each mouse were homogenized in 100 l complete lysis buffer ( mesoscale discovery , rockville , usa ) containing protease inhibitor .
the homogenates were then incubated 20 min on ice and tip - sonicated with 5 pulses for 23 s. samples were thereafter centrifuged at 10,000 g in 4 c for 20 min before the supernatants were collected .
protein concentrations were determined using the bradford method and samples were stored at 80 c until use .
the concentrations of different cytokines in the collected serum and homogenized brain samples ( 25 l / sample ) were measured in the msd mouse proinflammatory v - plex plus kit ( ifn , il-1 , il-2 , il-4 , il-5 , il-6 , il-10 , il-12p70 , cxcl1 , tnf ; k15012c , mesoscale ) using a sector imager 6000 ( mesoscale discovery , rockville , usa ) plate reader according to the manufacturer 's instructions .
samples the concentrations were normalized to the different protein concentrations measured in the bradford assay of each homogenized sample .
eight brain slices at the level of hippocampus from each mouse were homogenized in 160 l of ripa buffer ( r0278 , sigma - aldrich ) containing protease inhibitor ( 1% protease inhibitor cocktail , p8340 , sigma - aldrich ) samples were sonicated twice for 3 s before they were centrifuged at 8000 g for 10 min and the supernatants were collected .
briefly , proteins were loaded on 420% mini - protean tgx precast gels ( bio - rad ) then transferred to nitrocellulose membranes ( bio - rad ) using trans - blot turbo system ( bio - rad ) .
the membranes were then blocked with 10% casein ( sigma - aldrich ) diluted in pbs ( tablets , sigma - aldrich ) . after blocking
, the membranes were incubated with primary antibodies against nlrp3 ( mab , adipogen 1:4000 ) and galectin-3 ( m3/38 , made in - house , provided by professor hakon leffler , 1:500 ) at 4 c over night .
the membranes were then incubated with peroxidase secondary antibody ( vector labs ) and the blots were developed using clarity western ecl substrate ( bio - rad ) .
unfortunately , the forced treadmill exercise induced stress in the animals to such an extent that severe fighting behavior was induced in their home cages . therefore , several mice ( n = 16 ) had to be sacrificed during this exercise paradigm , which explains the uneven distribution of mice in the different groups . during the ischemic surgery , several mice ( n = 7 ) also died , as expected .
the number of mice in each group during the behavioral tests also decreased due to the expected mortality caused by evolution of secondary neuronal damage during the two weeks following induction of global cerebral ischemia .
furthermore , for the analysis of cytokines , all samples with lack of signal in the sector imager 6000 plate reader were excluded .
this altogether explains why the number of samples in each group varies for each experiment .
thus , the variations in number of samples between the experiments are not due to exclusion of outliers or conscious selection of subgroups .
anova were performed followed by fisher 's post hoc test for all comparisons between groups regarding the immunohistochemical evaluations , western blots , behavioral tests and cytokine levels .
two - tailed t - tests were used to compare the levels of corticosterone and anxiety in the two different groups ( exercising and non - exercising ) during the exercise program . for cytokines
ifn , il-6 and il-10 , as well as for galectin-3 and nlrp3 , the levels were converted to a logarithmic scale before analysis .
the correlations between immunohistochemical data , behavioral data and cytokine and corticosterone levels were analyzed using pearson 's correlation coefficients ( r ) .
r - values above 0.7 were regarded as very strong relationships and r - values between 0.4 and 0.69 were regarded as moderate to strong relationships . for survival analysis following ischemic surgery , kaplan - meier curves and subsequent log rank tests
survival during the ischemic surgery procedure for the exercised and non - exercised groups was evaluated by fisher 's exact test .
the results from the open field tests conducted before the induction of cerebral ischemia can be seen in fig . 2 .
at baseline , there were no significant differences in distance moved between mice subjected to exercise and those that were not ( fig .
2a , 3902 962 cm and 3496 502 cm respectively , t - test , p = 0.067 ) .
however , exercised mice spent more time close to the walls ( fig . 2b , 81.3 9.6% of time and 69.0 13.6% of time respectively , t - test , p = 0.002 ) of the open field box compared to those who were not subjected to any exercise .
after ischemia , there was no difference between the group subjected to exercise and the group that had not been exercising ( data not shown ) . however , there was a difference between the sham group and the groups subjected to ischemia in the behavior in the open field test ( see fig .
is known to be dependent on the patency and lack of posterior communicating artery ( zhen and dore , 2007 , murakami et al , 1998 ) . due to heterogeneity in the anatomy of the cerebral vascularization among the mice
the experimental induction of ischemia might cause neuronal damage in one or both hemispheres , and the respective hemispheres can therefore be analyzed separately ( olsson et al .
the global brain ischemia caused neuronal damage in the hippocampus both in the left and right hemispheres ( fig .
3a , b 1232 209 and 1210 220 viable - appearing neurons in sham compared to 652 487 and 795 297 living neurons in ischemic mice , anova p = 0.015 and p = 0.001 respectively , fisher 's post hoc test , p = 0.036 and p = 0.03 , respectively ) .
following ischemia , fewer neun positive neurons were detected in the right hippocampus of exercised mice compared to those who did not exercise ( fig .
3 , fisher 's post hoc test , 411 263 and 795 297 living neurons respectively , p = 0.039 ) .
no difference in neun positive cells could be found in the left hemispheres between the experimental groups .
the mortality during the surgical procedure did not differ between exercising or sedentary mice ( fisher 's exact test , p = 0.13 , in the ischemic groups 4/12 mice died in the exercising group and 1/17 mice died in the sedentary group ) .
furthermore , the mortality after ischemia did not differ between exercising or sedentary mice ( log rank test , p = 0.103 , in the sham group , no mice died , and in the ischemic groups 2/8 mice died in the exercising group and 7/16 mice died in the sedentary group ) .
we found no differences in microglial activity between the exercising and non - exercising mice in iba1 ( optical density were 13.7 8.4 and 14.8 8.6 for exercising and non - exercising mice respectively , p = 0.81 ) or galectin-3 ( 87 74 and 42 35 positive cells for exercising and non - exercising mice respectively , p = 0.13 ) immunohistochemical stainings following ischemia that could explain the differences in neuronal survival in the hippocampus . our behavioral data ( fig .
2 ) suggest that the mice in the treadmill training groups might be stressed due to the forced exercise .
we therefore measured the levels of corticosterone in the feces , which is an established method to measure the stress levels in mice ( kalliokoski et al , 2010 , siswanto et al , 2008 ) .
the corticosterone levels in feces collected during the open field test are shown in fig .
4 . to compare the effect of forced running on the corticosterone levels without ischemic insult as a confounding factor , mice only subjected to sham surgery are presented in this figure . in these sham groups ,
corticosterone levels were elevated in mice subjected to forced treadmill running with 61% compared to mice which had not been subjected to any exercise ( fig . 4a , 394 107
pg / ml and 244 73 pg / ml respectively , t - test , p = 0.016 ) .
these data suggest that the mice that were exposed to forced treadmill training were profoundly stressed the weeks before the ischemic insult , which can be related to the increased brain damage seen in this group . indeed , by correlating the corticosterone levels to the number of viable neurons in the hippocampus we found a negative correlation ( fig .
4b , pearson 's r = 0.567 , p = 0.004 ) . during the exercise program
, there was no significant difference in corticosterone levels between mice shown to be the most unwilling to run and mice that were more willing to run ( 520 208 pg / ml and 365 147 pg / ml respectively , t - test , p = 0.08 , for the mice most unwilling to run n = 6 and for the mice more willing to run n = 12 ) .
there was no difference in the levels of corticosterone in feces between those that had been exercising and those that had not been exercising in the group subjected to ischemia ( data not shown ) . however , both groups subjected to ischemia displayed high corticosterone levels , which is most probably due to the effect of global ischemia that has been previously shown ( de la tremblaye et al . , 2014 )
. the levels of galectin-3 and nlrp3 in the brain of the different groups were analyzed with western blots , and can be seen in fig . 5 , fig . 6a respectively .
the levels of nlrp3 and galectin-3 did not significantly differ between exercised and non - exercised mice following ischemia .
for galectin-3 , no differences were detected in the anova ( p = 0.054 ) .
however , a difference in galectin-3 was found in fisher 's post hoc test comparing non - exercised ischemic mice to sham ( p = 0.038 , fig .
the number of galectin-3 positively stained microglia also correlated negatively with the number of viable neun positive neurons in hippocampus ( fig .
5b , pearson 's r = 0.721 , p = 0.001 ) .
6a and b , anova p = 0.027 , fisher 's post hoc test p = 0.048 ) in non - exercised mice compared to sham .
the levels of nlrp3 correlated with the levels of corticosterone found in feces ( fig .
6c , pearson 's r = 0.475 , p = 0.046 ) following ischemia . collectively , this data support our microglial iba1 immunohistochemistry data , showing increased levels of inflammatory proteins after ischemia , but no significant effect of treadmill exercise prior to ischemia .
we measured several pro- and anti - inflammatory cytokines in the blood and the brain after global ischemia .
the levels of four of the cytokines for which significant differences could be observed between different groups are presented in fig
the levels of the other cytokines measured in the collected serum and brain tissue from the different groups can be seen in supplementary material 2 , tables 1 and 2 , respectively .
ischemic mice had elevated il-1 levels in the brain compared to sham ( fig . 7a , 0.040 0.023 and 0.014 0.002 pg / mg protein respectively , anova p = 0.045 , fisher 's post hoc test , p = 0.002 ) .
no significant difference between non - exercised and exercised mice following ischemia was seen for this specific cytokine .
strikingly , mice that had been subjected to exercise prior to ischemia showed much higher levels of ifn in the brain compared to non - exercised mice with ischemia ( fig . 7c , 8.93 * 10 8.1 * 10 and 1.64 * 10 0.71 * 10 pg / mg protein respectively , fisher 's post hoc test , p = 0.008 ) . for the il-10 levels in the brain
, no significant difference was seen between exercised and non - exercised mice after ischemia ( fig .
7b , 0.39 0.10 and 0.31 0.09 pg / mg protein respectively , p = 0.10 ) .
no difference was seen in the anova ( p = 0.084 ) for the serum levels of il-10 .
however , higher levels of il-10 were observed in serum from exercised mice compared to non - exercised mice following ischemia in fisher 's post hoc test ( fig . 7d , 5.68 0.55 and 4.68 1.08 pg / ml respectively , p = 0.042 ) .
the concentration of il-10 in serum correlated negatively with the immunoreactivity of the iba1 staining in the hippocampus ( fig . 8a , pearson r = 0.535 , p = 0.022 ) .
the amount of surviving neun positive cells in the hippocampus showed a negative correlation with the levels of several different pro - inflammatory cytokines such as il-1 ( fig . 8b , pearson r = 0.641 , p = 0.006 ) in the brain .
the corticosterone levels also correlated with the levels of il-1 ( fig . 8c ,
pearson r = 0.61 , p = 0.006 ) and il-10 ( fig .
8d , pearson r = 0.594 , p = 0.007 ) in the brain . in summary ,
our data suggest an expected , negative association between pro - inflammatory cytokines and neuronal survival ( fig .
8b ) and also a positive correlation between cytokine levels in the brain and corticosterone levels in feces indicating that the stress caused by the forced exercise affects the neuroinflammation in the injured brain .
there were no differences between the different groups in the y - maze test and the sucrose preference test ( see supplementary material 2 , table 3 ) .
mice subjected to voluntary wheel running did not display increased anxious behavior compared to their age - matched sedentary controls ( fig .
9a , 74.2 12.8% of time and 72.6 12.0% of time respectively , t - test , p = 0.86 ) .
9b , the corticosterone levels in feces from wheel running mice did not differ from their age - matched sedentary controls .
taken together , we found that running exercise per se does not cause a stress response with increased anxiety and corticosterone levels .
in the present study , we investigated the potential preventive effect of physical exercise prior to global cerebral ischemia in mice by subjecting one group of mice to a four - week long treadmill running program prior to induction of global cerebral ischemia .
our main findings show that forced treadmill exercise induced a stress response that can counteract the anti - inflammatory effects of exercise , and lead to increased neuronal damage in a mouse model of global cerebral ischemia . in the open field test during the exercise period prior to ischemic onset , we observed that exercised mice were significantly more anxious , spending more time close to the walls of the open field arena , compared to the non - exercised group .
these results confirm our observations during the running procedure , where the mice gave the impression of being anxious and stressed during and after the training . indeed
, we lost several mice ( n = 16 ) during the training period due to fighting between the mice that were housed together . also , many of the mice showed an increasing unwillingness to run and were therefore excluded from the exercise group .
mice that were most unwilling to run also seemed to have higher levels of the stress hormone corticosterone in feces collected during the open field test conducted during this exercise period , even though this difference was not statistically significant ( data not shown ) .
these stress levels represent the status of the mice the weeks before they were subjected to ischemia , suggesting that stress prior to an ischemic insult is detrimental .
indeed , the potential of stress hormones such as corticosterone to potentiate the neuronal damage in hippocampus following global cerebral ischemia was shown already in the 1980s ( sapolsky and pulsinelli , 1985 ) .
examination of the brain samples from our mice also showed that exercised mice had fewer surviving neurons in the hippocampus compared to non - exercised mice following ischemia .
furthermore , higher corticosterone levels correlated with fewer surviving neurons , indicating that the reduced neuronal survival in the exercised mice may be due to the increased stress caused by the enforcement to run .
this conclusion was further strengthened by the fact that this increase in corticosterone levels where not observed in mice subjected to voluntary wheel running when compared to age - matched sedentary littermate controls .
when it comes to the effect of exercise on the level of inflammation following ischemia , our results demonstrate the complexity of the immune response following exercise and cerebral ischemia .
it has been suggested to sustain microglial activation through binding and activating the toll - like receptor 4 ( burguillos et al . , 2015 ) .
interestingly , we found a strong negative correlation between galectin-3 positive cells in the hippocampus and the number of viable hippocampal neurons ( neun ) , suggesting that the presence of galectin-3 positive microglia is associated with increased neuronal cell death .
previous studies in rats have shown that running exercise can reduce microgliosis if the rats are exercised after ischemic induction ( lovatel et al . ,
however , no microglial effect of exercise prior to ischemic induction was observed . in our study , following ischemia , exercised mice had a subtle increase in the levels of the anti - inflammatory cytokine il-10 in serum compared to non - exercised mice . increased levels of il-10
is a well - known anti - inflammatory effect of exercise ( pedersen and febbraio , 2008 ) and can provide neuroprotection after cerebral ischemia ( ooboshi et al . , 2005 ) .
however , ischemic exercised mice had a 5 times elevation of the pro - inflammatory ifn in the brain compared to non - exercised counterparts .
as ifn is a canonical cytokine known to induce a pro - inflammatory phenotype in microglia ( prajeeth et al , 2014 , papageorgiou et al , 2016 ) , it is tempting to speculate that this could in part explain the increased neuronal damage seen in the exercised mice compared to the corresponding non - exercised mice following ischemic insult .
the effect of exercise on the levels of ifn has not yet been elucidated , several studies have shown that exercise can decrease as well as increases the levels of ifn ( tuon et al , 2015 , nichol et al , 2008 ) .
however , in mice , ifn has been shown to increase in response to stress ( fuertig et al . , 2015 ) .
this suggests the possibility that the elevated levels of ifn observed in our mice may be caused by the stress associated with the exercise rather than the exercise per se .
our data confirm the complex relationship between different cytokines , where functions are dependent on the inflammatory context and possible interplay between the different cytokines , and where the effect of pro - inflammatory cytokines can be counterbalanced by high levels of anti - inflammatory cytokines .
it is also possible that exercise and stress can alter the inflammatory interplay between cns and the periphery .
the connection between the various cytokines involved in neuroinflammation and their importance in the context of physical exercise remains to be elucidated .
a limitation of this study is that we measured the cytokines fairly late , and only at one time - point in the process , which made it impossible to determine their temporal dynamics .
we further show that high levels of pro - inflammatory cytokines , such as il-1 , in the brain is correlated with significantly lower number surviving neurons in hippocampus following ischemia , suggesting that these cytokines are related to a negative impact on neuronal survival .
this negative impact of pro - inflammatory cytokines on neuronal survival has been shown in ischemic rats previously ( li et al . , 2014 ) . in our study ,
mice with high levels of corticosterone also had higher levels of cytokines , such as il-1 and il-10 , in the brain .
these results are in accordance with other studies where it has been shown that stress can evoke a pro - inflammatory response in the brain , with increased expression of nlrp3 inflammasome involved in the cleavage and secretion of pro - inflammatory il-1 ( gadek - michalska et al , 2013 , frank et al , 2014 ) .
recently , it has been shown that a stress - induced increase in corticosterone levels lowers the threshold for microglia to release pro - inflammatory cytokines ( dey et al . , 2014 ) .
increased stress has even been suggested to exacerbate neuronal cell death by increasing neuroinflammation , which in turn is neurotoxic ( de pablos et al . , 2014 ) .
indeed , several studies have shown that stress increase the vulnerability to inflammation resulting in increased microglial activation and neuronal damage in different brain regions such as hippocampus ( espinosa - oliva et al . , 2011 ) ,
substantia nigra ( de pablos et al . , 2014 ) and prefrontal cortex ( de pablos et al . , 2006 ) .
furthermore , the same studies also showed that these effects of stress were dependent on glucocorticoid receptor signaling . in our study
we did not detect any differences between exercised and non - exercised mice in the levels of il-1 and nlrp3 in the brain , suggesting that the link between stress and the inflammatory response may also involve other mechanisms .
however , one possible confounding factor is that the corticosterone samples were collected more than a week prior to collecting the samples used for measuring cytokine levels , nlrp3 and assessing brain damage .
the forced treadmill running clearly induced a stress response that could explain the negative effects on the brains of the mice in our study .
the reason why many studies , including ours , are based on forced treadmill running as a method for studying the effects of exercise is that this method can be standardized compared to , for example , voluntary wheel running . with treadmills , the intensity , duration and timing of the exercise
can be standardized so that all mice in the same exercise group are subjected to exactly the same amount of exercise .
this is not possible when using voluntary wheel running as the wheel is placed in the home cages , allowing the mice to run as much , fast and often as they want .
however , one advantage of using voluntary wheel running is that it does not include the stressful element of forced exercise , and that the mice can choose to run during the night , which in itself is less stressful as they are nocturnal animals .
however , for the voluntary wheel running , we choose to cage the mice separately in order to better control the distance run by each mouse .
it has been shown in other study that social isolation per see could be very stressful to the mice , as it is used in standardized stress protocols ( de pablos et al , 2014 , espinosa - oliva et al , 2011 ) .
however , our single caged mice did not display any signs of anxiety in the open field test compared to the behavior we use to observe in our mice .
our treadmill study is not alone in demonstrating that forced exercise can have negative effects due to stress . indeed
, other studies have shown that forced treadmill running causes anxiety and stress in form of increased levels of corticosterone , effects that were not observed in animals subjected to voluntary wheel running ( leasure and jones , 2008 , ke et al , 2011 , griesbach et al , 2012 ) .
interestingly , another study showed that it might not be the enforcement to run per se that induces stress , but rather the fact that being pushed if not running ( greenwood et al . ,
, there were no differences in stress induction between voluntary wheel running and motorized , forced wheel running .
however , the animals subjected to forced treadmill running , being pushed by foot shocks when not running , were more stressed .
in fact , forced treadmill running is also used as a model to induce and study stress responses in mice ( hong et al . , 2015 ) .
furthermore , zheng and coauthors showed that following experimental stroke in rats , forced treadmill running leads to reduced motor recovery compared to rats subjected to voluntary wheel running ( ke et al . , 2011 ) .
it has also been shown that forced and voluntary exercise can have different effects on inflammatory response , where forced exercise increases and voluntary exercise decreases levels of pro - inflammatory cytokines after injury in mice ( cook et al . , 2013 ) .
moreover , it has been shown that voluntary running leads to higher levels of bdnf compared to forced running ( uysal et al , 2015 , griesbach et al , 2014 ) . however , there are also studies comparing the effect of forced versus voluntary running showing that both regimens can be of equal beneficial effect when it comes to improving cognition , neuronal survival and bdnf levels in different models of brain injury ( lin et al , 2015a , lin et al , 2015b ) .
furthermore , it has been shown that forced exercise can activate a stress response similar to restraint stress , reducing the ability to cope with an oxidative challenge .
for example , forced running has been shown to increase the cardiac infarct size in rats ( mancardi et al . , 2009 ) , and failed to improve recovery after experimental stroke in rats ( auriat et al . , 2006 ) .
however , several studies have shown beneficial effects of forced exercise on the recovery from cerebral hypoperfusion ( cechetti et al . , 2012 ) as well as experimental stroke ( park et al . , 2012 ) .
this study shows that forced treadmill running induces stress , which can cause increased neuronal damage following global cerebral ischemia in mice .
we detected 5-fold increased ifn levels in ischemic mice that had been subjected to treadmill running , even though we found a small increase in serum il-10 levels .
this stress response with increased anxiety and corticosterone levels were not seen in mice exposed to voluntary wheel running .
it is therefore tempting to conclude that running exercise can have beneficial neuroinflammatory effects , but that the stress induced by the enforcement to run is disadvantageous and exceeds the beneficial effects .
our study highlights the importance of taking into account the stress that the enforcement of running exercise can induce , which can counteract the otherwise positive effects of physical exercise .
hence , it is important to monitor chronic stress in experimental animals to be able to infer the right conclusions about the effects of physical exercise . | physical exercise is known to be a beneficial factor by increasing the cellular stress tolerance . in ischemic stroke ,
physical exercise is suggested to both limit the brain injury and facilitate behavioral recovery . in this study we investigated the effect of physical exercise on brain damage following global cerebral ischemia in mice .
we aimed to study the effects of 4.5 weeks of forced treadmill running prior to ischemia on neuronal damage , neuroinflammation and its effect on general stress by measuring corticosterone in feces .
we subjected c57bl/6 mice ( n = 63 ) to either treadmill running or a sedentary program prior to induction of global ischemia .
anxious , depressive , and cognitive behaviors were analyzed .
stress levels were analyzed using a corticosterone elisa .
inflammatory and neurological outcomes were analyzed using immunohistochemistry , multiplex electrochemoluminescence elisa and western blot . to our surprise , we found that forced treadmill running induced a stress response , with increased anxiety in the open field test and increased levels of corticosterone . in accordance , mice subjected to forced exercise prior to ischemia developed larger neuronal damage in the hippocampus and showed higher cytokine levels in the brain and blood compared to non - exercised mice .
the extent of neuronal damage correlated with increased corticosterone levels . to compare forced treadmill with voluntary wheel running , we used a different set of mice that exercised freely on running wheels .
these mice did not show any anxiety or increased corticosterone levels .
altogether , our results indicate that exercise pre - conditioning may not be beneficial if the animals are forced to run as it can induce a detrimental stress response . | Introduction
Material and methods
Results
Discussion
Conclusions
Conflict of interest |
PMC4874745 | a preterm birth is defined as occurring after 20 - 0/7 weeks gestation and before 37 - 0/7 weeks of gestation ( 259 days from the first day of the mother s last menstrual period ) , regardless of birth weight.4 historically , birth weight was also included in the definition , with a premature infant defined as weighing less than 2,500 g at birth , delivered before 38 weeks gestation , or both . a report by the expert committee on maternal and child health of the world health organization in the 1960s made a distinction between premature and low birth weight infants , which led to birth weight being removed from the definition of preterm birth.9 another important distinction is whether or not a preterm birth occurs following spontaneous initiation of parturition , as some preterm deliveries are medically indicated due to maternal / fetal complications .
worldwide , an estimated 15 million preterm births occur each year , with a global preterm birth rate of 11.1% ( among 184 countries).10 while more than 60% of these preterm births occurred in sub - saharan africa and south asia , this remains a significant problem in developed countries .
preterm birth rates remain as high as 9% and above for upper middle- and high - income countries .
preterm birth in the united states rose from 10.6% in 1990 to a peak rate of 12.8% in 2006 . since that time
, the rate has declined to 11.39% in 2013.1 the initial rise in preterm birth rate resulted from a combination of factors , including increased use of assisted reproductive technologies , improved pregnancy dating by ultrasound , and increased willingness of providers to deliver preterm for maternal / fetal indications.4 the subsequent decline in preterm birth rate has been credited to improved fertility practices to reduce the incidence of multiple gestation pregnancies and improved strategies to prevent recurrent preterm birth , as well as quality improvement programs to limit provider - initiated preterm delivery to those with appropriate medical indications.4 unfortunately , significant racial disparities remain , with a preterm birth rate of 16.27% among non - hispanic black mothers compared to 10.17% among non - hispanic white mothers.1
women with a prior spontaneous preterm delivery have a 2.5-fold increased risk of having a subsequent preterm delivery , with an even higher risk in women with a prior preterm delivery at less than 28 weeks.11 racial differences also exist , with recurrent preterm birth occurring in 26% of black women compared to 15%20% of caucasian women.3 other disparities that exist include an increased risk of preterm birth among low socioeconomic status , low education levels , residence in disadvantaged areas , and lack of access / late access to prenatal care.4 behavioral risk factors for recurrent preterm birth include low bmi ( < 19.8 ) , significant interpregnancy weight loss ( decrease in one bmi category or greater than 5 kg / m ) , initiation of cigarette smoking , and short interpregnancy interval ( less than 18 months).2,1214 additional risk factors include systemic and genital tract infections , periodontal disease , uterine anomalies including large fibroids , cervical dysplasia , and associated treatment , as well as previous cervical injury , shortened cervical length ( less than 25 mm on transvaginal ultrasound ) , conception using assisted reproductive technologies , persistent vaginal bleeding , and multifetal gestation.4,15 women carrying a singleton gestation with history of a prior spontaneous preterm birth should be offered progesterone supplementation ( weekly intramuscular 17 -hydroxyprogesterone caproate ) beginning at 1624 weeks gestation to reduce the risk of recurrent spontaneous preterm birth.1618 this therapy is continued until 36 weeks gestation . in women with a current singleton pregnancy , prior preterm birth at less than 34 weeks , and a short cervical length ( less than 25 mm ) before 24 weeks , cerclage placement should be considered , as placement in this population is associated with significant decreases in preterm birth and reductions in perinatal morbidity and mortality.19,20 in women without a history of preterm delivery who are diagnosed with a cervical length of less than 20 mm prior to 24 weeks , vaginal progesterone supplementation should be initiated for prevention of preterm birth.21
multiple interventions have been recommended to reduce the risk of recurrent preterm birth . historically bed rest has been recommended to women at high risk for a preterm delivery , despite a lack of evidence in the literature to support its use . a cochrane review by sosa et al22 to evaluate the effect of bed rest for preventing preterm birth in pregnancy women at high risk found only one trial of 1,266 women for meta - analysis.23 in the study , 432 women were prescribed bed rest and 834 women received a placebo or no intervention .
preterm birth prior to 37 weeks was similar between groups ( 7.9% vs 8.5% ; rr 0.92 , 95% confidence interval [ ci ] 0.621.37 ) .
a second study by elliott et al24 was included in the sosa et al review but not in the meta - analysis because data for singleton pregnancies were not available . in the study ,
73 women being treated for preterm labor with a negative fetal fibronectin test were randomized to activity reduction ( ar ) ( bed rest with bathroom and shower privileges while inpatient and modified on discharge to allow travel to prenatal visits ) or no ar that included resuming home and work activities .
no significant differences in preterm birth rate were found between groups ( preterm delivery rate 44.4% patients with ar and 35.1% patients without ar , p = 0.478 ) .
one randomized clinical trial studying 17- hydroxyprogesterone caproate and omega-3 supplementation for the prevention of preterm birth also investigated the effects of activity restriction on the preterm birth rate among nulliparous women with a short cervix ( less than 30 mm).25 in this study , activity restriction was defined as any of the following : pelvic rest , reduction of work activity , or reduction of nonwork activity .
of the 646 participants , 252 ( 39% ) were prescribed some form of activity restriction ( most commonly the combination of all three listed previously ) at a mean gestational age of 23.9 weeks .
preterm birth prior to 37 weeks , and prior to 34 weeks , was significantly more common among women placed on activity restriction .
this difference remained significant ( adjusted odds ratio [ or ] 2.37 for less than 37 weeks and 2.28 for less than 34 weeks ) after controlling for confounding factors , including demographics and ultrasonography findings .
all these studies demonstrate that bed rest and ar do not significantly reduce the risk for preterm birth and may in fact have numerous adverse effects , including emotional distress , venous thromboembolism , deconditioning and increased weight gain , and financial consequences.26,27 providers must consider these risks before prescribing bed rest and activity reduction ( ar ) to their patients .
many epidemiologic and observational studies have examined the effects of occupational activities such as prolonged standing , heavy lifting , and exertion , as well as regular exercise on preterm delivery rates and obstetric outcomes with mixed results .
mozurkewich et al28 performed a meta - analysis on 29 studies and concluded that physically demanding work , defined as heavy and/or repetitive lifting or load carrying , manual labor , or significant physical exertion , was associated with an increased risk for preterm delivery ( pooled or 1.22 , 95% ci 1.161.29 ) , as well as small for gestational age , hypertension , and preeclampsia . as stated by the authors ,
these results were modest and derived mainly from observational studies ; therefore , the results need to be interpreted with caution . a second review by bonzini et al29 included 49 studies and investigated the relationship between five occupational exposures ( prolonged work hours , shift work , lifting , standing , and heavy physical work load ) and three adverse obstetric outcomes ( preterm birth , low birth weight , and preeclampsia / gestational hypertension ) .
when evaluating risk for preterm birth , the findings ruled out a more than moderate effect ( defined as a rr > 1.4 ) for each exposure and pooled estimates of risk pointed to only modest or null effects ; therefore , no work activity restrictions could be justified .
a more recent prospective cohort study in the netherlands,30 evaluating associations between physically demanding work and obstetric outcomes among 4,680 pregnant women , identified no significant effects from long periods of walking , standing , lifting greater that 25 kg , nightshifts , and long work hours on the occurrence of preterm birth .
many of these reviews make an important note : women that work physically demanding jobs tend to be healthier prior to conception and may therefore be at lower risk for adverse obstetric outcomes . due to a lack of empirical evidence based on randomized controlled trials , recommendations for work activity during pregnancy
several studies have investigated the effects of exercise during pregnancy on preterm delivery and adverse obstetric outcomes .
clapp found that labor began 5 days earlier among the exercise group ( 277 6 vs 282 6 days ) with no difference in the incidence of preterm delivery in a study of 131 recreational athletes who continued their exercise regimen in pregnancy compared to recreational athletes who discontinued exercise during pregnancy.6 a randomized controlled trial by barakat et al,7 which randomized 290 pregnant women to either exercise ( 5560 minute general fitness classes three times per week , weeks 810 to 3839 ) or not exercise , found no significant differences in gestational age at delivery between groups ( eg = 39.7 1.3 vs cg = 39.6 1.1 weeks , p = 0.81 ) .
there were no significant differences in preterm deliveries and in fact fewer preterm deliveries among the exercise group ( 4.3% ) relative to the control group ( 7.2% ) .
additionally , jukic et al8 reported lower incidence of preterm delivery among women who performed vigorous recreational activities during pregnancy .
since the risk factors for preterm birth are exclusion criteria for many studies , the true risk for preterm birth due to performing short - term or chronic exercise greater than 85% of their maximum oxygen uptake ( vo2 max ) is not well studied .
human and animal studies demonstrate potential concerns when the athlete goes beyond the ventilator threshold or to the point of exhaustion.3133 these concerns are transitory contractions and fetal heart rate deceleration .
the woman at high risk for preterm birth may be at higher risk for complications with strenuous exercise taken to exhaustion .
the american college of obstetricians and gynecologists recently released a committee opinion on physical activity and exercise during pregnancy recommending women with uncomplicated pregnancies engage in at least 150 minutes of moderate intensity aerobic exercise per week.5 the american college of sports medicine ( acsm ) defines moderate intensity as any activity equivalent to brisk walking.34 more specifically , the acsm recommends that intensity should be 50%85% of vo2 max with a target of 60%70% vo2 max for women who were sedentary prior to pregnancy and above 70% vo2 max for pregnant women wanting to maintain fitness.34 research has shown exercise during pregnancy helps control weight gain,3537 reduces the risk of gestational diabetes and preeclampsia in obese women,3842 and improves psychological well - being.43,44 absolute contraindications to aerobic exercise in pregnancy include incompetent cervix or cerclage , multiple gestation at risk of premature labor , premature labor during the current pregnancy , and ruptured membranes.5 it is important to note that a history of preterm delivery is not listed among the absolute or relative contraindications . recommended activities include walking , swimming , stationary cycling , aerobics , modified yoga and pilates , running or jogging , racquet sports , and strength training .
activities to avoid include contact sports , activities with high risk of falling or impact ( downhill skiing , horseback riding , water skiing , etc . ) , scuba diving , sky diving , hot yoga , and hot pilates.5 additionally , women should avoid exercise in the supine position after 20 weeks gestation to prevent hypotension.45 it is important that pregnant women stay well hydrated during exercise and rest when needed .
there is no gestational age limit on exercise , and women may continue to exercise until they deliver .
based on the literature available , women with a history of preterm delivery who are receiving regular prenatal care and indicated progesterone therapy should be encouraged to continue working with minimal restrictions and participate in 30 minutes of moderate intensity exercise most days of the week .
this recommendation would not apply to women with a short cervix ( less than 25 mm ) who undergo cerclage placement , as exercise is contraindicated in that population . for patients who are hospitalized for preterm labor , regardless of whether they have a history of preterm birth , aerobic exercise is not recommended and work activity may need to be restricted depending on the patient s occupation .
many obstetricians may recommend women in that situation stop working but should be cautioned against prescribing bed rest .
given the modest occupational risks on preterm labor , it is reasonable for women with a history of preterm birth to reduce their amount of time standing and heavy lifting at work . in women with history of a
medically indicated preterm birth , exercise in the current pregnancy may help prevent a recurrent medically indicated preterm delivery in certain circumstances , particularly in the setting of poorly controlled hypertension and diabetes .
additional research is needed in this particular population , and physicians need to follow current guidelines while tailoring recommendations to each individual patient , considering their history and current health status .
in the united states , and worldwide , preterm birth remains a leading cause of neonatal morbidity and mortality . while a history of preterm birth is one of the strongest risk factors,2 significant racial disparities exist with a preterm birth rate of 16.27% among non - hispanic black mothers compared to 10.17% among non - hispanic white mothers.1 a variety of other disparities
exist , including an increased risk of preterm birth among low socioeconomic status , low education levels , residence in disadvantaged areas , and lack of access / late access to prenatal care.4 as continued research demonstrates the benefits of exercise during pregnancy , specific recommendations for high - risk populations need to be addressed .
historically , these women were placed on bed rest or a reduced activity regimen during pregnancy , despite a lack of evidence supporting this practice .
recent studies have demonstrated regular physical activity and exercise during pregnancy is safe and does not increase the risk of preterm delivery.58 women with a history of preterm delivery who are receiving regular prenatal care should be encouraged to continue working with minimal restrictions and participate in 30 minutes of moderate intensity exercise most days of the week . | preterm birth remains a leading cause of neonatal morbidity and mortality throughout the world .
numerous risk factors for preterm birth have been identified , including non - hispanic black race , a variety of social and behavioral factors , infections , and history of a prior preterm delivery . of these ,
a history of prior spontaneous preterm birth is one of the strongest risk factors .
traditionally , women with a history of preterm birth or those deemed at high risk for preterm delivery have been placed on bed rest or a reduced activity regimen during their pregnancy . however , there is little evidence to support this recommendation .
recent research has suggested that regular physical activity and exercise during pregnancy is safe and does not increase the risk of preterm delivery .
therefore , physicians should encourage women with a history of preterm birth to exercise throughout pregnancy according to guidelines published by the american college of obstetricians and gynecologists as long as they are receiving regular prenatal care and their current health status permits exercise
. however , there are no randomized controlled trials evaluating exercise prescription in women with a history of preterm birth , hence additional research is needed in this area . | Definition and Frequency
Prediction and Prevention
Activity Reduction and Bed Rest
Exercise and Risk of Preterm Birth
Current Recommendations
Conclusion |
PMC3675284 | retaining coronal restoration in endodontically treated teeth usually requires the use of intra - canal posts.1 - 6 although high success rates have been reported for fiber - reinforced composite ( frc ) post restorations,7,8 root fracture and loss of retention are still the most common reasons for failing these restorations.9,10 due to the similar elastic modulus of fiber posts and root dentin , distribution of loads in the root canal has an uniform pattern.11,12 so , less occurrence of root fracture has been reported for these restorations in comparison to metallic post retained restorations.7,8 the fiber post retention in root canal depends on the bond strength between different parts of the combined ' sandwich ' post - cement - dentin assembly.13 - 16 it has estimated that 60% of the fiber post failures occurred between the fiber post and resin cement.17,18 so , achieving effective bond strength between the frc post and resin cement is important.4,19 many post surface treatment protocols ( chemical and micro - mechanical modifications ) have been reported to improve surface energy characteristics of dental fiber reinforced posts.6,16,20 - 24 the bond strengths between post and resin cement can be improved chemically by post surface silanization.9,22,25,26 however , there is no definitive agreement on this subject.24,27 on the other hand , some studies concluded that roughening the post surface either with micro - mechanical methods like sandblasting20,24,28 - 34 or application of hydrogen peroxide35 - 37 can improve the retention of frc posts that are cemented with resin cements .
however , the effectiveness of these roughening techniques is not completely confirmed.6,27,28 since chemical surface treatments alone may not create a strong bond between fiber posts and resin cements,21 some micro - mechanical treatment methods have also been performed to remove a surface layer of fiber posts and exposes the quartz fibers for silanization.38 this type of surface treatments that combine both a chemical and micromechanical method is called as alternative etching techniques.21 although , sandblasting and hydrofluoric acid etching are used to improve the bonding of fiber posts to resin cement,18,24,27 these techniques can damage the glass fibers and affect the post integrity.18 hydrogen peroxide ( h2o2 ) is one of the materials that can selectively dissolve the epoxy matrix without interfering with the glass fibers and can expose the fibers to be silanated.6,20,39,40 the purpose of this study was to investigate the effects of some post surface conditioning methods before silanization on the bond strength between fiber post and resin cement .
the null hypothesis to be investigated was that the bond strength between fiber post and resin cement with the various surface conditioning methods are not significantly different .
in this in vitro study , 40 human maxillary incisor single - rooted teeth free of caries , cracks , fractures , resorption , with fully developed roots and without previous root canal treatments or posts were selected .
the teeth were cleaned of residues of soft tissue or calculus and disinfected by placing them in 2.5% naocl ( golrang , golrang co. , tehran , iran ) for two hours and then stored in 0.1% nan3 solution .
diamond discs ( ref.070 , d&z , berlin , germany ) were mounted in a dental lathe machine ( kavo polishing unit .
this job was done at low speed under water irrigation to achieve a 15 mm root length .
the pulp tissues were removed with barbed broach ( dentsply / maillefer , ballaigues , switzerland ) .
themaster apical file was the number 35 k file ( dentsply / maillefer , switzerland ) .
canals were irrigated with 2 ml of 5.25% solution of sodium hypochlorite during the canal preparation stages ( golrang , iran ) .
finally , paper points ( aria dent , asia chemiteb co , tehran , iran ) were used for drying the root canals .
the obturation was performed with vertical condensation method , using ah26 ( dentsply caulk , milford , germany ) and gutta - percha ( aria dent , iran ) . a provisional restorative material ( gc caviton ; gc dental products corp . ,
tokyo , japan ) was used for filling the coronal root canal orifices , and then to ensuring the setting of the used sealer , the teeth were stored for one week in 100% humidity at 37. after one week , the gutta - percha in the coronal aspect of each root was removed with a gates glidden drill # 3 ( dentsply / maillefer , switzerland ) so that , 4 mm gutta - percha was preserved in the apex to maintain the apical seal .
the all post spaces were prepared to a depth of 10 mm , measured from top of the sectioned root surface , with dedicated drills provided by a post manufacturer ( fibio , anthogyr , sallanches , france ) . to standardize the final shape of the post space , a no .
3 drill of this system was used as the last drill . finally , the canals were irrigated with distilled water and dried with paper points ( aria dent , iran ) . a glass reinforced fiber post ( hetco fiber post , hakim toos , mashhad , iran ) ( table 1 ) size # 3
was tried in the post spaces and then all posts were cut at a distance of 10 mm from the apical end with diamond discs ( ref .
this procedure was done for post length standardization and creation diameter similarity among the used posts.19 in all groups , the shortened hetcoposts ( hakim toos , iran ) were cleaned with 70% ethanol for 60 seconds , rinsed with distilled water and air dried .
the posts were randomly divided into four groups of 10 specimens each , according to surface treatments used : h2o2 + silane group . at the first , the 30% h2o2
were diluted to 20% h2o2 and then the posts in this group were immersed in diluted h2o2 for 20 minutes at room temperature . after 2 minutes rinsing with running water , they were air dried .
then the posts surfaces were painted with a single layer of a silane coupling agent ( ultradent porcelain etch and silane , ultradent products inc .
, ut , usa ) for 60 seconds and dried for 60 seconds with gentle air stream .
the posts in this group were sandblasted with 50 m aluminum oxide particles using an oral microblaster ( dento - prep , ronviga , daugaord , denmark ) for 10 seconds at 0.25 mpa pressure from a 10 mm distance . after rinsing posts with 96% ethanol
the posts surfaces were painted with a single layer of thesilane coupling agent ( ultradent porcelain etch and silane ) and were air dried for 60 seconds with gentle air stream .
only a single layer of thesilane coupling agent ( ultradent porcelain etch and silane ) was applied on the posts surfaces for 60 seconds and then air dried for 60 seconds with gentle air stream . control group .
the root canal dentin walls were conditioned with an autopolymerizing primer ( ed primer , kuraray medical inc . ,
one drop of each liquid ( a and b ) ( ed primer ) were mixed and applied to the root canal walls with a microbrush ( atlas microbrush ; atlas dandan co. , tehran , iran ) for 60 seconds .
then , the post space was air dried and the excess primer was removed with paper points ( aria dent , iran ) . for fiber post
cementation , equal amounts of base and catalyst pastes of an adhesive composite resin cement ( panavia f2.0 , kuraray medical inc . ,
japan ) were mixed and applied on the posts surface and into the root canals with a lentulo spiral instrument ( dentsply / maillefer , switzerland ) .
using gentle finger pressure , the fiber posts were inserted into the prepared post spaces to full depth .
finally , the remaining cement around the post was protected with oxygen inhibiting gel ( oxyguard ii , kuraray medical inc . ) . after the initial chemical polymerization , the resin cement was light cured for 60 seconds in such a way that the tip of the light unit ( coltolux50 , coltene , altstatten , switzweland ) was in close contact with the coronal end of the fiber post .
accurate light intensity of the light output was monitored before each light exposure by coltolux light meter ( coltene , altstatten , switzerland ) .
after the cementation procedures , all dental root specimens were placed in a light - proof box , containing 37 sterile saline for one week .
all the specimens were subjected to thermocycling treatment for 5,000 cycles at temperatures alternating between 5 and 55 for 30 seconds each , with an intermediate pause of 15 seconds .
the coronal part of each dental root was sectioned perpendicular toits long axis using a diamond disc ( ref.070 , d&z , germany ) mounted in a cutting machine ( tl-3000 , vafaeiindustrial , tehran , iran ) at low speed under constant distilled water irrigation to create 3 mm thick slices . in this manner ,
threepost / dentinsections ( coronal , middle and apical ) were obtained from each root . due to usingtaperedfiber posts , the post diameters were measuredusingadigital caliper ( mitutoyodigital caliper 500 - 714 - 10 , mitutoyo co. , tokyo , japan ) with 0.01 mm accuracy .
a universal testing machine ( walt + bai ag testing machines industriestrass 4 , lhningen , switzerland ) was used for applying tensile force at acrosshead speed of 1 mm / min .
the push - out pin was placed on the center of the apical end of the post surface and in an apico - coronal direction without inserting extra forces on the surrounding root canal walls .
therefore , three push - out pins were constructed in three diameters ( 0.7 , 0.9 and 1.0 mm ) that were used for each three root sections respectively ( apical , middle and coronal parts ) .
the peak force ( n ) required to extrude the fiber post from each root slice was recorded for all specimens . the bond strength in mpa
was calculated by dividing the load at failure ( n ) by the total area of the cemented post .
this calculation was done with the following formula : in this formula , is the constant 3.14 , r1 is the coronal post radius , r2 is the apical post radius and h is the slice thickness in mm .
the collected data were analyzed ( spss / pc16.0 ; spssinc . , chicago , il , usa ) using two - way anova and post hoc tukeytest at p<.05 level of significance .
table 2 shows the mean tensile bond strength ( tbs ) and standard deviation values for all experimental groups in different root canal regions .
the two - way anova showed that different surface treatments ( p=.004 ) and root canal dentin regions ( p<.001 ) had significant effects on tbs , but interaction between surface treatments and root canal regions ( p=.068 ) had no significant effects on tbs ( table 3 ) .
the h2o2 + silane group had the highest tbs mean value especially in the coronal region .
the lowest tbs mean value was seen in the control group and in the apical region .
post hoc tukeytest revealed that there was significant differences between h2o2 + silane group and other three groups ( p<.05 ) ( table 4 ) .
based on the results of this investigation , the null hypothesis that the various surface treatments do not have any effects on the tbs between fiber post and resin cement was rejected .
fiber posts are comprised of a matrix resin that surrounds different types of fibers . in the glass fiber posts , the fibers are made of glass and the matrix is made of epoxy resin.5 the epoxy polymers can not chemically bond with composite resin cements because of their highly cross - linked structure.41 therefore , some authors propose using silane coupling agent on the fiber post surface to increase the tbs between the fiber post and resin cement . however , it has been said that silane can not increase the bond strength of the fiber posts to resin cements or composite core materials.17,32,42 - 45 in fact this is due to this fact that the chemical bond is achieved mainly by creating covalent bonds between the silane coupling agent and the composite resin ; and between silane and the exposed glass fibers or filler particles of the post.46,47 in the present study , likes some other investigations , it was found that silane alone can not increase the tbs of the fiber posts to resin cements ( table 4 ) .
roughening the fiber post surface with micro - mechanical procedures can bring the glass fibers in better contact with silane coupling agent.6,20,40 although , sandblasting is used to roughen the fiber posts surface for better bonding , 18,24,27 also it can damage the glass fibers.18 instead , hydrogen peroxide ( h2o2 ) can selectively dissolve the epoxy matrix without damaging the glass fibers.6,20,39,40 the present study showed that using h2o2 before silanization can significantly improve the tbs in comparison to sandblast + silane , silane and control groups ( p<.05 ) ( fig .
1 ) , which is in accordance with some other studies.18,24,27 yet , radovic et al.48 showed that even if sandblasting can improve the bond strength between fiber post and resin cement but water aging can reduce the bond strength of sandblasted specimens .
it is said that by using h2o2 as a pretreatment for using silane coupling agent , a better chemical bonding between silane and fiber post was achieved.32,36,40,43,47 in the studies that bonding agent was applied immediately after using h2o2 ( without silane coupling agent as a mediator ) , the tbs mean value was reduced.1 however , some authors claimed that pre - treatments are not necessary before silane application.49 for root canal regions , our results are in accordance with other studies that reported higher bond strength for coronal region of the root than other tworegions.9,13,19 the tubular density and diameter of the coronal third of the root canal dentin are more than other regions .
furthermore , the dentinal hybridization is not uniform and lateral branches of resin tags are not seen in the apical regions of root canal dentin.50 furthermore , more access to the coronal portion of the canal makes etching and applying the adhesive agents17,50 and light transmission to canal walls50 become more effective in this area .
however , aksornmuang et al.9 found no significant differences in the bond strengths among different regions of the root canal walls .
for example , the specimens had no coronal tooth structure , only one type of fiber post and adhesive were evaluated and the influences of fatigue loading on the push out tbs of specimens were not investigated .
within the limitations of this in vitro study , the following conclusions were drawn : there were significant differences between the mean tensile bond strength values of different surface treatments and root canal regions .
application of hydrogen peroxide before silanization increased the bond strength of resin cement to the fiber posts .
the coronal region of the root canal showed significantly higher mean bond strength values than the other regions . | purposepost surface conditioning is necessary to expose the glass fibers to enable bonding between fiber post and resin cement .
the purpose of the present study was to evaluate the effect of different surface conditioning on tensile bond strength ( tbs ) of a glass fiber reinforced post to resin cement.materials and methodsin this in vitro study , 40 extracted single canal central incisors were endodontically treated and post spaces were prepared .
the teeth were divided into four groups according to the methods of post surface treatment ( n=10 ) : 1 ) silanization after etching with 20% h2o2 , 2 ) silanization after airborne - particle abrasion , 3 ) silanization , and 4 ) no conditioning ( control ) .
adhesive resin cement ( panavia f 2.0 ) was used for cementation of the fiber posts to the root canal dentin .
three slices of 3 mm thick were obtained from each root .
a universal testing machine was used with a cross - head speed of 1 mm / minute for performing the push - out tests .
two - way anova and tukey post hoc tests were used for analyzing data ( =0.05).resultsit is revealed that different surface treatments and root dentin regions had significant effects on tbs , but the interaction between surface treatments and root canal regions had no significant effect on tbs .
there was significant difference among h2o2 + silane group and other three groups.conclusionthere were significant differences among the mean tbs values of different surface treatments .
application of hydrogen peroxide before silanization increased the bond strength between resin cements and fiber posts .
the mean tbs mean values was significantly greater in the coronal region of root canal than the middle and apical thirds . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC4590906 | current estimates suggest approximately 35 million youth between the ages of 518 years participate in organized sports each year .
while a majority of these young athletes are playing sports for the aspects of comradery and fun , a growing segment of young athletes train to enhance their opportunity to make a career of sport . while elite sport has long seen the presence of young athletes ( nadia comaneci , 14 years of age ( 1976 olympic gold medalist ) , marjorie gestring , 13 years of age ( 1936 olympic gold medalist ) , and dimitrios loundras , 10 years of age ( 1896 olympic bronze medalist ) ) , the past few decades have experienced an expansion in the numbers of young athletes working to perform at higher levels as younger athletes .
this expansion can be seen in the establishment of the many facilities open focusing specialized training for sports performance on not only elite athletes but also largely youth athletes .
while the increase in physical activity of youth is important we currently do not fully understand the effects such training has on the growth and development of youth .
the american academy of pediatrics outlined potential risks associated with sports specialization in young athletes in a publication in 2000 .
james andrews , recently discussed the potential for negative effects of specialized training on developing bodies and the rise in youth sport injuries he experienced since around the same 2000 timeframe .
it is not within the scope of this review to discuss the ethical considerations of having youth focus their training on a singular sport , nor to discuss the potential for injury as related to overuse injuries .
however , with the continual trend in younger athletes training for high level performance it appears that our current options are to continue to underscore the potential risks while at the same time work with the participants providing as much assistance as possible to enhance safety .
proper nutrition ensures that an individual is amassing the fuels necessary for the energy production needs related to activity and recovery .
one of the areas needing to be addressed is the unique nutritional needs associated with intense exercise stress .
however , our understanding of the effects of strenuous physiological training and nutritional variations in combination with exercise stress in youth athletes is greatly limited .
this limited knowledge is most likely due to the ethical considerations of withholding nutrients and physiologically overstressing a vulnerable population such as children and adolescents still in the process of growth and development .
our knowledge regarding the nutritional needs of youth is based on the needs related to proper growth and development in healthy children or those suffering from illness .
most of the knowledge we possess related to the physiological adaptations to training , exercise performance , and sports nutrition is based on research conducted in college aged , middle aged , and older adult populations .
therefore , most sports nutrition recommendations promoted in youth sport are actually based on findings in adult populations . while this is a starting point ,
research has shown that adolescent energy expenditure and metabolism can differ from those of their adult counterparts so many of these recommendations may not provide ideal insight into the nutritional needs of the youth athlete [ 46 ] .
the goal of this review is to compile an overview of our understanding of the nutritional needs of the young athlete during training and competition .
we will also identify the knowledge gaps that currently exist in our understanding of this vulnerable population 's needs around physiologically stressing occasions .
due to the limited research on the young athlete population , in many instances the knowledge gained through research on adult populations is the only means to provide recommendations for the young athlete .
nutrition for healthy growth and maturation is governed by a variety of parameters , each essential in the development from child to adult .
this paper emphasizes the importance of adolescent nutrition by first examining gross total caloric intake to better understand the energy requirements of adolescents .
total caloric intake must be sufficient to meet the additional demands of growth , which vary at different stages of growth and maturation and between individual children .
likewise , the proportion of calories allocated to each macronutrient is heavily dependent on the situational constraints of the individual child , which is further complicated by the physiological constraints of a given level of development .
this paper emphasizes the importance of each macronutrient with specific focus on the physiological nuances of adolescent metabolism specifically focused around the young athlete .
similarly , micronutrient needs are driven by demands of growth and maturation as well as activity levels .
unique demands of the growing adolescent have highlighted a few select micronutrients in the literature which will be reviewed here following a general overview of micronutrient needs .
growth , maturation , and development are three constructs paramount in any discussion regarding youth . while these terms often manifest concurrently in youth , they refer to three different parameters .
growth simply refers to the quantifiable increase in size , whereas maturation refers to timing and tempo of progress toward the mature state .
timing and tempo refer to the age at which specific maturational events occur and rate at which an individual progresses through these events .
behaviors and attitudes developed during childhood and adolescence provide the basis for adult behaviors and attitudes .
refinement of accepted behaviors in a society requires the development of competencies in an array of interrelated domains that ultimately shape a given behavior and attitude toward that behavior .
taken together , growth , maturation , and development synergistically influence an individual 's general self - concept and self - esteem .
this holistic perspective is often overlooked when focused on specific pediatric topics , such as nutrition .
much like business , calorie supply ( i.e. , energy intake ) is dictated by demand ( i.e. , energy expenditure ) .
energy expenditure is represented by four major components in children and adolescents : basal / resting metabolic rate , thermic effect of food , thermic effect of activity , and the energy requirements of growth .
basal and resting metabolic rates ( bmr and rmr , resp . ) vary chiefly on assessment methodology , but only marginally in amount of calories . for the purposes of this discussion
however , when rmr is examined per unit of body mass , rmr decreases as children progress to their adult size , which demonstrates the contribution of growth to rmr in children and adolescents .
the thermic effect of food varies significantly by the proportion of macronutrients comprising the food consumed . on average ,
68% of ingested calories are used in the digestive , absorptive , and storage processes of food .
thermic effect of activity is the most variable component of energy expenditure and refers to the calorie cost of movement .
when estimating caloric requirements , activity levels are examined at three levels : light , moderate , and vigorous lifestyle physical activity . given the significant participation in high energy demanding activities ,
the energy cost of growth is examined in two parameters , the energy to synthesize tissue and the energy deposited in those tissues .
growth varies according to the tempo of maturational development and is very rapid during infancy and early childhood and , thus , accounts for a greater proportion of caloric expenditure .
conversely , during late childhood and adolescence , growth accounts for 1 - 2% , which reflects a slower rate of growth .
with consideration to each of these four components , the fao / who / unu expert panel used typical weight gains per year to develop age specific and gender specific caloric recommendations .
table 1 shows the caloric recommendations for boys and girls participating in vigorous lifestyles physical activity .
daily energy requirements increase with age and are similar between boys and girls until pubertal ages .
protein is needed for normal cellular functioning as well as synthesis of various bodily tissues .
athletes tend to have elevated demands for dietary protein intake compared to sedentary individuals . as a general recommendation for maintaining health ,
current recommendations are between 0.8 and 1.2 grams of protein per kg of body mass daily .
this recommendation is sufficient to meet the bodily demands of 97.5% of the population , which also accounts for variations in demographic bmi as well as gender .
a review by nemet and eliakim speculates these requirements are likely sufficient for children and youth athletes .
the american college of sports medicine and american dietetic association recommend intakes between 1.2 and 1.8 g / kg of body mass for active adults [ 14 , 15 ] , which appears to be an adequate requirement for youth athletes [ 16 , 17 ] .
protein synthesis is highest during infancy and , as such , during this time relative dietary protein intake is at an elevated demand .
the question of how much dietary protein is needed to maximize performance among athletes is a question that has been debated for more than 150 years [ 11 , 18 ] and still remains a debate .
recent evidence suggests two to three times the rda for protein intake may be optimal to enhance fat - free mass during periods of caloric restriction which may be commonly practiced among athletic populations to achieve a body composition more favorable for performance .
investigation of dietary intakes for various youth age groups suggests that intakes this high are often achieved in normal dietary patterns , which indicates intake is sufficient to meet the elevated demands .
many athletes make dietary modifications in attempt to maximize performance and meet body weight requirements for competitive classes .
several studies have shown increased dietary protein intake accompanied by exercise intervention may aid in weight loss as well as preservation of lean body mass typically associated with reduced body weight [ 2225 ] .
some suggest the mechanism may be partially attributed to increased thermogenesis and satiety associated with elevated protein intake . when compared to fat and carbohydrate , protein has a greater thermic effect that is likely only significant enough to result in weight loss when the high protein diet is maintained over the course of several months . additional research is needed to fully investigate this hypothesis .
several studies have demonstrated satiating effects of high protein diets [ 2729 ] , which may better elucidate a mechanism of weight loss with this dietary intervention . branched chain amino acids found in protein - rich foods
are known to assist in preservation of lean body mass , which has significant implications for athletes particularly during periods of weight loss .
leucine specifically is one branched chain amino acid that is strongly associated with protein synthesis .
this amino acid can be ingested in supplement form ; however , when determining the safe tolerable upper intake level for leucine intake , trials in youth are limited .
one study suggests the upper level for young males aged 2035 years is 500 mg / kg / day or 35 g / day based on plasma and urinary ammonia and leucine concentrations .
this recommendation has not been examined in youth and caution of leucine at these high levels is warranted .
however , food sources such as egg whites and dairy sources contain multiple amino acids and , as such , protein - rich foods should be emphasized to a greater extent than single amino acids alone .
the most significant question is whether or not youth athletes are obtaining the amount of protein needed for their elevated demands .
it has been documented that youth athletes in general are achieving protein intakes much greater than the rda . given this evidence , it is unlikely beneficial to promote increased protein intake in youth .
however , as a general recommendation , athletes should ingest balanced protein feedings throughout the day and emphasize whole foods as opposed to protein based supplements due to the lack of scientific support for protein based supplements in comparison to protein - rich whole foods .
additionally , research has shown the ingestion of 20 g of protein following exercise helps maintain positive protein balance following exercise .
nonetheless , protein supplements remain one of the most common dietary supplements purchased by athletes who seek to increase markers of performance such as speed , strength , power , and hypertrophy .
several reports have documented athletes ' perception that protein supplements are necessary to build muscle [ 37 , 38 ] and achieve peak performance .
this notion has been well investigated in adults but also appears true in the limited research regarding youth athletes , specifically high school football players .
this misconception among youth is partially driven by the lack of formal knowledge in nutrition .
youth athletes gain a significant amount of nutrition information from magazines , family members , and coaches and , thus , may not be able to make appropriate , evidence based decisions regarding the use of protein supplements . considering the lack of scientific support for protein based supplements being superior to natural protein containing foods ,
youth athletes should be advised to consume their protein from whole foods as opposed to supplements .
dietary lipids are essential for the absorption of vitamins a , d , e , and k , as well as synthesis of cholesterol and other sex hormones . in terms of caloric requirements ,
most sources recommend lipid intake should be limited to 2530% of total caloric intake , which is relatively the same for both sedentary and active individuals .
it is important to consider caloric demands are increased in athletic populations ; therefore , absolute lipid intakes are likely to be higher .
it is important to restrict lipid intake to avoid excessive caloric intake ; however , there is no health benefit in diets with less than 15% of calories from lipids . in terms of athletic requirements ,
an increase of dietary carbohydrate should account for a majority of the increased caloric demands , rather than an increase of dietary lipid .
adequate calorie consumption to support periods of rapid growth is of greatest concern when considering nutrition to maximize performance of adolescent athletes .
roughly fifty percent of adult body mass as well as skeletal mass is achieved during puberty .
large increases in lean and adipose tissue are also seen in males and females during the transition from child to adult as well . during this time
, dietary fat is especially important to aid in the synthesis of hormones and assist in normal bodily functioning as well as healthy growth and maturation .
dietary lipid intakes beyond 30% are not advised since this could contribute to excessive weight gain .
however , acutely , excessive lipid intake can also result in postprandial oxidative stress , which is associated with impaired vascular and metabolic functioning [ 48 , 49 ] .
elevated lipid intake is also potentially associated with the pathogenesis of cardiovascular disease which is particularly relevant for youth athletes , given that the origins of cvd begin at an early age and progress into adulthood [ 48 , 50 , 51 ] .
the organized group setting provides an ideal platform for discussion of nutrition and physical activity habits among individuals who already acknowledge their value .
adolescents are more efficient in terms of substrate utilization , which has been shown both at rest and during graded exercise tests since younger children derive a higher percentage of energy from lipids as indicated by lower rer values at submaximal intensities .
improved aerobic efficiency is related to increased dependency on lipids for atp production commonly noted in youth .
this could potentially be the result of an adaptive response since infants and toddlers ( under the age of 2 ) require a higher percentage of energy from lipids to support their increased caloric and growth demands .
alterations in dietary lipid intake could contribute to changes of enzymatic activity as well as elevated lipid metabolism .
a lack of glycolytic enzyme activity could be another reason for the aforementioned increased dependency on lipid metabolism . during exercise ,
carbohydrates and lipids are the main sources of skeletal muscle atp production , with lipids serving as an important source of energy during low and moderate intensity .
chronic exercise training results in favorable mitochondrial adaptations in adults , which favor enhanced lipid metabolism as well . upon investigation of differences in lipid oxidation among different age and gender groups in children , a review by aucouturier et al . reported only miniscule differences between age groups among male and female adolescents .
these miniscule differences are likely associated with a change in body size ( e.g. , acquisition of skeletal and muscle mass ) during periods of growth and maturation among different age groups and are more significant in males compared to females .
however , aucouturier et al . reported all children ( in general ) depend more readily on lipids in comparison to adults .
this metabolic characteristic could depend on pubertal status , since it has been shown that 12-year - old females demonstrate elevated lipid metabolism during exercise performed at 70% vo2 max compared to 14-year - old females .
similar findings have also been reported in boys aged 12 and 14 . however , to our knowledge there is little to no evidence showing prepubescent males and females differ significantly in terms of relative fat and carbohydrate oxidation during submaximal exercise . in comparison to adults , however , children lack the ability to sustain longer duration exercise , which may be related to a lack of the ability to store glycogen in children . generalizing substrate utilization during prolonged exercise is difficult given the paucity of experimental or quasi - experimental evidence which examines exercise testing in children for greater than one hour in duration , which is likely reflective of the general short and intermittent physical activity patterns and behaviors of that age group .
the composition of dietary fatty acids ( e.g. , chain length ) can affect fat oxidation during submaximal exercise .
however , this response may vary among maturational levels of the young athlete since prepubescent males tend to have a higher percentage of fatty acid oxidation .
it is also important to consider the potential adaptations resulting from modifications of lipid intake .
short term elevations in lipid intake are likely to result in positive energy balance which may not be immediately matched with an increase in beta - oxidation .
however , trials in adult populations show that exercise can enhance lipid metabolism by stimulating mitochondrial biogenesis as well as increasing activity of lipoprotein lipase and carnitine palmitoyltransferase 1
. the aforementioned adaptations can enhance lipid metabolism and contribute to an accommodated energy balance due to changes in fat metabolism .
this evidence is especially applicable to athletes since exercise training has been shown to accommodate the metabolic effects of short term high fat diets .
these adaptations , however , still do not constitute promotion of lipid intakes greater than 30% in youth athletes . as mentioned earlier , factors such as the type of lipid ingested ( i.e. , composition of the hydrocarbon chain ) can affect the subsequent metabolism and potential storage [ 65 , 66 ] . piers et al
. demonstrated the substitution of saturated fat with monounsaturated fatty acids can potentially have a favorable effect on body composition .
the contribution of saturated fatty acid intake to the development of cvd is important ; however , a meta - analysis by siri - tarino et al . included twenty - one studies and reported no significant risk of coronary heart disease with elevated saturated fatty acid intake .
it is important to note coronary heart disease is a chronic , progressive disease , the origins of which begin early in life .
thus , youth require appropriate dietary advice that may potentially become healthy behaviors in adulthood as a means of preventing or reducing disease risk .
further , these findings may not be applicable to all individuals since intensity of activity and lipid composition both affect lipid metabolism . considering the evidence to date , dietary unsaturated fatty acids
should not serve as exclusively the sole source of lipids . however , as a general recommendation , and in the interest of promoting long - term healthy behaviors , unsaturated fatty acids should be emphasized to a greater extent .
some athletes may believe certain lipid based supplements may allow for an ergogenic effect given the limited findings suggesting supplementation may enhance lipid metabolism by decreasing dependency on glycogen / glucose for energy metabolism .
commercial marketing is based on the premise some lipid based dietary supplements can thereby increase aerobic capacity and performance , improve lipid metabolism , and reduce inflammatory damage .
fish oil and conjugated linoleic acid ( cla ) are two lipid based supplements that have been investigated in relation to potential ergogenic effects .
fish oil contains two essential fatty acids ( eicosapentaenoic acid [ epa ] and docosahexaenoic acid [ dha ] ) .
increased dietary intake of these essential fatty acids has been associated with decreased prevalence of cardiovascular diseases [ 71 , 72 ] , as well as reduced markers of inflammation [ 73 , 74 ] . in regard to physical performance , a variety of trials failed to demonstrate an ergogenic effect of epa and dha [ 7577 ] ; tartibian et al .
reported improved pulmonary functioning in young wrestlers . in terms of the ability to improve athletic performance ,
the majority of data demonstrate a lack in ergogenic effect of fish oil ingestion on athletic performance in well trained athletes [ 76 , 79 , 80 ] .
the overwhelming scientific support for fish oil supplementation highlights improvements in cardiovascular health and decreases markers of inflammation which could contribute to decreased recovery time between exercises as speculated by macaluso et al . .
furthermore , the majority of clinical trials utilize extremely high doses ( > 3 g / day ) [ 69 , 77 , 81 ] , which is difficult to achieve without dietary supplementation of fish oil .
other benefits of fish oil consumption have been noted including improved cognitive function and reduced adhd symptoms in children .
a meta - analysis by yang et al . indicated dietary fish and/or fish oil supplementation is also associated with a reduced prevalence of asthma in children .
however , additional trials in youth ( specifically with athletic samples ) are warranted as this population has not been investigated to our knowledge .
given the gap in the literature examining youth athletes , clear recommendations for fish oil consumption can not be made and warrant further investigation .
cla is another lipid based dietary supplement that has been proposed to improve athletic performance .
cla is found naturally occurring in beef , lamb , and dairy products such as milk and cheese but is also available in supplement form .
animal studies utilizing cla administration have demonstrated potentially favorable effects on body composition . however , this may not be applicable to humans since zambell et al . failed to show a change in energy expenditure and lipid metabolism . a recent review by macaluso et al
. reported fish oil and cla supplementation can potentially have a favorable effect on anabolic effects of exercise which could be related to increased testosterone synthesis .
however , given the strong relationship between growth , maturation , and anabolic hormone levels in youth , the demands for youth athletes to intentionally manipulate hormone levels are not advised .
human metabolism relies primarily on the oxidation of fats and carbohydrates as its fuel sources . as physiological intensity increases from rest to vigorous
there is a transition from fat functioning as the primary fuel source to carbohydrate supplying a majority of the body 's fuel for energy .
the sources of carbohydrate for metabolism are glycogen stores in the muscle , glycogen stores in the liver , and exogenous carbohydrate entering the blood stream through the ingestion of carbohydrate .
some confusion exists in athletes understanding of the specific carbohydrate needs and it has been suggested by burke et al . to stem from the fact that many recommendations are based on percentage of total caloric intake which adds to the difficulty in understanding the dietary needs of carbohydrate in athletes that have caloric intakes which often exceed general recommendations .
general carbohydrate intake recommendations suggest adult athletes consume 512 grams of carbohydrate per kilogram per day dependent on their primary form of exercise / activity , activity intensity , sex , and environmental conditions .
the great variance that exists in the demands of sports , training , and level of play make it difficult to provide a single concise recommendation . as training duration and intensities
the recommendations for young athletes suggest at least 50% of young athletes diet should be in the form of carbohydrate or between 3 and 8 grams of carbohydrate per kilogram of body mass dependent primarily on exercise intensity .
early research into carbohydrate 's role in exercise performance examined the effect of blood glucose levels and physiological state following prolonged exercise [ 91 , 92 ] .
additional research investigated muscle glycogen 's role in muscle fatigue [ 93 , 94 ] .
subsequent research investigated the role of carbohydrate ingestion following exercise to restore muscle glycogen stores following the depletion related to exercise stress . more recently
the research focus has shifted to explore the role of carbohydrate ingestion during exercise stress in sustaining exercise intensity and improve performance [ 9698 ] .
it is commonly suggested that normal body stores of carbohydrate can be a significant fuel source for approximately 90120 minutes of moderate to vigorous exercise .
while this statement is accurate in many exercise settings where individuals are exercising at moderate - vigorous intensities typically associated with endurance exercise , more detailed investigation would demonstrate exercise intensity is a vital component in understanding glycogen depletion rates .
significant glycogen depletion can occur anywhere from ~10 minutes with supramaximal intensities to greater than 3 hours at low exercise intensities .
this oversimplification needs to be considered as we look at the findings from carbohydrate research .
many of the research investigations providing our understanding of carbohydrate needs around exercise are based on endurance type exercise . as we consider the sports typically involving young athletes some of our understanding may not fully translate . as stated previously part of our limited understanding in the nutritional needs of young athletes is the result of proper research ethics .
the research described above with adults involved muscle biopsies , exercise to failure , and exercise resulting in poor physiological states .
therefore most of our understanding of youth athletes comes from the utilization of less invasive techniques .
young athletes have been shown to have a lower respiratory exchange ratio ( rer ) during exercise at similar relative submaximal intensities ( % vo2 max ) as their adult counterparts .
based on the rer changes resulting from the shift from fat as a primary fuel source to increasing carbohydrate utilization , this would suggest young athletes are better able to utilize fat as a fuel or are potentially limited in their maximal performance as a result of not being able to utilize carbohydrate readily enough at higher intensities .
research has shown that increasing glycogen stores will enhance exercise performance and reductions in muscle glycogen content correspond with increasing levels of fatigue .
unfortunately , young athletes have been shown to store less glycogen than adults . during prolonged exercise and exercise at elevated intensities reduced glycogen levels
will lead to early onsets of fatigue . due to their lower glycogen stores , young athletes will likely experience accelerated rates of fatigue .
this accelerated fatigue is a result of the inability of the body to maintain sufficient blood glucose levels to meet the young athletes elevated glucose needs of the brain as compared to adults . with reduced muscle glycogen stores
the need for exogenous sources of carbohydrate becomes increasingly more important in the maintenance of exercise intensity .
much of our understanding regarding the role of carbohydrate ingestion in exercising youth is the result of the work of the researchers in the children 's exercise and nutrition centre at mcmaster university .
riddell demonstrated adolescent athletes utilized lower absolute amounts of exogenous glucose as compared to values reported in adults .
however , exogenous carbohydrate utilization has been demonstrated as a greater relative contributor to total energy utilization in the young athlete even with lower absolute utilization rates [ 58 , 101 ] .
current research foci have shifted more towards the improvement of health and wellness as opposed to performance
. however recent research continues to demonstrate the ergogenic effects of carbohydrate ingestion on youth sport .
dougherty et al . demonstrated performance improvements with basketball skills test with carbohydrate compared to water ingestion .
batatinha et al . demonstrated gymnasts experienced a reduction in the number of falls from a balance beam with carbohydrate ingestion during a session .
while the need for carbohydrate is recognized as important , the differences between youth and adult are still not fully understood .
research has shown carbohydrate ingestion spares endogenous carbohydrate stores in exercising youth while at the same time youth seem to be unable to utilize carbohydrate at rates similar to those seen in adults .
research in adults has led to the establishment of guidelines suggesting carbohydrate ingestion during activities lasting 45 minutes or longer provides an ergogenic effect with doses varying from small amounts including mouth rinse up to 90 g / h .
currently , specific recommendations similar to what exists for adults are not available for the youth athlete .
for performance enhancement young athletes will benefit from the ingestion of carbohydrate during exercise . without specific rates recommended for the young athletes , we must rely on the recommendations of adults and refine carbohydrate intakes during exercise based on trial and error methods .
these recommendations suggest athletes should ingest simple sugars at a rate of 3060 g / h for exercise lasting longer than 60 minutes .
g / kg of body mass in the 30 minutes following cessation of prolonged exercise .
the ingestion of carbohydrate during exercise should be considered an equally significant component of the training plan as the skill aspects of sport .
as with their adult counterparts hydration status during sport is important to performance in young athletes .
it could be argued that , due to their increased susceptibility to succumb to heat stressors , due to their greater body surface areas to body mass ratio , hydration is a more important consideration in young athletes .
in addition to an increased body surface area to body mass ratio , adolescents have been shown to have diminished sweat rates as compared to their adult counterparts .
diminished sweat rates are advantageous as a result of their protection of body water status but are disadvantageous due to the reduced ability to dissipate heat
. added importance of hydration is due to the fact that , in addition to performance decrements , hypohydration has been shown to lead to increased physiological strain , increased risk of heat injury / illness , and increased perceived exertion at similar workloads [ 15 , 107 ] . as a result of hypohydration , the body experience fluid shifts resulting in increased cardiovascular strain as plasma volume declines [ 108 , 109 ] .
additionally , the impaired cardiovascular function also leads to diminished skin blood flow resulting in a decline in the ability to dissipate heat to the environment .
hypohydration also leads to an increase in the perception of exertion required to maintain a steady work rate .
research investigating the role of hydration on exercise performance has shown fluid ingestion and the maintenance of proper hydration status improve performance [ 102 , 111 ] . to add to the physiological strain associated with dehydration inherent with physical activity , most athletes have been found to arrive to practice in a hypohydrated state [ 106 , 112 ] . even with education emphasizing the need for proper hydration ,
preactivity hydration assessments of athletes involved in sport have demonstrated significant percentages of the population to be hypohydrated .
kavouras et al . reported that educational interventions significantly improved preexercise hydration status , however following education regarding hydration 66% of the youth athletes reported to practice in a hypohydrated state .
while unlikely to be a broad risk in youth sport , attention must also be drawn to the potential to ingest fluids at excessive levels resulting in the risk of hypernatremia and potentially death .
since the first reports in the literature of hyponatremia in endurance events greater focus has continued to grow regarding the risks associated with over drinking .
hyponatremia has been reported to be as high as 51% in ultramarathons performed under hotter ambient temperatures .
the incidence of hyponatremia increases as duration of activity increases along with fluid ingestion . a potential means to reduce
the rate of plasma sodium concentration decline is through the ingestion of sodium containing beverages .
it should be noted that very few beverages actually contain sodium levels sufficient to maintain plasma sodium levels ; however the ingestion of sodium containing beverages will reduce the rate of decline .
the american college of sports medicine 's position stand on nutrition and athletic performance recommends athletes to consume 57 ml / kg of body mass 4 hours prior to exercise , enough fluid to reduce body mass changes to less than 2% during activity , and 450675 ml for every 0.5 kg of body mass lost during exercise .
these recommendations do not account for age but are likely a good place to begin for youth athletes since they are based on body mass rather than absolute volumes .
hydration goals should be to minimize body mass losses associated with dehydration while ensuring fluid ingestion does not exceed sweat losses .
this is easily assessed through body weight measures immediately prior to and immediately following activity .
increases in body mass would inform the athlete fluid ingestion was too high and significant decreases in body mass would inform the athlete fluid ingestion was insufficient .
additionally , in situations where repeated days of exercise are performed in the heat sodium ingestion rates should be increased to maintain plasma sodium levels .
flavored fluids have been repeatedly shown to aid in the maintenance of fluid intake reducing voluntary dehydration .
equation for determining sweat rate is as follows:(1)bw0+df0bwedfetime ( hrs),where bw0 is body mass before exercise , df0 is mass of exercise food and drink before exercise , dfe is body mass after exercise , and dfe is mass of exercise food and drink after exercise .
micronutrients categorically refer to vitamins and minerals used by the body during normal physiological functions .
generally , it is accepted that a well - balanced diet of sufficient caloric intake will provide the adequate micronutrients to support normal growth and maturation .
the american medical association ( ama ) and the american dietetic association ( ada ) recommend nutrients be obtained from food sources rather than supplements in healthy children .
likewise , the american academy of pediatrics ( aap ) does not endorse regular supplementation of vitamins and minerals in healthy children ( with the exception of fluoride in unfluoridated areas ) .
however , aap has noted that some children are at increased risk of nutrient deficiencies .
specifically , aap suggests that children and adolescents with anorexia or poor appetites , chronic diseases , and food insecurity are at greater risk for nutrient deficiencies .
youth who do not consume adequate amounts of dairy or have sufficient sun exposure may also be at risk for deficiencies .
the daily time constraints of an elite young athlete can make achieving a balanced diet difficult , thus putting these individuals at a potential increased risk for micronutrient deficiencies as well .
these deficiencies are most commonly observed in girls rather than boys and in mineral intake rather than vitamin intake .
athletic youth may actually be more likely to achieve the recommended intake of vitamins than nonathletic youth due to their increased total caloric intake .
adequate intake of minerals appears to be a bigger challenge for youth , particularly girls . specifically ,
iron deficiency and subsequent anemia are common in adolescents . increases in hemoglobin production , blood volume , and muscle mass are normal characteristics of growth and maturation and account for the majority of increased iron needs in developing adolescents .
iron - deficient anemia has significant , negative implications on performance in adults [ 121 , 122 ] and youth [ 123125 ] .
diminished performance is most apparent in iron - deficient anemic athletes participating in activities with higher aerobic demands ( i.e. , endurance event athletes ) .
the recommended intake of iron for boys and girls aged 913 years is 18 mg per day .
boys and girls aged 1418 years should consume 11 mg and 15 mg per day , respectively . in accordance with ama , ada , and aap guidelines
, youth should improve their iron status through consumption of iron - rich foods at meals such as red meat , beans , and green vegetables .
athletes will likely find additional benefit by including other iron - rich foods such as peanuts and dried fruits and iron - fortified cereals as regular snacks .
furthermore , the inclusion of foods higher in ascorbic acid with these nonheme iron sources will improve iron absorption from these snacks .
calcium requirements are greatest during adolescence , 1,300 mg per day for both boys and girls .
this higher requirement accommodates the prime opportunity for acquisition of bone mass that spans the pubertal ages .
availability of nutrients critical for bone development ( e.g. , calcium ) and opportunity for increased bone loading ( e.g. , physical activity ) prior to achieving skeletal maturation is critical in preventing osteoporosis later in life . despite the greater requirements and the clear benefits of consuming adequate amounts , united states children and adolescents ' average intake falls below the minimum recommendations . in the american diet
given the perishability of most dairy products , young athletes face practicality issues scheduling regular consumption throughout the day .
breakfast consumption is associated with greater calcium intake among this age group and should be strongly encouraged .
likewise , athletes should consume regular snacks that include rich sources of calcium ( e.g. , fortified orange juice , almonds , and broccoli ) , throughout the day .
thus , attention to sources of calcium fortified with vitamin d is warranted , particularly among individuals who are not likely to be exposed to sufficient sunlight to endogenously produce adequate vitamin d. while whole food sources are always preferred , the convenience of calcium supplements ( often fortified with vitamin d ) may make adequate intake a more viable possibility .
an additional consideration warranting attention in young athletes participating in large amounts of training and competition is the potential need to replenish sodium and potassium lost in sweat .
the summation of the electrolyte loss resulting from sweat loss has been shown to be equivalent to daily intakes , even in young athletes .
much of this additional loss in salt can be offset through the ingestion of sport drinks during practice and competition , which also partially addresses hydration concerns in the group .
research regarding the nutritional needs of young competitive athletes is sparse and is primarily composed of investigations of youth - adult differences .
in addition to the limited research , the majority of our current knowledge in the adult population is based on differences between typical adults compared to their more active counterparts .
research to date suggests similarities in the caloric and macronutrient needs of active adults and their younger counterparts ; however , youth - adult differences in fuel utilization have also been clearly demonstrated .
in addition to the energy needs of highly active youth , nutritional intake plays a critical role in the growth and development of young athletes and should be a principal emphasis at this stage in their lives .
these guidelines serve as recommendations that support healthy growth and development and also account for the additional caloric needs of active youth . as emphasis on performance outcome goals continues to increase in youth athletics , active youth quickly become young athletes . likewise
, nutritional considerations move beyond increased caloric needs and promotion of healthy growth and development to nutritional strategies that can optimize performance .
a summary of available performance - based nutritional strategies can be found in table 3 .
the paucity of data examining the unique needs of young athletes draws attention to increased need for further investigations on this subject which consider the distinct nutritional requirements of growth and maturation at all age and skill levels .
given the popularity of youth sports and the increasing demand for performance outcomes , increased attention on this topic is warranted by the scientific community . | nutrition is an integral component to any athletes training and performance program . in adults
the balance between energy intake and energy demands is crucial in training , recovery , and performance . in young athletes
the demands for training and performance remain but should be a secondary focus behind the demands associated with maintaining the proper growth and maturation .
research interventions imposing significant physiological loads and diet manipulation are limited in youth due to the ethical considerations related to potential negative impacts on the growth and maturation processes associated with younger individuals .
this necessary limitation results in practitioners providing nutritional guidance to young athletes to rely on exercise nutrition recommendations intended for adults .
while many of the recommendations can appropriately be repurposed for the younger athlete attention needs to be taken towards the differences in metabolic needs and physiological differences . | 1. Introduction
2. Growth and Development
3. Protein
4. Fat
5. Carbohydrate
6. Water
7. Micronutrients
8. Summary |
PMC2486402 | microsatellites or
ssrs ( simple sequence repeats ) are
sequences in which one or few bases are tandemly repeated for varying numbers of
times .
variations in ssr regions originate mostly from errors during the replication process , frequently dna
polymerase slippage , generating insertion or deletion of base pairs , resulting ,
respectively , in larger or smaller regions [ 2 , 3 ] .
ssr assessments in the human
genome have shown that many diseases are caused by mutation in these sequences .
ssrs can be found in different regions of genes , that is , coding sequences , untranslated
sequences ( 5-utr and 3-utr ) , and introns , where the expansions and/or
contractions can lead to gene gain or loss of function . also , there are
evidences that genomic distribution of ssrs is related to chromatin
organization , recombination , and dna repair .
ssrs are found throughout the
genome , in both protein - coding and noncoding regions .
genome fractions as low
as 0.85% ( arabidopsis thaliana ) , 0.37%
( zea mays ) , 0.21% ( caenorhabtis elegans ) , 0.30% ( sacharomyces cerevisae ) and as high as 3.0%
( homo sapiens ) and 3.21% ( fugu rubripes ) have been found .
some
bias for defined genomic locations has also been reported [ 6 , 7 ] .
this class of
markers is broadly applied in genetics and plant breeding , due to its
reproducibility , multiallelic , codominant nature , and genomic abundance .
its use
for integrating genetic maps , physical mapping , and anchoring gives geneticists
and plant breeders a pathway to link genotype and phenotype variations .
the protocols for
isolating ssr loci for a new species were always very labor - intensive .
currently ,
with the accumulation of biological data originating from whole genome sequence
initiatives , the use of bioinformatics tools helps to maximize the
identification of these sequences and consequently , the efficiency in the
number of generated markers .
the first in silico studies of ssrs were developed
using fasta and blast packages .
later , more specific algorithms , such as sputinick ,
repeatmasker , trf - tandem repeat find , troll , misa and
ssrit ( simple sequence repeat tool ) ,
were obtained .
ssr detection is generally followed by the use of another program for primer design , to be
anchored on flanking sequences .
also , in some applications , a third step using e - pcr
is added , with the goal of
verifying primer redundancy .
building such a pipeline can be a very difficult task
for research groups not familiar with programming tools . in the present work ,
the
application integrates the following functions : ( i ) detection and
characterization of ssrs and minisatellite motifs between 1 and 10 base pairs ; ( ii )
primer design for each locus found ; ( iii )
simulation of pcr ( polymerase chain reaction ) , amplifying fragments
with different primer pairs from a given set of fasta files ; ( iv ) global alignment
between amplicons generated by the same primer pair ; and ( v ) estimation of global
alignment scores and identities between amplicons , generating information on
primer specificity and redundancy .
the algorithms used for the searches , alignment , and homology estimates are described separately .
the algorithm used
for perfect and imperfect micro-/minisatellite searches was written in perl and
consists of the generation of a matrix that mixes a(adenine ) , t(thymine ) ,
c(cytosine ) , and g(guanine ) in all possible composite arrangements between 1
and 10 nucleotides .
the script instructions perform readings on fasta files ,
searching all possible arrangements in each database sequence .
several instructions in the algorithm used in ssrlocator resemble those from misa
and ssrit .
however , additional instructions have been inserted in ssrlocator 's code . instead of allowing the overlap of a few nucleotides
when two ssrs are adjacent to each other and one of them is shorter than the
minimum size for a given class as found in misa and ssrit , a module written in delphi language records the data and eliminates such
overlaps .
the ssr locator
software contains windows focused on the selection and configuration of ssr and
minisatellite types ( mono- to 10-mers ) and a
minimum number of repeats for each one of the selected types .
the algorithm
calls a perfect repeat when one locus is present with
adjacent loci at an up or downstream distance higher than 100 bp .
the algorithm calls
an imperfect repeat when the same motif is present on both sides of a fragment containing
up to 5 base pairs .
the algorithm
identifies a composite locus when two or more adjacent
loci were found at distances between 6 and 100 bp . in this study , only
these repeats
have been described as the most efficient loci for use as molecular markers
.
the software ssrlocator was configured to locate a minimum of 20 bp ssrs :
monomers(x20 ) , 2-mers(x10 ) , 3-mers(x7 ) , 4-mers(x5 ) , 5-mers(x4 ) , 6-mers(x4 ) , and
minissatellites : 7-mers(x3 ) , 8-mers(x3 ) , 9-mers(x3 ) , and 10-mers(x3 ) . in order to
validate the efficiency of ssrlocator in finding ssrs and minisatellites ,
the
same database was analyzed withmisa
and ssrit , using the same parameters for minimum number of repeats .
an algorithm written in delphi language performs calls to primer3 , which execute primer designs .
these
results are fed to a module that performs virtual - pcrs and allocates individual
identification , forward and reverse primer sequences , and a sequence fragment
corresponding to the region flanked by the primers ( original amplicon ) to each
ssr locus .
a window allows the selection of primer3 parameters , such as range
of primer and amplicon sizes , as well as optimum primer size , ranges of melting temperature
( tm ) ( minimum , maximum , and optimum ) and gc content ( minimum and optimum ) . for
primer searches ,
the software automatically looks for five base pair distances from
both ssr ( 5 and 3 )
flanking sites . in this study ,
the
following parameters were used : amplicon size
between 100 and 280 bp ; minimum , optimum , and maximum annealing temperature
( tm ) of 45 , 50 , and 55 , respectively , minimum , optimum , and maximum primer size
of 15 , 20 , and 25 bp , respectively .
the algorithm consists in reading the file generated by the previous module
( ssr locus , forward and reverse primers , and original amplicon ) , followed by a
search of sequences containing primer annealing sites .
when annealing sites are found for the two primers , the flanked
region and the primer sequences are copied to a new variable called paralog amplicon . for the global alignment between paralog and original amplicon sequences
and score calculations ( match , mismatch , gaps ) ,
a routine was written in delphi language using
the algorithms of needleman and wunsch ( 1970 ) and smith and waterman
( 1981 ) .
also , in the same module , amplicon identities were calculated
according to waterman ( 1994 ) and vingron and waterman ( 1994 ) .
the strategy of creating a two - language
hybrid program was established as a function of : ( i ) the higher speed achieved
by handling large text files with perl as compared to delphi , and ( ii ) the better
fitness of perl for generating combinatory strings to be located .
the perl
module was transformed into an executable file , making unnecessary to install perl libraries during program installing .
the graphic interface built , integrating input and output windows to the
windows operational system , was obtained using the suite turbo delphi , where a
menu system executes calls for each of the previously described modules .
nipponbare ) nonredundant full length nonredundant
cdna
sequences , sequenced by the rice full - length cdna consortium , mapped on the databases derived from the sequencing of japonica ( japonica draft
genome , bac / pac clones
irgsp ) and indica ( indica draft genome ) subspecies were used for the
analyses .
these sequences are deposited in ncbi as two groups , the
first comprising accesses from ak058203 to ak074028 , and
the second comprising accesses from ak98843 to ak111488 .
all these sequences
can be also found in kome ( knowledge - based oryza molecular biological
encyclopedia ) .
a flow chart representing the different steps performed by the software is shown in figure 1 .
a total of 3907 micro- and minisatellites were detected by ssrlocator in the 28 469
analyzed cdna sequences .
the same database searched with misa and ssrit presented
3913 and 3917 loci , respectively .
the mono- , 4-mer , 6-mer , 7-mer , 8-mer , 9-mer ,
and 10-mer repeats were identical for the three programs . in the case of 2-mer repeats ,
594 elements were detected by ssrlocator and 596 elements were detected by misa
and ssrit .
3-mer repeats were differently scored by ssrlocator ( 1990 ) and the
other two ( 1994 ) algorithms . for 5-mer repeats , ssrlocator and misa found the
same number of repeats ( 426 ) , while ssrit ( 430 ) found a different value .
the results obtained with ssrlocator
indicate that out of 28 469 cdna sequences , 3765 ( 13.22% ) presented one or
more micro-/minisatellite loci .
in other studies , microsatellites were found in
the following proportions in ests : 3% in arabidopsis , 4% in rosaceae , 8.11% in barley , 2.9% in sugarcane , and values ranging between 611% and 1.54.7%
for cereals in general ( maize ,
barley , rye , sorghum , rice , and wheat ) . considering the 3765 fl - cdna
sequences , in 3632 ( 92.96% ) only a single micro-/minisatellitelocus was detected . in 125 sequences ,
two loci were detected , in seven
sequences three lociandonly one sequence had four loci , adding
up to 3907 occurrences .
among the types analyzed , ssrs ( mono to 6-mer repeats )
and minisatellites ( 7- to 10-mer repeats ) comprised 96.98% and 4.12% of
detected loci , respectively .
the distribution of occurrences detected by ssrlocator was consisted of 138 monomers ,
594 2-mers , 1990 3-mers , 251 4-mers , 426 5-mers , 390 6-mers , 82 7-mers , 6 8-mers , 25 9-mers , and 5 10-mers , corresponding to rates of 3.53% , 15.20% , 50.93% ,
6.42% , 10.90% , 9.98% , 2.10% , 0.15% , 0.64% , and 0.13% , respectively ( see table 1 ) .
for the
remaining ssrs , average percentage values have been reported as between 17 and 40% for 2-mer ,
5478% for 3-mer , 2.66.6% for 4-mer , 0.41.3% for 5-mer , and less than 1% for 6-mer
repeats and 26.5%
for 2-mer , 65.4% 3-mer , 6.8% 4-mer , 0.77% 5-mer , and 0.45% for 6-mer repeats for barley , maize , wheat , sorghum , rye , and rice , respectively .
in nonredundant transcripts from the tigr database , 15.6% 2-mer , 61.6% 3-mer ,
8.5% 4-mer , and 14.4% 5-mer repeats were found in rice .
the frequency of micro / minisatellite
locus occurrence for each million nucleotides ( loci / mb ) in this study was 2.94 ,
12.64 , 42.34 , 5.34 , 9.06 , 8.30 , 1.74 , 0.13 , 0.53 , and 0.11 for mono to 10-mer
repeats / mb , respectively .
overall occurrences of 83.13 loci / mb were found ( see table 1 ) . in other studies ,
different taxa were described in analyses of est databases ,
such as 133 loci / mb ( barley ) , 161 loci / mb ( wheat , sorghum and rye ) , and 256 loci / mb
for rice .
also , for nonredundant ests in rice , sorghum , barley , wheat , and
arabidopsis , frequencies of 277 , 169 , 112 , 94 and 133 loci / mb were found ,
respectively .
frequencies closer to those found in this study were
described for cds regions of rosaceaespecies , with an average of 40.978 loci / mb for
rose , almond and peach , while 39 loci / mb were found for arabidopsis . on table 2 , the contents and percentage values for different micro-/minisatellite motifs
are shown . for monomer , 2-mer and 3-mer repeats ,
all possible arrangements are
shown , while for 4-mer to 10-mer repeats , only the ten most frequent motifs are
shown .
the a / t monomer
repeats were found in 125 loci , with 111 ( 88.80% ) and 14 ( 11.20% ) loci formed
by a and t nucleotides , respectively .
the c / g motifs were found in 13 loci ,
with ten ( 76.92% ) and three ( 23.08% ) loci formed by c and g , respectively .
motifs
ag / ct and ga / tc were the most frequent and added up to 8.52% of 2-mer ssrs , and
6.89% and 5.96% of all 3907 detected occurrences .
the motifs ct , ga , and tc were
the most abundant adding up to 172 , 143 , and 90 loci , respectively . in maize ,
barley , rice , sorghum , and wheat ests ,
the motif ag was described as the most
frequent [ 6 , 16 , 28 , 29 , 31 , 32 ] . however , in some studies , the most frequent
motif was ga [ 30 , 33 ] .
repeats composed by guanine and cytosine were the most
abundant among trimers , with occurrences of 18.44% , 17.89% , and 10.60% ,
respectively , for the motifs ccg / cgg , cgc / gcg , and gcc / ggc , adding up to 23.9% of
the overall frequencies of micro-/minisatellites in the analysis .
the motifs
cgc , ccg , and cgg were the most frequent comprising 218 , 197 , and 170 loci ,
respectively .
many reports indicate the 3-mer ccg as the most frequent in
maize , barley , wheat , sorghum and rye [ 6 , 16 , 28 , 32 ] , sugarcane and rice [ 29 , 31 ] . among 4-mers ,
100 different
arrangements were found , where the motifs gatc ( 7.17% ) , atta / aat ( 6.77% ) , and
atcg / cgat ( 5.98% ) were the most frequent .
these motifs add up to 19.92% of 4-mer
repeats found and represent 1.28% of the overall content of micro-/minisatellites .
in barley ests ,
acgt was reported as the most abundant motif [ 16 , 28 ] . for
other species ,
aaag / cttt and aagg / cctt in lolium
perene , aaag / cttt and aaac / gttt in arabidopsis utrs [ 6 , 35 ] , and aaat and
aaag in citrus [ 36 , 37 ] were described as most abundant . among 5-mers ,
188
different arrangements were detected and the most frequent were ctcct , ctctc , and
cctcc with 17 , 17 , and 12 occurrences , respectively . in the analysis of cds
regions , the acccg motif was the most frequent in arabidopsis , aaaag in s. cerevisae , c. elegans ,
and aaaac in different primates .
also , the motifs
aaaat , aaaac , and aaaag were described as the most frequent in eukaryotes .
in rice ,
repeats of
type 6-mer were detected in 230 different arrangements , where cgcctc and tcgccg
were the most frequent , occurring in 12 and 10 loci , respectively .
other
studies have shown higher frequencies for the motifs aagatg , aaaaat in arabidopsis
, aaaaag in citrus , aacacg in s. cerevisae , accagg in c. elegans and ccccgg in primates .
for
all remaining repeats ( minisatellites ) , the occurrences are widely distributed
with low - percentage values for each arrangement .
for 7-mer , 8-mer , 9-mer , and 10-mer
repeats , the totals of occurrences were 57 , 5 , 23 , and 5 , respectively .
the design of
primers for the 3907 detected micro-/minisatellites resulted in 3329 primer
pairs , covering 85.20% of loci .
the running of virtual pcr generated a total of
4610 amplicons . a module in ssrlocator checks for primer redundancy . a total
of 2397 primer pairs amplified only the fragment from its original locus ( specific
amplicons ) and 932 pairs amplified one or more regions besides the original
locus . from these ,
692 pairs amplified two fragments , one from the original
site and a second from another region ( paralogous ) . in this case , 692 specific amplicons
plus 692 redundant amplicons , were detected .
a total of 143 , 90 , 2 , and 5 primer
pairs generated three ( two redundancies ) , four ( three redundancies ) , five ( four
redundancies ) , and six ( five redundancies ) fragments , respectively .
the final
product of 932 primers with more than one anchoring region resulted in 932 specific
amplicons and 1281 redundant amplicons , adding up to 2213 fragments . to investigate the ability of these
primers in amplifying genomic sequences ,
an extra experiment was performed
against the whole rice genomic sequence available at ncbi .
the different groups
of redundant and nonredundant primer sets , that is , amplifying one , two , three ,
or more times in the cdna database , were tested against the genomic sequence .
from the 2397 nonredundant primers , only 924 amplified a locus in the genomic
sequence .
this difference was already expected because of difficulties in
amplifying genomic regions , that is , if some primers anneal to a boundary
region between two exons in the cdna , the presence of introns would make this
annealing site no more available .
it is interesting to note that from the 924
amplicons detected , 914 ( 99% ) did amplify only one locus in the genomic region ,
agreeing with the cdna results .
when the primer sets that amplified two
different cdnas were run against the genomic sequence , only 294/692 ( 42.5% ) did
amplify , having 14.5%
been able to amplify two different loci .
these results indicate that ssr
locator performance was consistent between the two databases regarding the
nonredundant loci , that is , from those loci that were able to be amplified in
both databases , their status of nonredundant was maintained . the changes
observed for the redundant loci can be attributable to many causes , including
redundancy in the cdna database , but also to biological reasons due to primer
positioning
. results of a global
alignment between amplicons from original and redundant sites are shown in table 3 . among
the 1281 redundant amplifications , 787 ( 61.44% ) resulted in a perfect
alignment between both loci ( identity equal to 100 ) .
for redundant amplicons
with identity levels of 9699% , and 9095% , 452
( 35.28% ) and 8 ( 0.62% ) loci were found , respectively .
the fact that such a high
percentage of redundant loci show high identity is probably a consequence of
the genome fraction chosen , that is , expressed sequences .
this fraction is
under tight selection pressure and should not accumulate variations such as
substitutions or indels at a high rate .
as expected , comparisons to whole
genome , generated a great deal of polymorphism , due to the inclusion of
intronic regions in the alignments ( data not shown ) .
the software ssrlocator was successfully implemented , adding steps for ( 1 ) ssr
discovery , ( 2 ) primer design , and ( 3 ) pcr simulation between the primers obtained
from original sequences and other fasta files .
also , the software produces
reports for frequency of occurrence , nucleotide arrangement , primer lists with
all standard information needed for pcr and global alignments . from the pcr
simulation ,
it was possible to point out which primer pairs were nonredundant ,
suggesting that these primers are more appropriate for mapping purposes . in
this case , however , wet lab experiments should be performed to confirm the
advantage of nonredundant over redundant primers for mapping .
it is possible that the results for micro-/minisatellite frequencies ( loci / mb ) obtained
in this study diverge from the results found in the literature .
this can be
explained by the different databases used ( redundant ests , nonredundant ests and/or
fl - cdna ) , different algorithm configurations and minimum requirements set for
counting motifs .
the results showed that 932 ( 27.99% ) primers presented
amplifications in more than one gene sequence
. this could be mostly due to the
fact that primer pairs derived from a specific gene ( cdna ) anchored in similar
sites in other duplicated genes , since 5,607/28,469 ( 19.70% ) genes were described as paralogs in the
annotation of the database used .
gene duplication along with polyploidy
and transposon amplification are the major driving forces in genome evolution .
also , a
second possibility is that some primers were generated from protein domain
regions within the analyzed cdnas .
these domains could be found in protein
families with many genome copies , resulting in the observed redundancies .
a
validation of the redundancies of cdna results was obtained through a virtual - pcr against the whole
rice genome sequence . from the nonredundant primers that generated an amplicon ,
ca .
finally ,
this tool can be used successfully for data mining strategies to find ssr
primers in genomic or expressed sequences ( ests / cdnas ) .
also , this software can
be a tool for microsatellite discovery in databanks of related species ,
anchoring primers in ortholog or paralog regions contained between databases
from two different species . | microsatellites or ssrs ( simple sequence repeats ) are ubiquitous short tandem duplications occurring in eukaryotic organisms .
these sequences are among the best marker technologies applied in plant genetics and breeding .
the abundant genomic , bac , and est sequences available in databases allow the survey regarding presence and location of ssr loci .
additional information concerning primer sequences is also the target of plant geneticists and breeders . in this paper , we describe a utility that integrates ssr searches , frequency of occurrence of motifs and arrangements , primer design , and pcr simulation against other databases .
this simulation allows the performance of global alignments and identity and homology searches between different amplified sequences , that is , amplicons . in order to validate the tool functions ,
ssr discovery searches were performed in a database containing 28 469 nonredundant rice cdna sequences . | 1. INTRODUCTION
2. MATERIAL AND METHODS
3. RESULTS
4. CONCLUSIONS |
PMC3318166 | chylous leakage can result from damage to the thoracic duct or its branches after surgical procedures of various types , such as neck or thoracic surgery .
although the majority of chylous leakage cases after breast surgery are managed conservatively , we chose to do a re - operation because of the presence of a high chyle output . herein , we reported our experience of a case of chylous leakage after axillary lymph node dissection , that was treated surgically .
a 38-year - old woman with invasive ductal carcinoma in the left breast underwent a modified radical mastectomy .
the surgical procedure was uneventful and two suction drains were left at the mastectomy site and axilla , in a routine manner .
analysis of the fluid was compatible with chyle ( milky color , protein , 3,400 mg / dl ; triglycerides , 784 mg / dl ; total cholesterol , 61 mg / dl ; and lymphocyte dominance ) .
initially , we tried conservative management with closed suction drainage , a compressive bandage , and limitation of oral intake ; however , the daily output of the drain increased to 700 ml by the seventh postoperative day . therefore , we re - explored the axilla and observed that the clear fluid was running from a single duct located just below the lateral pectoral bundle branch ( figure 1 ) .
we ligated the duct and reinforced the suspicious site 3 - 4 cm below the duct using a mass ligature .
after the re - exploration , the daily output of the drain decreased to 80 ml , although it remained slightly milky in nature .
the pathology report confirmed a 1.5-cm , poorly differentiated invasive ductal carcinoma and one metastatic lymph node of 15 lymph nodes evaluated .
chylous leakage seldom occurs after axillary surgery because the axilla is anatomically remote from the thoracic duct .
the reported incidence of chylous leakage after neck surgery is 1 - 3% and its incidence after surgery for breast cancer is ~0.5% . to prevent and control this complication , it is important to determine the location of the chyle leakage .
the anatomy of the thoracic duct is well documented and there are numerous variations in the pattern and site of the thoracic duct in the venous system .
it has been reported that more than two terminal ducts exist in 7 - 20% of cases and one case with a thoracic duct that emptied into the right internal jugular vein was described . a rare anatomical variation of the lymphatic trunk , posteroinferior to the axillary vein ,
it is very difficult to predict injury to the lymphatic trunks preoperatively or to recognize it during surgery because of its rare occurrence in axillary surgery and the absence of well - known risk factors .
chylous leakage is usually diagnosed by the typical " milky " drainage fluid after surgery and is confirmed by biochemical analysis of the electrolyte , protein , and fat content of the fluid , which , in these cases , are compatible with chyle .
nakajima et al . reported that they did not find any characteristic risk factors after analysis of four cases , although others mentioned previously the effects of wound seroma or obesity .
the initial management of chylous leakage should be conservative , including adequate drainage , diet control , and treatment with intravenous octreotide and oral injection of tetracycline hydrochloride .
however , some authors advocate that timely or early re - exploration should be used to manage chylous fistulas , considering the prolongation of the hospital stay and morbidity .
it is considered when pleural drainage is greater than 1 l per day for more than 5 days in cases of chylothorax or with continuous drainage over 500 - 600 ml per day without improvement .
however , an early surgical approach can easily be considered in breast surgery because the risk associated with re - exploration of the superficial chest wall and axilla is not very high .
surgical management consists of direct ligation of the injured lymphatic channel , if it can be seen during exploration , and plugging with gel foam , other packing materials , or with glue or local muscle rotation flaps .
awareness of this unusual complication after breast surgery should make it possible to avoid or recognize the injury to the thoracic duct during surgery .
thus , we suggest an even more careful dissection and ligation in cases where the patient has massive axillary metastasis . in addition , a timely surgical approach may represent an alternative , because of its low morbidity and because it avoids delaying subsequent oncologic treatments , even though many cases were treated conservatively . | chylous leakage is an extremely rare complication of surgery for breast cancer .
we experienced a case of chylous leakage after axillary lymph node dissection . a 38-year - old woman with
invasive ductal carcinoma in the left breast underwent a modified radical mastectomy after four cycles of neoadjuvant chemotherapy .
the postoperative serosanguinous drainage fluid became " milky " on the fourth postoperative day . after trying conservative management ,
we re - explored the axilla and ligated the lymphatic trunk .
although the success of many cases supports conservative management , timely surgical intervention represents an alternative in cases where leakage persists or where the output is high . | INTRODUCTION
CASE REPORT
DISCUSSION |
PMC3987881 | in 2005 , sato et al . identified a novel autoantibody recognizing a 140-kda protein in patients with dermatomyositis ( dm ) , particularly in those with clinically amyopathic dermatomyositis ( cadm ) .
the 140-kda autoantigen , which was identified as melanoma differentiation - associated protein 5 ( mda5 ) , is detected in 19% to 35% of the patients with dm . in the asian population
, this autoantibody seems to be associated with rapidly progressive interstitial lung disease and with severe cutaneous vasculopathy ( skin ulceration , tender palmar papules , or both ) [ 16 ] .
recently , the presence of anti - mda5 antibody - associated dermatopulmonary syndrome was described in the white population [ 79 ] .
mda5 , also known as interferon - induced helicase-1 ( ifih1 ) , is a member of the retinoic acid - inducible gene i - like helicase ( rig - i or rlh ) family of proteins , which function by recognizing single - stranded rna viruses and are involved in the innate immune response , including type i ifn production .
the main drawback to routine use of this antibody for clinical purposes is that its determination is limited to techniques that are only available in research laboratories , such as immunoprecipitation of radioactive - labeled protein or enzyme - linked immunoassay ( elisa ) using in - house fabricated recombinant proteins [ 12 , 13 ] .
our objective was to evaluate the prevalence and clinical manifestations of anti - mda5-positive patients in a large cohort of dm patients from a single center in barcelona , and to determine the feasibility of detecting this autoantibody with the use of more widely available techniques ( elisa and immunoblotting ) with commercially available recombinant mda5 as the antigen .
this study was performed in 117 adult patients ( 92 women ) with dm ( 15 with clinically amyopathic dm ) .
in addition , 45 patients with polymyositis ( pm ) , 30 with systemic sclerosis ( ssc ) , and 25 with systemic lupus erythematosus ( sle ) were included as controls .
twenty - five healthy controls were also included to determine the cut - off value for establishing the positive status by elisa .
the patients studied belong to a historical cohort diagnosed with idiopathic inflammatory myopathy at vall d'hebron general hospital in barcelona ( spain ) , between 1983 and 2012 .
our center is a single teaching hospital with approximately 700 acute care beds , attending a population of nearly 450,000 inhabitants .
all myositis patients in this population are referred to our hospital for diagnosis and therapy , regardless of the severity of the disease .
serum samples from these patients are routinely collected at diagnosis and during follow - up in our outpatient clinic , and stored at 80c .
patients and controls included in the study gave informed consent for the use of their serum for research purposes .
the diagnosis of dm and pm was based on the criteria of bohan and peter [ 14 , 15 ] .
the sontheimer criteria were used to diagnose amyopathic dm . interstitial lung disease ( ild ) was diagnosed according to the consensus classification of idiopathic interstitial pneumonias .
the diagnosis of ild was established by high - resolution ct findings , and rapidly progressive - interstitial lung disease ( rp - ild ) was defined as a worsening of radiologic interstitial changes with progressive dyspnea and hypoxemia within 1 month after the onset of respiratory symptoms .
cancer - associated myositis ( cam ) was defined as cancer occurring within 3 years of the myositis diagnosis .
patients received treatment with corticosteroids and immunosuppressive drugs ( methotrexate , azathioprine , calcineurin inhibitors [ cyclosporine a or tacrolimus ] , or cyclophosphamide pulses ) were added when needed ; intravenous immunoglobulin was used as an adjuvant therapy , and biological therapy ( rituximab ) was also instituted when possible in refractory cases .
a drug trial was defined as a single course from the beginning of the administration of a given drug to the time at which the drug was discontinued , or in the case of prednisone , as the time at which the dose was reduced to one quarter of the initial dose .
serum samples from each patient were screened by indirect immunofluorescence for antinuclear antibodies ( ana ) using hep-2 cells , and by a commercial elisa used in our routine laboratory setting for antibodies against extractable nuclear antigens ( ro , la , rnp , sm ) and anti - histidyl - trna synthetase ( anti - jo-1 ) .
anti - tif1 antibodies were detected by an in - house elisa and confirmed by immunoblot .
in addition , all samples were tested by protein and rna immunoprecipitation , which enabled detection of other synthetases and myositis - specific and myositis - associated antibodies ( anti - mi-2 , anti - srp , anti - ro52 , anti - ro60 , anti - la , anti - pm / scl , anti - p155 , and anti - u1rnp ) that may have been overlooked by elisa , and confirmed the elisa results .
briefly , 96-well elisa plates ( nunc , kamstrup , denmark ) were coated with 100 ng of purified recombinant mda5 ( origene , rockville , md ) , diluted in phosphate buffered saline ( pbs ) , and left to stand overnight at 4c .
wells were incubated for 1 hour at room temperature ( rt ) with blocking buffer ( 10% nonfat dry milk in pbs ) .
plates were then washed ( hrp wash , inova diagnostic inc . , san diego , ca ) ; human serum samples diluted 1 : 100 in blocking buffer were added in triplicate : two to mda5-coated wells and one to a pbs - coated well ( without antigen ) to determine the background absorbance .
after washing , hrp - labeled goat anti - human igg antibody ( inova diagnostic inc . , san diego , ca ) was added to each well , and plates were incubated for 1 h at rt and washed again .
color development was performed with peroxidase reagent tmb chromogen ( inova diagnostic inc . , san diego , ca ) and absorbances at 450 nm were determined . for each sample ,
the background absorbance from the pbs - coated well was subtracted from that of the corresponding mda5-coated wells ( the average of the two results ) .
the same positive serum ( from patient 11 , confirmed by ip by casciola rosen , from baltimore , usa ) was used as the reference in each assay .
briefly , 5 g of purified recombinant mda5 ( origene , rockville , md ) was run on 4% to 12% polyacrylamide - sds minigels with mops running buffer , and western blot was performed on a nitrocellulose membrane using the invitrogen nupage ( carlsbad , ca ) electrophoresis system .
mda5-transferred nitrocellulose was vertically cut into several strips and incubated for 1 hour at rt in pbs with 0.05% tween ( pbs - t ) containing 3% nonfat dry milk ( blocking buffer ) .
each strip was then incubated with the corresponding human serum sample diluted 1 : 100 in blocking buffer for 1 hour at rt .
after washing , phosphatase alkaline - labeled goat anti - human igg antibody ( dako , glostrup , denmark 1 : 2000 ) was added to each strip and strips were incubated for 1 hour at rt .
color development was performed by phosphatase reagent ( bcip / nbt , sigma - aldrich , st .
, the results were classified into negative , weak positive ( + ) , or positive ( + + , + + + ) ( figure 1 ) .
associations between anti - mda5 antibodies and qualitative variables were evaluated with the chi - square and fisher exact test .
the strength of the associations between variables was measured using odds ratios ( ors ) with 95% confidence intervals ( cis ) .
the corresponding area under the curve ( auc ) of the roc analysis of anti - mda5 antibody for detection of rp - ild , cadm , and total dm was analyzed with 95% of cis .
all tests were two - sided , and probability ( p ) values of < 0.05 were considered statistically significant .
all analyses were performed with spss , version 19.0 ( spss , chicago , il ) .
one - hundred and seventeen adult dm patients , 15 of whom had cadm , were included in the study .
anti - mda5 was determined by our in - house elisa and immunoblot techniques using a commercially available recombinant mda5 .
the cut - off value for a positive result on elisa was established at 0.188 absorbance units , which corresponded to 2 standard deviations above the mean value obtained for the 25 healthy controls .
the other control subjects included 45 patients with polymyositis ( pm ) , 30 with systemic sclerosis ( ssc ) , and 25 with systemic lupus erythematosus ( sle ) ( figure 2 ) .
only two patients diagnosed with dm , 2 with pm , and 1 with ssc showed weak anti - mda5 reactivity by elisa , which was not confirmed by immunoblot ; these results were considered false positives .
anti - mda5 antibodies detected by elisa and confirmed by immunoblot were only found in dm patients .
roc curve analysis for the positivity of anti - mda5 of all patients with dm against controls and cadm patients versus remaining dm disclosed an auc of 0.56 , 95% ci 0.490.63 and 0.74 ,
patients with the highest absorbance unit values on elisa also showed the strongest anti - mda5 positivity on immunoblotting .
fourteen patients , 8 with cadm , tested positive for anti - mda5 , which represents a prevalence of 12% of the dm patients from our cohort .
median ( range ) age at diagnosis of anti - mda5 positive patients was 47 ( 2860 ) years , which did not differ significantly from the remainder of the cohort .
seven patients also tested positive to anti - ro52 , but none of them was positive for any antisynthetase antibody .
interstitial lung disease was present in 9 of the 14 ( 64.3% ) patients with anti - mda5 autoantibodies , and the condition was rapidly progressive in 8 patients .
rp - ild was more frequent in patients with cadm , both in the anti - mda5 positive group ( 7 of 8 cadm versus 1 of 6 dm ; p < 0.05 ) and in the overall cohort ( 8 of 15 cadm patients versus 3 of 102 dm patients ; p < 0.05 ) .
when rp - ild was evaluated in relation to anti - mda5 positivity , a highly significant association was observed between the two parameters .
thus , 8 of the 14 anti - mda5-positive patients presented rp - ild versus 3 of the 103 anti - mda5-negative patients ( p < 0.05 ; or : 44.4 , 95% ci 9.3212 ; auc 0.84 , 95% ci 0.681 ) .
nevertheless , no association was found between anti - mda5 elisa titers at diagnosis of dm and development of a rp - ild .
moreover , 6 of the 8 ( 75% ) patients with anti - mda5 and rp - ild were ro52-positive in comparison to only 1 of the 6 ( 16% ) patients without rp - ild .
the cadm diagnosis was significantly associated with anti - mda5 positive status ( 8 of 14 mda5-positive patients versus 7 of 103 mda5-negative ( p < 0.05 ; or : 18.3 , 95% ci 4.967.6 ) ) .
no differences were observed in the frequency of gottron 's papules , heliotrope rash , photosensitivity , shawl or
v sign , cuticular overgrowth , calcinosis , or the presence of mechanic 's hands when the anti - mda5-positive and anti - mda5-negative groups were compared .
panniculitis was the only manifestation significantly associated with the presence of anti - mda5 ( 5 out of 14 anti - mda5-positive versus 13 out of 103 anti - mda5-negative ; p < 0.05 ; or : 3.85 , 95% ci 1.1113.27 ) ; nevertheless , a multivariate analysis was not possible to be performed due to methodological reasons .
cancer was diagnosed in 4 out of 14 ( 28.6% ) patients with anti - mda5 autoantibodies , and 3 ( 21% ) of them fulfilled criteria of cam .
however , no association was found between anti - mda5 autoantibodies and cam ( p > 0.05 ) .
a similar result was obtained when we repeated the analysis after excluding from the cohort the anti - tif1-positive patients ( 28 patients ) in order to avoid a possible confounding effect of the presence of these patients in the control group .
the cumulative 70-month survival rate was significantly lower ( 38% ) in the group of patients with anti - mda5 than in the remainder of the cohort ( 62% ) ( log - rank test , p < 0.05 ) ( figure 3(a ) ) .
comparison of cumulative 70-month survival between anti - mda5-associated ild and antisynthetase - associated ild also showed a statistical difference ( log - rank test , p < 0.05 ) ( figure 3(b ) ) .
no differences in 70-month survival were observed between the cadm group and the classic dm group in anti - mda5- positive patients .
all groups were comparable in age , gender , and number of immunosuppressive agents added to the corticosteroid treatment .
differences in survival could not be attributed to a higher proportion of deaths directly related to cancer between groups .
the results of this study prove the feasibility of detecting antibodies against mda5 in adult patients with dm by in - house elisa and immunoblot techniques using commercially available recombinant mda5 as the antigen .
in addition , the findings in our patients contribute to support the previously reported association of anti - mda5 antibody with rp - ild and cadm .
[ 16 ] , and the only studies performed in a white population have come from the united states [ 79 ] .
our results in a large series of mediterranean patients from a single reference center , together with those from other articles published on this topic , [ 18 ] indicate that anti - mda5 antibodies may be a hallmark of adult cadm patients with rp - ild regardless of their origin .
furthermore , some authors have suggested an association between this autoantibody and a specific , severe skin vasculopathy in adult dm , characterized by vascular fibrin deposition with variable perivascular inflammation [ 3 , 7 , 8 ] .
we found an association only with panniculitis , one of the mucocutaneous findings previously described by fiorentino et al . in white adults with dm and anti - mda5 antibody .
although we routinely test all serum samples from patients with inflammatory myopathies by immunoprecipitation assays using s - protein - labeled hela cells , until the development of our proposed method , we had not been able to clearly identify patients with anti-140 kda antibodies ( anti - cadm-140 ; i.e , anti - mda5 ) . in our analyses , this polypeptide migrated to an area in which almost 70% of sera , including those from normal subjects , immunoprecipitated a weak line around 140 kda .
[ 1 , 6 ] , and we tested these patients with a commercially available recombinant mda5 by our in - house elisa and immunoblot techniques , we were unable to recognize this autoantibody .
our first patient with cadm and rp - ild died nearly 20 years ago , and anti - mda5 was detected in a stored frozen serum sample by our in - house techniques .
thus , these methods could represent a significant advancement in identification of this autoantibody , even in laboratories with standard equipment .
anti - ro52 antibodies were present in most of our patients with rp - ild and anti - mda5 , an association that has only recently been reported in anti - mda5-positive ild patients .
this fact confers relevant significance on anti - ro52 as a costimulatory autoantibody , a concept reported in patients with antisynthetase syndrome [ 21 , 22 ] .
one patient was positive to both anti - mda5 and anti - tif1. to our knowledge , this is the first description of this association .
coexistence of two different myositis - specific antibodies has been rarely reported [ 19 , 23 ] .
the clinical course of anti - mda5-positive dm patients can be divided into three groups . first ( and most important from the prognostic perspective ) , is the group of patients with cadm and rp - ild , who usually have a poor prognosis and a mortality rate of nearly 50% despite aggressive immunosuppressive therapy and even lung transplantation .
second , the group with cadm and little lung involvement , who show skin manifestations , such as ulcerations , palmar pustules , and perhaps panniculitis , as was reported here .
the prognosis does not seem to be unfavorable in this group . and finally , the third group of patients , who have ild that is not rapidly progressive and shows a disease pattern similar to that of classic antisynthetase syndrome .
differences in the prognosis between the first and the third group may be due to genetic background , and either early immunosuppressive therapy or use of certain drugs , such as calcineurin inhibitors .
the existence of a clinical syndrome of rapidly progressive ild ( usually in antisynthetase - negative patients ) was recognized some years ago , but the absence of useful biomarkers made it difficult to characterize these patients .
the discovery of anti - mda5 antibodies will help to better define this population , facilitate an early diagnosis , establish the prognosis , and ultimately enable the development of randomized clinical trials to determine the optimal therapy in anti - mda5-positive patients with a poor prognosis .
moreover , as it has been recently described , anti - mda5 antibody measurement seems to be useful for monitoring disease activity [ 13 , 26 ] .
as our results show , elisa confirmed by immunoblot with commercially available recombinant mda5 antigen are useful techniques for anti - mda5 detection that can be reliably performed in a standard laboratory setting , with potential application in clinical practice . | a new myositis - specific autoantibody directed against melanoma differentiation - associated gene 5 ( anti - mda5 ) has been described in patients with dermatomyositis ( dm ) .
we report the clinical characteristics of patients with anti - mda5 in a large mediterranean cohort of dm patients from a single center , and analyze the feasibility of detecting this autoantibody in patient sera using new assays with commercially available recombinant mda5 .
the study included 117 white adult patients with dm , 15 ( 13% ) of them classified as clinically amyopathic dermatomyositis ( cadm ) .
clinical manifestations were analyzed , with special focus on interstitial lung disease and its severity .
determination of anti - mda5 antibodies was performed by a new elisa and immunoblot technique . in sera , from 14 ( 12% ) dm patients ( 8 cadm ) , mda5 was recognized by elisa , and confirmed by immunoblot .
eight of the 14 anti - mda5-positive patients ( 57.14% ) presented rapidly - progressive interstitial lung disease ( rp - ild ) versus 3 of 103 anti - mda5-negative patients ( 2.91% ) ( p < 0.05 ; or : 44.4 , 95% ci 9.3212 ) .
the cumulative survival rate was significantly lower in anti - mda5-positive patients than in the remainder of the series ( p < 0.05 ) .
patients with anti - mda5-associated ild presented significantly lower 70-month cumulative survival than antisynthetase - associated ild patients . among the cutaneous manifestations ,
only panniculitis was significantly associated with the presence of anti - mda5 antibodies ( p < 0.05 ; or : 3.85 , 95% ci 1.1113.27 ) .
these findings support the reliability of using commercially available recombinant mda5 for detecting anti - mda5 antibodies and confirm the association of these antibodies with rp - ild in a large series of mediterranean patients with dm . | 1. Introduction
2. Patients and Methods
3. Results
4. Discussion |
PMC4877408 | paenibacillus antibioticophila strain gd11 (= dsm 28228 = csur p1358 ) is the type strain of paenibacillus antibioticophila sp .
nov . it is a gram - positive , aerobic , indole - negative rod - shaped bacterium isolated as part of a culturomics study , .
this bacterium was isolated from a 63-year - old woman with multidrug - resistant tuberculosis who was receiving a broad - spectrum antibiotic regimen at the time of stool collection , as recently reported .
the current classification of prokaryotes is based on a combination of phenotypic and genotypic characteristics , which includes 16s rrna gene phylogeny , g+c content and dna dna hybridization .
although these tools are considered to be the reference standard , they have several pitfalls , . as a result of the declining cost of sequencing ,
there has been rapid growth in the number of bacterial genomes being sequenced , and we thus recently proposed to add genomic information to phenotypic criteria for the description of new bacterial species , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . the genus paenibacillus initially included gram - variable , facultative anaerobic endospore - forming bacteria that were reclassified as a separate genus in 1993 after being grouped with the genus bacillus . to date , the genus consists of 170 described species and four subspecies that have been isolated from a variety of environments such as soil , water , rhizosphere , vegetable matter , forage and insect larvae , , .
one paenibacillus species was cultured for the first time from human faeces as a part of a culturomics study . here
we present a summary of the classification and set of features for paenibacillus antibioticophila sp .
a stool sample was collected from a 63-year - old woman with a pulmonary form of multidrug - resistant tuberculosis .
the study was approved by the ethics committee of the institut fdratif de recherche ifr48 , faculty of medicine , marseille , france , under agreement 09 - 002 .
strain gd11 ( table 1 ) was isolated in march 2012 by cultivation on 5% sheep 's blood agar in aerobic conditions at 37c after a 21-day preincubation in a blood culture bottle with sterile cow rumen fluid and sheep 's blood .
strain gd11 exhibited a 97.6% 16s rrna sequence identity with p. puldeungensis ( genbank accession no .
nr117451 ) , the phylogenetically closest bacterial species with standing in nomenclature ( fig . 1 ) .
this value was lower than the 98.7% 16s rrna gene sequence threshold recommended by stackebrandt and ebers to delineate a new species without carrying out dna - dna hybridization .
growth at different temperatures ( 25 , 30 , 37 , 45 and 56c ) was tested ; no growth was observed at 45c or 56c .
enriched columbia agar ( biomrieux , marcy letoile , france ) under anaerobic and microaerophilic conditions using the genbag anaer and genbag microaer systems , respectively ( biomrieux ) , and under aerobic conditions , with or without 5% co2 .
cells grown on agar were soft and translucent after 24 hours and had a mean width of 0.49 m and mean length of 2.67 m ( fig .
using an api zym strip ( biomrieux ) , positive reactions were observed for esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase and -glucosidase . using rapid id32a ,
positive reactions were observed for -glucosidase , -arabinosidase , -glucuronidase , n - acetyl--glucosaminidase , nitrate reduction , glutamic acid decarboxylase , fermentation of mannose and raffinose . using an api 50 ch strip ( biomrieux ) ,
positive reactions were recorded for esculin hydrolysis and fermentation of l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - rhamnose , d - mannitol , n - acetylglucosamine , amygdalin , arbutin , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , inulin , d - melezitose , d - raffinose , starch , glycogen and d - lyxose . using an api zym strip ( biomrieux ) , negative reactions were observed for acid phosphatase , alkaline phosphatase , leucine arylamidase , valine arylamidase , cystine arylamidase , lipase ( c14 ) , trypsin , -chymotrypsin , -glucosidase , -mannosidase , and -fucosidase . using rapid api 32a , negative reactions were observed for arginine dihydrolase , urease , production of indole , leucine arylamidase , histidine arylamidase , phenylalanine arylamidase , tyrosin arylamidase , alanine arylamidase -mannosidase , -glucosidase , -fucosidase . an api 50 ch strip ( biomrieux ) , negative reactions were recorded for fermentation of erythritol , d - arabinose , l - xylose , d - adonitol , l - sorbose , dulcitol , inositol , d - sorbitol , xylitol , d - turanose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate , potassium 5-ketogluconate and potassium-5-ketogluconate .
clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim sulfamethoxazole , but resistant to metronidazole . by comparison with p.
puldeungensis strain cau 9324 , its phylogenetically closest neighbor , p. antibioticophila differed in alkaline phosphates , acid phosphatase , oxidase and -glucosidase ( table 2 ) .
matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof ) protein analysis was carried out as previously described . briefly , using a pipette tip , one isolated bacterial colony from a culture on agar plate was transferred and spread as a thin film on a msp 96 maldi - tof target plate ( bruker daltonics , leipzig , germany ) .
each smear was overlaid with 2 l of matrix solution ( saturated solution of alpha - cyano-4-hydroxycinnamic acid ) in 50% acetonitrile , 2.5% trifluoroacetic acid , and dried for 5 minutes .
microflex spectrometer ( bruker ) was used for measurements , and spectra were then recorded in the positive linear mode for the mass range of 2000 to 20 000 da ( parameter settings : ion source 1 ( isi ) , 20 kv ; is2 , 18.5 kv ; lens , 7 kv ) .
the 20 gd11 spectra were imported into maldi biotyper 3.0 software ( bruker ) and analysed by standard pattern matching ( with default parameter settings ) against the main spectra of 7379 bacteria , including 129 spectra from 70 paenibacillus species .
the method of identification included the m / z from 3000 to 15 000 da .
a maximum of 100 peaks were compared with spectra in the database for every spectrum .
the resulting score enabled the identification of tested species ( or not ) : a score of 2 with a validly published species enabled identification at the species level ; a score of 1.7 but < 2 enabled identification at the genus level ; and a score of < 1.7 did not enable any identification .
no significant maldi - tof score was obtained for strain gd11 against the bruker database , suggesting that our isolate was not a member of a known species .
finally , the spectral differences with other members of the genus paenibacillus were highlighted by gel view ( fig .
as part of a culturomics study isolating all bacterial species from the human digestive flora from patients with multidrug - resistant tuberculosis and treated with broad - spectrum antibiotics , this organism was isolated and selected for sequencing on the basis of its phenotypic differences , phylogenetic position and 16s rrna sequence similarity to other members of the genus paenibacillus .
the genbank bioproject number is prjeb1962 and consists of 131 large contigs in nine scaffolds .
table 3 shows the project information and its association with minimum information about a genome sequence ( migs ) 2.0 compliance .
paenibacillus antibioticophila strain gd11 (= dsm 28228 = csur p1358 ) was cultured aerobically on columbia agar ( biomrieux ) .
a total of 200 l of bacterial suspension was diluted in 1 ml tris - edta ( te ) buffer for lysis treatment . after a lysozyme incubation of 30 minutes at 37c ,
the lysis was performed with laurylsarcosylby 1% final and rnasea treatment at 50 g/l final concentration during 1 hour at 37c , followed by an overnight proteinase k incubation at 37c.the dna was purified three times by phenol
the concentration was measured by the quant - it picogreen kit ( invitrogen ; life technologies , carlsbad , ca , usa ) on the genios_tecan fluorometer at 48 ng/l .
genomic dna of paenibacillus antibioticophila strain gd11 was sequenced on 454_roche_titanium ( roche , basel , switzerland ) .
this project was loaded twice on a region for the 5 kb insert libraries on ptp picotiterplates .
the library was constructed with 2.5 g of dna according to the 454_roche_titanium paired end protocol and manufacturer .
it was mechanically fragmented on the covaris device ( kbioscience lgc genomics , teddington , uk ) through minitube - red 5 kb .
the dna fragmentation was visualized through the agilent 2100 bioanalyzer on a dna labchip 7500 , with an optimal size of 4.7 kb .
circularization and nebulization were performed and generated a pattern with optimal at 490 bp .
after pcr amplification through 17 cycles followed by double size selection , the library was then quantified on the quant - it ribogreen kit ( invitrogen ) on the genios_tecan fluorometer at 759 pg/l .
the 5 kb paired end library was clonal amplified with 0.25 and 0.5 cpb in 4 empcr reactions per condition , with the gs titanium sv empcr kit ( lib - l ) v2 .
the yield of the empcr was 5.21% and 5.69% , respectively , according to the quality expected by the range of 5 to 20% from the roche procedure .
twice , 790 000 beads were loaded on the gs titanium picotiterplates ptp kit 70 75 and sequenced with the gs titanium sequencing kit xlr70 .
the runs were performed overnight and then analysed through the gsrunbrowser and newbler assembler _ roche .
the global 411 382 passed filter sequences generated 150.40 mb with a length average of 354 bp .
these sequences were assembled on the gsassembler from roche with 90% identity and 40 bp as overlap .
it led to nine scaffolds and 131 large contigs ( > 1500 bp ) , generating a genome size of 5.6 mb , which corresponds to a coverage of 19.96 genome equivalent .
open reading frames ( orfs ) were predicted using prodigal with default parameters , but the predicted orfs were excluded if they were spanning a sequencing gap region .
the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups ( cogs ) database using blastp .
the trnascanse tool was used to find trna genes , whereas ribosomal rnas were found by using rnammer and blastn against the genbank database .
lipoprotein signal peptides and the number of transmembrane helices were predicted using signalp and tmhmm , respectively .
orfans were identified if their blastp e value was lower than 1e-03 for alignment length greater than 80 aa .
if alignment lengths were smaller than 80 aa , we used an e value of 1e-05 .
here , we compared the genome sequence of p. antibioticophila strain gd11 with those of paenibacillus barengoltzii strain g22 ( genbank accession no .
assd00000000.1 ) , which were identified using the proteinortho software ( version 1.4 ) using a 30% protein identity and 1e-05 e value .
the average percentage of nucleotide sequence identity ( table 4 ) between corresponding orthologous sets was determined using the needleman - wunsch algorithm global alignment technique .
artemis was used for data management , and dna plotter was used for visualization of genomic features .
the genome of p. antibioticophila strain gd11 is 5 562 631 bp long with a 49.1% g+c content ( fig .
6 ) . of the 5155 predicted genes , 5084 were protein - coding genes and three were rnas .
three rrna genes ( one 16s rrna , one 23s rrna and one 5s rrna ) and 68 predicted trna genes were identified in the genome . a total of 3814 genes ( 73.98% ) were assigned a putative function .
the properties and the statistics of the genome are summarized in table 5 , table 6 .
the genome of p. antibioticophila strain gd11 was compared to each of paenibacillus barengoltzii strain g22 ( genbank accession no .
the draft genome of p. antibioticophila has a larger size than that of p. barengoltzii , p. sanguinis and p. zanthoxyli ( 5.56 , 4.75 , 4.8 and 5.05 mb , respectively ) .
the g+c content of p. antibioticophila is higher than those of p. massiliensis , p. panacisoli , p. polymyxa , p. senegalensis and p. terrae ( 49.1 , 48.5 , 48.3 , 44.9 , 48.2 and 46.8% , respectively ) but less than that of p. barengoltzii , p. sanguinis and p. zanthoxyli ( 51.9 , 49.3 and 50.9% , respectively ) . the gene content of p. antibioticophila is larger than those of p. barengoltzii , p. sanguinis , p. senegalensis and p. zanthoxyli ( 5155 , 4394 , 4209 , 4422 and 4878 , respectively ) .
however , the distribution of genes into cogs categories was similar in all nine compared genomes ( fig .
in addition , p. antibioticophila shared 5084 , 4307 , 5055 , 5059 , 5068 , 4093 , 4278 , 5525 and 4676 orthologous genes with p. barengoltzii , p. massiliensis , p. panacisoli , p. polymyxa , p. sanguinis , p. senegalensis , p. terrae and p. zanthoxyli , respectively ( table 6 ) .
finally , no sequences coding for nonribosomal peptide synthetases or polyketide synthases were found within the p. antibioticophila genome .
the analysis of virome revealed the presence of one intact phage of 20.8 kb , and other three incomplete phages , of , respectively , 17 , 15.4 and 14.3 kb with 43.96 , 50.60 and 49.84% g+c content ( table 7 ) .
on the basis of phenotypic , phylogenetic and genomic analyses , we formally propose the creation of paenibacillus antibioticophila sp .
the strain has been isolated from the stool sample of a 63-year - old woman with multidrug - resistant tuberculosis in marseille , france .
several other previously undescribed bacterial species were also cultivated from different faecal samples through diversification of culture conditions , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , thus suggesting that the human faecal flora of humans remains partially unknown .
paenibacillus antibioticophila strain gd11 (= dsm 28228 = csur p1358 ) is the type strain of the genus paenibacillus .
it was isolated from the stool samples of a 63-year - old woman with a pulmonary form of multidrug - resistant tuberculosis hospitalized in an infectious diseases ward in marseille , france .
the main scope of the culturomics study is to cultivate all the species within the human faeces .
p. antibioticophila is a gram - positive bacilli that does exhibits neither catalase nor oxidase activity .
colonies were 0.5 mm in diameter , and cells have a mean width of 0.49 m and a mean length of 2.67 m .
esterase ( c4 ) , esterase lipase ( c8 ) , naphthol - as - bi - phosphohydrolase , -galactosidase , -galactosidase and -glucosidase , -arabinosidase , -glucuronidase , n - acetyl--glucosaminidase , nitrate reduction , glutamic acid decarboxylase , fermentation l - arabinose , d - ribose , d - xylose , methyl-d - xylopranoside , d - galactose , d - glucose , d - fructose , d - mannose , l - rhamnose , d - mannitol , n - acetylglucosamine , amygdalin , arbutin , salicin , d - cellobiose , d - maltose , d - lactose , d - melibiose , d - saccharose , d - trehalose , inulin , d - melezitose , d - raffinose , starch , glycogen and d - lyxose were positive . acid phosphatase , alkaline phosphatase , leucine arylamidase , valine arylamidase , cystine arylamidase , lipase ( c14 ) , trypsin , -chymotrypsin , -glucosidase , -mannosidase , -fucosidase , arginine dihydrolase , urease , production of indole , leucine arylamidase , histidine arylamidase , phenylalanine arylamidase , tyrosin arylamidase , alanine arylamidase -mannosidase , fermentation erythritol , d - arabinose , l - xylose , d - adonitol , l - sorbose , dulcitol , inositol , d - sorbitol , xylitol , d - turanose , d - tagatose , d - fucose , l - fucose , d - arabitol , l - arabitol , potassium gluconate , potassium 2-ketogluconate , potassium 5-ketogluconate and potassium-5-ketogluconate were negative .
clavulanic acid , ceftriaxone , imipenem , vancomycin , rifampicin , erythromycin , gentamicin , ciprofloxacin and trimethoprim sulfamethoxazole and resistant to metronidazole .
the 16s rrna gene sequence and whole - genome shotgun sequence of p. antibioticophila strain gd11 is deposited in genbank under accession number kc158472 . | paenibacillus antibioticophila strain gd11 t sp . nov . is the type strain of a new species within the genus paenibacillus .
this strain , whose genome is described here , was isolated from human faeces of a 63-year - old woman with multidrug - resistant tuberculosis who was receiving numerous antibiotics at the time of stool collection .
p. antibioticophila is a gram - positive aerobic bacterium .
we describe here the features of this bacterium , together with the complete genome sequence and annotation .
the 5 562 631 bp long genome contains 5084 protein - coding and 71 rna genes . | Introduction
Organism information
Extended features descriptions
Genome sequencing information
Genome properties
Insights from genome sequence
Description of |
PMC3963722 | there are few issues in medicine less controversial than treatment of acute myocardial infarction . unlike in stable coronary syndromes
there is no discussion whether patients should be better off in terms of prognosis and long term relief of symptoms after bypass surgery or coronary angioplasty or simply remaining on medical therapy , a difficult decision in individual cases that often leads to fierce discussions in our multidisciplinary meetings . for primary angioplasty
there is unanimous consent that a direct immediate mechanical opening of the occluded artery with angioplasty is preferable to fibrinolysis and far preferable to no reperfusion treatment , as often occurs when patients have contraindications to fibrinolysis .
this article reports the indications coming from guidelines supported by large trials , meta - analysis and large registries and reviews the reasons why this clear message has not led to a generalised application in most european countries , addressing the changes in clinical practice required to implement this winning strategy .
the first esc guidelines clearly indicating that primary angioplasty was the treatment of choice when performed in good time , by an experienced team , were published in 2003 .
retrospectively , we can ask ourselves whether it was timely enough since the first two large randomised trials using streptokinase . or rtpa .
were published 10 years before , followed by a compelling meta - analysis of more than 3000 patients showing advantages in mortality .
we must remember that at the time there was still heated discussion on the value of third generation fibrinolytics and questions coming from poorly interpreted registry data .
the 2003 guidelines should be considered as highly innovative , because they gave the strongest possible recommendation ( class i , level of evidence a ) to a practice that many still felt to be inapplicable in most situations . in countries with an advanced health service
it was seen as a distraction , or as an excuse to delay or prevent the application of fibrinolysis , the other treatment truly available everywhere within minutes from diagnosis . with minimal changes in subsequent updates ,
we must gratefully thank professor van der werf and his coauthors for having given an official approval to a therapy still felt experimental , or not practically applicable , by many at the time .
this guideline faced the dilemma of defining an acceptable time delay of pci in order to remain superior to fibrinolysis .
the issue is complicated by the basic knowledge that a minimal delay in the first hours after symptom onset causes much greater damages than the same delay after 6 or 9 h. an incorrect interpretation of some early pci trials suggested similar mortality , irrespective of the time delay after symptom onset when pci was used , a very different outcome than after fibrinolytics , that also become less effective on old thrombi .
more recently this mistake has been corrected , based on the evidence of a worsening in outcome according to the time delay between symptoms onset and pci , more evident in high - risk patients .
the 2008 guidelines have identified a cohort of patients with large anterior infarctions and low bleeding risk in whom a lower threshold ( no more than 90 minute delay ) should be used , while the general time delay has been prolonged to 2 h , including also the delay between first medical contact and the transport to the hospital door . despite its clear formulation , the application of primary angioplasty has been very slow , to the point that an immediate past - president of the esc , jean pierre bassand , felt the need to create a task force for promoting its implementation .
the results of the survey , conducted in 2005 , observed a large gap between guideline recommendations and clinical practice , with none of the large european countries having greater than 50% application of primary angioplasty .
the treatment looked suitable for small , well - organised countries such as the netherlands , belgium , czech republic or switzerland , but impractical elsewhere .
this was the driving force of the first stent for life ( sfl ) initiative and a repeated survey led by petr widimsky in 2008 , showed that germany , poland , sweden , hungary , croatia , slovenia and others had come on board and much progress was made in france and italy too .
no data could be obtained on the quality of the pci performed and , in particular , on the respect of the 90-minute door - to - balloon time suggested at the time . a large
, ongoing survey is being coordinated by dr kristensen on behalf of the sfl group and data are expected to be concluded in 2013 .
a major increase in penetration of primary angioplasty will emerge , probably showing the fastest pace of progress since its introduction .
the biggest positive surprise is certainly the uk and we will not express more than admiration for a stunning performance moving from < 40% in 2006 to > 90% pci in 2011 .
the uk has a unified national health care system and decisions taken in whitehall apply to the entire country , a situation very different from italy or france , where the patchy application mirrors different regional directives .
hospitals in the uk respond to authorities with the power to decide which hospital should perform pci and regulate patient transfer accordingly .
secondly , the uk had already developed an excellent system to timely administer fibrinolysis based on dedicated nurses in a&e .
the system worked equally well when these resources have been allocated upstream , in the ambulance , with the personnel performing and interpreting the ecg and informing the closest primary pci centre of the arrival of a stemi patient .
thirdly , auditing was fully implemented via the minap acs and bcis pci databases , offering the advantage of constant monitoring of quality indicators such as time delays and outcomes such as mortality .
this advantage is shared with sweden and it is interesting to know that data from both countries indicate impressive mortality benefit after the implementation of the universal primary pci program , together with cost reduction mainly generated by shorter hospital stays and immediate patient triage .
equally impressive results have been achieved in most of the 13 countries participating to the sfl initiative .
it is obviously impossible to distinguish the natural process of diffusion of a new therapeutic treatment from the additional push the participation of sfl gave to the program . in italy and spain , wide regional differences
remain , with large regions such as lombardy in italy and catalonia in spain close to universal application of primary angioplasty and others much further behind .
similar considerations can be repeated for france with the additional complication in interpreting figures that pre - hospital thrombolysis is still occasionally applied in the country .
it is seen not as an alternative to , but as a preparation for , angioplasty .
characteristics specific to the country are the reluctance of the samu , the very efficient equipped ambulances with an anaesthetist on board , to play a passive role simply shipping the patient to the closest angioplasty centre where mechanical recanalization is performed .
, at odds with all the other thrombolysis vs primary pci randomised comparisons because they saw superiority of thrombolysis within 2 h from symptoms onset must be interpreted in the context of a very atypical 80% use of angioplasty in the hours after fibrinolysis . unlike in assent iv .
there were no signals of higher morbidity and mortality when pci immediately followed the administration of lytics , possibly because of the timely use with fibrinolysis of dual antiplatelet treatment . unlike almost all the other trials of facilitated pci ( finesse . ,
etc ) there was no excess of bleeding , possibly because of the frequent adoption of a radial approach .
the real surprise , however , comes from the other countries participating in the sfl program , starting from a very low penetration and expected to be limited by an insufficient number of 24/7 primary pci centres and equipped mobile units for pre - hospital diagnosis .
serbia , bulgaria , romania , turkey made impressive steps forward , thanks to the ability of their country champions to make primary angioplasty an absolute priority in receiving ample resources to meet the goals .
the process of integration between ems and 24/7 hospitals to provide a seamless delivery of care throughout a geographical area irrespective of the time of the day , or day of the week , was already strongly advocated in the 2008 stemi guidelines .
this point acquired dignity of a specific recommendation , however , only in the 2010 esc myocardial revascularisation guidelines . and in the last update of the acc / aha / scai stemi guidelines . again a class 1 level of evidence a was indicated , based on large series showing how individual changes in service organisation have impacted morbidity and mortality .
the principle is the identification of a geographical area where delivery of care for stemi patients is provided by a single ems system , connected to well - identified 24/7 primary centres .
the practical application sees enormous differences from country - to - country and region - to - region , in terms of dimension of population served , organisation of ems service and modalities of offer of the primary angioplasty service .
first medical contact : a unified telephone number for emergency calls with an operator promptly available and able to identify symptoms possibly related to acute myocardial infarction , is the first essential requirement for a timely delivery of p - pci . in europe
there is large variability in the education of the public to use such an emergency service , which is key to ensure the smoothest and most rapid access to primary angioplasty .
occasionally patients rely on their gp and waste precious time to wait for him to be available to give advice , or come to see them .
more often , relatives or neighbours believe they can speed up diagnosis and treatment by bringing the patient directly to the a&e .
this is not only a dangerous exercise because of the possible complications arising en route , but also hardly the best way to immediately get the full attention of overcommitted staff in a crowded a&e department .
it is almost certainly bound to lead to delays when compared to the ideal path described below .
the operator on the line will probably have the most difficult job in the many steps leading to a successful recanalization : identify the symptoms of an acute cardiac ischaemic syndrome from the broken voice of an emotional relative or from the words of an old patient trying to minimise the severity of his / her symptoms to the outside world .
constrictive chest pain with classical irradiation lasting more than 10 minutes is not the way symptoms always manifest . as shown in the article by chieffo et al .
older women often only complain of the dyspnoea secondary to left ventricular failure , and many patients have pain limited to the jaw or the epigastrium and/or profuse vomiting and diaphoresis .
all these symptoms , especially when they develop in a patient within an age group at risk , or in a patient with clear risk factors for cad , or with known cardiac pathology , should trigger the dispatch of an ambulance with the equipment and personnel able to perform and interpret / transmit an ecg , perform resuscitation , transfer him if needed to the closest primary pci centre available , not stopping in the a&e but bringing the patient directly to the cath lab .
emergency medical system : the organisation of an efficient , yet affordable , emergency system is a complex task .
the guidelines stress the word integration , which means that the intensive care specialists , nurses and drivers of the ambulance offering first physical contact with a doctor or a paramedic after the phone call , should feel like part of the team involved in a very special diagnosis and treatment facing one of the relatively few medical emergencies where a timely and appropriate treatment can make the difference between life and death .
after having checked vital signs and pressure , when the suspect of an stemi is present , an ecg should be performed immediately and the interpretation obtained either directly by the ambulance personnel , using clear thresholds of abnormality to trigger a primary pci call , or via teletransmission to a dedicated specialist giving feedback as a first priority .
there are advantages in having an expert central operator coordinating the entire metropolitan area , because he is aware of possible unusual circumstances ( road blocks , unavailability of the closest centre engaged in other calls ) and may suggest transfer to a different primary pci centre . if the ambulance personnel has an appropriate background knowledge , or has attended a well - structured training course on ecg interpretation targeted to the recognition of st - segment elevation , then this is a simple , but appealing , alternative and ensures the full attention of the ambulance crew towards this demanding choice .
the experience in london is that courses organised and repeated in the main p - pci hospitals achieve their educational goal and help in creating the team approach and the strive for excellence these sick patients desperately need .
a correct stemi diagnosis of 94% , as achieved in london , is even more valuable when you consider that most of the other diagnoses ( pericarditis , aortic dissection , etc ) still require urgent cardiological input . besides pain control
, many other treatments can be started in the spacious modern well - equipped ambulances , provided protocols are well defined and the ambulance personnel is authorised to administer drugs . while nobody will argue for aspirin , decisions on the association of clopidogrel , prasugrel or ticagrelor and the use of pre - hospital thrombolysis when the anticipated delay is greater than 2 h , are obviously dependent on the duration of the transfer .
24/7 primary angioplasty hospital : in principle , the population served must be sufficient to maintain competency of the primary angioplasty centre .
there has been a progressive and , at times , rapid decrease in the incidence of stemi , attributed to better control of risk factors
.
the decline of stemi is a quite recent phenomenon and is probably related to the almost complete abolition of the smoking habit in high - risk groups in some european countries .
early recognition and treatment of new onset angina and nstemi is probably equally responsible , but more difficult to quantify .
epidemiology of acute coronary syndrome ( acs ) is outside the scope of this paper , but it is important to realise that these rapid changes require equally rapid adjustments . a gross estimate of 1000 stemi per million inhabitants was initially used as average european prevalence to set the goal of the stent for life initiative to offer primary angioplasty to at least 600 patients out of a million inhabitants served .
this estimate is probably applicable only in some eastern european countries such as ukraine or russia .
northern european countries , with a historically very high prevalence of coronary artery disease ( cad ) , see only pockets of epidemics in more deprived areas , while densely - populated regions such as south - east england are down to 500 - 600 stemi cases / million / year .
the rapid rise of obesity and diabetes limits the contraction of cad and determines a change in the groups at risk with women > 70 years of age becoming a more frequent target .
assuming an average stemi population of 500 patients / million inhabitants which is probably closer to the current incidence in many european countries , a single hospital providing service should not have less than 250300,000 inhabitants in its served territory , in order to be able to provide 125150 stemi cases per year , as in some lucky areas of southern europe such as catalonia or some regions in southern france . in rural or mountainous areas lower basins can be an acceptable choice .
the esc guidelines do not indicate minimal numbers to maintain competency of hospital and operators , and this is probably a reflection of the extreme geographical variations across europe that makes difficult to set meaningful thresholds .
are of help but their targets are also very loose , with a minimum of 36 primary pci / year to maintain competency as a centre .
more importantly , they reaffirm that a primary pci centre should have a reasonable volume of at least 400 pcis / year with each operator performing a minimum of 75 pci / year .
these are numbers nobody can argue with : a pci centre with a volume below 400 cases / year makes no sense economically and is unlikely to offer adequate safety to patients when its personnel is exposed to an average of < 8 pci / week .
this is especially so when the patient in question has an occluded artery that requires rapid recanalization and is prone to arrhythmias and haemodynamic complications .
also the number per operator of 75 pci / year ( all pcis , not only primary angioplasties ) fits well these calculations because you need a minimum of 4 to 6 operators to maintain a sustainable rota and 400 - 450 pcis are sufficient to maintain this level , if evenly split among the operators . if this is a meaningful general rule , exceptions have to be accepted and sometimes encouraged .
the operators ' and possibly the paramedics ' competency can be maintained performing angioplasties in a different centre and you may accept a centre with a suboptimal number of 75100 primary angioplasties / year if this is the only solution to ensure a timely offer of primary angioplasty in a homogeneous region sufficiently remote and this is compatible with the economic resources available for the health system .
where possible , however , and this includes all the urban / metropolitan areas of europe and probably 2/3 of its population , the encouragement should be to move in the opposite direction and encourage the development of large primary angioplasty hubs with 300 - 500 patients / year .
obviously , there are no possible randomised trials comparing outcomes in low volume and high volume centres but all data coming from matched comparisons suggest success increases and complications fall in larger volume centres .
clearly there is an inherent cost in maintaining an on - call service which is relatively inactive , and this also plays a role in suggesting to limit the number of active primary pci centers .
impersonal service , with the operator seen during the acute treatment not showing up thereafter , and occasional instances of excessive pressure to retransfer or discharge the patient are potential drawbacks to consider . much more difficult to accept , is that the hard work of the ems to quickly bring the patient for primary angioplasty is wasted because of unacceptable delays to the cath lab because of several simultaneous cases of primary angioplasty .
this is a problem likely to occur especially at nights or during weekends if only one lab is used for primary angioplasty .
a good balance can be maintained when various centres are active within the same metropolitan areas and there is a central allocation unit able to accommodate patients to the nearest hospital with a cath lab available to immediately start angioplasty .
there is great creativity in the way the primary angioplasty service is organised across europe and the solutions are often a compromise to satisfy all of the hospitals involved , not necessarily those offering the most efficient or cost - efficient solution . in most european cities , all centers able to offer a round the clock service are allowed to offer primary angioplasty .
the advantage is that ambulances are spoilt with choices and can shorten the drive to the hospital and the competition creates an incentive to raise standards .
the disadvantage is that this often drives numbers below the minimal acceptable figures indicated above .
a solution which is possibly acceptable in remote areas , but not in the heart of a large city .
rotation of the hospitals on call for providing service is a possible alternative to maintain all hospitals involved , but is applicable only when the hospitals in question are strategically placed to meet the needs and not creating another problem of insufficient experience being built up in some of the participating centres . in order to maintain an active 24/7 service , centres may need to attract operators not routinely working in the hospital , which may create difficulties dealing with acute cases in a foreign environment .
provisions after angioplasty : primary angioplasty has drastically changed the treatment and prognosis of stemi patients . the risks of acute arrhythmias and late ventricular tachycardia , mechanical complications , pericarditis and dressler 's syndrome , haemodynamic compromise , recurrence of angina or reocclusion have all become rare events .
patient triage is performed during primary angioplasty and patients with multivessel disease are identified and scheduled for further treatment .
the use of radial approach and the elimination of the most heroic antithrombotic and antiplatelet cocktails , substituted by weight - adjusted doses of less dangerous drugs , potentially allow rapid transfer after p - pci .
transfer can be positive when it offers access to departments where the patient completes up - titration of beta - blockers and ace - inhibitors , receives detailed instructions on diet , pressure and cholesterol control , and the importance of physical activity .
transfer can help to balance patient flow , compensating hospitals that have accepted not to promote p - pci facilities because they are redundant for the territory in question or they have transferred the patient after having performed diagnosis .
conversely , staying in the same unit where angioplasty was performed , speaking with the operator and receiving reinforcement messages on the importance of double antiplatelet treatment , can work equally well or better if the situation allows .
the important message to convey is that the stent is only the first of a series of measures to implement in order to transform the risk profile .
again , multiple models have been developed , but it is essential that the patient can not be transformed into a postal package to move when and where convenient and that the patient 's safety and well - being must remain the centre of the treatment strategy .
primary angioplasty can be performed in the vast majority of patients with st elevation myocardial infarction , with the possible exception of patients living in remote regions who are unable to reach the hospital within the first 90120 minutes after diagnosis . in all other areas , covering more than 85% of the european population ,
the poor penetration of primary angioplasty is caused by the lack of a coherent organisation , revolving around a coordination centre and a network of cleverly placed primary pci hospitals . |
abstract : this manuscript is focused around two key messages from the current guidelines of the european society of cardiology on st elevation myocardial infarction ; the need to use primary angioplasty whenever timely and effectively applicable and the importance of organisational changes in the emergency system to implement this indication . based on a review of the trials motivating these guidelines and the successful experience of many european countries ,
practical indications are provided on the methods to overcome resistances and malpractices that prevent the delivery of optimal care in these critically ill patients . | Introduction
Primary angioplasty is the preferred treatment if performed by an experienced team <90 minutes after first medical contact: Class I A
Implementation of a well functioning network based on pre-hospital diagnosis and fast transport to the closest available primary PCI capable centre: Class I A
Conclusions |
PMC3092509 | toxic levels of aluminum ( al ) in acid soils inhibit root growth and cause substantial reduction in yields of al - sensitive crops [ 1 , 2 ] .
its toxicity mechanisms include interference with nutrient and water uptake and translocation , disruption of calcium homeostatis , disruption of cytoskeleton [ 5 , 6 ] , callose deposition in apoplast that affects movement of substances from cell to cell , lipid peroxidation and reactive oxygen species production , and interference with cell division and elongation [ 9 , 10 ] .
in concert , these disorders thwart root growth and development that is typically manifested in stunted and swollen root system at the morphological level [ 11 , 12 ] .
al disrupts cellular components and processes by high binding affinity to phosphate , sulfate , and carbonyl functional groups of cellular components in apoplast and symplast .
perhaps as a direct and parallel evolutionary response to the nature of al - ligand interaction , plants secret substances that possess these functional groups namely , organic acids , phenolics [ 1416 ] , and phosphate and polypeptides [ 17 , 18 ] to bind and detoxify al in the rhizosphere .
sequestration of al in the rhizosphere with root secreted organic acids mainly citrate , malate , and oxalate is a common and well - documented physiological mechanism of al - tolerance in a wide variety of plants [ 11 , 13 ] . in soybean ,
al - tolerance is a quantitatively inherited trait and , physiologically it is correlated with root secretions of citrate [ 20 , 21 ] and phenolics .
even though the physiological and cellular responses induced by al stress are direct consequences of change in gene expression and cell metabolism , the genetic components of al - tolerance pathways are not well understood .
monitoring protein expression changes under al stress is one possible way of identifying the genetic and molecular components underlying al - tolerant phenotype .
a few research groups have conducted proteome analysis in plant roots under al stress [ 2224 ] .
these studies consistently identified antioxidation and detoxification enzymes and proteins as important determinants of plant al - tolerance trait .
their limitation , however , is that nonutilized tolerant - sensitive genotype pair that would ensure the specificity of the identified proteins to al - tolerance mechanism .
we previously examined al - induced protein profile changes in roots of al - tolerant and al - sensitive soybean genotypes , but did not pursue functional annotation of the differentially expressed proteins . in the present study , we combined 2d dige differential expression , mass spectrometry , and bioinformatics tools to characterize the proteome profile changes in al - tolerant and al - sensitive soybean genotypes with the aim of identifying potential biomarkers for soybean al - tolerance .
our results suggest that organic acid biosynthesis and antioxidation and detoxification enzymes play important role in soybean al - tolerance .
two soybean genotypes pi 416937 ( al - tolerant ) and young ( al - sensitive ) were used in the study .
seeds were surface sterilized with 20% household bleach ( clorox ) in water for 12 min , rinsed with distilled - deionized water several times and were germinated in standard germination paper at 25c in an incubator for 72 h. three - day - old seedlings uniform in tap root length were transferred to black - painted pots filled with approximately 4 l of 800 m cacl2 background solution with 10 m al added ( treated ) or no al added ( control ) in a conviron growth chamber ( 16/8 h light / dark cycle with respective temp .
the ph of the culture solution was adjusted to 4.3 and maintained at that level for the entire duration of the experiment .
after 6 , 51 , or 72 h of al treatment primary roots and laterals of ( see supplementary figure 1 , available online at doi:10.1155/2011/282531 , for representative root picture ) approximately 15 plants / pot were harvested and immediately flash frozen in liquid nitrogen , stored at 70c until protein extraction .
protein was extracted using the phenol method , and protein concentrations of samples were determined using the bradford method .
protein samples were dissolved in 300 l of 2d cell lysis buffer ( 30 mm tris - hcl , ph 8.8 , containing 7 m urea , 2 m thiourea and 4% chaps ) and sonicated at 4c followed by shaking for 30 min at room temperature .
samples were then centrifuged for 30 min at 14,000 rpm and the supernatant collected . an internal standard for assay normalization
was created by mixing equal amounts of protein from each sample . for each sample ,
30 l of protein was mixed with 1.0 l of diluted cydye and kept in the dark on ice for 30 min .
proteins from the control and treated samples were labeled with cy3 and cy5 , respectively , whereas the internal standard was labeled with cy2 .
the labeling reaction was stopped by adding 1.0 l of 10 mm lysine and incubation in the dark on ice for an additional 15 min .
the labeled samples from the control and treated groups and the internal standard were blended for a run of three samples per gel .
the 2x 2d sample buffer ( 8 m urea , 4% chaps , 20 mg / ml dtt , 2% pharmalytes , and trace amount of bromophenol blue ) , 100 l of destreak solution , and rehydration buffer ( 7 m urea , 2 m thiourea , 4% chaps , 20 mg / ml dtt , 1% pharmalytes , and trace amount of bromophenol blue ) were added to the labeling mix to a total volume of 250 l .
ief ( ph 3 - 10 linear ) was run following the protocol of amersham biosciences .
after ief , the ipg strips were incubated in fresh equilibration buffer-1 ( 50 mm tris - hcl , ph 8.8 , containing 6 m urea , 30% glycerol , 2% sds , trace amount of bromophenol blue and 10 mg / ml dtt ) for 15 min with gentle shaking .
the strips were then rinsed in fresh equilibration buffer-2 ( 50 mm tris - hcl , ph 8.8 , containing 6 m urea , 30% glycerol , 2% sds , trace amount of bromophenol blue and 45 mg / ml dtt ) for 10 min with slow shaking .
afterwards , the ipg strips were rinsed with the sds - gel running buffer and transferred to 12% sds - gel .
sds - gels were run at 15c until the dye front began running out of the gel .
gel images were scanned immediately following the sds - page using typhoon trio ( amersham biosciences ) .
the scanned images were then analyzed by image quant software ( version 6.0 , amersham biosciences ) .
the fold changes of protein expression levels were obtained from biological variation analysis ( bva ) using the decyder software version 6.5 ( amersham biosciences ) .
the spots of interest were picked by ettan spot picker ( amersham biosciences ) based on the in - gel analysis and spot picking design by decyder software .
the gel spots were washed few times and digested in - gel with modified porcine trypsin protease ( trypsin gold , promega ) .
peptides were eluted from the zip - tip with 0.5 l of matrix solution of cyano-4-hydroxycinnamic acid ( 5 mg / ml in 50% acetonitrile , 0.1% trifluoroacetic acid , 25 mm ammonium bicarbonate ) and spotted on the maldi plate ( model abi 01 - 192 - 6-ab ) .
maldi - tof ms and tof / tof tandem ms / ms were performed on an abi 4700 mass spectrometer ( applied biosystems , framingham , ma ) .
maldi - tof mass spectra were acquired in reflectron positive ion mode , averaging 4000 laser shots per spectrum .
tof / tof tandem ms fragmentation spectra were acquired averaging 4000 laser shots per fragmentation spectrum on each of the 5 to10 most abundant ions present in each sample , excluding trypsin autolytic peptides and other known background ions .
the peptide mass and the associated fragmentation spectra were submitted to gps explorer workstation equipped with mascot search engine ( matrix science ) to search the database of national center for biotechnology information non - redundant protein database ( ncbinr ) .
searches were performed without constraining protein molecular weight or isoelectric point , with variable carbamidomethylation of cysteine and oxidation of methionine residues , and with one missed cleavage also allowed in the search parameters .
mass tolerances of 100 ppm for the precursor ion and 0.3 da for fragment ion were used as constraints to control false positives .
candidates with ci% greater than 95 either for protein or ion score , or e - value less than e and % identity greater than 30 when blasted against soybean protein database ( http://www.phytozome.net/soybean ) were considered significant . for peptide data summary and protein prediction significance criteria ,
three criteria were used to identify al - responsive proteins among hundreds of protein spots resolved by 2-dige gel to assure data quality : ( 1 ) expression fold change of at least 1.3 , either up- or downregulation , at least at one of the time points in al - treated versus control comparison , ( 2 ) presence in all replicates , and ( 3 ) standard error less than the mean . a total of 49 proteins in al - tolerant pi 416937 ( table 1 ) and 47 proteins in al - sensitive young ( table 2 ) met these criteria and were subsequently identified by mass spec ( see figures 1 and 2 , e.g. , 2d profile of the identified proteins ) . in pi 416937 , 1 , 29
, 30 proteins were upregulated at 6 , 51 , and 72 h , respectively ( figure 3 ) , whereas in young 4 , 7 , 36 proteins were upregulated at 6 , 51 , and 72 h , respectively ( figure 4 ) .
downregulated proteins were 5 each at 51 and 72 h in pi 416937 ( figure 3 ) and 5 , 3 , 7 in young at 6 , 51 , and 72 h , respectively ( figure 4 ) . in both genotypes
, there were more upregulated than downregulated proteins , and more proteins were detected at later stages of al stress than during the early hours of exposure to al .
there were more common proteins between time points in pi 416937 than in young ( figure 5 ) .
in young , a major change in protein profile was observed 72 h posttreatment , whereas in pi416937 major change in protein expression was observed starting at 51 h of treatment . in addition to the difference in the timing of the molecular response to al stress between the two genotypes , there was a clear difference in the identity of the expressed proteins as well .
20 ; cysteine synthase , spot no.39 ; isoflavone biosynthesis enzyme - nadph : isoflavone reductase , spot no .
. 89 ) ( table 1 ) , in the al - tolerant pi 416937 .
conversely , aluminum induced mainly non - al - specific general stress tolerance proteins in the al - sensitive young .
nonetheless , vacoular atpase ( v - atpase)a known al - tolerance transport protein ( spot no .
155 , table 2 ) was upregulated 1.3-fold at 72 h in young indicating that young may possess some basal tolerance mechanism to aluminum toxicity as suggested by bianchi - hall et al . .
the identified proteins were grouped into 13 classes based on cellular function according to the classification scheme in houston et al . .
the distribution of the proteins over functional classes revealed that al - responsive proteins in pi 416937 are more of proteins of primary metabolism , disease / defense , energy and signal transduction with 16% in the unknown class ( figure 3 ) ; and in young al - responsive proteins are more of proteins of primary metabolism , disease / defense and protein destination with 26% in the unknown class ( figure 4 ) . in discussion of this paper , the disease / defense category was renamed as antioxidation and detoxification and the energy category as organic acid biosynthesis enzymes to easily connect these proteins to physiologically known plant al - tolerance mechanisms .
al toxicity induces production of reactive oxygen species ( ros ) in cells beyond the level generated by normal metabolism as one mode of its toxicity [ 8 , 12 , 23 ] .
plants have elaborate enzymatic and nonenzymatic mechanisms for ros scavenging and variation in plant antioxidant capacity has been shown to correlate with variation in plant al - tolerance level .
several known antioxidation and detoxification enzymes including thioredoxin ( spot 15 ) , lactoyl glutathione lyase ( spot 81 ) , three isoforms of glutathione - s - transferase ( gst ) ( spots 75 , 82 , and 85 ) , dehydroascorbate reductase ( spot 89 ) , cysteine synthase ( spot 39 ) , aldo / keto reductase ( spot 43 ) and nadh : isoflavone reductase ( spot 46 ) were upregulated in al - tolerant pi 416937 ( table 1 ) .
none of these enzymes except glutathione - s - transferase ( gst ) was upregulated in al - sensitive young .
earlier proteome analysis in al - tolerant varieties of rice , soybean , and tomato [ 12 , 23 ] suggest that antioxidation and detoxification enzymes play an important role in internal al - tolerance mechanism . pi 416937 accumulates less al than young in symplast and , as revealed by the current study , appears to have better antioxidant and detoxification capacity implying that it employs both the exclusion and internal detoxification mechanisms of al - tolerance . among the antioxidation and detoxification enzymes differentially expressed between pi 416937 and young , it is interesting to highlight the al detoxification role of cysteine synthase , gst , and nadph : isoflavone reductase .
there is increasing evidence for the synergetic effect of cysteine synthase and gst in conferring plant al - tolerance [ 12 , 22 , 24 ] .
cysteine , the byproduct of cysteine synthase catalyzed reaction , is a precursor for glutathione synthesis ( gsh ) which in turn is a key substrate for biosynthesis of metallothionin ( mt ) and phytochelatin- ( pc- ) low molecular weight peptides known to confer heavy metal tolerance in plants .
mt has been reported to be induced by al stress probably as a tolerance mechanism .
in addition to its role in mt and pc biosynthesis , gsh is directly involved in detoxification of al - elicited toxins in concert with gst .
gst transfers gsh to such substances forming s - gluthionaylated reaction products that are nontoxic to the cell .
further , reduced glutathione has an antioxidant property capable of scavenging al - triggered ros .
therefore , the concomitant upregulation of cysteine synthase and glutathione - s - transferases in al - tolerant pi416937 is suggestive of the significance of antioxidation and detoxification proteins in protecting this soybean type from al - induced oxidative stress and endogenous toxins .
at the physiological level , a positive correlation between root flavonoids and phenolics secretion and plant al - tolerance has been documented [ 1416 ] .
aluminum stabilizes , depolymerizes , and alters the orientation of cytoskeleton networks resulting in inhibition of cell division , elongation and consequently root growth [ 5 , 6 , 33 , 34 ] . whether this phenomenon is a direct outcome of al - interaction with cytoskeletal structure or by its indirect influences on factors that control cytoskeletal assembly and disassembly
the expression of cytoskeleton proteins has been proposed to be one of the primary targets of al toxicity .
al - induced increase in abundance of mrna level for actin - bundling / rigidifying protein fimbrin and actin protein has been reported .
in the present study , actin protein was upregulated at 51 h of treatment in al - tolerant pi 416937 , whereas alpha tubulin was downregulated at 51 and 72 h ( table 1 ) . in al - sensitive young ,
alpha tubulin was upregulated at 51 h and downregulated at 72 h and tubulin beta-3 was upregulated at 72 h ( table 2 ) .
these results show that the two genotypes differ in expression of cytoskeletal proteins under al stress .
however , how this dynamics relates to al - induced cytoskeleton stabilization and reorientation that leads to root growth inhibition is yet to be determined .
it has been shown that al - induces mechanical rigor / stability in actin networks of soybean root cells .
thus , the upregulation of actin protein in al - tolerant pi 416937 could be an adaptive response to al stress .
new actin networks could form from newly synthesized actin protein and compensate for networks stabilized by al . apart from cytoskeleton proteins , one important protein related to cell division ,
brac1 is a dna damage and cell cycle control protein and its homolog is a breast cancer inhibitor in human .
dna damage is one mechanism of al mediated cellular injury , suggesting brac1 might be an important novel candidate biomarker for plant al - tolerance . in response to al stress ,
root accumulation and secretion of organic acids increase particularly in al - tolerant plant genotypes [ 21 , 3739 ] , which in most cases is preceded by an increase in expression and activities of enzymes involved in their biosynthesis [ 12 , 39 ] . however , the particular tca cycle enzyme(s ) activated by al and the organic acid anions modulated are species specific [ 39 , 40 ] .
for example , in al - tolerant microbe ( pseudomonas fluorescens ) that depends on oxalate for al detoxification , al reconfigures the enzymatic reactions of the tca cycle in favor of the synthesis of glyoxylate - a precursor for oxalate , by upregulating isocitrate layse and downregulating isocitrate dehydrogenase and ketoglutarate dehydrogenase complex . at the physiological level , soybean al - tolerance is correlated with increased levels of tissue citrate accumulation and secretion [ 20 , 21 ] . nonetheless , the specific tca cycle enzymes that modulate organic acid metabolism in soybean in response to al stress have not been identified .
here , we provide the first evidence at protein expression level that show al simultaneously induces enolase 1 ( spot 20 ) , malate dehydrogenase ( spot 42 ) , malate oxidoreductase ( spot 18 ) , and pyruvate dehydrogenase e1 ( spot 35 ) ( table 1 ) , in roots of al - tolerant soybean pi 416937 .
none of these enzymes was triggered by al in the al - sensitive young underscoring the specificity of the response to al - tolerance mechanism .
all of these enzymes contribute to citrate biogenesis ( figure 6 ) , which could explain the increased citrate accumulation and secretion observed in al - tolerant pi 416937 under al stress by silva et al . .
engineering enzymes of the tca cycle have been the focus of one aspect of plant al - tolerance research for over a decade .
transgenic upregulation of individual tca cycle enzymes such as malate dehydrogenase , citrate synthase and phosphoenolpyruvate carboxylase ( pepc ) have had some success in producing transgenic plants with enhanced al - tolerance [ 4144 ] .
we speculate that concomitant over - expression of several of these enzymes , as exemplified by natural plant defense response in our study , might be a plausible genetic engineering strategy for developing transgenic crops adapted to high al soils .
a net increase in cellular citrate level is a balance of its formation and degradation .
therefore , transgenic downregulation of enzymes involved in its breakdown is an alternative scenario for improving plant al - tolerance .
while there is no experimental data available regarding how such transgenic plants would perform under nonstressful environment , improved al - tolerance level has been observed in yeast mutants null for citrate degrading enzymes .
also in al - tolerant maize , pieros et al . observed al - induced reduction in the activity of isocitrate dehydrogenase ( a citrate degrading enzyme ) , which paralleled an increase in root citrate level .
perception of stress signal and subsequent changes in gene expression , cell metabolism , and physiology are key events in plant adaptation response to a stress factor .
al - signaling pathway has not been elucidated yet with the exception of the identification of one protein ( cell wall associated kinase 1 , wak 1 ) as a possible component of al signal transduction pathway .
serine / theorine protein kinase and protein phosphatase 2a , both with signaling function , were identified in the present study .
both were upregulated in al - tolerant pi 416937 and serine / theorine kinase in al - sensitive young .
earlier proteome analysis studies suggest that g - protein or gtp binding protein is involved in al signaling [ 22 , 24 ] .
no change in the expression level of this protein was observed in the present study .
however , the activity of g - protein coupled receptor is controlled by its phosphorylation state , which in turn is attenuated by protein kinase and phosphatase .
the upregulation of serine / theorine protein kinase and protein phosphatase 2a might therefore have relevance in al stress signaling .
ros signaling 14 - 3 - 3-like protein ( spots 55 & 64 ) was upregulated in pi416937 , but downregulated in young .
one transcription factor creb binding ( camp element binding ) protein ( spot 23 ) was upregulated in pi 416937 .
creb binds to cre ( camp responsive element ) and regulates the transcription of several genes .
it modulates several physiological functions including brain memory in human , but its role in plant system has not been well documented . nevertheless , the fact that it regulates the expression of several genes makes it an interesting putative gene for al stress tolerance .
these proteins are crucial for survival of biota under adverse conditions and respond to various biotic and abiotic stress factors including al .
they play a role in protein folding , translocation , degradation , and assembly both under normal and stress conditions .
stress conditions increase the activity of these proteins by altering the intricate balances in cellular protein synthesis and turnover .
aluminum induced a distinct protein profile changes in al - tolerant and al - sensitive soybean genotypes .
the al - tolerant genotype pi 416937 expressed multitude of al - tolerance - specific proteins when mildly intoxicated with al , whereas the sensitive genotype young lacks such al specific response . the comparative proteome analysis enabled us to identify two enzyme categories namely , enzymes of the antioxidation and detoxification systems and organic acids biosynthesis pathway as important players in soybean al - tolerance .
we also identified two novel proteins , brac1 : a dna damage control and cell cycle regulator , and creb : transcription factor , which might contribute to soybean adaptation mechanisms to al toxicity .
increased organic acid synthesis , accumulation , and root secretion are the well - known physiological mechanisms of plant al - tolerance . at the molecular level
, attempts to engineer al - tolerant plants by modulating the activities of individual organic acid biosynthesis enzymes have produced encouraging results [ 4144 ] . in the present study ,
we observed a concurrent upregulation of several organic acid biosynthesis enzymes in al - tolerant soybean pi 416937 in response to al stress which suggests that simultaneous transgenic over - expression of these enzymes might be a more robust genetic engineering strategy for developing al - tolerant plants than the engineering one enzyme at a time paradigm . | toxic levels of aluminum ( al ) in acid soils inhibit root growth and cause substantial reduction in yields of al - sensitive crops .
aluminum - tolerant cultivars detoxify al through multiple mechanisms that are currently not well understood at genetic and molecular levels . to enhance our understanding of the molecular mechanisms involved in soybean al tolerance and toxicity , we conducted proteomic analysis of soybean roots under al stress using a tandem combination of 2-d - dige , mass spectrometry , and bioinformatics tools and al - tolerant ( pi 416937 ) and al - sensitive ( young ) soybean genotypes at 6 , 51 or 72 h of al treatment .
comparison of the protein profile changes revealed that aluminum induced al tolerance related proteins and enzymes in al - tolerant pi 416937 but evoked proteins related to general stress response in al - sensitive young .
specifically , al upregulated : malate dehydrogenase , enolase , malate oxidoreductase , and pyruvate dehydrogenase , in pi 416937 but not in young .
these enzymes contribute to increased synthesis of citrate , a key organic acid involved in al detoxification .
we postulate that simultaneous transgenic overexpression of several of these enzymes would be a robust genetic engineering strategy for developing al - tolerant crops . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion |
PMC4311954 | the wet - chemical synthesis
of colloidal nanocrystals ( ncs ) is a
well established method providing highly monodisperse quantum dots for applications such as bright and stable fluorophores . among these ,
vi lead chalcogenides ( pbx , x = s , se ,
or te ) ncs provide efficient emission over a large spectral range
in the infrared .
they are promising for applications , e.g. , where
pbs ncs act as optical active materials in organic photodiodes for
the near - infrared .
the applications for
pure lead salt ncs are limited , however , because of their instability
in the emission quantum yield over time caused by surface oxidation
processes .
this problem can be overcome by growing a protective shell
around the ncs for chemical passivation
with the advantage of an additional electronic passivation preventing
the transfer of excitonic energy to surface trap states .
the core / shell architecture for nanostructures also allows semiconductor metal
hybrid nanoparticles with enhanced functionalities to be realized
that are otherwise not accessible . in
contrast to other postsynthetic shell growth techniques like epitaxial
shell growth on top of the core or galvanic replacement
applied to nobel metal ncs
, we investigate in this
study the core / shell growth driven by cationic exchange .
the replacement of pb by cd is achieved by adding an excess of cd
oleate to the pbx nc suspension resulting in a well - defined cdx shell
around the pbx core as it was shown for pbse , pbte , and pbs .
shell
ncs show strongly enhanced photoluminescence ( pl ) quantum yield as
compared to pure pbs ncs , whereas the passivating hard protective
cds shell allows additionally the pbs / cds ncs to be stabilized in
water or to be encapsulated in an amorphous
glass matrix .
the pbs / cds ncs also show
a higher efficiency and stability within photodetectors and within solar cells .
the maximum quantum yield was found for a thin layer of around 0.7
nm and is reduced again for shell thicknesses exceeding 2 nm . this was explained by defect formation caused
by the 2% lattice mismatch between the rock salt ( rs ) crystal structure of the core and zinc blende ( zb ) structure of the shell . in this study , we investigate the cationic exchange process
for
three different initial pbs nc diameters of 4.7 nm ( small ) , 6.3 nm ( medium ) , and 8.7 nm ( large ) , resulting in three different final cds shell thicknesses tshell of 0.9 , 1.5 , and 2 nm after maximum exchange
time .
the chemical composition profiles of the individual core / shell
nc ensembles as a function of reaction time are derived from anomalous
small - angle x - ray scattering ( asaxs ) using synchrotron radiation .
the crystalline structure of the ncs is retrieved from x - ray diffraction
( xrd ) experiments combined with transmission electron microscopy ( tem )
analysis .
we relate this received chemical and structural information
to the measured pl intensities of the core / shell ncs to probe if the
crystal structure of cds - shell affects the optical properties .
pristine
pbs nanocrystals ( ncs ) were prepared according to the
method proposed by hines and scholes . briefly , in a typical synthesis of 9 nm pbs nanocrystals , pbo ( 0.45
g ) , oleic acid ( oa , 10 ml ) , and 1-octadecene ( ode , 10 ml ) were loaded
in a three - neck flask and heated up to 110 c under vacuum . during
this time , pb oleate is formed , indicated by the discoloration of
the reaction mixture .
then , the reaction flask was backfilled with
ar , and the temperature was increased to 150 c . at 150 c ,
the injection mixture , containing 0.21 ml of hexamethyldisil - thiane
and 10 ml of anhydrous ode , was injected , after which the temperature
dropped to about 130 c .
the reaction flask was allowed to cool
down to 100 c within 3 min .
the temperature of 100 c was
kept for another 5 min before quenching the reaction by means of a
water bath .
samples were washed by a 1:1 hexane / ethanol mixture three
times and redispersed in hexane .
smaller sizes of pbs nanocrystals
were prepared by decreasing the amount of oleic acid , while keeping
a constant total volume of the solution .
the pbs / cds core / shell
ncs were synthesized following the method
of pietryga et al .
a pbs nc solution ,
containing 90 mg pbs ncs , was dried , and ncs were redispersed in 5
ml of anhydrous oa . after the pbs / oa solution was heated to 80 c ,
20 ml of 0.4 m cd oleate solution in anhydrous diphenylether was added .
the reaction temperature was kept constant and after several time
step aliquots were taken , which were cooled rapidly to room temperature .
these samples were washed and dissolved again in toluene or hexane
with a concentration of 12 wt % .
alternatively , the cd oleate
solution ( 0.4 m , 20 ml ) was added every hour , which provided thicker
cds shells at shorter reaction times .
this method was applied to the
growth of the large core / shell ncs , whereas for the
growth of the medium and small ncs
only in the beginning of the cationic exchange process was a sufficiently
large amount of cd oleatic solution added .
the cd solution was prepared
as described in ref ( 18 ) . a jeol 2011 fastem transmission electron microscope ( tem )
operated
at an acceleration voltage of 200 kv
was used to obtain high resolution
tem images . the direct electron beam intensity used for the imaging
mode
the tem images were created and
analyzed by a supplementary digital micrograph software package .
tem
image contrast differences can be created by many types of amplitude
and phase contrasts . in this work ,
the
darkfield images were created by using the diffraction contrast method ,
where either the rock salt bragg peaks or the zinc blende peaks alone were used to create the tem image .
the excitation source for the photoluminescence ( pl ) measurements
was a spectra physics continuous wave argon - ion laser , model 163a
5216 , emitting at a wavelength of 514 nm .
the exciting beam was chopped
by a chopper wheel and focused on the cuvettes containing the ncs
in solution .
the photoluminescence ( pl ) was collected by two caf2 lenses and spectrally resolved by an acton research corporation
spectra pro 150 monochromator .
the monochromatic light was detected
by a nitrogen cooled judson j10-m204-r10 m insb photodiode .
this signal
was amplified with a judson pa-7 preamplifier and a stanford research
system sr510 lock - in amplifier , which used the excitation source chopper
frequency as a reference . a self - written program running on a personal
computer operated the monochromator over the desired spectral range
and evaluated the amplified signals at the different wavelengths to
obtain the photoluminescence spectra .
to quantify the luminescence
enhancement , the optical density of all samples was adjusted to have
the same value at a wavelength of 500 nm .
the contrast variation
in anomalous small - angle x - ray scattering
( asaxs ) is due to the energy dependency of the atomic scattering factor f(z , e ) in the vicinity
of x - ray absorption edges , where e is the x - ray energy
and z the atomic number .
it can be shown that the total measured intensity is composed of a nonresonant and
a resonant scattering contribution .
the nonresonant scattering term
depends only on the total electron density within a material and is
related to the overall elementary composition and the bulk density ,
whereas the resonant scattering term depends on e. thus , the total amplitude deviates significantly from the atomic
number z by varying the energy around an elemental
absorption edge . by tuning the x - ray energy just below the pb - liii - edge at e = 13.035 kev ,
the contribution
of pb as the strongest scatterer with z = 82e to the total scattering amplitude f(z , e ) and hence to the
total scattering intensity i(q , e ) can be varied significantly . hence , for asaxs experiments
synchrotron x - ray sources have to be used where the x - ray energy can
be continuously varied .
the asaxs spectra for the large and small sample series have been measured at the
7t - mpw - saxs beamline at the bessy ii synchrotron ( hzb berlin , germany ) ,
whereas the medium sized ncs have been recorded at
the beamline id01 at the european synchrotron radiation facility esrf
( grenoble , france ) . in asaxs
the scattered intensity is a function
of the scattering
vector q and the x - ray energy e , with q = 4 sin / and the x - ray wavelength .
for a minimum set of three scattering curves measured at different
energies
the pb - electron density and the total electron density within
the core and the shell can be derived independently .
this is achieved
by applying a unique spherical core / shell model to fit up to five
different scattering curves .
all scattering curves have been calibrated
into absolute units ( i.e. , macroscopic scattering cross sections in
units of cross - section per unit volume cm / cm = cm ) .
for all energies the scattering of the
solvents toluene and hexane was measured with the same statistical
accuracy and was removed from the scattering curves of the sample
before applying the fitting procedure . by varying randomly the scattering
data within the experimental error band and repeating the fitting
procedures up to 20 times , the stability of our results is tested
and the errorbars for the density profiles are obtained .
the
rutherford backscattering spectrometry ( rbs ) method is based
on elastic collisions between light , energetic ions ( h , he with a
few hundred kev up to 2 mev ) and the atomic nuclei in a stationary
sample .
scattering
kinematics permits the elements present in a thin layer to be identified
from the yield of particles backscattered from the sample in a large
angle .
rbs measurements were performed , employing the van de graaff
accelerator an700 ( 30700 kev ) .
the monte carlo program simnra was used to evaluate the sample compositions
by fitting the peaks corresponding to the individual elements in the
measured spectra .
x - ray diffraction ( xrd ) experiments in transmission
geometry were
performed with the nanostar instrument from bruker axs , in the wide - angle
x - ray scattering ( waxs ) configuration with a sample to detector distance
of 6 cm .
this allows diffraction peaks up to a 2 angle of around
44 to be recorded with the cu k x - ray wavelength
of 1.5418 .
twelve of the 14 individual nc ensembles used for
the asaxs experiments were measured sealed in quartz - glass capillaries
of 1.5 mm diameter .
the 2d detector ( bruker histar ) images showed
x - ray diffraction powder rings , and thus for the data analysis line
scans were extracted from the 2d images by azimuthal integration .
for further analysis of the peak width q the
detector resolution of this setup was determined using the peak widths
of a diffraction standard ( al2o3-powder ) . from the q - value
we derive directly d as a measure
for the crystalline diameter using the scherrer formula d = k2/q with a k - value close
to one for the ncs without a shell .
these
values are only 10% to 20% smaller as compared to asaxs , which can
be related to an additional peak broadening due to imperfections in
the crystal structure .
we modified the previously
reported method for the synthesis of
pbs / cds ncs .
in particular , we applied
multiple injections of the cadmium precursor , which leads to a faster
cds shell growth and consequently to shorter growth times .
after heating
the pbs nc solution in toluene up to 80 c , cd
aliquots were taken after defined time steps as
shown in the reaction scheme in figure 1a .
the shell formation for the large core / shell ncs
was proven by high resolution tem ( hr - tem ) studies . in figure 1b the structure of one single pbs / cds
nc is shown
after 3 h of exchange time , where the core ( indicated by a red dashed
line ) depicts different lattice spacings as compared to the shell
region . for the core with about 7 nm diameter
we derive the rock salt ( rs ) crystal structure ( fm3 m ) , whereas within the 1 nm
shell the lattice plane configuration indicates the zinc blende ( zb ) structure ( f43 m ) .
these two crystalline phases are also visible in dark field hrtem
images , where either the rs or the zb electron diffraction peaks were used for highlighting the corresponding
crystalline phase within hr - tem images ( see figure s1 , supporting information ) . from the aliquot samples
pl spectra were recorded and compared to that of the pure pbs ncs
( see figure 1c ) .
the peak maximum at around
1800 nm is increased by a factor of 4 for the samples after 1 and
2 h reaction time , but after 3 h this enhancement in the peak maximum
is again slightly decreased .
the resolution of our tem analysis , however ,
did not allow differences to be revealed in the thickness or the composition
of the cds shells between 1 and 3 h exchange time .
furthermore , tem
analysis alone is restricted to a small sample set of ncs ( typically
around 100 individual ncs ) deposited on a flat substrate , but the
pl spectra are produced by a large ensemble of individual ncs in solution .
as complementary methods
we have chosen x - ray scattering techniques
that allow mean structural parameters of a large ensemble of ncs with
subnanometer resolution to be determined .
the scattering experiments
were directly performed on the nc solutions sealed in glass capillaries
of 1.5 mm diameter resulting in nc ensembles of 10 individual particles probed during one single x - ray measurement .
therefore , we can probe in detail the influence of the mean structural and compositional core / shell parameters of the ncs on
their optical output .
this analysis was done for the three samples
series ( large , medium , and small ) with initial particle diameters from 9 to 5 nm at
various reaction time steps , resulting in 14 different nc ensembles .
( a ) scheme
of the synthesis of pbs / cds core / shell nanocrystals
driven by cationic cd for pb exchange starting with pure pbs ncs ( red
sphere ) in the rock salt crystal phase and resulting in core / shell
particle with the same diameter , but surrounded by a cds shell ( blue ) .
( b ) high resolution tem image of a single 9 nm sized core / shell nc
after 3 h of exchange time , where the dashed circles indicate the
pbs core ( red ) and the cds shell region ( blue ) . ( c )
pl spectra of large sized pbs and pbs / cds ncs with diameters d of around 9 nm , measured after different reaction time steps . small angle x - ray scattering ( saxs )
is a method widely used in the
analysis of nanoscale structures .
an
extension of saxs , asaxs allows element specific contrast
variation and hence the possibility to
determine the chemical compositions of the core and the shell of a
macroscopic ensemble of ncs .
the contrast variation in asaxs is due
to the energy dependency of the atomic scattering factor f(z , e ) in the vicinity of x - ray
absorption edges . from a minimum set of three asaxs patterns measured
at different energies we can separate independently the total electron
density from the pb - electron density inside the core / shell ncs .
this
was achieved by applying a step - like spherical core / shell model to
fit all scattering curves ( see the section methods and the supporting information for a detailed description
of the asaxs method ) . in figure 2a the experimental
scattering curves are shown together with the model fits for the large core / shell ncs measured after 1 h of exchange time .
all five curves were fitted with the same unique spherical core / shell
model with the following fitting parameter : the inner radius rcore , the shell thickness tshell , the pb density , and the total electron density
in the core and the shell , as well as the total size distribution
of the spherical core / shell ncs ( is assumed to be
equal for the core and the shell ) .
the accordance between data and
fit is generally excellent ; small deviations are only visible at large q - values , where the error band of the scattering data is
getting larger due to statistics . to illustrate the energy dependent
effect , the position in q of the first minimum in
the asaxs curves ( see figure 2a ) is plotted
over e in the inset .
the resonant shift is in the
range of 2% , whereas the reference minimum - position of pb
ncs without cds - shell ( dashed red line ) is constant within 0.3% .
this
shift can be directly related to a difference in the pb electron densities
between core and shell .
( a ) experimental asaxs curves ( symbols ) fitted
with a unique spherical
core / shell model ( lines ) for the large core / shell
pbs / cds nc - sample after 1 h of reaction time .
the absolute intensity i(q , e ) plotted over the
scattering vector q was recorded at five different
x - ray energies below the pb - liii - edge . the curves , except
the black one , are shifted vertically by a factor of 2 for clarity .
inset : the squares show the shift in q of the first
minimum in the asaxs curve as a function of e , whereas
the red dashed line shows the position of the pure pbs nc as reference .
in ( b ) , ( c ) , and ( d ) the resulting density profiles for the large , medium , and small sized pbs / cds samples are depicted . the pb - density ( red line ) is
plotted on the left axis and the total electron density ( blue line )
on the right axis as a function of the distance from the particle
center .
( e ) scheme of the final shell formation for all three initial
particle sizes , i.e. , large , medium , and small , after maximum exchange time .
the received structural parameters
from figure 2a result in the density profile
shown in figure 2b .
the red line depicting
the pb - density ( left axis ) gives
a value of 19.9 0.25 pb - atoms / nm within the core
with a diameter of 7.34 0.03 nm ( rcore = 3.67 nm ) .
also the total electron density shown on the right
axis in figure 2b with 1568 13 e / nm matches the expected
value of 1566 e / nm for pbs .
no pb can be traced in the 0.69 nm shell , whereas the e - density reaches 86% of that expected
for bulk cds in the zb crystal phase .
this slight
discrepancy can be related to a small deviation of the nc from the
spherical shape due to surface roughness or facetting , which is not
included in our model . from the composition profile
we can conclude ,
however , on an atomically sharp interface between pbs - core and a pure
cds - shell as was already reported for the pbx / cdx core / shell systems .
a similarly well - defined
but much thicker cds - shell is formed around
the medium sized pbs - ncs as shown in figure 2c . this density profile is derived from measurements
and fits shown in figure s2a in the supporting
information .
we start from pure pbs ncs with an initial diameter
of around 6 nm applying a similar synthesis procedure as for the large core / shell ncs .
here we took more samples at times
even below 1 h reaction time and we monitored the shell growth up
to 18 h. within a total diameter dtotal of 6.2 nm a 3.4 nm diameter pbs - core is formed surrounded by a 1.5
nm thick cds - shell . the pb - density ( red line ) and the total e - density ( blue line ) of the core meet
quite closely the expected values for pbs , and the electron density
of the shell that for cds , respectively . again
a different profile results
from the asaxs data of the small pbs ncs ( see figure
s2b , supporting
information ) with initial 5 nm size as shown in figure 2d . in the core / shell ncs with a dtotal of 4.7
nm only a 0.8 0.05 nm pbs - core remains ,
whereas a 1.9 0.02 nm thick shell is formed containing 4.5
0.25 pb / nm within the cds shell .
the e - density of the shell , however , reaches only 65%
of the value for pure cds ( blue line in figure 2d ) .
the low total shell electron density is a strong indication that no cd1xpbxs alloy is formed , because such a ternary compound
should have a significantly larger density value with respect to pure
cds and not a lower one .
a similar amount of pb is detectable for
all small nc samples taken at different time steps
during the pb cd exchange up to
29 h reaction time . in our simple core / shell model
we can not unambiguously
determine where exactly the detected pb is located within the shell
to give the observed result .
two extreme pb positions within the shell
are possible : ( i ) the pb can be located close to the remaining pbs - core ,
or ( ii ) the pb - atoms can be bound on top of the cds shell surface
forming an additional pb - surface shell .
the last scenario ( ii ) may
be related to the proposed growth mechanism for a cationic shell growth
thicker than one monolayer : the pb ions have to diffuse toward the particle surface , where
they are subsequently exchanged by the cd ions of the
cd
oleatic compound within the solution ( see more details in
the supporting information and in ref ( 36 ) ) .
if this last reaction
step is not fully completed , e.g. , due to a too low cd oleate
concentration , a submonolayer ( ml ) of pb - atoms should remain on the
surface .
the 4.5 pb / nm found within the cds shell would
correspond to an 75% ml - coverage of pb on top of the cds shell .
thus , an additional second surface shell with a reduced
electron density should be detectable with a thickness in the range
of two times the ionic radius of pb with 0.133 nm and the -ml thickness of pbs of around 0.34 nm .
analyzing
the data with an extended model consisting of three density steps ,
i.e. , a core plus two shells , the fit at a fixed x - ray energy gives
indeed a surface shell with 0.38 nm thickness and a reduced electron
density of around 800 e / nm .
moreover , the derived e - density value of the thick intermediate cds - shell of 1.5 nm thickness
matches now to 96% of the cds value ( see figure s3 , supporting information ) . the alternative model ( i ) ,
where the total pb amount in the small core / shell
ncs is concentrated close to the particle
center , would require a pbs - core with nearly 3 nm diameter .
the simulated
data using this large pbs core compared to the experimental saxs data
shows , however , that the model ( i ) for the pb - distribution can be
ruled out ( see figure s3a , supporting information ) .
thus , our detailed saxs analysis suggests that the small core / shell ncs are surrounded by a second surface shell consisting
of a sub - ml of pb atoms remaining after a not fully completed cd for
pb exchange ( see figure s3d , supporting information ) . to test the reliability of the retrieved
pb - density distributions ,
we calculate from these core / shell profiles the total pb - concentrations
within the ncs
we compare these values with the pb concentrations
that are derived from an independent method , which is not sensitive
to the internal pb - distribution .
for this purpose , the elementary
ratio of pb : cd : s was probed by rutherford backscattering spectroscopy
( rbs ) for all investigated large and small pbs / cds ncs ( summarized in table s1 ,
see supporting information ) .
the amounts
of pb derived from both methods , rbs and asaxs , are in very good agreement
as can be seen in figure 3d , where the pb - concentration
is plotted as a function of the exchange time .
the small core / shell ncs contain a large amount of cd and only a small amount
of pb in the range of 13 atom % to 18 atom % , which is in good accordance
with a profile with a thick cds shell , a small pbs core , and some
remaining surface pb atoms .
it can bee seen for all three sample series that the total pb amount decreases while the cds - shell
increases and remains constant when the maximum shell thickness is
reached , corresponding to a decreasing pbs core during the cd for
pb exchange . in figure 2e
we summarize schematically
the final core / shell parameters as obtained from asaxs for all three
sample series .
a clear trend is visible : the cds shell increases and
hence the pbs cores decrease with decreasing initial
pbs - nc diameter . the shrinkage of the pbs core is also reflected in
a shift of the peak wavelength in the pl spectra to shorter wavelengths ( see figure 1c and figure
s5 , supporting information ) .
a detailed
investigation of the peak shift as a function of the core diameter
and of the cds shell thickness is presented in the supporting information and shown in figure s6 .
additional important conclusions can be
drawn from the time dependence of the core / shell structural parameters ,
when relating them to the optical performance of the large , medium , and small ncs . in figure 3a the time evolution of the cds - shell thickness tshell
is plotted over the square root of the
exchange time in minutes to cover the whole range of the investigated
time steps , starting from 20 min up to 29 h. the values at growth
time zero originate from the pure starting pbs - ncs .
the initial diameter
and the total core / shell diameter dtotal remains constant within the error bars during the cationic exchange
process that means the cds - shell increases with the same rate as the
pbs - core shrinks .
the large cds - shell of the small
ncs ( green symbols ) is already fully formed after 60 min exchange
time and remains quite constant up to the maximum growth time of 29
h. for the medium sized ncs ( cyan symbols ) we can
observe between 40 and 120 min after cd - injection small variations
in the tshell - value , whereas after 2 h
the final shell thickness of 1.5 nm is reached .
for the large ncs ( black symbols ) the cds - shell increases from 0.7
nm up to 0.9 nm after 3 h. whether this is already the final shell
thickness can not be answered here , since no data above 3 h are available .
( a ) cds - shell
thickness tshell vs exchange
time for the large ( black ) , medium ( cyan ) , and small ( green ) core / shell
ncs .
( b ) the time dependence of the pb amount
within the shell is shown , whereas in ( c ) the normalized integrated
pl emission of the ncs vs time is depicted .
( d ) the total pb - amount
within all ncs derived from the asaxs profiles ( full circles ) together
with the rbs values ( open squares ) are shown over the exchange time .
( e ) the integrated pl
emission normalized to the values of the pure pbs ncs is plotted over
the cds - shell thickness .
no significant increase in the total size distribution as
reported
for pbte / cdte core / shell ncs is found
during the whole exchange time .
the initial size distributions of
12% , 14% , and 15% for the large , medium , and small ncs is found to be constant within 1% . over the whole time
series no pb can be traced within the cds - shells
of the large and medium sized core / shell
ncs ( black and cyan symbols in figure 3b ) ,
whereas the amount of pb on top of the thick cds shell of the small ncs ( green symbols ) varies between 3.5 0.2
and 4.7 0.3 pb - atoms / nm . the pb - amount within the
pbs - core is found to be also constant within 0.7 pb / nm around the theoretical value for rock salt pbs
of 18.9 pb / nm for all three sample series . in figure 3c we depict the pl emission of
all three sample series also as a function of the exchange time and
can thus relate the optical performance of the core / chell ncs to their
different structural and chemical compositions .
the integral pl emission
is derived from the total area under the pl emission spectra with
peak wavelengths between 1200 and 1900 nm increasing from the small
to the large nc samples ( see figure 1c and
figure s5 , supporting information ) .
the
integral intensity values are all normalized to one at the corresponding
value of the initial pure pbs ncs .
the strongest relative increase
in the normalized pl , and hence the relative increase in the quantum
yield , is observed for the thin shell growth around the large pbs / cds ncs .
the intensity is enhanced by a factor of over 4 with
respect to the pure pbs ncs sample .
after the strong enhancement after
1 h growth time the further increase after 2 h is moderate and constant
after 3 h , although the cds - shell increases in the last growth step
from 0.7 to 0.9 nm ( black symbols ) .
the medium and small core / shell
ncs reveal a smaller enhancement of the pl by a factor of 2 ,
but only after short growth times ( cyan and green symbols ) . for both
series in the further reaction time
steps a decrease in the pl and hence in the quantum yield can be observed .
this decrease
can not be directly related to a pronounced change in the chemical
composition profile of the core / shell ncs as shown in figure 3a , b , d , where only small variations in the shell
thickness are visible .
this effect is better visualized in figure 3e , where the integrated pl intensity is plotted
as a function of the shell thickness tshell , where initially an increase of the pl - enhancement with growing
shell thickness can be observed .
after this initial increase , however ,
the relative pl intensity levels off and even decreases from a maximum
pl value with longer exchange times . since there is no evidence for a compositional
origin of this unexpected optical behavior , we have additionally investigated
the crystal structure of the core / shell ncs by recording powder x - ray
diffraction patterns . in figure
4a the diffraction
patterns of the large , medium , and small initial pbs ncs are shown together with their fits
using gaussian peak shapes on top of a linear background .
the vertical
dashed lines mark the positions of the bragg peaks for rock
salt ( rs ) pbs ( apbs = 5.936 ) and zinc blende ( zb ) cds ( acds = 5.818 ) bulk structures .
the peak positions for the pure pbs ncs matches very well the theoretical
values indicating that the ncs are fully relaxed . from the fits
we
derive the peak maximum value , the center position , the full width
half - maximum , and the area under the peaks giving the integrated crystal
peak intensity iint .
( a ) experimental diffraction
pattern of the large , medium , and small initial pbs
ncs are shown ( open circles ) together with their fits ( lines ) .
the
vertical dashed lines mark the positions of the bragg peaks for rock salt ( rs ) pbs and zinc blende ( zb ) cds bulk structures .
( b ) the xrd pattern for
pbs / cds core / shell ncs after maximum exchange time . in ( c )
the ratio
between the integrated peak intensity of the ( 200 ) and ( 111 ) diffraction
peaks is shown over the exchange time , in ( d ) the position in q of the ( 220 ) peak maximum .
an interpretation of the core / shell diffraction patterns
shown
in figure 4b can not be done in a straightforward
manner due to the strong overlapping of two broad peaks related to
the small lattice constant difference of 2% between pbs and
cds .
one can observe , however , directly a change in the peak intensity
ratio between the ( 111 ) and the ( 200 ) peak , as well as a shift in
the peak position toward the cds bulk position .
these effects are
most pronounced for the medium and small core / shell samples , where the diffracted intensity mainly originates
from the thick cds shells ( see figure 4b ) .
the intensity ratio change can be interpreted in terms of a change
in the crystals structure from rocksalt to zinc blende , because the zb structure of
bulk cds depicts a strongly reduced ( 200 ) bragg peak with respect
to the rs structure of pbs ( blue and red squares
in figure 4a , b ) .
the shift of the peak positions ,
especially that of the ( 220 ) bragg peak , where we are most sensitive
to changes in the lattice spacings , can be caused by both a different
crystal phase and strain effects .
the assumption of a metastable rs phase fraction
in the cds shell is motivated by the fact that due to cationic exchange
in ncs the chemically new compound can preserve the crystal structure
of the starting nc . in our case
the cds
shell should keep to a certain extent the rs structure
of the pbs core .
zb phase
ratio within the shell as a function of the exchange time and the
shell thickness . for this
we calculate the individual diffraction
patterns for all pure pbs ncs and the pbs / cds ncs by means of the
debeye formula using the individual chemical
composition data for 12 different sample sets as retrieved from asaxs
( see the supporting information for a detailed
description ) .
we assume for the pbs core always a pure rs structure , but for the cds - shell either the rs or
the zb structure .
fitting the simulated diffraction
data with the same procedure as the experimental data ( see figures
s8s10 , supporting information )
we retrieve for each single chemical composition the theoretical intensity
ratio and peak shift for the cds shell being either 100% in the rs ( red dashed lines in figure 4c , d )
or 100% in the zb phase ( blue dashed lines ) .
this
allows the relative amount of the two crystalline phases within the
cds shell to be determined , where a value of one means pure rs - phase and a value of zero a pure zb - phase ,
respectively . in figure 4c the ( 200)/(100 )
intensity ratio of all samples and hence a measure for the rs
the reliability of our model is reflected by the
values for the pure rs pbs ncs ( black dashed line )
in the range between 0.95 and 1 .
for the large core / shell
ncs ( black symbols ) the peak intensity ratio for the two slightly
thinner cds shells formed after 1 and 2 h gives values of around 0.70 .
after 3 h the signal drops , however , significantly to a lower value
corresponding to 20% of a remaining rs phase .
a similar behavior , decreasing amount of the rs phase with exchange time , is visible for the medium ( cyan symbols ) and small ( green symbols ) sized
core / shell samples , but here already the first formed shells depict
a significantly lower amount of cds in the rs phase .
the shell of the medium sized ncs with 1.4 nm thickness
exhibits 50% of rs phase fraction , whereas the nearly
2 nm shell of the small ncs contains only 30% of
this metastable phase .
furthermore , only the 2 nm thick cds shell
reaches the zb phase finally after 29 h of exchange
time , whereas the medium sized ncs keep within their
shell 30% of the metastable rs crystal phase . the same trend is also reflected in the shift of the ( 220 ) bragg
peak depicted in figure 4d .
the cds shells
of the large ncs show a quite linear shift from the pbs lattice constant
to the cds value , whereas the shell lattice constant in the medium
sized core / shell ncs finally seems to remain constant in the middle
between pbs and cds .
both sample series are chemically stable during
the growth time as derived from the asaxs profiles , so the peak shift
can be related to different crystalline phases within the shell . to
conclude from the lattice constant values alone on the amount of crystalline
phases within the shell is not unambiguous , due to the fact that the
lattice spacing can be altered by strain .
this is reflected in the
lattice constant values of the small sample series ,
where after maximum exchange time the lattice constant of the shell
is even smaller than that of the bulk cds value .
this can be explained by a compressive strain caused
by a surface relaxation due to the high surface / volume ratio for the
small sized ncs .
this can be already seen for the small pure pbs ncs
depicting a 0.4% smaller lattice constant with respect to the bulk
pbs value .
the lattice spacing for the 2 nm thick cds shell after
the longest growth time is even 0.9% smaller .
this effect was already
reported for zns nanocrystals , where
the magnitude of the compressive strain for 5 nm
nanocrystals is in the same order of magnitude .
the different
crystal structures , the rs and zb - phase , can be realized by keeping the anionic s position
in both lattice types constant , whereas only the positions of the
cations differ .
the pb - ions in the rs structure have
six nearest neighbors and occupy a face - centered - cubic ( fcc ) sublattice
displaced by ( 1/2 , 1/2 , 1/2 ) of the cubic lattice constant apbs . the more covalent bound cd ions are fourfold
coordinated , which occupy fcc positions that are displaced by ( 1/4 ,
1/4 , 1,4)acds ( see figure 5a ) .
hence , we explain the appearance of the metastable rs structure within the shell by a fast cd for pb exchange
process , where the cd cations occupy in the beginning the same lattice
positions as the expelled rs core pb - ions .
the change
from this metastable rs phase to the zb - phase is caused by a subsequent cd - ion transition to their equilibrium
positions within the zb lattice , whereas the anionic
s - sublattice in both crystal structures is unchanged .
such a stable
anionic sublattice was recently found for the homogeneous cationic
exchange in cdse ncs .
a continuous anionic
sublattice between the rs and the zb structure is also identified by hr - tem and verified by ab
initio simulations for pbte quantum dots embedded in a cdte
matrix .
( a ) scheme of the crystals phases of cds
occurring during the cationic
cd for pb exchange .
first the cds - shell mimics nearly fully the core rock salt ( rs ) structure of pbs , and later
both the rs ( cyan ) and the equilibrium zinc
blende ( zb ) phase ( blue ) of cds can be found
within the shell .
the indicated distribution of the metastable rs phases within the shell is only a suggestion .
( b ) integrated
pl intensity normalized to the cds layer thickness plotted over the zb phase fraction within the shell .
the cd structural transition starts to a certain extent directly
after the cd for pb exchange ; the full rearrangement of the cd - ions
in the zb structure , however , takes up to 30 h at
a temperature of 80 c as can be deduced from figure 4c , d .
we could not observe any change of the phase
fractions with time after cooling the samples to room temperature .
the proposed scheme is sketched in figure 5a , where it is also indicated that the highest amount of rs - phase is found for the thinnest cds shell , i.e. , for
the largest initial sphere diameter .
the influence of the shell thickness
on the rs phase fraction is shown by comparing rs phase fraction values at 1 h exchange time .
we found
values of 70% , 50% , and 30% for the rs phase for
the large , medium , and small ncs , respectively ( see figure 4c ) .
we expect
that these amounts of metastable crystal phases are not homogeneously
distributed along all crystallographic directions , but our current
xrd data do not allow preferred crystallographic directions to be
identified , in which this metastable phase is enriched .
an additional
indication for the coexistence of the zb and rs phases within one single shell is found in the dark field
hrtem images of a large core / shell nc after 3 h of exchange time .
in figure s1d , e ( supporting information ) either the rs or the zb electron
diffraction peaks are used for highlighting the corresponding crystal
phase .
the bright rs core phase in figure s1d extends in the shell region , whereas in figure s1e the bright zb phase
depicts an inhomogeneous distribution within the shell .
this kind of polytypism
of crystal phases within one shell was also found for shells grown
epitaxial on top of the core .
the driving force
for the thickness dependence of the amount of
the rs phase fraction can be related to the strain
energy built up within the cds - shell due to the lattice mismatch of
around 2% between pbs and cds .
the piled up tensile
strain with increasing number of mls of the
thicker shells can be fast relaxed by creating stacking faults that can trigger the transition from the rs to
the zb crystal structure .
the relation between phase
fraction and shell strain is summarized in figure s11 ( supporting information ) .
these defects in the
shell should be reflected in a reduced pl emission due to a partial
extension of the exciton wave function into the shell material .
indeed , the core / shell ncs with medium and thick
shells depict only half of the pl increase with respect
to the thin - shell samples .
to investigate only the influence of the rs - zb phase fraction on the pl we relate
in figure 5b the ratio of the rs to the zb phase to the normalized pl emission values .
here , the
pl intensity values of the samples are additionally normalized to
the individual shell thickness values .
two distinct features
are visible : a decrease of the pl with decreasing rs phase fraction is visible for all three samples series . additionally ,
a pronounced jump in the relative pl increase between
the large samples with shells below 1 nm and the medium and small ncs with shells above
1.3 nm is visible .
these thick - shell samples depict more than three
times lower relative pl values as the thin - shell samples compared
at the same phase fraction value of around 0.5 .
the slope of the decrease ,
however , is constant for all three sample series .
this is demonstrated by the two black lines in figure 5b having the same slope as derived from a linear fit to the
data of the thin - shell samples only . within the three thin - shell samples
the metastable rs phase fraction changes from 70%
to 20% while the relative pl is decreased by around 16% .
the thick - shell
samples start already with 75% decreased relative pl value at 50% rs phase fraction and reach only 10% of the initially relative
pl increase .
these experimental findings suggest that two different
mechanisms
are involved to mediate the transition from the metastable rs phase to the equilibrium zb structure
of the cds shell .
one mechanism for transitions between fourfold and
sixfold coordinated crystal structures was reported for ncs that implies a collective sliding of crystal planes accompanied
by the creation of stacking folds .
this martensitic - like phase transition
from a fourfold to a sixfold phase is induced by high external applied
pressures .
in contrast , in our pbs / cds core / shell ncs the sliding
of the cd - sublattice
planes from the sixfold rs phase to the fourfold zb structure , i.e. , in the reverse direction , is driven
by the tensile stress within the cds shell . during
this phase transition
an alternative transition - path mechanism can avoid the creation of
stacking faults but is based on the uniaxial compression of crystal
planes and results in an increase of the surface energy . in theoretical studies by grnwald et al .
it was shown that the fourfold to a sixfold phase transition starts
via the compression path mechanism nucleating on the crystal surface
and is continued by the sliding plane mechanism , but depending on
the size and surface structure also a mixture of both is possible . moreover , very recently it was shown by the
same group that the transformation mechanisms for metastable phases
within core / shell ncs depends strongly on the shell thickness . for shells below 3 mls thickness
taking these studies into
account we interpret our observations
summarized in figure 5b as follows : for the
thin - shell samples the transformation between the metastable rs phase and the zb phase , and hence the
relaxation of the shell - strain , is realized by the surface nucleation
mechanism , which results in an only moderate decrease of the pl due
to surface defects .
for the thick - shell samples the initial transformation
proceeds via the sliding plane mechanism resulting in stacking faults
that considerably decrease the pl intensity . due to the stacking fault
formation , thin shell layers below 3 mls are created , in which the
compression mechanism mediates a further rs to zb phase transition with reduced influence on the pl .
the
physical origin of this pronounced influence of the shell on the pl
intensity is related to the delocalization of charge carriers over
the whole core / shell volume .
in conclusion , we have demonstrated that , during the cds shell
growth around pbs nanocrystals due to a cd for pb cationic exchange ,
the shell initially maintains the metastable rock salt crystal structure of the core within a chemically pure cds shell .
such a metastable phase within a core / shell system , which does not
occur in the bulk material under ambient pressure , was quite recently
theoretically predicted .
for the first
time we have determined the quantitative amount of the metastable
phase in the shell as a function of the exchange time and the shell
thickness . moreover ,
the core / shell ncs show a pronounced pl enhancement
after the cds shell formation with the pl showing a clear dependence
on the rs / zb phase fraction : it
decreases with decreasing rs phase and increasing zb structure .
this revealed correlation is related to the
formation of lattice distortions during the phase transitions .
the
control of this metastable phase within the shell to optimize the
core / shell interface will have significant impact on tailoring the
optical properties of core / shell ncs also in related material systems . | we reveal the existence of two different
crystalline phases , i.e. ,
the metastable rock salt and the equilibrium zinc blende phase within the cds - shell of pbs / cds core / shell
nanocrystals formed by cationic exchange .
the chemical composition
profile of the core / shell nanocrystals with different dimensions is
determined by means of anomalous small - angle x - ray scattering with
subnanometer resolution and is compared to x - ray diffraction analysis .
we demonstrate that the photoluminescence emission of pbs nanocrystals
can be drastically enhanced by the formation of a cds shell .
especially ,
the ratio of the two crystalline phases in the shell significantly
influences the photoluminescence enhancement .
the highest emission
was achieved for chemically pure cds shells below 1 nm thickness with
a dominant metastable rock salt phase fraction matching
the crystal structure of the pbs core .
the metastable phase fraction
decreases with increasing shell thickness and increasing exchange
times .
the photoluminescence intensity depicts a constant decrease
with decreasing metastable rock salt phase fraction
but shows an abrupt drop for shells above 1.3 nm thickness .
we relate
this effect to two different transition mechanisms for changing from
the metastable rock salt phase to the equilibrium zinc blende phase depending on the shell thickness . | Introduction
Experimental Section
Results and Discussion
Conclusions |
PMC3336849 | a questionnaire - based study was conducted from march to december 2006 at the out - patient department of vallabhbhai patel chest institute , delhi , a tertiary care referral hospital .
patients from national capital , delhi and nearby towns are referred to this public hospital .
patients aged 18 yr who presented to the chest clinic for the first time with history suggestive of asthma were included . they were evaluated and the diagnosis of bronchial asthma was confirmed based on the symptoms and reversible spirometry as per the global initiative for asthma ( gina ) guidelines16 .
patients with an acute exacerbation of asthma and those with another concurrent respiratory disease or any systemic disease were excluded .
spirometry was carried out on a dry rolling seal spirometer of computerized pulmonary function equipment ( benchmark model , pk morgan , uk ) according to standardized guidelines of the american thoracic society / european respiratory society , 20051819 .
information on quality of prior treatment , patients knowledge about asthma and quality of life was obtained by using three questionnaires administered by one of the authors ( ak ) .
the two questionnaires , asthma knowledge and asthma quality of treatment were developed on the basis of the gina guidelines16 .
the content of knowledge a patient is expected to have served as the basis for development of the questionnaire .
repeatability was tested by re - administration on another occasion and comparing the two sets of responses .
the asthma quality of life questionnaire20 is a standard tool extensively used for this purpose and its repeatability and validity have been well studied .
quality of treatment questionnaire ( appendix 1 ) : a questionnaire was developed based on the gina guidelines for asthma management , both in english and a local language , hindi .
it contains 20 questions which have been divided into the following four domains information about the health care provider ( 3 questions ) , diagnosis and assessment of severity ( 4 questions ) , treatment ( 6 questions ) and patient education ( 7 questions ) .
many questions in this questionnaire made inferences about the doctor talking to his / her patient about the diseases , treatment , education the patient received , and whether the patient had been given information about self - management .
these questions gave insight into doctor - patient interaction , such as how doctors explain spirometery , techniques and precautions to be taken on the use of inhalers , the purpose of each inhaler , written action plans provided to the patient and how doctors explain the early signs of worsening of asthma and its management .
subject responses were categorical ( yes / no ) as well as structured multiple - choice . the percentage of subjects with different responses to each question was calculated .
quality of treatment questionnaire for asthmatics asthma knowledge questionnaire ( appendix 2 ) : this was developed in both english and hindi and designed to obtain information about different aspects of the disease that a patient is expected to be given to make him / her an active partner in self - management .
the 28 questions were divided into the following six domains aetiology ( 3 questions ) , pathophysiology ( 3 questions ) , symptoms and assessment of severity ( 8 questions ) , medication ( 8 questions ) , prevention ( 4 questions ) and natural history ( 2 questions ) .
subject responses were scored on a categorical scale where 0 represented no knowledge / no response and 1 represented knowledge of the subject addressed in a particular question . a final score was obtained by summing these scores .
the scores were expressed as percentage of the maximum possible score . the percentage of subjects with different responses to each question was also calculated . asthma knowledge questionnaire asthma quality of life questionnaire ( aqlq ) : aqlq ( provided kindly by professor e. juniper ) is a standardized , widely available and validated instrument to assess quality of life in asthmatic patients .
the 32 questions of aqlq are divided into the following four domains symptoms ( 12 questions ) , activity limitations ( 11 questions ) , emotional function ( 5 questions ) , and environmental stimuli ( 4 questions ) .
patients respond to each question on a seven - point scale and recall their experiences during the previous two weeks .
each domain score also ranges from 1 to 7 with 1 representing the greatest impairment possible or most of the time and 7 represents the least or none of the time .
statistical analysis : scores obtained from aqlq and asthma knowledge questionnaire were expressed as mean sd .
correlation between quality of treatment , asthma quality of life , the pulmonary function test parameter ( forced expiratory volume in one second , fev1 ) and asthma knowledge was determined by using the pearson correlation coefficient .
fifty adult patients ( 26 men and 24 women ) completed the three interviewer administered questionnaires .
of these , 22 , 18 and 10 patients , respectively had mild , moderate and severe persistent asthma .
twenty eight patients said that they had asthma for over 10 yr , and four patients had for more than one year but less than two years .
quality of treatment ( table i ) : information about health care providers - of the 50 patients , 33 ( 66% ) had consulted more than one doctor before visiting the referral chest clinic . up to 22 ( 44% ) patients had no knowledge about their doctor 's qualification . among those who knew the qualifications of their doctor ,
12 ( 24% ) patients consulted mbbs doctors , 10 ( 20% ) went to doctors with md degree and 6 ( 12% ) consulted practitioners from the alternative systems of medicine ( homeopathy , unani , etc . ) or unqualified medical practitioners .
asthma quality of treatment questionnaire analysis ( n=50 ) diagnosis and assessment of severity - only 30 ( 60% ) patients were informed by their doctors that they were suffering from asthma ; of these , only 11 ( 22% ) patients were told about the severity of their disease .
lung function test and peak flow measurement were done in only 5 ( 10% ) and 9 ( 18% ) of patients , respectively .
treatment - of the total 50 patients , only 22 ( 44% ) were prescribed inhaled therapy by their doctors . of these 22 patients ,
only 10 were aware of the medicine in the inhalers and the purpose of inhalation .
of these 10 patients , eight were prescribed both reliever and preventer inhalers whereas two patients received only reliever therapy .
twelve patients revealed that they learnt the use of inhalers from the package insert , other patients or hospital staff other than their doctor .
patient education - none of the subjects were provided with any educational material about the disease process . only 6 ( 12% ) patients were given a written plan to manage asthma in the event of a worsening condition but the content was never discussed by the doctor so these patients could not recall .
only 3 ( 6% ) patients recalled having been given information regarding the early signs of worsening of asthma .
one patient had an objective mean of assessing his asthma as he was advised to use a peak flow meter at home .
four patients were told about the measures to be taken if asthma symptoms increase . up to 33 ( 66% ) of patients revealed that they were not given any instructions regarding the preventive measures to lessen the symptoms of asthma .
asthma knowledge : scores obtained in different domains revealed that a large proportion of patients lacked basic knowledge about asthma and its treatment ( fig . ) .
aetiology - when patients were asked to identify the causative factors for asthma , 25 ( 50% ) patients believed that asthma is a contagious disease and could be contracted by living with another asthma patient .
up to 74 per cent of patients ( 37 ) were not aware of the fact that drugs can also cause asthma symptoms .
pathophysiology - between 36 and 68 per cent patients ( 18 - 34 ) had knowledge of the underlying pathologic process during an asthma attack .
symptoms and assessment of severity - between 80 and 90 per cent patients ( 40 - 45 ) were aware of the symptoms of asthma . however , only 14 ( 28% ) patients knew about the early signs of worsening of asthma .
merely 5 ( 10% ) patients knew that the severity of asthma can be judged by a peak flow meter and the remaining 45 ( 90% ) patients had no understanding of the concept .
ten per cent of patients did not think that asthma can be dangerous and can result in death during an attack .
medication - sixty per cent of patients did not know whether the drug prescribed to them was a
up to 30 per cent of patients did not believe that the best way of taking asthma medicines is by inhalation .
seventy eight per cent patients did not think steroids were the most effective drugs for controlling asthma and 68 per cent believed that steroids are in fact harmful even when given by inhalation route .
prevention - up to 68 per cent of patients knew that asthma could be prevented by avoiding trigger factors and by taking medication regularly .
natural history - thirty ( 60% ) patients believed that asthma can not be cured but 44 ( 88% ) patients mentioned though asthma can not be cured but can usually be controlled .
the overall quality of life impairment due to asthma was moderate with a mean value of 3.68 0.78 .
asthma quality of life questionnaire analysis activity limitation - asthma limited the activities of all patients except one with moderate to severe limitation observed in 80 per cent of the patients .
strenuous activities such as running and walking upstairs led to symptoms in 94 and 88 per cent of patients respectively .
up to 60 per cent of patients reported moderate to very severe problems during sleep due to asthma .
symptoms - moderate to severe symptoms were observed in up to 36 ( 72% ) patients . up to 34 ( 68% )
nocturnal asthma occurring most of the time over the past two weeks was reported by 27 ( 54% ) patients .
emotional function - about 82 per cent of the patients felt frustrated as a result of asthma and 84 per cent were concerned about having asthma in the past two weeks .
thirty eight per cent patients revealed that they felt worried most of the time when they did not have their asthma medicines with them .
environmental stimuli - environmental triggers such as dust , weather , air pollution , and strong smells were reported to have provoked recent asthma symptoms in 47 patients .
more than 80 per cent patients ( 40 ) reported avoiding situations because of potential exposure to these triggers .
pulmonary function and correlation : correlations between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , and asthma knowledge ( ak ) were studied .
a significant correlation was seen only between the quality of treatment and asthma knowledge ( p<0.05 ) ( table iii ) .
pearson correlation coefficients between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , pulmonary function test ( fev1 ) and asthma knowledge ( ak )
the overall quality of life impairment due to asthma was moderate with a mean value of 3.68 0.78 . mean scores in each domain are as shown in table ii .
asthma quality of life questionnaire analysis activity limitation - asthma limited the activities of all patients except one with moderate to severe limitation observed in 80 per cent of the patients .
strenuous activities such as running and walking upstairs led to symptoms in 94 and 88 per cent of patients respectively .
up to 60 per cent of patients reported moderate to very severe problems during sleep due to asthma .
symptoms - moderate to severe symptoms were observed in up to 36 ( 72% ) patients .
up to 34 ( 68% ) patients recalled waking up with asthma symptoms most of the time .
nocturnal asthma occurring most of the time over the past two weeks was reported by 27 ( 54% ) patients .
emotional function - about 82 per cent of the patients felt frustrated as a result of asthma and 84 per cent were concerned about having asthma in the past two weeks .
thirty eight per cent patients revealed that they felt worried most of the time when they did not have their asthma medicines with them .
environmental stimuli - environmental triggers such as dust , weather , air pollution , and strong smells were reported to have provoked recent asthma symptoms in 47 patients .
more than 80 per cent patients ( 40 ) reported avoiding situations because of potential exposure to these triggers .
pulmonary function and correlation : correlations between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , and asthma knowledge ( ak ) were studied .
a significant correlation was seen only between the quality of treatment and asthma knowledge ( p<0.05 ) ( table iii ) .
pearson correlation coefficients between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , pulmonary function test ( fev1 ) and asthma knowledge ( ak )
the overall quality of life impairment due to asthma was moderate with a mean value of 3.68 0.78 . mean scores in each domain are as shown in table ii .
asthma quality of life questionnaire analysis activity limitation - asthma limited the activities of all patients except one with moderate to severe limitation observed in 80 per cent of the patients .
strenuous activities such as running and walking upstairs led to symptoms in 94 and 88 per cent of patients respectively .
up to 60 per cent of patients reported moderate to very severe problems during sleep due to asthma .
symptoms - moderate to severe symptoms were observed in up to 36 ( 72% ) patients .
up to 34 ( 68% ) patients recalled waking up with asthma symptoms most of the time .
nocturnal asthma occurring most of the time over the past two weeks was reported by 27 ( 54% ) patients .
emotional function - about 82 per cent of the patients felt frustrated as a result of asthma and 84 per cent were concerned about having asthma in the past two weeks .
thirty eight per cent patients revealed that they felt worried most of the time when they did not have their asthma medicines with them .
environmental stimuli - environmental triggers such as dust , weather , air pollution , and strong smells were reported to have provoked recent asthma symptoms in 47 patients .
more than 80 per cent patients ( 40 ) reported avoiding situations because of potential exposure to these triggers .
pulmonary function and correlation : correlations between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , and asthma knowledge ( ak ) were studied .
a significant correlation was seen only between the quality of treatment and asthma knowledge ( p<0.05 ) ( table iii ) .
pearson correlation coefficients between quality of treatment ( qot ) , asthma quality of life ( aqlq ) , pulmonary function test ( fev1 ) and asthma knowledge ( ak )
our study has identified substantial deficiencies in the management of asthma in an urban community in india .
other studies from different parts of the world have also reported under - treatment or under - use of essential asthma medication8913142122 . in india , a majority of asthma patients in the community are managed by general practitioners in the private sector and non - specialized public sector physicians in primary / secondary health care facilities .
our previous study indicated that in many states of india essential inhalation medicines for asthma were not available in public sector7 .
earlier studies from india and other countries have documented that general practitioners have poor knowledge about preventive therapy for asthma and its management8122123 which contributes to increased morbidity and mortality .
guidelines recommend that the diagnosis and follow up of patients should be based on the evaluation of the frequency of symptoms and on the level and variability of the peak expiratory flow rate .
however , in the present study , a spirometer or peak flow meter was used for diagnosis in only 10 and 18 per cent of patients , respectively . non adherence to recommended treatment guidelines
results from these studies revealed that only 43 per cent of australian general practitioners and 33 per cent of spanish pediatricians routinely measured airways function with spirometers and peak flow meters25 .
inhalers which are considered to be the mainstay of asthma therapy were prescribed to only 44 per cent of patients in our study .
these findings are consistent with several other studies that also reported the underutilization of inhalers222427 .
the primary reason reported in earlier studies was the non - availability of free inhalers ( medicines ) in public facilities , where a large proportion of populations received prescribed oral medications , and low affordability at private sector facilities due to the high cost of inhalers2728 .
further , a survey conducted in pakistan revealed that a few practitioners felt inhalers were addictive and should be reserved only for severe cases22 .
it is of concern that many subjects were not aware of their doctor 's qualification and the majority had consulted more than two doctors at primary and secondary healthcare before visiting the referral ( tertiary ) chest hospital .
another disappointing fact was that up to 40 per cent of patients were not given the diagnostic label of asthma by their doctors .
this finding is consistent with an earlier study from australia which reported that most practitioners believed that informing a patient that he or she has asthma is not important9 .
this fact also shows that most doctors at primary and secondary care facilities do not make a diagnosis based on lung function tests , as the equipment is not available in these facilities and they prescribe only symptomatic treatment .
the findings of this study also gave an insight into doctor - patient interaction ; only 10 patients were told by the doctor about the purpose of each inhaler and were advised how to use inhaler .
only 10 per cent of the patients were told about the peak flow meter and none of the patients were given any written education material .
hence , there is a pressing need for developing good partnerships between patients and doctors for the best practice in the treatment of asthma .
our study revealed that a majority of the patients were misinformed about the aetiology of the disease , and its pathophysiology and management , whereas higher scores were observed in the domains of symptoms and natural history .
patients probably knew enough about the symptoms of asthma because of their own personal experience .
earlier studies also document that patients are ignorant or have distorted knowledge of their disease and the prescribed treatment which leads to poor compliance263132 .
non adherence to the standard treatment leads to uncontrolled symptoms , limitation of daily life activities , unscheduled urgent healthcare visits and progression of the disease resulting in poor quality of life33 .
the findings of our study indicate that prescriber as well as patient awareness and education is needed for effective management of asthma in the community . a systematic review by the cochrane airways group showed that written self - management action plans had significant benefits in reducing morbidity34 .
effectiveness of patient education for asthma management has been shown in several studies from india and elsewhere3537 .
there is an enormous gap between evidence - based standard guidelines for asthma management and clinical practice
. limitations of our study include a small sample size from one chest hospital and a potential for recall bias .
generalization of the results should be done with caution as this study was done in at one referral hospital of delhi . in conclusion
, this study provides evidence of unsatisfactory asthma management , poor doctor - patient interaction and poor patient education at primary and secondary care levels .
prescriber education at all levels of healthcare and proper patient education will enable patients to comply with treatment regimens and consequently improve the quality of life of asthma patients . | background & objectives : high prevalence and poor control of asthma make its management a major public health issue worldwide , especially in developing countries
. optimum review of asthma management in the community is essential to improve asthma control .
this study was conducted to investigate the quality of asthma management , knowledge about asthma and quality of life of asthma patients referred to a public tertiary care chest hospital in delhi.methods:diagnosis of asthma was confirmed by symptoms and reversible spirometry in 50 referred patients on their first visit .
patients were interviewed using three questionnaires on quality of asthma management before visiting referral hospital , asthma knowledge and asthma quality of life ( aqlq ) .
correlation amongst quality of treatment , asthma quality of life , and asthma knowledge was also determined.results:findings revealed that only 60 per cent of patients were informed about their disease , and 10 per cent had undergone lung function tests previously .
only 44 per cent of patients were prescribed inhalers .
none were provided with any educational material .
patients had poor knowledge of aetiology , pathophysiology , medication and how to assess the severity of their asthma .
the mean scores in aqlq indicated a moderate degree of impairment in quality of life.interpretations & conclusions : this study provides evidence of unsatisfactory asthma management and patient - doctor interaction as patients had limited knowledge of asthma disease , its management and had poor quality of life as measured by a standardized questionnaire . thus , there is need to implement suitable interventions to improve asthma management according to standard treatment guidelines in the community . | Material & Methods
Results
None
Asthma Quality of Life
Discussion |
PMC2957588 | currently one in five ( 4.1 million ) australians have at least one allergy ; with prevalence estimated to be growing at a rate of 0.09% per annum .
most suffer from more than one condition at a time ( an average of 1.74 comorbid allergies per person ) and over half ( 51.4% ) are 2049 years of age .
the prevalence of allergic rhinitis is increasing ; approximately 16% of australians have allergic rhinitis , including about 19% of working aged adults .
the relative risk of mortality due to allergic rhinitis is low ; explaining why it has previously been regarded as a trivial condition .
however , it is widely accepted that allergic rhinitis can have a significant negative impact on several areas of health - related quality of life ( hrql ) [ 35 ] .
daily activities are impaired in more than 80% of patients with moderate or severe allergic rhinitis and 40% of those with mild disease .
health impairment often leads to work impairment in the form of both absenteeism and presenteeism .
the ability of allergic rhinitis sufferers to engage in productive work has been shown to be influenced by sleep , hrql , specific symptoms , and antihistamine use .
our survey examined the impact of self - reported allergic rhinitis on the emotional and psychological wellbeing of australian adults .
in august 2008 , an online survey was conducted amongst participants from a permission based panel ( galaxy dp pty ltd , australia ) . in a permission based panel ,
all panellists give prior permission to receive email invitations to participate in research . with each invitation
they have the opportunity to accept or decline and are paid $ 2.00 to participate in each survey . a minimum sample size of 200 participants per state was sought to ensure a representative sample .
the total sample was then weighted to national population proportions ( issued by the australian bureau of statistics ( abs ) ) based on state , age , and gender .
the survey tool was not validated , but was based on that previously used by long in a similarly conducted research carried out in the us in 2005 .
the survey comprised five screening questions , six demographic questions and 13 questions relating to the respondents ' self - reported experience of allergies and their subsequent impact on quality of life .
male and female respondents , aged 20 to 49 years , were included if they had self - reported moderate to severe hay fever , hives / skin allergies , or allergies triggered by air - borne factors ( pet dander , pollen / grass , dust mites , mould ) .
in addition , patients were asked to report how often they suffer from allergies ?
the possible options for this question were based on the aria guideline classification of intermittent versus persistent allergic rhinitis .
the options listed corresponded to the classification as such : intermittent allergic rhinitis = less than four days a week and seasonally for less than four weeks at a time and persistent allergic rhinitis = more than four days a week and seasonally for more than four weeks at a time .
respondents were excluded if they had self - reported mild allergies , only asthma , or had allergies triggered by any factors other than those listed above .
one thousand , four hundred fifty three people responded to the survey , of whom 1060 met the study criteria , completed the survey and were included in the study results .
overall demographic characteristics are listed in table 1 . of the respondents who met the study criteria ,
82% ( n = 865 ) self - reported having moderate symptoms and 18% ( n = 195 ) self - reported having severe symptoms .
the most frequently self - reported type of allergy was hayfever ( n = 879 ) , followed by pollen / grass allergies ( n = 665 ) , hives / skin allergies ( n = 450 ) , dust mite allergies ( n = 420 ) , pet allergies ( n = 399 ) , and mould allergies ( n = 199 ) . of the respondents who self - reported as having moderate symptoms ,
55% ( n = 479/865 ) stated having allergy symptoms that would be classified as being persistent allergic rhinitis sufferers . whereas , 76% ( n = 148/195 ) of those who reported having severe allergy symptoms would be classified as being persistent allergic rhinitis sufferers . of the respondents who reported having moderate symptoms , 54% ( n = 466/865 ) reported that they
always or mostly used medication to treat their allergy symptoms , whilst 46% ( n = 399/865 ) reported that they
this is in contrast to those who reported having severe symptoms , where 82% ( n = 159/195 ) reported that they
always / mostly used medication and 19% ( n = 36/195 ) reported that they sometimes used medication . over half of all the participants ( 597 ; 56% ) reported that their allergy symptoms were more severe in the morning than at any other time of the day ( figure 1 ) .
more females reported that their allergy symptoms were at their worst in the mornings ( females : 60% [ 328/545 ] versus males : 52% [ 269/515 ] ; p < .01 )
. when asked which symptoms were worse in the morning , sneezing ( 630 ; 59% ) , itchy / watery eyes ( 621 ; 59% ) , running nose ( 542 ; 51% ) , and blocked nose ( 459 , 43% ) were the most frequently reported .
( 630 ; 63% ) and blowing nose ( 610 ; 58% ) were the most frequently reported symptoms which made respondents feel embarrassed or unattractive around others .
the majority of respondents reported that their symptoms had an impact on their lives , either by waking them up ( 88% ; n
= 931/1060 ) or by affecting their mood for the day ( 97% ; n = 1025/1060 ) . when asked how their symptoms affected their mood for the day ( table 2 ) , being irritable and being tired and exhausted were most commonly reported .
almost all respondents ( 970 ; 91% ) indicated that their symptoms had some impact on their day ahead , 868 ( 82% ) reporting a negative impact on relationships and/or family life , and 969 ( 91% ) reporting that their symptoms had prevented them from doing something they would usually have done ( e.g. , exercised or gone to work ) . with the exception of reporting being tired and exhausted ,
those with self - reported severe allergy symptoms had a significantly higher impact on their mood compared to those with self - reported moderate allergy symptoms ( p < .05 ) .
a similar trend was seen with responses to the impact on the day ahead ( table 2 ) .
females reported a significantly higher impact than males for several measures relating to appearance and or interpersonal interactions .
in contrast , males were significantly more likely to report that their professionalism at work would be compromised ( 24% [ 125/515 ] versus 17% [ 93/545 ] , p < .05 ) .
the impact of morning symptoms on mood was analysed by the time of day that the symptoms were most severe ( figure 2 ) .
respondents who reported that their symptoms were most severe during the morning were more likely to report that their condition made them grumpy , moody , and frustrated .
conversely , people who suffered more at other times of the day ( including the night time ) were more likely to report feeling
the negative impact of morning allergy symptoms also extended to how people behaved during the day ( figure 3 ) , with a significant impact on usual morning behavioural patterns ( dreading public transport , feeling the need to explain that you are not sick , and being less affectionate with partner and children ) .
much work has already been conducted to determine the impact of allergic rhinitis on hrql , however , most is focussed on individuals with verified allergy leaving out those with self - reported allergy symptoms .
our study has shown that self - reported symptoms of moderate to severe allergic rhinitis have a significant negative impact on emotional and psychological wellbeing .
almost two out of every three respondents reported being either irritated or tired and exhausted as a consequence of their symptoms .
morning symptoms , in particular , have a substantial impact on mood for the day ( sufferers report feeling more grumpy , moody , and frustrated ) as well as on their usual daily activities .
these findings are similar to those published previously , which demonstrated that morning allergy symptoms not only have a negative impact on the quality of life of individuals with moderate to severe allergic rhinitis but that that these symptoms substantially impact the person for the rest of the day .
the results obtained in our study have extended these findings , demonstrating how symptoms in people who report self - awareness of allergic rhinitis negatively impact on home and work life and relationships with partners .
both seasonal and perennial allergic rhinitis sufferers may develop a group of psychological complaints related to symptom severity .
perceptions of symptom severity have been shown to be positively associated with personality factors ( hypochondriasis and somatic awareness ) whilst being independent of skin prick test results .
neuroticism , the tendency to experience negative emotions and to be highly self - conscious and somatically concerned , has been positively correlated with allergy .
furthermore , findings suggest that less adaptive coping strategies are associated with higher levels of psychological distress amongst allergy sufferers .
gender differences were apparent in our study , particularly in areas of the questionnaire related to appearance .
this could be explained by sociocultural attitudes , females have a greater willingness to acknowledge and report symptoms .
gender - specific differences in the interpretation of symptoms and their impact on the individual have previously been reported [ 14 , 15 ] .
moreover , gender is known to play a strong role in depression and other psychological disorders . amongst a sample of self - reported adult allergy sufferers in the us
, there was a significant relationship between allergy and depression in women but not in men . in the attempt to better understand the links between allergy and psychological status
the most notable limitation of our study is that the questionnaire used was not formally validated .
the questions were developed based on review of prior published instruments and conformed closely to that utilised by long .
our questionnaire extended focused predominantly on the emotional and psychological impact of typical allergic rhinitis symptoms .
as such , our questionnaire had an increased focus on how the symptoms made the respondent feel and which particular symptoms made them feel embarrassed or unattractive while at work or in the presence of others .
the absence of a numerical response scale in the questionnaire limits the ability to compare our results with those obtained in other studies .
although the sample sizes were weighted with respect to abs data , a permission - based panel was used such that the study population may not be representative of the general allergy population .
nasal congestion has been described as a cardinal symptom of allergic rhinitis . in our study
only 43% of respondents reported that nasal congestion was worse in the morning , which is lower than was expected .
one explanation for this discrepancy is that it is a direct result of a problem with the data collection tool .
the question asked was for you , which allergy symptoms are worse in the mornings ? it may be that nasal congestion is always present and therefore does not get worse in the mornings .
alternatively , our result may simply reflect that the study respondents did not have to have a prior physician diagnosis of allergy .
this is consistent with prior research ; it has previously been shown that nasal congestion is the symptom most likely to prompt a person with allergic rhinitis to seek medical help and that blocked nose
is not the dominant symptom in people who self - report awareness of allergic rhinitis .
the widespread availability of nonprescription allergy medications means that many people with symptoms suggestive of allergic rhinitis self - diagnose their condition and present to the pharmacy for treatment . in many cases they are then able to self - select a medication without recourse to health professional advice
it has previously been established that self - management of allergy symptoms is enhanced when healthcare professional intervention is sought and tailored goals established . we have shown that symptoms , particularly those that are most severe in the mornings , can substantially impact the overall emotional and psychological wellbeing of consumers who are self - aware of allergic rhinitis and who are self - medicating . encouraging these consumers to seek formal diagnosis and then combining appropriate treatment strategies , such as those offering sustained effectiveness over 24-hours , with an individually tailored
, goal - oriented , and self - management program may aid in optimising symptom management . |
background . allergies can substantially impact health - related quality of life ( hrql ) .
we investigated the psychosocial impact of morning symptoms amongst australian adults with self - reported allergic rhinitis ( ar ) .
method .
an online survey comprising 24 questions was conducted in august 2008 .
inclusion criteria were age ( 2049 years ) and self - reported moderate to severe symptoms of ar . results .
one thousand sixty respondents met the inclusion criteria . amongst consumers with self - reported ar ,
symptoms were more severe in the morning in 597 ( 56% ) and affected mood in 1025 ( 97% ) .
nine hundred seventy ( 91% ) indicated that their symptoms had some impact on their day ahead and 868 ( 82% ) reported a negative impact on relationships .
morning symptoms in particular had a substantial affect on mood for the day .
hrql impact was more pronounced in those who reported severe symptoms and in females .
discussion . encouraging consumers with self - diagnosed ar to seek formal diagnosis and offering appropriate treatment strategies , such as those offering sustained effectiveness over 24-hours , may aid in negating the negative impact of morning symptoms . | 1. Introduction
2. Methods
3. Results
4. Discussion |
PMC3499296 | the 2nd annual antibodies for cancer therapy symposium was opened by the chairman , mitchell ho ( national cancer institute ) .
he welcomed the attendees and thanked the cambridge healthcare institute , inc . for organizing this symposium .
he also thanked his fellow scientific advisory board members , soldano ferrone ( university of pittsburgh ) and horacio g. nastri ( pfizer , inc . ) for nominating and selecting this year s speakers .
he dedicated the opening keynote session to the celebration of the 15th anniversary of rituximab , the first approved cancer antibody drug , and then he introduced two keynote speakers on day one , ivor royston ( forward ventures ) and alain beck ( pierre fabre ) .
ivor royston gave the first keynote speech , monoclonal antibodies : the origins of cancer targeted therapy .
they are major components of the oncologist s armament in treating cancer and the forerunners of today s targeted therapy of cancer .
his talk focused on the historic views of cancer antibody therapy by discussing the evolution and ups and downs of these magic bullets .
royston generated mabs to t cell leukemia lines , but not the epstein - barr virus- transformed lymphoblastoid b cell lines from the same patient , when he was on the oncology faculty of the university of california at san diego in 1977 , two years after the nobel prize winning work was published in nature by kohler and milstein . in 1986 ,
rituximab ( rituxan ) , which is a chimeric anti - cd20 mab , was originally developed by idec under the name idec - c2b8 . with his post - doctoral fellow and later colleague bob dillman ,
dr . royston began pilot clinical studies in patients with t cell lymphoma and chronic lymphocytic leukemia ( cll ) in the early 1980s .
one of their patients with cutaneous t cell lymphoma ( ctcl ) showed a great response to the murine mab , but after several cycles of treatment , the response was abrogated by the production of human anti - murine antibody ( hama ) . at the same time , drs .
ronald levy and richard miller at stanford were developing anti - idiotypic mabs against ctcl and b cell lymphoma .
lee nadler , jerome ritz and stuart schlossman at the dana - farber cancer institute were running clinical trials on lymphoma and acute myeloid leukemia ( aml ) .
bismarck lozzio at the university of tennessee was also working on aml , and dr .
stefan thierfelder at the university of munich in germany was evaluating mab therapy on acute lymphoblastic leukemia ( all ) .
given that murine mabs have immunogenicity , induce hama in humans , and have a short half life , it became clear that human mabs were necessary for cancer therapy . in 1983 , dr .
royston published a paper on the creation of a drug - resistant human lymphoblastoid b cell line ( uc 7296 ) that could be used to make human - human hybridomas , which secrete antibodies . in this paper , dr .
royston and colleagues described the human - human hybridomas derived from lymphocytes isolated from regional draining lymph nodes ( ln ) of cancer patients .
uc 7296 can be fused with human lymphocytes to generate stable human - human hybridomas , some of which secrete antibodies reactive to human cell surface antigens .
royston discussed how 30 y ago , the first cancer patient in the world was treated with a fully human mab .
royston s post - doctoral fellow from japan , learned of a japanese patient with advanced cervical cancer and asked if he could bring the patient s tumor draining ln to the lab to make hybridomas . with dr .
they made a number of hybridomas and found a few that made antibodies that reacted with cervical cancer cell lines , as well as the ln dr .
the japanese patient was eventually treated with human mabs derived from this human - human hybridoma , and the event was featured in 1983 on a cbs evening news with dan rather episode .
the video also showed a cll patient being treated with an early murine mab ; this patient had a good clinical response and no hama response .
royston and others also noted this effect later in lymphoma patients , and they quickly realized that b cell lymphoma patients or cll patients only infrequently had an immune response to murine mabs , which is why rituximab was so successful in b cell lymphomas .
the human mabs to the japanese patient s tumor were later described in 1987 . in 1981 , drs .
richard miller and ronald levy were able to put a patient with follicular b cell lymphoma into complete remission for a number of years with a murine anti - idiotype antibody .
such an antibody was truly tumor specific since it only reacted with the tumor b cell idiotype .
ivor royston , as a co - founder of idec , tried to commercialize the idea of producing a customized anti - idiotype mab for lymphoma patients . however , the idea was not economically feasible , so the company turned its attention to a pan - b cell mab and subsequently produced the chimeric anti - cd20 rituximab . in 1997 , this product became the first antibody approved by the us food and drug administration ( fda ) to treat cancer .
the differential expression of cd20 on lymphoma cells provides the rationale for targeted agents against the antigen .
additionally , cd20 is an ideal target for therapy because it is neither shed nor modulated .
rituximab is a genetically engineered antibody that consists of the variable light- and heavy - chain regions from the murine anti - cd20 antibody idec - c2b8 grafted on human immunoglobulin ( ig ) g1 and constant regions .
rituximab is indicated for the treatment of cd20 + , relapsed or refractory , low - grade or follicular non - hodgkin lymphoma ( nhl ) , but the exact mechanism of cd20 mab induced antitumor or anti
it is generally accepted that host immune effector mechanisms , which include antibody - dependent cell - mediated cytotoxicity ( adcc ) and complement - mediated cytotoxicity ( cdc ) , are involved .
first , while rituximab ( igg1 ) effectively depleted cd20 + b cells , an equivalent igg4 version of rituximab was unable to deplete b cells in vivo in primates .
this is consistent with the role played by complement or adcc effector cells , given that they mediate cytotoxic effects best via the igg1 constant region .
second , rituximab showed a significant decrease in its antitumor effect in fcriii receptor deficient mice .
this argues that an fc receptor dependent mechanism ( i.e. , adcc ) contributes substantially to rituximab s antitumor effect .
third , in vitro studies showed that lymphoma cell lines and freshly isolated follicular lymphoma cells are targets for adcc and cdc in the presence of rituximab .
in addition to immune - mediated mechanisms via adcc and cdc , a recent study has shown that rituximab can induce apoptosis of some lymphoma cell lines via the caspase pathway .
it is likely that each of these mechanisms plays a role in the clinical activity of rituximab , although the relative contribution of each , and whether certain mechanisms are more important in selected histologies , is not yet clear . since the landmark approval of rituximab in 1997 , there have been seven additional unconjugated mabs , including trastuzumab ( herceptin ) , bevacizumab ( avastin ) , alemtuzumab ( campath ) , cetuximab ( erbitux ) , panitumumab ( vectibix ) , ofatumumab ( arzerra ) , and ipilimumab ( yervoy ) , that have been approved by the fda for cancer therapy .
four conjugated mabs , ibritumomab tiuxetan ( zevalin ) , tositumomab - i131 ( bexxar ) , brentuximab vedotin ( adcetris ) , and gemtuzumab ozogamicin ( mylotarg ) , have also been approved by the fda .
gemtuzumab ozogamicin , an igg4 conjugated to the drug calicheamicin , has been removed from the market because further studies failed to show efficacy .
the unconjugated antibodies are quite effective , have long half - lives in the blood stream , and can interact with human complement and effector cells in the patients s immune system .
the two radiolabeled anti - cd20 mabs ( ibritumomab tiuxetan and tositumomab - i131 ) are murine , which expedites clearance from the circulation .
the latest approval was seattle genetics brentuximab vedotin ( sgn-35 ) for refractory hodgkin disease and anaplastic large cell lymphoma ( alcl ) .
overall , there are 11 approved mabs ( plus one approval withdrawn ) for cancer therapy .
dr . royston concluded by stating that modern recombinant techniques have made it possible to rapidly produce chimeric , humanized and totally human antibodies .
these mabs are changing the face of cancer therapy and have ushered in the era of targeted therapy .
there are now more than 300 antibody drugs being evaluated in clinical trials . along with the targeted small molecule drugs that can act on mutated or aberrant gene products , the anti - cancer mabs are resulting in more remissions and better outcomes with far less toxicity . as the second keynote speaker , alain beck focused on the strategies and challenges for the next generation of therapeutic antibodies . in the first part of his talk , dr .
beck discussed selection of targets for mab therapy ( clinical relevance vs. biological functionality ) , optimization of mab structures ( pharmaceutical properties , functions , antibody - drug conjugation or antibody - drug conjugates ( adc ) , bispecifics , oligoclonals , and different scaffolds ) , and emerging research on biosimilars and biobetters .
he reviewed the regulatory approvals of antibodies and related structures and selection technologies ( chimerization , humanization , phage display and transgenic mice ) .
among 165 anti - cancer antibodies currently in clinical study , 84 ( 51% ) are unmodified igg , 25 ( 15% ) are adc , 10 ( 6% ) are bispecific antibodies , 17 ( 10% ) are engineered , and 16 ( 10% ) are fragments .
beck discussed how to select targets based on competition , safety , proof of concept and intellectual property .
he also analyzed targets in three categories : ( a ) clinically validated targets ( e.g. , cd20 , her2 , egfr , vegfa , epcam , and ctla-4 ) , ( b ) pre - validated targets ( e.g. , igf-1r , igf1/2 , hgf , c - met , her3 , vegf / vegf - r , trail - r , il6/il6r , il4/il13 , cd19 , cd22 , cd30 , cd33 , cd44 , cd80 min cxcr4 , and icam-1 ) , and ( c ) new functional targets ( e.g. , raag12 , cd9 , jam - a , cd151 , tsn-1 ) .
beck first reviewed the crystal structures of her2/egfr mabs and explained how functional mabs against her2 or egfr can be achieved by targeting different sub - domains on the receptors .
for example , functional mabs to the her2 extracellular domain target three different sub - domains ( i , ii , iv ) .
functional mabs to egfr target sub - domain ( iii ) except for mab 806 , while small tyrosine kinase inhibitors are directed at enzyme catalytic intracellular domains .
it partially occludes the ligand binding region on this domain and sterically prevents the receptor from adopting the extended conformation required for dimerization .
interestingly , when used as first - line treatment for her2-positive metastatic breast cancer in a study with 808 randomly assigned patients , the combination of pertuzumab , trastuzumab , and docetaxel ( pertuzumab group ) significantly prolonged progression - free survival with no increase in cardiac toxic effects , compared with the placebo , trastuzumab , and docetaxel ( control group ) .
the median progression - free survival was 12.4 mo in the control group , as compared with 18.5 mo in the pertuzumab group .
beck also briefly described the ongoing development of next generation mabs targeting igf1-r , hgf / sf - met , cxcr4 and a novel human junctional adhesion molecule a ( hjam - a ) .
beck then discussed the optimization of igg structure for antibody therapy with an emphasis on physicochemical and functional analysis of glycosylation .
he compared main glycoforms found in human and recombinant fc produced in cho , ns0 , and perc.6 mammalian cell lines , analyzed immunogenicity and half - life related to glycoforms produced in ns0 , sp2/0 , and plants , and discussed glyco - engineering to increase cytotoxicity or anti - inflammatory properties of mabs .
for example , he gave one example showing that genetically removing the c - terminal lysine residues in the constant region of igg yielded more homogeneous igg molecules .
he also compared igg1 and igg4 , and he showed that the single amino acid mutation of the igg hinge region ( s225p ) could avoid half antibody of igg4 in vivo .
he started with two adc drugs , inotuzumab ozogamicin ( pfizer / wyeth ) , a calicheamycin conjugated antibody targeting cd22 being evaluated in nhl patients and trastuzumab emtansine ( genentech ) , a maytansinoid ( dm ) 1conjugated antibody targeting her2 for breast cancer .
brentuximab vedotin ( seattle genetics ) , consisting of bretuximab and auristatin e ( 4 cytotoxic drugs per igg molecule ) , was developed to treat relapse cd30-positive lymphomas . in a phase 2 trial for hodgkin lymphoma , 94% ( 96 of 102 ) of patients achieved tumor reduction .
it combines both biologic activity by blocking downstream her2 signaling to inhibit proliferation of cells and intracellular delivery of the dm1 cytotoxic agent .
tratuzumab - dm1 is currently being evaluated in phase 3 clinical trials for metastatic breast cancer .
the reactive thiol in cysteine is used for coupling maleimide linkers in the generation of adcs . to make 2nd generation thiomabs ,
efforts have been made to engineer site - specific thio - trastuzumab variants for coupling to thiol - reactive linkers without perturbing antibody structure and function . based on antibody structural modeling ,
cysteines were engineered into the igg molecule at three sites differing in solvent accessibility and local charge .
the higher stability and superior in vivo efficacy of the lc - v205c variant conjugate may be due to faster maleimide ring hydrolysis , which prevents drug loss through the maleimide exchange from antibody to thiol - reactive constituents in the plasma . in the final part of his talk , dr .
beck discussed biosimilars , biobetters ( or biosuperiors ) , and next - generation antibody derivatives .
he started with the definitions of biosimilars and biobetters because there is confusion in the field .
he defined biosimilars as copies of an approved mab with the same amino acid sequence .
biosimilar antibodies can be produced from a different clone , and the manufacturing process can result in differences in glycosylation and other microvariations .
biobetters are mabs with a very close amino acid sequence , but optimized glycosylation profile ( e.g. , low fucose to enhance adcc , less immunogenic ) .
biobetters may have engineered fc domains to increase the serum half - life ( usually two or three amino acid mutations ) .
next - generation antibodies are follow - up mabs with different amino acid sequences and improved variable domains ( e.g. , better affinity ) or improved fc domains ( glyco- or amino acid - engineered to increase effector functions or half - life ) .
the first review in the european union of the marketing application of a biosimilar mab is in progress , and a draft of regulatory guidance is in the works .
dr . beck then noted that there is an urgent need to analyze the structures of biosimilar and biobetter antibodies qualitatively and quantitatively .
he and colleagues recently used a mass spectrometry method to characterize structures of trastuzumab and cetuximab .
cetuximab produced in sp2/0 ( a murine myeloma cell line ) contains > 30% of mouse glycoforms such as gal--1,3 gal .
the gal--1,3 gal epitope in cetuximab induces anaphylaxis in patients and their iges were specific to the gal--1,3 gal epitope .
however , iges are not able to bind other chimeric mabs ( rituximab and infliximab ) produced in cho cells .
a recent phase 1 study of anti - egfr mab in patients with solid tumors was completed in china . a recombinant anti - egfr mab ( cmab009 ) that has the same amino acid sequence as cetuximab was made but produced in cho cells .
therefore , unlike cetuximab , the new mab does not have the gal--1,3 gal epitope .
beck ended his talk by discussing the challenges to be faced in the next 10 y , such as identification and validation of new targets , addressing the resistance to current drug treatments , understanding target cross talk and regulation .
in the meantime , efforts must be made to decrease the costs of industrial production by increasing the productivity of the current cell lines , developing alternative production systems and purification processes , and optimizing the design of more homogeneous and stable iggs .
beck also pointed out that the availability of regulatory pathways to register biosimilar antibodies ( european medicines agency , fda ) might provide further motivation to achieve these goals . in the next section of the symposium , a featured panel presentation chaired by mitchell ho , ivor royston and alain beck discussed the future of mabs for cancer therapy .
the panel focused on three topics : ( a ) strategies to identify or select good targets ( clinical relevance vs. biological effects ) , ( b ) strategies to develop good antibodies ( monoclonal , polyclonal , bispecific , alternative scaffolds ) and ( c ) biosimilar antibodies and emerging markets ( e.g. , china , india ) .
beck s points about how some targets are clinically relevant ( for example , overexpression of the target in a large percentage of patients with the same tumor type ) and how some have biological effects ( for example , inhibition of cell proliferation or migration , induction of apoptosis ) .
royston and beck how the selection of therapeutic targets in cancers has evolved since the 1970s and whether major advances in genomics or proteomics may play a role in target identification .
the panel believes that functional genomics has already played a role in antibody therapy and will play a more important role in target identification in the future .
for example , tumor specific mutations have already provided opportunities for novel cancer therapies . on the other hand , quick analysis of the clinical relevance of novel functional targets remains challenging .
the panel then discussed strategies to develop better antibodies ( monoclonal , polyclonal , bispecific , alternative scaffolds ) .
beck emphasized the target ( such as igf and egf ) crosstalk . in conclusion ,
the panel discussed drug resistance and how the current trend in bispecific or multifunctional antibodies could potentially work better than single antibodies in cancers that are more resistant to single agents .
mitchell ho ( nci ) started by discussing the discovery of human mabs targeting mesothelin in mesothelioma , ovarian cancer , cholangiocarcinoma , and other cancers .
dr . ho described the new human mab hn1 specific to cell surface mesothelin in various tumor types and showed that the new antibody and recombinant immunotoxin based on the hn1 fv exhibited potent anti - tumor activity .
he then discussed how tumor microenvironments may present significant barriers to anti - tumor agents including mabs .
molecules involved in multicellular tumor microenvironments are difficult to study ex vivo . in collaboration with v. courtney broaddus ( university of california , san francisco ) and shuichi takayama ( university of michigan ) , dr .
he generated a tumor spheroid model using the nci - h226 mesothelioma cell line and patient cells .
microarray analysis revealed that 142 probe sets were differentially expressed between tumor spheroids and monolayers .
gene ontology analysis revealed that upregulated genes were primarily related to immune response , wound response , lymphocyte stimulation and response to cytokine stimulation , whereas downregulated genes were primarily associated with apoptosis .
these genes , including mmp2 , baff / blys / tnfsf13b , rantes / ccl5 and tnfaip6/tsg-6 , which are specific to the three - dimensional biological structure of tumors , may also have potential as targets for cancer therapy and diagnostics .
both tumor penetration and acquired apoptotic resistance cause the major barriers for antibodies , including antibody - drug / toxin conjugates in solid tumors .
dr . ho identified a key cell contact protein , e - cadherin , related to tight junctions in tumor penetration using mesothelioma spheroids .
he showed that the downregulation of e - cadherin by sirna and an e - cadherin - specific functional blocking antibody could sensitize cancer cells to anti - mesothelin immunotoxin killing .
andre lieber ( university of washington ) discussed junction opener 1 ( jo-1 ) , which is a small , recombinant adenovirus serotype 3-derived protein specific for the epithelial junction protein desmoglein 2 ( dsg2 ) . in mouse xenograft models employing her2/neu- and egfr - positive human cancer cell lines ,
jo-1 mediated cleavage of dsg2 dimers and reduced e - cadherin expression in tight junctions .
he found that jo-1-triggered changes that allowed for increased intratumoral penetration of the anti - her2/neu mab trastuzumab and increased therapeutic efficacy of trastuzumab in mouse xenograft models using breast , gastric , and ovarian cancer cells that were her2/neu - positive .
his group also combined jo-1 with the egfr - targeting mab cetuximab and found improved therapeutic outcomes in a metastatic model of egfr - positive lung cancer .
the afternoon session on day one entitled novel formats against cancer : antibody - drug conjugates and bispecifics was chaired by horacio g. nastri ( pifzer ) .
johnson described the pre - clinical development of several dart proteins that recruited effector cells to tumor targets .
berengere vire ( national heart , lung , and blood institute ) discussed the development of a novel antibody - drug conjugate ( adc ) derived from the igm - fc portion for cll therapy .
she noted that the fc receptor for igm ( also known as fcr ) was recently identified , and that adrian wiestner s group at the nih showed that fcr was overexpressed in cll cells .
aggregation of fcr on cll cells by igm prompted rapid internalization of both igm and fcr , reaching half - maximal internalization of cell - bound igm within one minute .
upon internalization , fcr transported igm through the endocytic pathway to the lysosome , where it was degraded , indicating a potential pathway for the delivery of therapeutic mab - drug conjugates into cll cells . in collaboration with christoph rader ( national cancer institute ) ,
vire generated an adc derived from the fc portion of igm to selectively target cll cells using a selenocysteine - based site specific conjugation with cemadotin and monomethylauristatin f. jin - san yoo ( pharmabcine ) started with tanibirumab ( ttac-0001 ) , a human mab specific for both human and murine vegfr-2 .
vegf binds to its receptor ( vegfr ) and stimulates angiogenesis , an important step in tumor growth and metastasis .
they found that tanibirumab inhibited the binding of vegf to vegfr-2 ( also known as kdr ) and , therefore , inhibited angiogenesis .
they compared tanibirumab with bevacizumab in a human malignant glioblastoma ( u-87 mg gbm ) orthotopic model , as well as lung cancer , breast cancer , colon cancer , and liver cancer models in mice and showed that tanibirumab had better efficacy in all of the models .
yoo then discussed a bispecific antibody format called dig - body and a specific example : dig - kt , a dig - body inhibiting the binding of vegf and angiopoietin to their receptors vegfr-2 and tie-2 .
dig - kt can inhibit both vegf- and angiopoietin- mediated cancer cell migration in vitro .
dig - kt has better efficacy than tanibirumab in u87-mg gbm and hep3b ( hepatocellular carcinoma ) orthotopic models .
yoo discussed pig - km , a bispecific antibody for kdr ( vegfr-2 ) and cmet and showed that pig - km had better efficacy than tanibirumab in a u-87 mg gbm orthotopic model .
christoph spiess ( genentech ) presented a knobs - into - holes technology to generate bispecific antibodies with non - common light chains .
each heavy chain is expressed with its corresponding light chain in separate escherichia coli cultures .
the first forum entitled effective penetration of tumor targets was moderated by mitchell ho ( nci ) .
it focused on : ( a ) penetration of solid tumors and the blood - brain barrier : challenges and opportunities , ( b ) role of cell junction proteins in tumor microenvironments and the identification of novel targets , and ( c ) 3d tumor culture technologies and applications .
the second forum entitled clinical potential of immunotherapy against advanced cancers was moderated by richard a. morgan ( nci ) .
it discussed immunotherapy categories ( antibody - based therapy , cell - based therapy , vaccines / gene therapy , what cancers to target and clinical trial design / end - points ) .
the third forum entitled analyzing trends for success of mabs chaired by alain beck ( pierre fabre ) discussed ( a ) target selection and validation , ( b ) antibody structure optimization , ( c ) alternative formats , ( d ) synergistic mechanisms of action , ( e ) biomarker identification and patient selection , ( g ) biosimilar and biobetter mabs .
the fourth forum entitled anticalins : diagnostic and therapeutic applications was moderated by laurent audoly ( pieris ag ) .
the second day symposium was chaired by soldano ferrone ( university of pittsburgh ) , who reviewed the origin of hybridomas by kohler and milstein and the substantial challenges that faced the field of therapeutic antibodies in the 1990s .
ferrone suggested that a lesson from that time is that it is critical to discuss important problems in the field so that solutions can be found .
ira h. pastan ( nci ) gave a keynote presentation entitled immunotoxin with low immunogenicity for cancer treatment .
recombinant immunotoxins are hybrid proteins containing an fv that reacts with a cancer cell and a bacterial or plant toxin that can induce antibody responses and limit the number of treatment cycles .
dr . pastan and colleagues have developed approaches to identify human b cell and t cell epitopes and produced active immunotoxins in which both types of epitopes have been removed .
examples of the recombinant immunotoxins currently being developed include ha22 ( cat-8015 ; moxetumomab pasudotox ) , which targets cd22 , and ss1p , which targets mesothelin .
each of these molecules contains pe38 , a truncated form of pseudomonas exotoxin a ( pe ) containing amino acids 253364 and 381613 .
cd22 is a cell surface protein only expressed on b cells and b cell malignancies .
it is not present on stem cells ; thus normal b cells can be regenerated after treatment stops .
phase 1 studies of moxetumomab pasudotox in patients with hairy cell leukemia ( hcl ) are completed . among the patients who failed standard chemotherapies , the overall response rate for moxetumomab pasudotox was 86% , and
therefore , moxetumomab pasudotox at doses up to 50 g / kg every other day ( qod ) 3 has activity in relapsed / refractory hcl and has a safety profile that supports further clinical development for treatment of this disease .
mesothelin is a cell surface glycoprotein overexpressed in mesothelioma , ovarian cancer , pancreatic cancer , lung cancer and many other cancers .
phase 1 clinical studies of ss1p in patients with advanced mesothelin - positive cancers are completed .
ss1p , given as an intravenous infusion qod for six or three doses , was administered to 34 patients with advanced mesothelioma ( n = 20 ) , ovarian ( n = 12 ) , and pancreatic ( n = 2 ) cancer .
seventeen patients were treated on the qod x 3 schedule , and the maximum tolerated dose ( mtd ) was 45 microg / kg / dose .
however , immunogenicity , i.e. , the formation of neutralizing antibodies , prevents retreatment and better responses in patients .
bl22 , an early version of anti - cd22 immunotoxin , has been reported to induce complete remissions in 4761% of patients with chemoresistant hairy cell leukemia .
unfortunately , in the phase 2 trial of bl22 , four ( 11% ) patients produced levels of neutralizing antibodies sufficiently high to prevent retreatment , and others made lower levels of antibodies . in solid tumors , immunogenicity is much higher ; nearly all patients treated with ss1p produced significant levels of neutralizing antibodies .
therefore , it is critical to reduce the immunogenicity of immunotoxins for cancer therapy . to reduce immunogenicity , dr .
pastan and colleagues identified b and t cell epitopes on pseudomonas exotoxin and removed them while retaining toxin activity . to determine b cell epitopes on pe38 , dr . pastan and colleagues hypothesized that mice and humans may have similar b cell epitopes .
therefore , initially they made 60 murine mabs against pe38 and identified seven major epitope groups .
the locations of many of these epitopes were validated by mutating large surface - exposed residues to alanine .
a mutant of moxetumomab pasudotox containing eight epitope - eliminating mutations ( ha228x ) was prepared , and this greatly reduced immunogenicity in mice . in parallel , two large sections of pe38 containing lysosomal protease cleavage sites were removed , leaving only amino acids 274284 and 394613 of the toxin . the resulting molecule ( named ha22-lr ) with the deletion of domain ii of pseudomonas exotoxin , retained cytotoxicity toward cd22 + cell lines , killed primary cll cells more potently than moxetumomab pasudotox , was much less toxic to mice , and
they also made a new molecule called ha22-lr-8 m to destroy the epitopes on domain iii of pseudomonas exotoxin by mutating eight bulky amino acids to alanine , glycine or serine .
m is fully cytotoxic against malignant b - cell lines , has high cytotoxic activity against cells directly isolated from patients with cll , and has strong antitumor activity in mice .
the immunotoxin does not induce antibody formation in mice when given repeatedly by intravenous injection , and it does not induce a secondary antibody response when given to mice previously exposed to ha22 . ha22-lr-8 m also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to ha22 .
in addition to mouse b cell epitopes , dr . pastan and colleagues have also investigated human b cell epitopes on pseudomonas exotoxin .
they recently generated several phage display libraries from the b cells in the patients treated with immunotoxins and identified major human b cell epitopes in order to remove them in immunotoxins . in the last part of his talk , dr .
pastan discussed their ongoing research in t cell epitope mapping of pseudomonas exotoxin and mutants that induced less t cell activity .
richard a. morgan ( nci ) discussed the development of a chimeric antigen receptor ( car ) , a hybrid protein composed of the antigen recognition domain from a fv fused to an intracellular t cell activation domain(s ) .
gross et al . first reported the generation of cars as functional receptors with antibody - type specificity .
since then , three generations of cars have been developed by adding additional t cell signaling domains .
the molecules targeted antigens such as cd19 ( b cell lymphoma and leukemia ) , cd20 ( mantle cell lymphoma and relapsed / refractory b - cell lymphoma / leukemia ) , gd2 ( neuroblastoma ) , cea ( adenocarcinoma , stomach carcinoma , breast cancer , colorectal carcinoma ) , psma ( prostate cancer ) , her2/neu ( lung malignancy , metastatic cancer , advanced osteosarcoma ) and il-13r2 ( glioblastoma ) . dr .
morgan discussed results obtained from ongoing anti - cd19 car gene therapy protocol to treat follicular lymphoma , cll , splenic marginal zone lymphoma , and large b cell lymphoma .
the protocol consisted of chemotherapy followed by an infusion of anti - cd19-car - transduced t cells and a course of il-2 ( clinicaltrials.gov , nct00924326 ) .
six of eight patients treated on their protocol obtained remissions of their advanced , progressive b cell malignancies .
four out of eight patients treated on the protocol had long - term depletion of normal polyclonal cd19(+ ) b - lineage cells .
he believes that anti - cd19-car - transduced t cells have great promise to improve the treatment of b cell malignancies because of a potent ability to eliminate cd19(+ ) cells in patients .
an anti - egfr variant iii ( egfrviii ) car vector based on human mab 139 is specific for egfrviii and has no reactivity against wild - type egfr .
egfrviii - car transduced glioblastoma multiforme ( gbm ) patient t cells recognize egfrviii - expressing targets , including glioma stem cell lines .
a phase 1/2 study led by steven rosenberg ( nci ) of the safety and feasibility of administering t cells expressing anti - egfrviii car to patients with malignant gliomas expressing egfrviii is currently ongoing ( clinicaltrials.gov , nct01454596 ) .
morgan also discussed the development by his nci colleagues of anti - mesothelin car targeting pancreatic cancer . in his collaboration with ira pastan
, steven rosenberg is leading a phase 1/2 study of metastatic cancer using anti - mesothelin car engineered peripheral blood lymphocytes in patients ( clinicaltrials.gov , nct01583686 ) .
hisataka kobayashi ( nci ) described a new antibody conjugate with a photosensitive near infrared ( nir ) phthalocyanine dye , ir700 , which can be used as an optical imaging agent at low doses of light , but becomes a photoimmunotherapeutic ( pit ) at higher doses of light .
kobayashi previously developed numbers of targeted ' activatable ' fluorescent imaging probes using rational chemical designs .
these agents are activated after cellular internalization by sensing the ph change in the lysosome or catabolizing by acidic enzymes using clinically feasible materials . among them , a novel acidic ph - activatable probe based on the boron - dipyrromethene fluorophore was synthesized .
it was then conjugated to a cancer - targeting mab and tested ex vivo and in vivo imaging of human egfr type 2-positive lung cancer cells in mice .
massive necrotic cell death was induced immediately after irradiating mab - ir700-bound target cells with nir light .
the mab - ir700 conjugates were most effective when bound to the cell membrane , and they produced no phototoxicity when not bound .
cancer stem cells ( cscs ; also known as tumor initiating cells ) were first demonstrated in leukemia .
the notch pathway mediates intercellular signaling in stem cell self - renewal , proliferation , and differentiation ; the ligand dll4 for the notch receptor is required for embryonic vascular development , and it is upregulated in tumor angiogenesis .
colleagues generated demcizumab , which has subnanomolar affinity for human dll4 but not murine dll4 , and found that the antibody was a potent and complete blocker of the interaction of dll4 and its notch receptor .
treatment with an anti - human dll4 mab inhibited the expression of notch target genes and reduced proliferation of tumor cells .
they found that specifically inhibiting human dll4 in the tumor , either alone or in combination with the chemotherapeutic agent irinotecan , reduced csc frequency .
they also found that an anti - murine dll4 antibody reduces tumor growth by de - regulating angiogenesis .
furthermore , they showed that anti - dll4 , not anti - egfr , is active in k - ras mutant colon tumors in mice .
they believe that mechanisms of action for anti - dll4 antibody are based on : ( a ) reduction of csc frequency and ( b ) disruption of tumor angiogenesis .
hoey summarized the antibodies currently being developed at oncomed that target : 1 ) notch pathway [ dll4 ( demcizumab , phase 1 as a single agent and combination therapy ) , dll4/vegf ( bispecific ) , notch2/3 ( phase 1 ) , notch 1 ] ; 2 ) wnt pathway [ fzd7 ( phase 1 ) , fzd8fc , smols ] ; and 3 ) rspo - lgr pathway .
laurent audoly ( pieris ag ) gave the presentation entitled path to clinical development and completion of a phase i clinical trial for a new class of therapeutic protein anticalins .
audoly described the safety and efficacy of anticalins in humans , the identification and characterization of a highly specific and purely antagonistic cmet - targeting anticalin , and the manufacturing of bispecific anticalins .
the results of the phase 1 trial demonstrated prs-050 was well - tolerated , and no mtd was reached .
the data supported the advancement of prs-050 into phase 2 studies in patients with solid tumors .
wayne a. marasco ( harvard medical school ) gave the presentation entitled human anti - ccr4 mab immunotherapy for the treatment of cutaneous t cell lymphoma .
ctcl is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin - homing malignant t cells .
these cells express a high level of chemokine receptor ccr4 that is associated with their skin - homing capacity .
marasco described a humanized anti - ccr4 mab and showed its potent anti - tumor effects in a mouse ctcl tumor model . in vitro studies
the anti - ccr4 mab was also able to reverse treg - induced suppression on effector t cell ( teff ) proliferation .
this latter property may allow this anti - ccr4 mab to be used to augment natural anti - cancer immunity against other solid tumors and hematologic malignancies where a contribution of treg to immune evasion by the tumor cells has been established .
david m. hilbert ( zyngenia ) discussed a new format of multi - specific , multi - valent mabs , called zybodies . this technology recombinantly fuses short target - binding peptides to the n- or c - termini of the ig heavy and light chains .
the resulting proteins can be bi- , tri- , tetra , or penta - specific , with each specificity represented bivalently .
he discussed the production , stability , binding attributes , and biological function of several lead therapeutic candidates for the treatment of breast cancer .
zyngenia made a trastuzumab igg - based penta - specific zybody simultaneously bound to five targets ( ang2 , v3 , her2 , igf-1r , and egfr ) and showed that the zybody was a more potent inhibitor of cell proliferation than trastuzumab alone .
hillbert showed that a bi - specific zybody ( her - ang2 ) targeting both her2 and ang2 showed more efficacy than trastuzumab alone in a sk - ov-3 xenograft model .
they also developed an adalimumab - based bispecific antibody called hum - ang2 , which targets tnf- and ang2 .
interestingly , treatment with the bi - specific hum - ang2 zybody inhibits both nf-b and akt pathways .
treatment with adalimumab inhibited only the nf-b pathway , and treatment with a control ang2 antibody inhibited only the akt pathway .
they found that hum - ang2 is more potent than adalimumab in an htnf- transgenic mouse model of autoimmune polyarthritis and colitis .
hans de haard ( argen - x ) gave the presentation entitled new insights into modulation of c - met function in cancer through a novel human antibody platform .
haard discussed argen - x s simple antibody platform with a focus on the c - met antibody program .
marc a. van dijk ( 4-antibody ag ) discussed a b cell based antibody display technology called retrocyte display .
this technology allows the stable expression of full - length human igg antibodies on the cell surface of b lymphocytes with monoclonal diversity > 1 x 10 for flow cytometry sorting of antigen - binding cells . using the library , they found human antibodies for a cell surface protein with affinity less than 2 nm and cross - reactivity with non - human primate and rodent homologs .
they can also screen multiple targets ( e.g. , tnf , egfr ) simultaneously by flow cytometry . | the 2nd annual antibodies for cancer therapy symposium , organized again by cambridge healthtech institute as part of the protein engineering summit , was held in boston , usa from april 30th to may 1st , 2012 . since the approval of the first cancer antibody therapeutic , rituximab , fifteen years ago ,
eleven have been approved for cancer therapy , although one , gemtuzumab ozogamicin , was withdrawn from the market .
the first day of the symposium started with a historical review of early work for lymphomas and leukemias and the evolution from murine to human antibodies .
the symposium discussed the current status and future perspectives of therapeutic antibodies in the biology of immunoglobulin , emerging research on biosimilars and biobetters , and engineering bispecific antibodies and antibody - drug conjugates .
the tumor penetration session was focused on the understanding of antibody therapy using ex vivo tumor spheroids and the development of novel agents targeting epithelial junctions in solid tumors .
the second day of the symposium discussed the development of new generation recombinant immunotoxins with low immunogenicity , construction of chimeric antigen receptors , and the proof - of - concept of photoimmunotherapy. the preclinical and clinical session presented antibodies targeting notch signaling and chemokine receptors . finally , the symposium discussed emerging technologies and platforms for therapeutic antibody discovery . | April 30, 2012: Day 1
Keynote Session
Tumor Penetration
Novel Formats against Cancer: Antibody-Drug Conjugates and Bispecifics
May 1, 2012Day 2: Opening Remarks
Immunotherapies in the Fight against Cancer
Pre-clinical and Clinical Data
Emerging Companies and Trends in Oncology |
PMC3334949 | in preparation for implementation of a central water processing system at a dental department , we analyzed the costs of conventional decentralized disinfection of dental units through daily continuous decontamination , purging and regular intensive decontamination against a central water treatment concept based on electrochemical disinfection .
this was studied at six departments running dental clinics in germany with a total of 44 dental units and approximately 35,000 patient contacts per year .
the necessity for preventive microbiological maintenance of dental units water line is given because of the ambient water temperature in the water lines , the synthetic material of water lines supporting biofilm formation together with curves and kicking leading to stagnation , , , because of dead spaces , and because of water stagnation during non - operational times , .
the combination of the above conditions is an ideal prerequisite for biofilm formation in case of bacterial contamination of the indwelling water , which is always the case .
biofilms , harboring non - pathogenic and pathogenic micro - organisms , are a source for continuous contamination of the cooling water in dental units , , , , .
aside of the total bacterial count , which also includes non - pathogenic microorganisms , also facultative and obligate pathogenic bacteria such as pseudomonas aeruginosa , , , legionella pheumophilia , , , mycobacteria and amoeba , may be present in contaminated cooling water .
if a microbial contamination with high colony counts of pathogenic bacteria is present , patients may be exposed to the risk of infection , particularly because of aerosol generation during treatment . because of this , the cooling water in modern dental units is continuously processed with hydrogen peroxide during patient treatment .
this , however , will not eliminate already existing biofilms , but may only be feasible to prevent initial biofilm formation , if continuously applied , , .
therefore , continuous purging and intensive purging is required and recommended by the manufacturers of dental units .
alternatively , instead of a decentralized , in the dental unit based purging system , a centralized decontamination concept may be applied , whereby the indwelling water is continuously treated before entering the dental unit s water line .
which of the both methods , however , is more cost effective , was not systematically investigated so far .
for calculation of the costs of the conventional decentralized water treatment concept , the intervals of the treatment and the volume of water usage during the decontaminating process were determined .
water costs are based on the price of 1 m water , during the time of the investigation at 1.96/m of water .
the unit price of decontamination chemicals was 23.34 respectively 13.34 per 1,000 ml of disinfectant .
the added concentration of hydrogen peroxide during the routine operation and the costs of the individual water usage for a dental unit could not be calculated because of technical restrictions .
the concentration of hydrogen peroxide was monitored continuously during patient treatment . for this , 1 ml of a sample was added to 100 ml using deionized water and mixed .
the hydrogen peroxide concentration was measured by titration against potassium permanganate until the color changed to pink over 30 seconds .
the hydrogen peroxide concentration was calculated using following formula : h2o2 + 2 kmno4 + 3 h2so4 2 mnso4 + 3 o2 + 4 h2o 1 ml kmno4 = 1.701 mg h2o2 for calculation of the costs of the blue safety technology concept , the costs of the required resources for generating hypochlorous acid , fundamentally nacl and water were calculated .
the system delivers a maximum of 10 liter hypochlorous acid per hour at a ph of 7.0 .
the 44 investigated dental units consisted of two different models from different manufacturers each . hence , the water line processing programs varied among the models , but also between the 6 encompassed dental departments ( table 1 ( tab
two hydrogen peroxide based products of two different manufactures were used . for sirona units ,
a product with 1.41% hydrogen peroxide ( recommended dilution of hydrogen peroxide : 0.0141% ) , and for kavo - units with 6% hydrogen peroxide ( recommended dilution of hydrogen peroxide : 0.02% ) was used . for maintenance , the sirona - unit has a purge rinsing function .
the concentration of the decontamination product corresponds to a dosage of 1:100 hydrogen peroxide during the normal operation of the unit .
this program is also available as auto - purge which consists of two single rinse procedures .
the typical flow rates range between 150 ml / min and for motor , turbine and ultrasound 60 ml / min , each . during intensive
additionally , one rinse is run before and after intensive purge with a total water volume of 500 ml .
the concentration of the decontaminating agent during the rinsing operations corresponding to the normal operation for the kavo - unit 12 litres of water and 60 ml of the 6% decontamination product ware used for intensive purge .
the concentration of the decontamination product ranges at 0.25% during intensive purge ( 1.5 litres decontamination product / water mixture ) .
the kavo - unit does not possess a comparable purge function like the sirona - unit .
in total annual costs of 4,448.37 were determined for the 6 investigated departments for routine maintenance of the dental units water lines ( table 2 ( tab .
the hydrogen peroxide concentration ranged above the 1:100 dilution recommended by the manufacturer for 8 sirona - units at 36.5% 23% above the target concentration . for 4 kavo - units , the target concentration for hydrogen peroxide at 0.02% as recommended by the manufacturer
was not reached . in total , the mean hydrogen peroxide dosage for constant dosage ranged at 2.5% 16.5% above the target concentration .
the blue safety technology device generates hypochlorous acid through membrane electrolysis of a 0.2%0.3% nacl solution . according to the manufacturer , the anolyte at a concentration of 200 mg / l contains ~158 mg / l hypochlorous acid , ~2 mg / l hypo chlorite ions , < 1 mg / l ozone , < 2.5 mg / l chlorine dioxide , < 1.5 mg / l chlorous acid , and <3 mg / l chlorine acid at a ph of 7.0 and a half life time of up to 48 hours .
the redox potential of the anolyte solution is + 914 mv ( 1 mv ) .
the anolyte solution is stored in a storage tank and a concentration of 0.6 mg / l hypochlorous acid is injected directly into the water - works network centrally .
therefore , issues with manufacturer s warranty of dental units do not arise , since the active compound in injected into the water work of the indwelling water before it enters the dental unit itself . based on the calculated annual water consumption of approximately 1,012 m water ( table 2 ( tab .
2 ) ) , which is added additionally 0.6 mg / l of allowed free chlorine , the system must generate 3,036 liters of a 200 mg / l concentrated anolyte solution .
a 25 kg bag of nacl costs approximately 6 . calculated on an annual consumption ,
since the water consumption of 4 m will incur anyway , the water costs are not to be included further into this calculation .
the cost of the consumables for the blue safety technology accounts for 0.05% of the costs for the decentralized , in - built disinfection procedure .
this , however , is only based on the required consumables and disinfection products during maintenace , since for both methods the additional costs of electricity could not be calculated because of missing information not provided by the manufacturers .
also , the costs for investment could not be calculated , since the disinfection program in the dental - units are part of the device , making it difficult to assess the sole price of this feature without the price for the dental unit alone . because the blue safety technology was newly developed at the time of this study , the manufacturer was not able to provide commercial prices for purchasing the technology , installation , and validation . however , because the blue safety technology fundamentally uses salt as medium to generate antimicrobially active hypochlorous acid as chief active agent , it can be concluded that the one - time only investment costs of possibly 10,000 to 15,000 will quickly amortize during routine usage .
if the avoided costs for sanitizing heavily contaminated water lines , loss of income due to closure of a unit during sanitation etc . are added , then the breakeven will occur even faster . finally ,
because of no requirement for purchasing , storage , manual preparation and maintenance of decontamination products , an additional saving of staff time , and hence money , may be assumed .
after installation of the blue safety technology , a continuous dosage of hydrogen peroxide is dispensable and can be omitted as cost factor .
a comparable procedure to the blue safety technology is the electrochemical generation of ecasol. the efficacy of this was demonstrated during a 2-years clinical study .
the authors concluded that no higher concentrations than 12 mg / l free chlorine were required .
the necessary constant water conditions were achieved by a cascade of lavishly processing steps , starting with particle filtration , water softening , an active charcoal module , and kdf ( kinetic degradation fluxion )
80 medium , and finally a kdf-55 filter . by means of gas chromatographic mass spectrometry
the processed water was regularly checked for unwanted side products such as chloroform , bromodichloromethane , dibromochloromethane , bromoform , trichloroethane , tetrachloroethane , and 1,2-dichloroethane . for all , the levels ranged below < 0.01
mg / l , the level of 1,2-dichloroethane was below 0.001 mg / l . at these concentrations ,
however , a comparison of the corrosivity related to the hydrogen peroxide - based decontamination has not been made so far .
although the concentrations of hydrogen peroxide partly surpassed the manufacturer s recommendations , the mean concentration of 0.021% 0.007% was not exceeded . because of the high d - value particularly of candida spp . even at planktonic state
, such a hydrogen peroxide concentration may not be able to inhibit biofilm formation , particularly in segments with stagnation or dead spaces , if used only during patient treatment .
this is further supported , if the unit s routine preventive maintenance using purge and intensive purge is not regularly performed . | background : in preparation for implementation of a central water processing system at a dental department , we analyzed the costs of conventional decentralized disinfection of dental units against a central water treatment concept based on electrochemical disinfection.methods : the cost evaluation included only the costs of annually required antimicrobial consumables and additional water usage of a decentralize conventional maintenance system for dental water lines build in the respective dental units and the central electrochemical water disinfection system , blue safety technologies.results : in total , analysis of costs of 6 dental departments reviled additional annual costs for hygienic preventive measures of 4,448.37 .
for the blue safety technology , the additional annual total agent consumption costs were 2.18 , accounting for approximately 0.05% of the annual total agent consumption costs of the conventional maintenance system .
for both water processing concepts , the additional costs for energy could not be calculated , since the required data was not obtainable from the manufacturers.discussion : for both concepts , the investment and maintenance costs were not calculated due to lack of manufacturer 's data .
therefore , the results indicate the difference of costs for the required consumables only .
aside of the significantly lower annual costs for required consumables and disinfectants ; a second advantage for the blue safety technology is its constant and automatic operation , which does not require additional staff resources .
this not only safety human resources , but add additionally to cost saving.conclusion : since the antimicrobial disinfection capacity of the blue safety was demonstrated previously and is well known , this technology , which is comparable or even superior in its non - corrosive effect , may be regarded as method of choice for continuous disinfection and prevention of biofilm formation in dental units water lines . | Introduction
Methods
Results
Discussion
Notes
Competing interests |
PMC4226061 | the cd4 count is a measure of immune status in hiv - infected individuals which measures the severity of immunosuppression more accurately than clinical staging of disease .
it is therefore included in international ( who / unaids ) and national aids programme guidelines as a threshold marker to indicate the initiation of antiretroviral therapy ( art ) [ 1 , 2 ] .
however , an accurate determination of cd4 cell count requires flow cytometry which is often unavailable in resource - limited settings for a variety of reasons . if cd4 testing is not available , who guidelines have previously recommended using clinical staging either alone or in combination with an absolute lymphocyte count for determining art eligibility . in fiji ,
a cd4 cell count is recommended to determine eligibility for initiating art but is often unavailable .
an absolute lymphocyte count is therefore used as an alternative , with a count of 2300 cells/l being used to determine eligibility for art in an hiv - infected individual with clinical stage 1 or 2 .
furthermore , fiji is now implementing the recent world health organization ( who ) guidelines which use a higher cd4 count ( 500 cells per mm ) than previously ( 350 cells per mm ) to determine eligibility .
the implication of this change for the practice of using an absolute lymphocyte count as an alternative is not known .
this study aimed to describe the cd4 counts and absolute lymphocyte counts in hiv - infected fijians prior to initiation of art , to determine the correlation between them in individual patients , and to evaluate the accuracy of a range of threshold values of absolute lymphocyte counts as an alternative to the recommended cd4 count threshold .
this was a retrospective study which evaluated clinical records from all hiv - infected individuals in fiji up until 31 december 2012 .
fiji is a low to middle income country with a population of 837,271 ( 2007 census ) .
the ministry of health administers three major divisions and each division has a reproductive health clinic that provides care for all hiv - infected individuals in fiji .
the first case of hiv infection was diagnosed in fiji in 1989 and art was introduced in 2004 . following the diagnosis of hiv infection , assessment for eligibility for art
blood samples were taken at the reproductive health clinics and sent within 624 hours to the divisional hospital laboratory for full blood count , including total white blood cell and lymphocyte counts , and to the fiji centre for communicable disease control for cd4 cell count ( alere pima analyser ) .
cd4 testing became available in fiji in 2010 but has been unavailable since early 2012 due to stock - out of laboratory reagents . at the time of initial clinical and laboratory assessment of the hiv - infected individuals included in the study ,
the guidelines in fiji recommended that art be initiated in hiv - infected individuals categorised as who clinical staging 3 or 4 and in those with a cd4 cell count of 350 cells / mm regardless of the clinical stage . when cd4 count was not available , the senior hiv clinician ( dab ) used an absolute lymphocyte count value of 2300 cells/l as a surrogate for a cd4 cell count of 350 cells / mm on the basis of studies from other settings .
the national guidelines have recently been revised to be consistent with the new who guidelines that now recommend initiation of art in hiv - infected individuals that are categorized as who clinical stage 1 or 2 with a cd4 cell count of 500 cells / mm [ 3 , 5 ] .
only those with complete data available for results of both absolute lymphocyte count and cd4 count for baseline and follow - up were included in the analysis .
variables included age , gender , ethnicity , weight , the who clinical staging , and laboratory markers that included absolute lymphocyte count , haemoglobin , and cd4 cell count taken before and 3 months after initiation of art .
data were collected and double - entered directly into an electronic data collection sheet ( epi data 3.1 ) .
data were compared for discordance and corrections were made by verification with the patient folders to produce a finalized data set .
positive predictive value , negative predictive value , and sensitivity and specificity of varying alc cut - offs were computed for cd4 counts 350 cells / mm and 500 cells / mm. the paired t - test was used to compare the differences in clinical markers in response to art .
ethics approval for the study was obtained from the ethics advisory group of the international union against tuberculosis and lung disease , the fiji national health research committee , and national research ethics review committee .
the data from 101 hiv - infected patients were available and were included in the study .
the mean age of the patients was 34 years ( range : 18 to 61 years ) and 50% were males . prior to initiation of art ,
the median absolute lymphocyte count was 1429 cells/l ( range : 135 to 3825 cells/l ) and the median cd4 cell count was 157 cells / mm ( range : 6 to 722 cells / mm ) .
the correlation between absolute lymphocyte count and cd4 cell count was weak ( r = 0.497 ) and this is illustrated in figure 1 .
table 1 shows the frequency distribution and corresponding who clinical stage of patients using various cd4 cut - offs that have been applied in recommendations for the initiation of art .
the majority of the patients had a cd4 count of 200 cells / mm at the time of initial assessment , and 86% had a cd4 count of 350 cells / mm which would indicate eligibility for art irrespective of clinical staging applying the guidelines in use at the time .
of the 101 hiv - infected individuals , 88 ( 87% ) had an absolute lymphocyte count of < 2300 cells/l and 59 ( 58% ) were either clinical stage 1 or 2 and so required cd4 count data to determine eligibility for art . however , the majority of the 59 patients assessed as clinical stage 1 or 2 had cd4 counts of 350 cells / mm or of 500 cells / mm , 46 ( 78% ) and 55 ( 93% ) , respectively .
of the 46 clinical stage 1 or 2 patients eligible to initiate art on the basis of a cd4 count of 350 cells / mm , 6 ( 13% ) had absolute lymphocyte counts of greater than 2300 cells/l and so would have been wrongly denied initiation of art if absolute lymphocyte count alone was used . on the other hand , all four ( 4% ) hiv - infected patients that had a cd4 count of > 500 cells / mm and were clinical stage 1 , and so ineligible for art , had an absolute lymphocyte count of less than 2300 cells/l .
table 2 lists the calculated positive predictive values , negative predictive values , and sensitivities and specificities for a range of different absolute lymphocyte counts cut - offs that might be used as a surrogate for a cd4 cell count of 350 cells / mm. as expected , increasing the absolute lymphocyte count cut - off increased the sensitivity of the test for determining those with a cd4 count of 350 cells / mm , but with the cost of reduction in specificity .
the sensitivity of the absolute lymphocyte count in use in fiji that has been used ( i.e. , < 2300 cells/l ) was 88.5% for a cd4 of 350 cells / mm in all patients , reflecting that the majority of patients had low numbers of both counts ( figure 1 ) .
however , the specificity was only 13.5% as the majority ( 12 of 14 ) of patients with a cd4 of > 350 cells / mm had an absolute lymphocyte count level of < 2300 cells/l ( figure 1 ) .
absolute lymphocyte counts for a cut - off of 500 cells / mm showed similar sensitivity to that found for a cd4 count of 350 cells / mm ( data not shown ) .
an absolute lymphocyte count of 2300 cells/l had a sensitivity of 87.6% for a cd4 count of 500 cells / mm but a specificity of 0% because all four patients with a cd4 of > 500 cells / mm had an absolute lymphocyte count level of < 2300 cells/l .
the response to art for weight and laboratory markers after 3 months of initiation is shown in table 3 .
there was significant weight gain and increase in alc and cd4 counts after 3 months of art but no change in haemoglobin values .
this study provides original data from fiji of clinical staging , cd4 counts , and absolute lymphocyte counts in hiv - infected individuals at the time of assessment for initiation of art .
of the 101 individuals , 42% were indicated to receive art on the basis of clinical staging alone . of the remaining individuals of clinical stage 1 or 2 that required a cd4 count of 350 cells / mm to determine eligibility , as per recommendations at the time
, the use of an absolute lymphocyte count of 2300 cells/l as a surrogate cut - off would have resulted in incorrect management in 10 ( 17% ) of 59 individuals : 6 eligible for art that would not receive art and an additional 4 not yet eligible for art that would have started art . with adoption of the recent recommendations of using a cd4 count cut - off of 500 cells / mm to determine eligibility , using an absolute lymphocyte count of 2300 cells/l as a surrogate cut - off
would have resulted in all individuals receiving art including the 4 individuals with clinical stage 1 disease that had cd4 counts > 500 cells / mm. these findings are important and currently relevant for two main reasons .
firstly , in line with who recommendations , the fiji national guidelines have recently increased the cd4 cell count to initiate art to 500 cells / mm from the previous 350 cells / mm [ 1 , 2 ] .
this will increase the number of hiv - infected individuals that are eligible to receive art .
we found that almost all ( 96% ) hiv - infected individuals had a cd4 count at the time of assessment that was 500 cells / mm. secondly , there is only one pima cd4 machine for cd4 testing in fiji and the cd4 test continues to be frequently unavailable due to the lack of laboratory reagents for testing .
however , using an absolute lymphocyte count as a surrogate would have resulted in all individuals receiving art . due to the low absolute lymphocyte counts and cd4 counts in the majority of this population and a weak correlation between counts within individuals , it was difficult to determine an optimal cut - off that provides an acceptable balance between sensitivity and specificity .
for example , if a low absolute lymphocyte count level with a high specificity was used , such as < 1500 cells/l , then nearly half of the hiv - infected individuals eligible for art by cd4 criteria would not receive treatment .
an important limitation is the small number of hiv - infected individuals with complete data available for retrospective analysis .
this study population of 101 individuals represented a convenience sample that was included on the basis of having complete test results available , when cd4 testing was only available for a limited time from 2010 .
it is not certain therefore how representative this group might be for the wider population of hiv - infected individuals in fiji .
further , clinical staging data were not collected prospectively and so it is difficult to validate accuracy of the clinical categorisation .
it is also not possible to know the wider relevance of these findings to other populations .
previous similar studies from a range of settings including early studies from the united states , england , and south africa [ 69 ] and more recent studies from low - resource settings such as india and nigeria [ 1013 ] have found variable results and have interpreted similar results differently .
there are many factors that are likely to influence the results of absolute lymphocyte counts and cd4 counts in different populations apart from the effect of hiv infection , which include prevalence of other infections as well as other biological and genetic factors .
these groups have different guidelines for art initiation which do not rely on cd4 counts and previous studies have shown that alc would not be a substitute for cd4 count in these populations [ 14 , 15 ] .
we did find that absolute lymphocyte count increased in response to 3 months of art as did cd4 count and weight .
however , we did not determine whether alc could be a suitable alternative to cd4 to monitor response after 3 months of art or would provide additional benefit over clinical improvement and weight gain . in conclusion , the results of this study would not support the continued use of absolute lymphocyte count to determine art initiation in hiv - infected individuals in fiji .
rather , when cd4 count is unavailable , the introduction of a recommendation to initiate all hiv - infected individuals on art should be considered . |
introduction .
an absolute lymphocyte count is commonly used as an alternative to a cd4 count to determine initiation of antiretroviral therapy for hiv - infected individuals in fiji when a cd4 count is unavailable .
methods .
we conducted a retrospective analysis of laboratory results of hiv - infected individuals registered at all hiv clinics in fiji .
results . paired absolute lymphocyte and cd4 counts were available for 101 hiv - infected individuals , and 96% had a cd4 count of 500 cells / mm3 .
correlation between the counts in individuals was poor ( spearman rank correlation r = 0.5 ) .
no absolute lymphocyte count could be determined in this population as a suitable surrogate for a cd4 count of either 350 cells / mm3 or 500 cells / mm3 .
the currently used absolute lymphocyte count of 2300 cells/l had a positive predictive value of 87% but a negative predictive value of only 17% for a cd4 of 350 cells / mm3 and if used as a surrogate for a cd4 of 500 cells / mm3 it would result in all hiv - infected individuals receiving art including those not yet eligible .
weight , cd4 count , and absolute lymphocyte count increased significantly at 3 months following art initiation .
conclusions .
our findings do not support the use of absolute lymphocyte count to determine antiretroviral therapy initiation in fiji . | 1. Introduction
2. Methods
3. Results
4. Discussion |
PMC3839072 | for quite some time , especially the practical courses in biochemistry were deemed to be difficult amongst hamburg medical students .
biochemistry is a very abstract subject many medical students have difficulties with . in order to increase the students interest and
therewith the motivation for self - organised learning , we expanded the e - learning offer for biochemical courses at the university medical center hamburg - eppendorf ( uke ) in 2007 to include audio podcasts .
for this , we relied on the most used journalistic format from radio broadcasting , the radio documentary ( see figure 1 ( fig .
1 ) ) . what is characteristic of the radio documentary is the interplay between host and original sounds .
the focus is on the text of the host , running through the feature like a red thread .
because of their authenticity , they are particularly credible and elaborate on what the host only touched upon .
thanks to the new voice and often times a new acoustic environment , the feature becomes more lively and invites to continue listening , .
many e - learning projects are initialised with the help of generous funding subsidies and can often not be continued once the funding is terminated .
we are here presenting a cost - efficient project along with means of how to continue it with a long - term perspective .
at the same time we are trying to answer the question whether audio podcasts in the shape of radio documentaries have some kind of expiry date .
is the quality of the features still recognized in the same way after a longer useful time ?
usually , students use scripts and textbooks to prepare for conducting and interpreting experiments in practical courses . the uke podcasts are intended to provide them with another interesting insight into the topic of the practical course and , in addition , strengthen their intrinsic motivation for learning .
different groups of people are responsible for conducting the course , and they are all included with original sounds in the podcasts ( see figure 2 ( fig .
2 ) ) . the teacher explains the theoretical background , a technical assistant helps with the practical implementation .
another group , not an integral part of the course but just as important as the others , are the clinicians , who illustrate the relevance of the conducted experiments with regards to patient care .
the part of the host , which is central to the feature , is taken over by a student , who receives a 100 expense allowance per podcast at the uke . producing the podcasts
, we paid attention to keeping the time burden as low as possible for all involved . at first , a student , a teacher , a technical assistant , and a clinician meet for an editorial conference in order to compile the themes of the text blocks assigned to the different speakers ( see figure 3 ( fig .
each speaker role comprises two to three text blocks per feature , two to four minutes each .
following the editorial conference , the text blocks are elaborated on independently on the basis of the guiding keywords assigned .
the manuscript is double - checked by the teacher and then approved for recording which can be done independently at any time by the speakers .
a conventional office desktop pc , the audio software audacity ( freeware ) , and a usb microphone ( around 50 ) are sufficient means for recording . finally , the host compiles the recorded text blocks to represent the completed feature using the software audacity .
the audio podcasts were published on the e - learning platform mephisto hosted by the uke as well as in the cross - university podcast platform
between 2007 and 2010 , eight podcasts have been framed for practical courses in biochemistry ( see table 1 ( tab .
1 ) ) . to address the question whether over time there is a change in quality to be noticed with regards to audio podcasts in the shape radio documentaries ,
the quality of the audio podcast is evaluated in a descending range from one to six with regards to listening experience , structure ( i.e. the distribution of speaker roles ) , topicality , feature length , and overall satisfaction .
apart from their gender , probands indicated whether they listened to the podcast during the corresponding course . proposals and criticism as well as general recommendations concerning the use of podcasts
the questionnaire introduced in 2012 contained additional questions concerning usage and benefits of audio podcasts ( see tables 2 ( tab .
, the questionnaire was distributed amongst third semester students in practical courses in biochemistry at the uke .
the independent samples t - test is used as a method of inference statistics to test for differences in podcast quality ( =0.05 ) between 2008 and 2012 .
the individual podcasts have been extensively used by both students of the uke and the interested public .
1 ) ) exceed by far the number of medical students in hamburg ( close to 400 per year ) .
unfortunately , it is impossible to distinguish between accesses from students and from other users .
thus , from these figures we can not draw any conclusions on our primary target group .
we have no explanation for the extremely high access numbers for the blood glucose podcast .
maybe it was used by many diabetics as a means of informing themselves about their disease . despite their very specialised contents , the features on
lipoproteins , enzymes , and blood glucose have been among the five best liked ( accessed most often ) and five best ( according to listener evaluation ) on podcampus . in 2008 , the biochemistry podcast
practical course on lipoproteins at the university medical center hamburg - eppendorf was elected from a group of 50 audio and video podcasts among the 10 finalists of a nation - wide podcast competition for universities and scientific institutions via online voting . in 2008 , 323 third semester students participated in the anonymized audio podcast evaluation ; in 2012 it was 360 students ( see table 2 ( tab .
, a much higher percentage of males ( 52% ) had listened to the podcasts compared to females ( 32% ) . in 2012 , usage clearly declined in both genders : of the 360 students questioned only 8% stated that they had listened to the podcasts offered on practical courses in biochemistry .
37% of the participants stated explicitly in their free - text comments that they had not been aware of the podcast offer , probably this applied to most other participants as well .
of the 331 students who in 2012 had not listened to the podcasts , 57 wished for podcasts on additional topics from biochemistry , 24% wished for a clearer announcement of the podcast offer in the future .
evaluation of the podcasts in the shape of the radio documentary was both times very positive ( see figure 4 ( fig .
the total satisfaction was in both 2008 and 2012 m = 2.1 , which is to be interpreted as good . comparing the marks of quality looking at the two times of survey
, only the item topicality shows a significant difference at a p - level < 0.05 .
the evaluation of the uses of podcasts in 2012 showed that 57% of those questioned use the podcasts to prepare for the practical course .
at least 27% use them for a general understanding of biochemical contents . with just 7% ,
usage for recapitulating the content of the practical course was a rather minor motivation ( see table 3 ( tab .
the use of podcasts for preparation of the course , the following written examination , and the understanding of lesson contents was evaluated similarly by the students ( see table 4 ( tab .
involving students , podcasts in the shape of the radio documentary can be produced to support classroom teaching with little expenses in terms of costs and time for the faculty .
they are very popular with part of the students , which can be attributed to the emotional impact of the spoken word , but as well to the medium s being free from restrictions to time and place . even after a couple of years
nonetheless , the number of listeners strongly declined after four years , probably due to lack of awareness amongst the students . upon their introduction
, the podcasts had been promoted in lectures and seminars as well as via messages on the e - learning platform .
this advertisement was missing in the following years , but seems to be necessary in order to advise interested students of the offer . in case of changes in the curriculum
, podcasts can , thanks to the modular structure , easily be updated by just replacing outdated text blocks of individual speakers with new ones . beside practical courses in biochemistry , many other topics are imaginable , such as other courses , portraits of individual teachers ( http://www.podcampus.de/channels/67 ) , or interdisciplinary aspects of the faculty ( http://www.podcampus.de/channels/60 ; http://www.podcampus.de/channels/91 ) .
in addition to their usage in blended learning , the podcasts are surprisingly often downloaded by the interested public , despite their specialised contents . in this way
, they contribute to the positive visibility of biochemistry as a subject and of the medical faculty in hamburg , as is illustrated by the very good evaluations on podcampus and the mentioning by other universities , .
we would like to thank all involved teachers , technical assistants , clinicians , and students for their support . at the beginning of the project ,
norvin kubik and annemarie seidel have especially contributed a lot to its success . in addition
, we would like to thank anette stber and patrick peters of the multimedia kontor hamburg for their technical support in the early years . | introduction : audio podcasts are an e - learning format that may help to motivate students to deal with the contents of medical education more intensely .
we adopted a well - proven format from radio broadcasting , the radio documentary , to direct the listeners attention to information about practical courses in biochemistry over a period of 20 minutes at most .
information , original sounds , and a specific atmosphere allow listeners to perceive the contents intensely.method : in order to organise the production of the podcast as cost - efficient and least time - consuming as possible , a student , a teacher , a clinician , and a technical assistant compile the core themes of their respective text blocks in an editorial conference first .
after that , the speakers can elaborate on and record their blocks independently .
coordination is widely handled by the student . at two points of time ,
the podcasts were evaluated by the medical students by means of a questionnaire.results : with little cost and time expenses , eight podcasts were produced .
they have been used by the students extensively and have also been evaluated very positively by non - student listeners . for long - term usage , a regular reference to the podcast offer
is required in the courses.conclusion : involving students , successful podcasts can be produced to support classroom teaching with little expenses and contribute to the external presentation of the medical faculty . | Introdcution
Setting up audio podcasts for practical courses in biochemistry
Method
Results
Discussion and Conclusion
Acknowledgement
Competing interests |
PMC1852381 | persistent biliary fistulas can be associated with local skin morbidity , malabsorption of fat soluble vitamins , steatorrhea , impaired wound healing , and sepsis as a result of fistula tract obstruction.1 we describe a rare scenario in which a liver transplant recipient developed a biliary cutaneous fistula secondary to hepatic artery thrombosis . through a novel approach ,
the fistula skin opening was deepithelialized and , using this transabdominal fistula tract as a conduit , anastomosed to a jejunal loop , thereby internally draining the fistula .
this operative procedure , which has provided a successful long - term result , is discussed .
a 60-year - old man of chaldean descent underwent orthotopic liver transplant for cirrhosis secondary to chronic alcohol use .
his early postoperative course was complicated by hepatic artery thrombosis with necrosis of the extrahepatic biliary system .
surprisingly , his graft remained viable with good metabolic function despite the absence of normal extrahepatic drainage . for 7 years subsequent to transplantation ,
his biliary drainage was managed with a u - tube connected to a bulb drain exiting the skin in the right subcostal region ( fig . 1 ) .
as a result , he suffered from severe malnutrition , pain , and skin breakdown .
the persistent external drainage markedly affected his lifestyle , as the continuously draining bile required dressing changes over the exit site two to four times per day .
the u - tube , frequently becoming obstructed with biliary sludge , required bimonthly replacement .
2 ) demonstrated the continuity of the cutaneous opening with a large channel through the mid portion of the liver , but with no evidence of a true ductal system .
figure 1(a ) longstanding ( 7-year - old ) biliary cutaneous fistula draining through right upper quadrant scar .
( b ) close - up of u - tube exit site.figure 2preoperative fistulogram in which contrast was injected through the existing u - tube .
the cutaneous opening at the upper left side of the frame is in continuity with the large contrast - filled channel that enters the substance of the liver .
( a ) longstanding ( 7-year - old ) biliary cutaneous fistula draining through right upper quadrant scar .
preoperative fistulogram in which contrast was injected through the existing u - tube . the cutaneous opening at the upper left side of the frame is in continuity with the large contrast - filled channel that enters the substance of the liver .
note the complete absence of an extrahepatic biliary system . with the patient under general anesthesia
, the abdominal cavity was entered through a right subcostal incision along the scar of the previous transplant incision .
the incision was fashioned so as to preserve a 1 " diameter button of full - thickness skin surrounding the opening to the fistula tract ( fig .
extensive adhesions between the fascia , liver surface , and small intestine were carefully lysed .
the fistula tract was dissected retrogradely from surrounding subcutaneous tissue and muscle , preserving a rectus fascial ring .
dissection of the tract was stopped proximally at the level of the liver surface to preserve its integrity and vascularity .
a 60-cm long roux - en - y jejunal limb was constructed 50 cm distal to the ligament of treitz using stapled technique .
figure 3(a ) at operation , the u - tube was prepped into the surgical field .
( b ) dissection of the epithelialized chronic biliary cutaneous fistula tract . in entering the abdomen through the previous right subcostal incision
, a 1 " diameter button of full - thickness skin containing the fistula tract was preserved .
( a ) at operation , the u - tube was prepped into the surgical field .
( b ) dissection of the epithelialized chronic biliary cutaneous fistula tract . in entering the abdomen through the previous right subcostal incision ,
a 1 " diameter button of full - thickness skin containing the fistula tract was preserved .
a 4-mm - wide skin edge was sharply deepithelialized circumferentially around the dissected fistula opening .
this conduit was then anastomosed to the roux limb using two layers of running 4 - 0 prolene suture ( fig . 4 ) .
the inner layer approximated the deepithelialized skin edge of the fistula tract to full - thickness jejunum .
the outer layer approximated the conduit s scar tissue and the preserved rectus fascial ring to seromuscular jejunum .
the anastomosis was performed over an 8-french feeding tube , which was exteriorized through a previous u - tube exit site and connected to a bile bag .
figure 4(a ) anastomosis of roux - en - y jejunal limb to deepithelialized fistula tract .
( a ) anastomosis of roux - en - y jejunal limb to deepithelialized fistula tract .
a 60-year - old man of chaldean descent underwent orthotopic liver transplant for cirrhosis secondary to chronic alcohol use .
his early postoperative course was complicated by hepatic artery thrombosis with necrosis of the extrahepatic biliary system .
surprisingly , his graft remained viable with good metabolic function despite the absence of normal extrahepatic drainage . for 7 years subsequent to transplantation ,
his biliary drainage was managed with a u - tube connected to a bulb drain exiting the skin in the right subcostal region ( fig . 1 ) .
as a result , he suffered from severe malnutrition , pain , and skin breakdown .
the persistent external drainage markedly affected his lifestyle , as the continuously draining bile required dressing changes over the exit site two to four times per day .
the u - tube , frequently becoming obstructed with biliary sludge , required bimonthly replacement .
2 ) demonstrated the continuity of the cutaneous opening with a large channel through the mid portion of the liver , but with no evidence of a true ductal system .
figure 1(a ) longstanding ( 7-year - old ) biliary cutaneous fistula draining through right upper quadrant scar .
( b ) close - up of u - tube exit site.figure 2preoperative fistulogram in which contrast was injected through the existing u - tube .
the cutaneous opening at the upper left side of the frame is in continuity with the large contrast - filled channel that enters the substance of the liver .
( a ) longstanding ( 7-year - old ) biliary cutaneous fistula draining through right upper quadrant scar .
preoperative fistulogram in which contrast was injected through the existing u - tube . the cutaneous opening at the upper left side of the frame is in continuity with the large contrast - filled channel that enters the substance of the liver .
with the patient under general anesthesia , the abdominal cavity was entered through a right subcostal incision along the scar of the previous transplant incision .
the incision was fashioned so as to preserve a 1 " diameter button of full - thickness skin surrounding the opening to the fistula tract ( fig .
extensive adhesions between the fascia , liver surface , and small intestine were carefully lysed .
the fistula tract was dissected retrogradely from surrounding subcutaneous tissue and muscle , preserving a rectus fascial ring .
dissection of the tract was stopped proximally at the level of the liver surface to preserve its integrity and vascularity .
a 60-cm long roux - en - y jejunal limb was constructed 50 cm distal to the ligament of treitz using stapled technique .
figure 3(a ) at operation , the u - tube was prepped into the surgical field .
( b ) dissection of the epithelialized chronic biliary cutaneous fistula tract . in entering the abdomen through the previous right subcostal incision
, a 1 " diameter button of full - thickness skin containing the fistula tract was preserved .
( a ) at operation , the u - tube was prepped into the surgical field .
( b ) dissection of the epithelialized chronic biliary cutaneous fistula tract . in entering the abdomen through the previous right subcostal incision
, a 1 " diameter button of full - thickness skin containing the fistula tract was preserved .
a 4-mm - wide skin edge was sharply deepithelialized circumferentially around the dissected fistula opening .
this conduit was then anastomosed to the roux limb using two layers of running 4 - 0 prolene suture ( fig . 4 ) .
the inner layer approximated the deepithelialized skin edge of the fistula tract to full - thickness jejunum .
the outer layer approximated the conduit s scar tissue and the preserved rectus fascial ring to seromuscular jejunum .
the anastomosis was performed over an 8-french feeding tube , which was exteriorized through a previous u - tube exit site and connected to a bile bag .
figure 4(a ) anastomosis of roux - en - y jejunal limb to deepithelialized fistula tract .
( a ) anastomosis of roux - en - y jejunal limb to deepithelialized fistula tract .
this patient has now been followed for 7 years since the described procedure and is doing well without external drainage of bile .
the condition of his periincisional skin improved dramatically shortly after the procedure . at this time
his liver function tests and nutritional parameters are normal and his graft function remains adequate on a low dose immunosuppressive regimen .
chronic biliary cutaneous fistulas are challenging to manage from many standpoints : metabolic , nutritional , hygienic , and quality of life
first , malignant change was reported in chronic biliary cutaneous fistulas 2 ; this possibility is of real concern in light of this patient s immunosuppressed state .
second , this patient had severe malabsorption and resulting difficulty in maintaining acceptable cyclosporin blood levels . finally , the ongoing presence of his u - tube poses the threats of ascending cholangitis , hemobilia , intrahepatic biloma , and biliary - venous fistula , which are all well - documented complications that have been associated with u - tubes.3 the decision for operative intervention was made out of these concerns and for progressive difficulty in maintaining adequate external biliary drainage .
late retransplantation was considered ; however , the patient s graft function had been excellent over the 7 years subsequent to his transplant , and the patient and his family refused to consider it . in both the posttraumatic and postbiliary surgery settings , existing literature advocates initial nonsurgical management of biliary cutaneous fistulas , as a significant number will close either spontaneously or with nonsurgical intervention.1,4,5 in the series by zer , four of seven biliary fistulas sealed spontaneously.5 endoscopic approaches to reducing intrabiliary pressure and thereby encouraging drainage along a path of least resistance include endobiliary stenting , sphincterotomy , and nasobiliary drainage.1,4 finally , selective biliary embolization , percutaneous transhepatic catheter drainage , sclerosis with tetracycline , and oral nitrates were also described to promote closure.4,69 operative procedures for refractory biliary cutaneous fistula were dictated by the anatomic site of biliary tract disruption .
roux - en - y hepaticojejunostomy , in which a jejunal loop is sutured directly to the liver capsule , was employed in the context of intrahepatic biliary injury after trauma.11,10,4 reports in which an actual fistula tract is used as a conduit and sutured to a loop of small bowel , however , are rare .
smith described the anastomosis of a jejunal loop to a divided fistulous tract arising from the lateral surface of the liver in a patient with a penetrating injury to the upper abdomen.12 that same author described the internal drainage of a biliary cutaneous fistula secondary to blunt liver trauma into the gallbladder.12 in both cases the fistula tract was divided and then the proximal end of the divided tract was anastamosed to an intestinal loop over a tube .
deepithelialization of the skin opening and its direct usage in an anastomosis has not , to our knowledge , been previously described .
furthermore , this procedure is unique in that the internalization was done in the case of a functioning liver after hepatic artery thrombosis .
the existing anatomy that had been established over 7 years of fistulous drainage dictated the type of procedure that was performed .
in contrast to fistulas that arise after biliary tract surgery ( for example , after cholecystectomy with common bile duct exploration ) , there was no remnant of a previous biliary ductal system .
the preoperative fistulogram revealed flow of contrast from the skin directly to the liver surface with no evidence of an extrahepatic ductal system .
an alternative surgical option would have been hepaticojejunostomy , in which the jejunum would have been sutured to a fibrous ring at the origin of the fistula tract from the liver surface .
this , however , would have required more extensive dissection with possible disruption of collateral vasculature to the previously ischemic liver and to the tract itself , as well as dissection of the transverse colon , which in part bordered the tract . at operation , the superficial portion of the fistula tract was well established and appeared well vascularized . the potentially harmful deep dissection necessary for a hepaticojejunostomy
we emphasize the decision to have fashioned a tongue of omentum over the anastomosis . by nature
we feel that the well - vascularized , adherent omental tongue was an important aspect of the operation in reinforcing the anastomosis .
as of 7 years of follow - up , the patient has not developed signs of biliary obstruction .
his immunosuppressant levels are easier to maintain and he does not have evidence of malabsorption .
it is unlikely that he will develop cicatricial narrowing of the tract , as it had remained well epithelialized for the 7 years before this operation .
there is a theoretical concern about formation of squamous cell carcinoma at the anastomotic site , which we continue to keep in mind during long - term follow - up .
in summary , through a novel approach this patient s biliary cutaneous fistula was internalized , which has eliminated his associated skin morbidity and malabsorption . furthermore
, his well - functioning graft was salvaged , thus saving the cost of retransplantation and sparing an additional liver for another patient .
internalization of a long - standing biliary cutaneous fistula through deepithelializing the skin opening and using the tract as a conduit is technically feasible .
we believe it to be a valid approach in the patient in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal biliary drainage by conventional approaches . | biliary cutaneous fistulas are uncommon sequelae after biliary surgery and can be a source of significant morbidity .
we describe a liver recipient who developed a biliary cutaneous fistula secondary to hepatic artery thrombosis ; this subsequently drained for over 7 years . through a novel approach , using the transabdominal fistula tract as a conduit , the fistula skin opening was deepithelialized and anastomosed to a jejunal loop , internally draining the tract . for over 7 years
postoperatively , this internal drainage procedure has continued to function effectively .
this approach may have value in internalizing longstanding biliary cutaneous fistulas in well - selected patients in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal drainage through conventional approaches . | Introduction
Materials and Methods
Case Report
Surgical Procedure
Result
Discussion
Conclusion |
PMC3333783 | many individuals , in the absence of organic injuries or orthopedic pathology , show a slight misalignment of body segments.1,2 this misalignment is associated with detectable asymmetries in the tonic and phasic activation of symmetrical muscular groups in the lower limbs that produce a stable misalignment of one s left and right patellar margins1 during motions that are intended to be symmetrical , such as moving in a sit - up motion from a supine to a sitting position.2 this phenomenon , which we call functional dysmetria3 ( fd ) , may be related to the effects of neuropsychomotor attitude on postural control and may be the consequence of an adaptation to environmental stress411 in animals1214 as well as humans.1519 as for finger - to - nose dysmetria , fd is difficult to evaluate as an absolute measurement.20 therefore , the evaluation of fd must be made by medical experts and consists of observation of the presence or absence of fd . in assessing fd , a specific caliper specially designed for this type of evaluation21 ( dismetrometro , asmed , florence italy )
the radioelectric asymmetric conveyer reac22,23 is an innovative medical device originally designed to use the effects produced by the interaction between the electromagnetic field of the human body and those produced by the instrument .
reac is an innovative technology for biostimulation and bioenhancement that uses weak radioelectric fields to modulate brain activity .
reac treatment has previously been reported to ameliorate several stress - related disorders,2427 depression,26,28,29 anxiety,26,29 bipolar disorders,30 some forms of dementia,31 and impaired motor control.32,33 in a recent study , we examined the effects of reac administered according to a precisely defined neuro - postural optimization3 ( npo ) protocol on brain activation patterns during a finger tapping motor task .
we found that changes in activity patterns in motor cerebral and cerebellar regions were evident following the reac - npo treatment.34 the aim of the present preliminary study was to examine whether the reac - npo treatment that was previously shown34 to affect brain activity patterns in motor regions during the aforementioned finger tapping task would affect fd evident during a lower limb motor task in which subjects are asked to perform a bilateral flexion - extension movement of the legs , bending the legs at the knees . based on our clinical observations2,3 of improvements in balance and coordination following reac - npo in our patients , we hypothesized that the reac - npo treatment would reduce fd signs in the present experimental study group .
each participant gave signed informed consent to participate in the study , which is registered in the australian new zealand clinical trial registry ( actrn12611000366954 ) .
physically active , right - handed students who regularly engaged in running were recruited to ensure a cohort of subjects in good health , particularly with respect to motor performance ability .
after screening ten volunteers for fd , a total of ten healthy graduate students ( six females , four males ) , ranging in age from 2532 years ( mean , 28 years ) , were confirmed by two clinicians to show fd and volunteered to participate in this study without financial compensation .
fd was analyzed by two expert clinicians as volunteers moved from a supine to a sitting position on a rigid surface .
this action reveals the presence of fd as a misalignment of the patella ( red line in figure 1a ) . as summarized in figure 1 ,
the volunteers were subjected to two fd assessments and two functional magnetic resonance imaging ( fmri ) scans . before reac - npo , each subject was assessed for fd as described above ( figure 1a ) and then scanned while performing the alternating leg flexion - extension motor task described below ( figure 1b ) . immediately following the scan ,
the subject was brought to a designated treatment room for delivery of the reac pulse ( figure 1c ) .
forty minutes after the reac - npo , the subject was brought back to the magnet room and scanned while performing the alternating leg flexion - extension motor task , as described below ( figure 1d ) . following the second scan ,
a post reac - npo assessment was performed ( figure 1e ) . the motor task consisted of a simple block of leg - bending movements with alternating flexion and extension of the knee with the subject lying in a supine position ( figure 1b ) . within each block
a convogliatore di radianza modulante reac instrument ( asmed , florence , italy ) specific for noninvasive brain stimulation techniques was used in this study .
the reac - npo protocol used consisted of a single 250-millisecond radiofrequency burst administered at 5.8 ghz .
the reac probe is placed at the level of the scapha28,32 when the radiofrequency pulse is delivered ( figure 1c ) .
the reac procedure is painless , and no adverse effects from its use have been reported .
the density of the radio - electric current flowing to the subject ( 150 cm from the emitter ) during the single radiofrequency burst is 7 a / cm and the electromagnetic field surrounding the device is approximately 20 w / m .
brain fmri was performed with a high - field unit ( 1.5 t philips intera nt , the netherlands ) , shown in figure 1b .
the survey was obtained with sequences of centering axial , sagittal , and coronal planes .
volumetric sequences ( with gradient echo t1 ( 3d tfe ; tr = l3 , te = 3 , fa = 30 ) and t2 ( epi - ffe ; tr = 3000 , te = 50 , fa = 90 ) ) were oriented upon these planes . the total duration of the fmri acquisition , conducted while the subject performed the alternating leg flexion - extension motor task , was about 40 minutes .
two radiologists who were not present for the reac - npo treatments performed the scans . acquired dicom images were sent to a computer running on a linux operating system and then exported as com pressed nifti files .
the nifti files were then processed using feat ( fmri expert analysis tool ) software in the fmrib software library ( fsl ) .
brain tissue was isolated from surrounding tissues using the brain extraction tool ( bet ) in the fsl .
the mcflirt motion correction tool was used to correct for misalignment due to subjects inadvertent movements using an algorithm based on the flirt technique of intra- and intermodal brain image registration . the output
was then processed with fmrib s improved linear modeling ( film ) tool in accordance with the block diagram of the study and taking into account the spatial parameters of the head motion detected by the mcflirt tool .
the results were coregistered ( ie , the different sessions were registered to each other with flirt ) .
the relative z statistic image was designated with a z - threshold of 4.6 to show which clusters were activated significantly ( p = 0.05 ) .
the activation images were rendered with a pseudocolor scale ( z value range , 4.69 ) and then superimposed onto a standard brain template . to assess inter - subject and inter - group variance and average activation within each phase of the experiment ( before and after reac - npo )
, the output of the first - level statistical analysis was processed using fmrib s local analysis of mixed effects algorithm , which eliminates outlier data .
an arbitrary z - threshold of 2.3 was set to enable rendering in a pseudocolor scale using a contiguous - clusters technique ( significance level p = 0.05 ) .
each participant gave signed informed consent to participate in the study , which is registered in the australian new zealand clinical trial registry ( actrn12611000366954 ) .
physically active , right - handed students who regularly engaged in running were recruited to ensure a cohort of subjects in good health , particularly with respect to motor performance ability .
after screening ten volunteers for fd , a total of ten healthy graduate students ( six females , four males ) , ranging in age from 2532 years ( mean , 28 years ) , were confirmed by two clinicians to show fd and volunteered to participate in this study without financial compensation .
fd was analyzed by two expert clinicians as volunteers moved from a supine to a sitting position on a rigid surface .
this action reveals the presence of fd as a misalignment of the patella ( red line in figure 1a ) .
as summarized in figure 1 , the volunteers were subjected to two fd assessments and two functional magnetic resonance imaging ( fmri ) scans . before reac - npo , each subject was assessed for fd as described above ( figure 1a ) and then scanned while performing the alternating leg flexion - extension motor task described below ( figure 1b ) . immediately following the scan ,
the subject was brought to a designated treatment room for delivery of the reac pulse ( figure 1c ) .
forty minutes after the reac - npo , the subject was brought back to the magnet room and scanned while performing the alternating leg flexion - extension motor task , as described below ( figure 1d ) . following the second scan ,
the motor task consisted of a simple block of leg - bending movements with alternating flexion and extension of the knee with the subject lying in a supine position ( figure 1b ) . within each block
a convogliatore di radianza modulante reac instrument ( asmed , florence , italy ) specific for noninvasive brain stimulation techniques was used in this study .
the reac - npo protocol used consisted of a single 250-millisecond radiofrequency burst administered at 5.8 ghz .
the reac probe is placed at the level of the scapha28,32 when the radiofrequency pulse is delivered ( figure 1c ) .
the reac procedure is painless , and no adverse effects from its use have been reported .
the density of the radio - electric current flowing to the subject ( 150 cm from the emitter ) during the single radiofrequency burst is 7 a / cm and the electromagnetic field surrounding the device is approximately 20 w / m .
brain fmri was performed with a high - field unit ( 1.5 t philips intera nt , the netherlands ) , shown in figure 1b .
the survey was obtained with sequences of centering axial , sagittal , and coronal planes .
volumetric sequences ( with gradient echo t1 ( 3d tfe ; tr = l3 , te = 3 , fa = 30 ) and t2 ( epi - ffe ; tr = 3000 , te = 50 , fa = 90 ) ) were oriented upon these planes .
the total duration of the fmri acquisition , conducted while the subject performed the alternating leg flexion - extension motor task , was about 40 minutes .
two radiologists who were not present for the reac - npo treatments performed the scans .
acquired dicom images were sent to a computer running on a linux operating system and then exported as com pressed nifti files .
the nifti files were then processed using feat ( fmri expert analysis tool ) software in the fmrib software library ( fsl ) .
brain tissue was isolated from surrounding tissues using the brain extraction tool ( bet ) in the fsl .
the mcflirt motion correction tool was used to correct for misalignment due to subjects inadvertent movements using an algorithm based on the flirt technique of intra- and intermodal brain image registration . the output
was then processed with fmrib s improved linear modeling ( film ) tool in accordance with the block diagram of the study and taking into account the spatial parameters of the head motion detected by the mcflirt tool .
the results were coregistered ( ie , the different sessions were registered to each other with flirt ) .
the relative z statistic image was designated with a z - threshold of 4.6 to show which clusters were activated significantly ( p = 0.05 ) .
the activation images were rendered with a pseudocolor scale ( z value range , 4.69 ) and then superimposed onto a standard brain template . to assess inter - subject and inter - group variance and average activation within each phase of the experiment ( before and after reac - npo )
, the output of the first - level statistical analysis was processed using fmrib s local analysis of mixed effects algorithm , which eliminates outlier data .
an arbitrary z - threshold of 2.3 was set to enable rendering in a pseudocolor scale using a contiguous - clusters technique ( significance level p = 0.05 ) .
the fd ( evidenced by misalignment of the patellae during a sit - up movement ) observed before reac - npo treatment was alleviated in all ten subjects at the time of the second , post reac - npo fmri scan .
that is , two expert clinicians reported that the subjects showed proper alignment of the patellae at the second scan ( see figure 1e ) . due to the presence of artifacts that degraded the functional images ,
the averaged fmri datasets from the remaining nine subjects before and after reac - npo are illustrated in figure 2 .
in particular , note that there was markedly less activation in motor cortical areas , disappearance of right thalamic activation , and reduced activation in the cerebellar vermis and pontomesencephalic regions in the post reac - npo averaged images ( figure 2a ) relative to the pre reac - npo averaged images ( figure 2b ) .
the current study showed that a 250-millisecond reac - npo pulse was able to produce changes in brain activity that were apparent 40 minutes after delivery of the reac stimulation .
this observation implies there are stable changes in brain activation patterns , including activation of brain areas involved in motor control . additionally , following the reac - npo treatment , we observed improved patellar symmetry when subjects were asked to perform a sit - up motion , indicating they experienced a concomitant amelioration of fd .
we suggest that the presently observed reduction in fd may be due to brain activity changes enabling a more efficient motor control and motor strategy,35 similar to that described following other brain stimulation techniques.36 the modulation of cerebellar function is the most important finding of this study , considering the importance of the cerebellum in motor control.3740 moreover , there is growing evidence implicating the cerebellum in stress - influenced affective41 and cognitive4244 functions . interestingly , numerous studies have related stress to various forms of dysmetria , including motor dysmetria and behavioral and cognitive dysmetria phenomena such as cognitive dysmetria,45,46 dysmetria of thought,4750 and cerebellar cognitive affective syndrome.48,5153 these forms of dysmetria do not appear to result from any anatomical or physiological lesion , but rather seem to reflect a cerebellar dysfunction.47,50 hence , fd may be the result of dysfunctional adaptive phenomena that involve the complex interrelationships between cerebellar , cerebral motor , cognitive , emotional , and behavioral functions .
since in our clinical experience the positive effects of reac - npo on coordination and fd tend to persist for long periods of time , even years,3 we have an interest in examining the potential of reac - npo for remodeling cerebellar activation related to coordination and precision of movement .
additional research is needed to delineate the physiological mechanisms underlying the establishment and alleviation of fd .
furthermore , longer - term controlled studies are needed to elucidate the long - term stability of changes in the brain activation pattern and fd alleviation observed following reac - npo .
correction of fd may be particularly helpful in the rehabilitation of patients showing fd in addition to a motor physiological pathology , as improved coordination may facilitate recovery .
additional studies to compare the brain activities of an experimental stimulation group with those of a sham stimulation group are needed . | purposethis study assessed changes in functional dysmetria ( fd ) and in brain activation observable by functional magnetic resonance imaging ( fmri ) during a leg flexion - extension motor task following brain stimulation with a single radioelectric asymmetric conveyer ( reac ) pulse , according to the precisely defined neuropostural optimization ( npo ) protocol.population and methodsten healthy volunteers were assessed using fmri conducted during a simple motor task before and immediately after delivery of a single reac - npo pulse . the motor task consisted of a flexion - extension movement of the legs with the knees bent . fd signs and
brain activation patterns were compared before and after reac-npo.resultsa single 250-millisecond reac - npo treatment alleviated fd , as evidenced by patellar asymmetry during a sit - up motion , and modulated activity patterns in the brain , particularly in the cerebellum , during the performance of the motor task.conclusionactivity in brain areas involved in motor control and coordination , including the cerebellum , is altered by administration of a reac - npo treatment and this effect is accompanied by an alleviation of fd . | Introduction
Material and methods
Study population
Study design
Motor task
REAC-NPO
Image acquisition
Image processing
Statistical analysis
Results
Discussion
Conclusions |
PMC3272553 | robot - assisted laparoscopic radical prostatectomy ( ralp ) has been rapidly accepted as a viable option for the minimally invasive surgical treatment of localized prostate cancer . since the introduction of the da vinci robotic surgical system ( intuitive inc . , surgical , ca , usa ) in 2001 , various reports on ralp have claimed comparable or superior outcomes to open and laparoscopic surgery in oncological and functional aspects .
the recovery of potency and continence are important factors for the quality of life of patients undergoing ralp .
the recovery of continence depends on various factors , and among these , the surgical technique is considered most critical .
the recovery rate is satisfactory , with rates of 85% to 97% by 1 year after radical prostatectomy , but complete recovery may require a much longer time in some patients [ 2 - 6 ] . in this study , we compared the outcomes of bladder neck preservation ( bnp ) using lateral bladder neck dissection ( lbnd ) and the posterior urethral reconstruction ( pur ) techniques for early recovery of continence during ralp .
the effects on the early recovery of continence and positive surgical margins ( + sms ) were evaluated and compared when the bnp technique using lbnd and the pur technique were used .
the study subjects included 107 patients who underwent ralp owing to localized prostate cancer within the period from january 2008 to august 2010 . the following techniques for early recovery of continence
were performed chronologically , and recovery of continence was observed during this period . in group a , standard ralp techniques with ven velthoven continuous suturing for urethrovesical anastomosis ( uva ) were performed in 31 patients . in group b ,
standard ralp techniques with pur and ven velthoven continuous suturing for uva were performed in 28 patients . in group c ,
standard ralp techniques using lbnd as the bnp technique with pur and ven velthoven continuous suturing for uva were performed in 48 patients .
the ven velthoven technique requires continuous suturing of the urethra using monofilament 3 - 0 double arm sutures from 6 to 12 o'clock of the urethral lumen .
the pur technique requires identification of the posterior portion of the rhabdosphincter and the anterior leaflet of denovilliers fascia . using one arm of the continuous suture , the posterior part of the rhabdosphincter is anchored with continuous sutures of three to four bites to the remaining portion of the denovilliers fascia .
the other arm of the continuous suture is then placed to approximate between the posterior bladder neck and the posterior urethra and then tied to the other arm of the continuous suture ( fig .
another arm of the continuous suture is used to perform ven velthoven continuous suturing for uva .
lateral bladder neck dissection for the bnp technique requires precise identification of the detrusor muscle fibers at the critical junction of the lateral bladder neck , prostate base , and the fat pad .
meticulous dissection is performed just medially to the fat pad of whitmore encountering adipose tissue and the lateral border of the bladder neck , while appreciating the ventrolateral and dorsal longitudinal muscles , until the dissection reaches the prostate base and the anterior surface of the seminal vesicle .
after the bilateral dissection of the lateral bladder neck , which exposes the anterior surface of the seminal vesicle , the detrusor muscles in the posterior bladder neck are well appreciated medially from the anterior surface of the seminal vesicles and the bladder neck is then approached at the midline .
the dissection is continued until distinct musculus sphincter vesicae are identified and preserved , and then the bladder neck is divided ( figs . 2 , 3 ) .
the medical records of the patients were collected , and the preoperative and postoperative clinical course and recovery of continence in the three groups were compared . digital rectal examination , prostate biopsy , magnetic resonance imaging , and bone scan
perioperative parameters such as operative time , blood loss , catheterization time , pathology data , and positive surgical margin rate ( + smr ) were reviewed from our surgical database .
postoperative continence was assessed through a questionnaire survey administered 1 day after catheter removal , and at intervals of 1 month , 3 months , and 6 months after ralp .
postoperative continence was defined as lack of incontinence or the use of less than one diaper ( 50 ml ) per day for security .
postoperative cystograms were performed in all patients on the 7th postoperative day . in the case of leakage ,
catheterization was maintained for an additional 5 to 10 days and the cystogram was repeated .
the variables before and after the operation among the patient groups were compared by using one - way analysis of variance test and fisher 's exact test , and p values of less than 0.05 were considered statistically significant .
the mean age of the patients in groups a , b , and c was 65.9 , 65.2 , and 64.9 years , respectively ( p=0.41 ) , and the mean preoperative prostate - specific antigen values were 9.4 , 10.6 , and 9.2 ng / ml , respectively ( p=0.63 ) .
the mean body mass index values were 24.3 , 23.9 , and 24.8 kg / m , respectively , ( p=0.36 ) , and the mean prostate sizes were 33.8 , 39.2 , and 37.2 g , respectively ( p=0.22 ) ( table 1 ) .
the gleason grade was similar in the 3 groups : 6.89 in group a , 6.68 in group b , and 6.61 in group c ( p=0.37 ) .
also , the international prostate symptom score was comparable in the 3 groups : 18.6 in group a , 18.8 in group b , and 17.9 in group c ( p=0.47 ) .
the preoperative stages were as follows : 26 patients in t2 , 3 in t3a , and 2 in t3b for group a ; 25 patients in t2 , 2 in t3a , and 1 in t3b for group b ; and 42 patients in t2 , 5 in t3a , and 1 in t3b for group c. the mean operative times were 226.3 , 192.1 , and 170.9 minutes , respectively ( p=0.021 ) , and the mean blood loss was 188 , 121.3 , and 106.8 ml , respectively ( p=0.004 ) .
the catheterization time was 8.9 , 7.8 , and 7.1 days in each group ( p=0.047 ) .
in the postoperative cystogram , two cases of urine leakage were reported in group a , whereas no cases were observed in groups b and c. the postoperative + smrs were 10 patients in group a ( 31.2% ) , 5 patients in group b ( 17.9% ) , and 3 patients in group c ( 6.3% ) ( table 2 ) . in group
a , seven patients had + sms in the lateral , two in the base , and one in the apical area of the prostate . in group b ,
one patient had + sms in the lateral , three in the base , and one in the apical area of the prostate . in group c ,
two patients had + sms in the lateral and one in the apical area of the prostate ( table 3 ) .
continence was assessed by recording pad usage 1 day after catheter removal and 3 , 6 , and 12 months postoperatively .
recovery of continence was reported as follows : 11 patients by 1 month ( 35.5% ) , 22 patients by 3 months ( 71.0% ) , and 27 patients by 6 months ( 87.5% ) in group a ; 16 patients by 1 month ( 57.2% ) , 25 patients by 3 months ( 89.2% ) , and 26 patients by 6 months ( 92.8% ) in group b ; and 30 patients by 1 month ( 62.5% ) , 36 patients by 3 months ( 90.6% ) , and 29 patients by 6 months ( 92.3% ) in group c ( table 4 ) . early return of continence at 3 months of follow - up was observed in groups b and c compared with group a. continence at 1 year was comparable in the 3 groups : 92.1% in group a , 94.5% in group b , and 96.0% in group c.
recent advances in video - endoscopic equipment and the development of minimally invasive surgical techniques have reduced the rates of mortality and complications from radical prostatectomy . in particular , the da vinci robotic surgical system ( intuitive inc . ,
sunnyvale , ca , usa ) , which was introduced in 2001 , offers a stable and magnified operative field , enables meticulous dissection and the preservation of the bladder neck , and enhances nerve - sparing and suturing for uva during ralp . despite these improvements , however
, temporary postoperative incontinence is blamed for lowering the quality of life of patients after ralp .
therefore , to achieve early return of continence , various authors have introduced numerous techniques with variable outcomes . in the current study
, patients treated with bnp using lbnd and the pur techniques showed an advantage with respect to early continence .
nevertheless , no statistically significant difference was observed at 6 and 12 months postoperatively among the 3 groups . since the adaptation of the running , bidirectional monofilament anastomosis to uva in 2003
, several modifications have been introduced to enhance watertight cooptation of uva . a technique reported by rocco et al
. arose from a theory based on the functional anatomy and restores the posterior aspect of the rhabdosphincter with radical prostatectomy , from which recovery of continence is expected .
in addition , in a study that followed up 100 patients for 13.3 months , duthie et al . reported 68% recovery of continence by 12 months with the improvements in the surgical technique . in the current study , we experienced the additive effects of pur when compared with the ven velthoven continuous suturing technique .
this technique allows approximating the bladder neck closer to the urethra and thus enables tension - free uva and prevents leakage at the uva site .
compared the outcomes of ralp with the ven velthoven continuous suturing technique and reported a reduction in incontinence from 26.2% to 14.3% to 2.3% with the progress in the surgical technique , as shown in the cystogram .
in addition , the recovery time was shortened as the staff gained more experience in this technique .
he also observed a decreased rate of urine leakage and the early recovery of continence when the highly experienced staff used the technique . in our prior experience
, we moved to the continuous suturing technique for uva from the interrupted sutures in the early days of laparoscopic radical prostatectomy because the ven velthoven continuous suturing technique was shown confer a better continence rate .
the bnp technique was first described by curto and gaston in 2006 . using the bnp technique in 1400 cases from 2001 to 2010 , asimakopoulos et al .
reported 80% recovery of continence immediately after catheter removal and 73% recovery of continence in the remaining patients by 3 months . in south korea ,
jeong et al . reported the methodology and outcome of typical ralp using both rocco 's and the bnp technique . in his study , 91% recovery of continence was observed in the patient group that underwent surgery using the bnp technique . in our study ,
also , more than 90% recovery of continence was observed in all the groups 6 months after ralp .
however , when the outcomes were compared at 1 , 3 , and 6 months after ralp , early recovery of continence was confirmed in group c in which both rocco 's and the bnp techniques were used .
we assume that the pur technique restores the normal anatomic position and reinforces the backbone in the axis of the pelvic anatomy .
regarding + sms , some reports showed that the + smr was high in the group with the bnp technique , but our study showed no differences . when performing the bnp technique by lbnd , it is crucial to identify the detrusor muscle fibers at the critical junction of the lateral bladder neck , the prostate base , and the fat pad laterally .
the meticulous dissection must be utilized just medially to the fat pad of whitmore to avoid inadvertent bleeding from the lateral venous plexus while appreciating the ventrolateral and dorsal longitudinal bladder muscles medially , until the dissection reaches the prostate base and the anterior surface of the seminal vesicle .
clear visualization and identification of the surgical landmarks and meticulous dissection will reduce the + smr . when both rocco 's and the bnp techniques were used as in group c , the early return of continence was observed .
in addition , there was no sign of leakage at the site of uva when both techniques were used .
it is possible that recovery was achieved earlier due to the absence of leakage , which is thought to have an adverse effect on the healing of the uva and recovery of continence .
the pur technique helps to reduce the tension between the urethra and the bladder , leading to more stable and watertight uva .
the study was performed in a retrospective manner and the patients were not randomly assigned . also , because operative methods were performed chronologically , there may have been some contribution of the learning curve , because cases accumulated with the bnp and the pur techniques .
however , patients with large prostates or prominent median lobes were not spared from performing the bnp technique and a single surgeon performed all the procedures in this study .
even though our study did not address the impact of these factors on postoperative outcomes , continence and + sms did not vary across the study groups .
posterior urethral reconstruction reduced the risk of urine leakage and proved to have good outcomes on early continence .
when both bladder neck preservation using lateral bladder neck dissection and the posterior urethral dissection techniques were used , a much better outcome was observed in the early recovery of continence at the 3 month follow - up .
also , bladder neck preservation did not influence + sms . further studies of these surgical techniques are expected to achieve better quality of life for patients undergoing ralp . | purposeto report our results on urinary continence after bladder neck preservation ( bnp ) and posterior urethral reconstruction ( pur ) during robot - assisted laparoscopic radical prostatectomy ( ralp).materials and methodsdata from 107 patients who underwent ralp were compared
on the basis of whether the patients underwent bnp and pur , bnp only , or the standard technique ( st ) . in group
a ( n=31 patients ) , st was performed by using ven velthoven continuous suturing for urethrovesical anastomosis . in group b ( n=28 patients ) , st with only pur was performed . in group c ( n=48 patients ) , both the bnp and pur techniques were used . "
recovery of continence " was defined as the use of 1 pad ( 50 ml ) or less within 24 hours.resultsthe three groups were comparable in terms of patient demographics . the mean operative time and
the mean blood loss decreased significantly from group a to group c ( p=0.021 for mean operative time and p=0.004 for the mean blood loss ) .
mean catheterization time was 8.9 , 7.8 , and 7.1 days in each group ( p=0.047 ) .
early return of urinary continence at 3 months was observed in group b ( 89.2% ) and group c ( 90.6% ) compared with group a ( 71% ) .
however , continence at 6 months was comparable in the 3 groups ( 87.5% in group a , 92.8% in group b , and 92.3% in group c ) .
rates of positive surgical margins decreased from 30.2% in group a to 20% in group b and 12% in group c.conclusionsbnp and pur during ralp showed a favorable impact on the early postoperative recovery of continence while not affecting positive surgical margins . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
PMC3180076 | measuring the radiation dose a patient accumulates through life is an important matter that has been receiving much attention recently , in particular for growing children ( e.g. , in the new england journal of medicine 's recent critique of ct use , and the adoption of the image gently program by the society of pediatric radiology , the american society of radiologic technologists , the american college of radiology , the american association of physicists in medicine , and others ) . while directly measuring dose to individual organs is impractical , the development of computational phantoms containing dosimetric information ( e.g. , ) , such as the extended cardiac - torso ( xcat ) phantom used in this study have begun to be a reliable substitute .
a key shortcoming of this strategy is that standard phantoms can not adequately reflect variability between patients , especially for children of different sizes and ages , and defining new phantoms for each patient manually would be unfeasible .
the strategy used here consists of manually segmenting a small subset of organs from pediatric ct data and calculating a full body mapping to a similarly segmented adult xcat phantom .
the resulting transformation is used to map rich anatomical and dosimetric information to the child 's body . to map dense image data as well as point - based manifold data between adult and child ,
this application requires a smooth invertible transformation ( a diffeomorphism ) to be defined everywhere on the background space of the ct scan .
such transformations are an important focus of computational anatomy , where anatomical variability is understood by studying diffeomorphisms mapping anatomical manifolds to one another .
formally , anatomy is modelled as the quadruple ( , , , ) , where is the background space ( i.e. , subsets of ) , is a group of diffeomorphisms on , is the orbit of a template i0 under , and is a family of probability measures on . geodesic paths , t for t , are used to evolve a template according to i0t , and a mapping to a target i1 is defined when i1 = i01 .
large deformation diffeomorphic metric mapping ( lddmm ) generates such mappings ( 1(x ) ) by integrating a smooth time dependent velocity field vt(x ) ,
( 1)t(x)=0tvt(t(x))dt ,
with the initial condition being identity , 0(x ) = x. a functional of the velocity field , which enforces image matching as well as smoothness and ensures the path is a geodesic , is minimized as discussed below .
there are existing algorithms for full body image registration , which are used ( e.g. ) in registering pet to ct data [ 911 ] and compensating for deformations such as breath holds . however , these tend to use elastic models , which are suitable for describing the small deformations that register two images of the same patient but are unable to accurately describe the widely varying deformations between adults and children of various ages .
in addition to the constraints on smoothness and invertibility , transformations generated by lddmm are well suited to this application , because its fluid model ( rather than elastic ) allows for large deformations to be generated and because the submanifold preserving property of diffeomorphisms allows a transformation calculated from a handful of segmented structures to be accurately applied to the thousands of anatomical structures defined in the xcat phantom . moreover , additional properties are well suited to future exploration .
for example , lddmm allows metric distances to be defined between template and target anatomies [ 8 , 14 ] and allows statistical codification of anatomy [ 15 , 16 ] . in this work ,
we use multichannel lddmm ( mc - lddmm ) , an algorithm which treats each segmented organ as a separate image linked by a common background space to calculate diffeomorphisms .
this is accomplished by calculating the velocity field minimizing the energy functional
( 2)e=dt||lvt||22+i=1m1i2||i0it=11i1i||22 ,
where i1 and i0 are the ith ( out of m ) channels ( organs ) of the target and template images , t=1 is a diffeomorphism generated by integrating the velocity field vt from t = 0 to 1 , and i describes the contribution of the ith channel to the overall energy . the operator l = id + , where = 1 is fixed and is varied , i d is identity , and is the laplacian operator , ensures smoothness of the velocity field and resulting deformations , with larger corresponding to smoother deformations , and smaller corresponding to more accurate transformations .
the energy gradient can be computed as
( 3)vet=2vtk[i=1m2i2|dt,1|jt0i(jt0ijt1i ) ] ,
where k is the operator inverse of ll , || denotes determinant and d denotes the jacobian .
the transformation generated by integrating ( 1 ) from time t = s to time t = t is denoted s , t ( i.e. , s , t = ts = t , s ) .
the quantity jt is the ith template channel transformed up to time t ( i.e. , jt = i0t = i0t,0 ) , jt is the ith target channel transformed backwards from time 1 to time t ( i.e. , jt = i1t,1 ) , and is simply the spatial gradient . it can be seen that the transformation and its inverse must be defined at all times , which was discretized here into 11 equally spaced time points from t = 0 to t = 1 . calculating
integration in time is performed using semi - lagrangian advection , a technique used in numerical weather prediction .
we use an implicit method for numerical integration , with 3 iterations per voxel at each timestep .
we use trilinear interpolation , which corresponds to another large computational load . to optimize calculations ,
the images for each channel were computed in the same loop ( loop fusion ) .
finally , application of the operator k is implemented by multiplication in the fourier domain .
the fft calculations were performed and parallelized using intel math kernel library 's ( mkl ) fft routines . since many steps of this algorithm involve independent calculations on a regular 3d voxel grid , it is well suited to parallelization . in our c++ implementation of the lddmm algorithm , openmp ( open multiprocessing ) library routines were used . as stated in , the openmp application program interface ( api ) supports multi - platform shared - memory parallel programming in c / c++ and fortran on all architectures .
openmp is a portable , scalable model that gives shared - memory parallel programmers a simple and flexible interface for developing parallel applications for platforms ranging from the desktop to the supercomputer . in our algorithm , at each iteration of gradient descent , different operations defined on data over the voxel grid were parallelized using work - sharing constructs , and loop iterations were split among the threads .
the program was compiled using intel c++ compiler version 12.0 , with automatic compiler optimizations .
it was run on a dell r900 , a 4 socket node with 6 cores per socket , with an intel xeon cpu e7450 at 2.40 ghz .
in previous work , the feasibility of using multi - mc - lddmm for this purpose was explored .
a mapping to a single pediatric patient was calculated , and a reasonable subset of segmented organs was determined . however , generalizing this algorithm to a population of patients proved difficult . for example , where initial overlap of organs or bony details between template and target was poor , the diffeomorphism tended to shrink organs close to a point .
furthermore , when structures were shrunk by the diffeomorphism details were lost , and when structures were expanded , their initial voxelized character was spuriously reproduced at the larger scale .
these difficulties are illustrated by showing a deformed adult template in figure 1 , where abdominal organs are seen contracting to a very small size , nearby structures in the neck and thorax are distorted , and features in the face and skull are lost .
further investigation resulted in the algorithm being made more robust but at the expense of increased computation time . in the modified mc - lddmm algorithm , ( 2 )
is minimized by initializing the velocity field to 0 and using a gradient descent routine with a large value of . at convergence , the value of is decreased , and minimization resumes , starting with the previously calculated velocity field .
this sequential reduction of the parameter ( denoted cascading ) allows for a coarse to fine registration and is responsible for the increased robustness as well as increased computation time of the modified algorithm .
beginning with a large value of is analogous to tikhonov regularization , encouraging a desirable solution to an ill posed problem . the final small value for is chosen to give the desired level of accuracy in our mapping .
decreasing the value for abruptly often results in nondiffeomorphic transformations due to numerical instability .
so , we include 2 intermediate values to mitigate this effect and unfortunately must bear the price of considerably increasing computation time .
the mc - lddmm algorithm with cascading was used to generate mappings between one of two adult templates ( one male and one female ) , and pediatric patients ( 24 male and 18 female ) .
each was defined on an 256 256 520 2 mm voxel grid .
the patients varied in size between 0.072 and 0.472 times the volume of the adult , with an average of 0.233 times .
males ranged from 0.072 to 0.472 times the adult volume with a mean of 0.246 , while females ranged from 0.076 to 0.372 times the adult volume with a mean of 0.215 .
the images were segmented into 8 channels with corresponding organs and weightings defined in table 1 , and 87 landmarks were placed automatically mainly on easily reproducible bony structures .
images were initially aligned with an affine transformation minimizing distances between corresponding landmarks , followed by nonlinear landmark lddmm . following this , cascading mc - lddmm
, we found this particular sequence to give qualitatively good results in 2d simulations and 3d full body data .
the sequence of transformations used to generate the final mapping is illustrated in figure 2 .
each transformations for each pediatric patient were combined to yield a double precision displacement vector at each voxel of the adult template images .
this transformation was trilinearly interpolated to map nurbs ( nonuniform rational b - spline ) surfaces defined in the xcat phantoms to the coordinate system of the child .
the bulk of the computational work was performed during cascading mc - lddmm , and as such , its performance was investigated more thoroughly .
four patients were selected , 2 males and 2 females , corresponding to the largest , smallest , and 1/3 interquartile sizes , denoted small , med - small , med - large , and
mappings were calculated on these patients using each of 1 , 2 , 4 , 8 , 16 , and 24 ( the maximum readily available ) processors .
the total computation time excluding input - output ( io ) operations was analyzed for each case as well as the time spent in specific functions .
this allowed us an understanding of how computation time scales with the number of processors used , and in particular identify at what point computation time begins to increase beyond what would be expected . to be more thorough , the portions of the program that were affected by parallelization , including io operations , were analyzed .
speedup , cn , due to parallelization on n processors was calculated ( using amdahl 's law as in ) to be
( 4)cn = t(1)t(n)=a+ba+b / n ,
where t(n ) is the total computation time for n processors , and for a single processor , a is the time spent that can not be parallelized , and b is the time spent that can be parallelized .
these two quantities are easily estimated from a two parameter fit to the above equation , which allows determination of the fraction of the total computational time that can be parallelized .
furthermore , the efficiency of parallelization was calculated according to finally , the quality of the mappings produced was validated . for each segmented organ ,
for each template ( target ) vertex , the minimum distance to a vertex on the target ( template ) surface was measured .
distances for template and target vertices were combined , and their distributions were analyzed . breaking down this analysis into categories allows an understanding of the robustness of the algorithm . as such , distributions were analyzed separately for males , females , as well as for each segmented organ .
a summary of the 4 subjects used to analyze computation performance is included in table 2 . the number of voxels in each image is shown in the second column , giving more precise meaning to the labels small , med - small , med - large , and
the total number of iterations of gradient descent across the 4 applications of mc - lddmm is shown in the third column .
due in part to adaptive stepsize selection in gradient descent , the number of iterations until convergence can not be known before hand . in the fourth
column , the product between number of voxels and number of iterations is shown as a rough approximation of the number of calculations used .
this value can be used to better understand the timing results that follow . in particular
the med - small case required the most iterations to converge , and the approximate number of calculations was much less for the small patient than for the other three .
we stress that these four patients were chosen with interquartile spacing of their total number of voxels , as opposed to uniform spacing across number of voxels , or uniform spacing across number of calculations .
such a choice is reflective of the pediatric population to be examined , rather than properties of the algorithm itself .
the total computational time in hours , excluding io operations , is shown in table 3 .
the two largest components of calculations are also shown . numerically integrating the velocity field using semi - lagrangian interpolation
is shown in table 4 , and trilinearly interpolating the images is shown in table 5 .
surprisingly , the longest amount of time was spent on the med - small case .
while this is partially explained by the large number of iterations for this case shown in table 2 , other factors such as the specific implementation of the fast fourier transform on a grid of this size , contribute as well . to better understand this behavior ,
the same data is shown graphically , on a log - log axes in figure 3 .
figure 3(a ) shows the total time , figure 3(b ) shows the time spent calculating semi - lagrangian advection , and figure 3(c ) shows the time spent interpolating images .
it appears that computation time scales with number of processors up until around 8 , when efficiency starts to break down .
again , this data must be interpreted with caution , because the images used are different sizes and a different number of iterations of gradient descent is required to converge in each case , as shown in table 2 .
therefore , the timing data was also plotted after being normalized by total number of voxels times total number of iterations in figure 4 .
it should be noted that the smallest image actually takes the most time per voxel per iteration , while the largest image takes the least .
the speedup factor and efficiency were calculated according to ( 4 ) and ( 5 ) and are plotted in figure 5 .
this analysis confirms and quantifies the sharp drop in efficiency beyond 8 processors . from a 2 parameter fit to the data in figure 5(a ) , it was determined that 93.84% of the computation time is parallelized , demonstrating the effectiveness of our implementation . to give a qualitative understanding of the mappings produced , an example of triangulated surfaces , for target and mapped template ,
are shown in figure 6 with the body in figure 6(a ) , the bones in figure 6(b ) , and the other organs in figure 6(c ) .
one can see the quality of the mappings is good in most areas , the exceptions being the inferior - most regions , where the extent of template and target images vary , the scapula , where sliding motions between the nearby ribs and body surface are difficult to generate given the diffeomorphism constraint , and the sharp borders of some abdominal organs , whose curvature varies markedly from that of the template .
the mappings produced were used to generate customized dosimetry phantoms based on the adult xcats .
the adult male xcat is shown in figure 6(d ) and an example pediatric dosimetry phantom is shown in figure 6(e ) .
previous work has shown dosimetry measurements generated with these phantoms to agree within 10% percent of ground truth .
they are shown for all patients pooled together as well as for males and females separately in figure 7(a ) .
the differences in accuracy , on average , between male and female patients is negligible .
the results show that the majority of surfaces ( a fraction of 1/e ~ 1 standard deviation of the vertices ) agree within 24 mm or 1 - 2 voxels .
moreover , accuracy for females tends to be more variable than that for males , likely due to larger differences in body proportions between child and adult .
surprisingly , the least accurate case , apparent in figure 7(c ) , is an average seeming patient of intermediate size between the med - small and med - large test cases .
furthermore , differences in accuracy for each organ are observed , where the brain is matched with the most fidelity and the stomach followed by lungs with the least fidelity . while these differences are small when compared to the voxel size , it is worth noting that the relatively poor performance for the stomach was likely due to its internal location and close proximity with many other abdominal structures , and the relatively poor performance of the lungs was likely due to large differences in curvature between the adult and child at the apexes and inferior borders
this work presented an interesting application of diffeomorphic image registration , generating pediatric patient specific detailed dosimetry phantoms , made feasible on large scale due to parallel computing .
the need for parallelization in deformable image registration is well recognized [ 23 , 25 , 26 ] , and other authors have investigated parallelization of diffeomorphic registration from maspar to gpu implementations .
the algorithm used here for generating full body maps involves a sequence of increasingly detailed transformations between adult templates and child images .
this procedure ensures robustness to automate calculations across a wide range of pediatric patients but comes at the price high computational cost . to overcome this cost ,
running times for the various patients examined ranged from over 30 hours on a single processor to under 1 hour on 24 processors in parallel .
an analysis of speedup and parallelization efficiency shows that performance begins to rapidly decline when implemented on more than 8 processors . as applications for lddmm become more numerous and larger scale ,
it is likely that the effects of memory to cpu communication bandwidth , load balancing overhead ( due to workload not evenly distributed across the available processors ) play a major role .
the full body mapping algorithm is quite accurate for all the patients examined , with the majority of vertices defined on organ surfaces agreeing between template and target to within 2 voxels .
overcoming a main drawback of the diffeomorphism constraint , namely , forbidding sliding motions in the deformation , is the subject of ongoing research .
one strategy we are currently investigating involves relabelling a strip of the segmented image , between two structures where sliding would be expected , as background .
the xcat phantoms generated are being further investigated for their accuracy and clinical utility . while generating mappings using a sequence of transformations results in a robust algorithm for this application , it detracts from some of the theoretical appeal of lddmm .
describing transformations by a single time - dependent vector field allows a rigorous study of anatomical variability .
future work will involve combining these transformations , for example , as described in , and beginning to engage in shape analysis of full bodies . | this paper describes an automated procedure for creating detailed patient - specific
pediatric dosimetry phantoms from a small set of segmented organs in a child 's ct
scan .
the algorithm involves full body mappings from adult template to pediatric
images using multichannel large deformation diffeomorphic metric mapping ( mc - lddmm ) . the parallel implementation and performance of mc - lddmm for this application
is studied here for a sample of 4 pediatric patients , and from 1 to 24
processors .
93.84% of computation time is parallelized , and the efficiency of parallelization remains high until more than 8 processors are used .
the performance of the algorithm was validated on a set of 24 male and 18 female pediatric patients .
it
was found to be accurate typically to within 1 - 2 voxels ( 24 mm ) and robust across
this large and variable data set . | 1. Introduction
2. The Multichannel LDDMM Algorithm
3. Methods
4. Results
5. Conclusions |
PMC3474548 | it is the result of the resin monomer infiltration into the demineralized collagen network and their polymerization in situ .
this interaction forms a new micromorphologic structure referred to as a hybrid layer or interdiffusion zone.12 a fundamental condition for the formation of the hybrid layer is the maintenance of the dentin organic matrix hydrated after demineralization , which supports the expansion of the collagen fibrils and preserves the integrity of the interfibrillar spaces .
this disposition is compatible with the bonding process , since it allows an appropriate infiltration of the resin monomers and may be achieved through the application of the total - etch wet - bonding technique associated with resin monomers dissolved in non - aqueous organic solvents or an aqueous solution of hydrophilic primers.3 alternatively , the self - etch bonding technique uses acidic monomers that combine tooth surface etching and priming in a single procedure , minimizing the risk of technical imperfections.4 when the dentin is demineralized by means of the wet - bonding technique , there is a substantial reduction in the mechanical properties of the remaining structure,5 particularly when the collagen network remains expanded in the aqueous medium.67 this condition of transitory fragility is partially reversed with the infiltration of resin monomers inside the collagen network , as originally demonstrated in an experimental macro - model of the hybrid layer5 and a hypothetical device.8 the results of these studies demonstrated a positive correlation between the ultimate tensile bond strength of the adhesive resin and that of the resin - infiltrated demineralized dentin , suggesting that stronger resins may result in hybrid layers that are more resistant to mechanical efforts . based on these evidences , a tendency exists to incorporate inorganic fillers in adhesive systems in the attempt to improve the bonding effectiveness .
however , they are much larger when compared with the interfibrillar spaces of the demineralized dentin,9 which have been reported to be about 12 nm in width.10 so , the fillers were normally distributed in the adhesive layer and around the tubular orifices at the bonding interface , but not inside the hybrid layer.9 with the progressive development of the dentin bonding agents , an innovative simplified - step adhesive , prime & bond nt ( dentsply , caulk , milford , of ) , was introduced that incorporates the use of inorganic fillers comprised of fumed silica with primary particles of nanometric dimensions .
even with the reduced primary particle size of 7 nm , the nanofillers did not infiltrate the collagen network of the etched dentin.11 on a nanometric scale , nanofillers have the natural tendency to aggregate / agglomerate , forming micrometric electro - dense clusters that are larger than the interfibrillar spaces.11 as a result , the strengthening of the hybrid layer claimed by the manufacturer was not observed and the resulting bond strength was described as being at the same level as other simplified - step adhesives without nanofillers.1213 more recently , a new simplified - step adhesive , adper single bond 2 ( 3 m espe dental pruducts division st .
the nanoparticles are treated in situ with a silane stabilizing agent during manufacture . according to the manufacturer
, the improvement in the technology used for surface treatment of the nanofillers provides higher stability and avoidance of aggregation / agglomeration .
this way , the nanofillers could remain uniformly dispersed and would be able to infiltrate the interfibrillar spaces .
as little information is available on this topic , the aim of the present study was to investigate the interaction morphology and bond strength of four simplified - step adhesives applied to acid etched dentin : two of them with nanofillers , one with microfillers and the other one without fillers ( control ) .
the tested hypothesis was that there is no significant difference between the filled and the unfilled adhesives evaluated regarding the interaction morphology and bond strength to dentin .
thirty - three caries - free , human third molars were collected according to the local institutional review board ( # 073/2007 ) , with the informed consent of the donors .
they were stored in a 1% chloramine t solution at 4c and used within one month following extraction .
the three simplified - step filled adhesives examined were optibond solo plus ( os ) , prime & bond nt ( nt ) and adper single bond 2 ( sb2 ) .
in addition , the simplified - step unfilled adhesive adper single bond ( sb ) was used as a control .
the components , batch numbers and manufacturers recommended protocols of the tested adhesives employed are summarized in table 1 .
prior to bonding procedures , the exposed middle dentin surfaces were wet - polished with 600-grit silicon carbide paper under running water to create a standard smear layer . the 35% phosphoric acid with silica thickener ( scotchbond etchant , 3 m espe dental products , st .
after that , the adhesives were applied following the respective manufactures instructions ( table 1 ) and light - cured using a quartz - tungsten - halogen curing unit ( xl 3000 - 3 m espe ) with a power density of 600 mw / cm .
after removal of the occlusal enamel and the roots , each tooth received two longitudinal sections perpendicular to each other ( mesiodistally and buccolingually ) in order to produce four quarters using a diamond - impregnated disk ( extec , enfield , ct , usa ) under water cooling in a specific cutter machine ( isomet 1000 , buehler , lake bluff , il , usa ) .
after that , the dentin surface of each quarter from the same tooth was individually treated with one of the adhesives evaluated .
subsequently , three 2-mm - thick increments of a resin composite ( filtek z250 , 3 m espe ) were built up on each bonded surface and individually light - cured for 20 s , constituting a crown of 6.0 mm in height .
the restored tooth - quadrants were stored in distilled water at 37c for 24 h. after this period of storage , they were serially sectioned in a longitudinal direction into 0.8-mm - thick slabs , using a diamond saw .
each slab was further sectioned to produce bonded specimens or sticks of approximately 0.8 mm in cross - sectional area .
the sticks were individually fixed to a custom - made testing jig ( geraldeli s device)14 with a cyanoacrylate glue ( model repair ii blue , dentsply - sakin , japan ) and subjected to tensile load at a crosshead speed of 0.5 mm / min until failure ( instron 4411 , instron corporation , canton , ma , usa ) . after testing , the dentin and composite sides of the failed specimens were sputter - coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers , schaan , leichtenstein ) and examined through scanning electron microscopy ( sem ) ( jsm 5600lv ; jeol , tokyo , japan ) with 85x magnification , operating at 15 kv , high vacuum and secondary electron mode . no pre - testing failures occurred .
failure modes were classified in one of the four following categories:15 type i : cohesive failure in the adhesive ; type ii : cohesive failure in the dentin ; type iii : cohesive failure in the hybrid layer ; and type iv : mixed failure : cohesive in the adhesive and hybrid layer . in cases of uncertainty ,
examination under higher magnifications ( 15003000x ) was performed in order to confirm the nature of the failure .
statistical differences between the mean bond strength of the four bonding systems tested were submitted to one - way anova and tukey s hsd post - hoc test , at a pre - set alpha of 0.05 .
the roots were severed along the cement - enamel junction using a diamond - impregnated disk under water cooling , followed by the removal of the occlusal enamel through a second parallel section .
two discs , of 1 mm ( 0.5 ) in thickness , were prepared by means of two parallel sections of the remaining coronary portion .
the discs were laminated into disc - pairs using a thin layer of the flowable composite filtek flow ( 3 m espe ) , according to the sandwich - technique first described by inokoshi et al.16 the dentin - resin - dentin sandwiches were light - cured for 160 s in four different directions and stored in distilled water at 37c for 24 h. following that , each sandwich was mesio - distally sectioned to produce 4 resin - dentin bonding interfaces for evaluation .
common procedures were employed to specimen preparation for sem examination of the bonding regions , including demineralization in 50% phosphoric acid for 5 s , deproteinization by immersion in 10% naocl for 15 min , dehydration in ascending ethanol series ( 20% , 30% , 40% , 50% , 70% , 90% and 100% for at least 20 min per step ) and immersion in hexamethyldisilazane ( hmds ) for 10 min.17 after chemical dehydration , half of the processed bonding interfaces were sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers ) in order to examine the resin - dentin interfaces in a sem ( jsm 5600lv ; jeol ) operated in high vacuum and secondary electron mode .
the other half of the processed bonding interfaces were submitted to carbon evaporation ( scd 050 ; balzers ) for energy - dispersive x - ray ( eds ) analysis using a sem ( jsm 5600lv ; jeol ) equipped with an x - radiation detector eds ( voyager , noran instruments ) , operated in low vacuum and backscattered electron mode .
this eds equipment contains an ultra - thin norvar window and works with a windows nt - based ( vantage ) digital micro - analysis system .
a qualitative and semi - quantitative elemental analysis was performed in four different points of each specimen ( point 1 adhesive layer ; point 2 hybrid layer ; point 3 - initial portion of the resin tag ; point 4 - medium third of the resin tag ) .
the working distance ( wd ) was 20 mm and the accelerating voltage was 15 kv for both sem and eds analysis .
after the bonding procedures were performed , a thin layer ( 0.5mm - thick ) of flowable composite ( protect liner f , kuraray medical inc . ,
the resin - dentin bonding interfaces were exposed and processed according to the protocol previously described by tay et al11 the resulting epoxy - resin embedded , 90nm - thick nondemineralized unstained sections , were examined by means of transmission electron microscopy ( tem ) ( em10 zeiss , oberkochen , germany ) operating at 80 kv .
the three simplified - step filled adhesives examined were optibond solo plus ( os ) , prime & bond nt ( nt ) and adper single bond 2 ( sb2 ) .
in addition , the simplified - step unfilled adhesive adper single bond ( sb ) was used as a control .
the components , batch numbers and manufacturers recommended protocols of the tested adhesives employed are summarized in table 1 .
prior to bonding procedures , the exposed middle dentin surfaces were wet - polished with 600-grit silicon carbide paper under running water to create a standard smear layer . the 35% phosphoric acid with silica thickener ( scotchbond etchant , 3 m espe dental products , st .
after that , the adhesives were applied following the respective manufactures instructions ( table 1 ) and light - cured using a quartz - tungsten - halogen curing unit ( xl 3000 - 3 m espe ) with a power density of 600 mw / cm .
five teeth were used . after removal of the occlusal enamel and the roots , each tooth received two longitudinal sections perpendicular to each other ( mesiodistally and buccolingually ) in order to produce four quarters using a diamond - impregnated disk ( extec , enfield , ct , usa ) under water cooling in a specific cutter machine ( isomet 1000 , buehler , lake bluff , il , usa ) .
after that , the dentin surface of each quarter from the same tooth was individually treated with one of the adhesives evaluated .
subsequently , three 2-mm - thick increments of a resin composite ( filtek z250 , 3 m espe ) were built up on each bonded surface and individually light - cured for 20 s , constituting a crown of 6.0 mm in height .
the restored tooth - quadrants were stored in distilled water at 37c for 24 h. after this period of storage , they were serially sectioned in a longitudinal direction into 0.8-mm - thick slabs , using a diamond saw .
each slab was further sectioned to produce bonded specimens or sticks of approximately 0.8 mm in cross - sectional area .
the sticks were individually fixed to a custom - made testing jig ( geraldeli s device)14 with a cyanoacrylate glue ( model repair ii blue , dentsply - sakin , japan ) and subjected to tensile load at a crosshead speed of 0.5 mm / min until failure ( instron 4411 , instron corporation , canton , ma , usa ) . after testing , the dentin and composite sides of the failed specimens were sputter - coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers , schaan , leichtenstein ) and examined through scanning electron microscopy ( sem ) ( jsm 5600lv ; jeol , tokyo , japan ) with 85x magnification , operating at 15 kv , high vacuum and secondary electron mode .
failure modes were classified in one of the four following categories:15 type i : cohesive failure in the adhesive ; type ii : cohesive failure in the dentin ; type iii : cohesive failure in the hybrid layer ; and type iv : mixed failure : cohesive in the adhesive and hybrid layer . in cases of uncertainty ,
examination under higher magnifications ( 15003000x ) was performed in order to confirm the nature of the failure .
statistical differences between the mean bond strength of the four bonding systems tested were submitted to one - way anova and tukey s hsd post - hoc test , at a pre - set alpha of 0.05 .
the roots were severed along the cement - enamel junction using a diamond - impregnated disk under water cooling , followed by the removal of the occlusal enamel through a second parallel section .
two discs , of 1 mm ( 0.5 ) in thickness , were prepared by means of two parallel sections of the remaining coronary portion .
the discs were laminated into disc - pairs using a thin layer of the flowable composite filtek flow ( 3 m espe ) , according to the sandwich - technique first described by inokoshi et al.16 the dentin - resin - dentin sandwiches were light - cured for 160 s in four different directions and stored in distilled water at 37c for 24 h. following that , each sandwich was mesio - distally sectioned to produce 4 resin - dentin bonding interfaces for evaluation .
common procedures were employed to specimen preparation for sem examination of the bonding regions , including demineralization in 50% phosphoric acid for 5 s , deproteinization by immersion in 10% naocl for 15 min , dehydration in ascending ethanol series ( 20% , 30% , 40% , 50% , 70% , 90% and 100% for at least 20 min per step ) and immersion in hexamethyldisilazane ( hmds ) for 10 min.17 after chemical dehydration , half of the processed bonding interfaces were sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers ) in order to examine the resin - dentin interfaces in a sem ( jsm 5600lv ; jeol ) operated in high vacuum and secondary electron mode .
the other half of the processed bonding interfaces were submitted to carbon evaporation ( scd 050 ; balzers ) for energy - dispersive x - ray ( eds ) analysis using a sem ( jsm 5600lv ; jeol ) equipped with an x - radiation detector eds ( voyager , noran instruments ) , operated in low vacuum and backscattered electron mode .
this eds equipment contains an ultra - thin norvar window and works with a windows nt - based ( vantage ) digital micro - analysis system .
a qualitative and semi - quantitative elemental analysis was performed in four different points of each specimen ( point 1 adhesive layer ; point 2 hybrid layer ; point 3 - initial portion of the resin tag ; point 4 - medium third of the resin tag ) .
the working distance ( wd ) was 20 mm and the accelerating voltage was 15 kv for both sem and eds analysis .
after the bonding procedures were performed , a thin layer ( 0.5mm - thick ) of flowable composite ( protect liner f , kuraray medical inc . ,
the resin - dentin bonding interfaces were exposed and processed according to the protocol previously described by tay et al11 the resulting epoxy - resin embedded , 90nm - thick nondemineralized unstained sections , were examined by means of transmission electron microscopy ( tem ) ( em10 zeiss , oberkochen , germany ) operating at 80 kv .
the means and standard deviations ( sd ) of the microtensile bond strength ( -tbs ) test for the adhesives evaluated are presented in table 2 .
no significant differences were found between the results of sb , os and nt ( p>.05 ) .
the -tbs mean value of sb2 was significantly higher than those obtained for the other adhesives ( p<.05 ) .
the failure pattern distribution ( % ) as analyzed by sem can be observed in figure 2 .
sb2 presented the smallest percentage of the type iii failure mode and the largest incidence of the type i failure mode .
representative sem micrographs of the bonding region of the adhesives studied are illustrated in figure 3 , where authentic hybrid layers about 5 m - thick were observed .
the bonding region of sb ( figure 3a ) , os ( figure 3b ) and nt ( figure 3c ) showed similar micromorphological aspects , characterized by uniform hybrid layers and resin tags distributed along the interface .
circular formations , which probably represent a grouping of the polialkenoic acid molecules , could be identified in the adhesive layer .
another particularity of this bonding region was the presence of round grains in the medium third of some resin tags .
a qualitative and semi - quantitative elemental analysis of a point selected at the bonding region formed by the adhesive systems is shown in figure 4 .
figure 4a shows a representative elemental distribution of the selected point 2 at the bonding region formed by sb .
chlorine and oxygen were identified in reduced amounts . as was expected , other chemical elements , which could suggest the presence of inorganic filler ,
the chemical elements calcium and phosphorus were predominant in the initial portion of the resin tags of the bonding region formed by os ( figure 4b - selected point 3 ) .
the eds analyses do not provide images of high resolution , some micrometric white structures could be observed within the adhesive layer and at the initial portion of the resin tags .
the detection of silicon in these regions ( selected points 1 and 3 ) suggests that these structures are the microfillers of the adhesive .
however , silicon was not detected in the hybrid layer ( selected point 2 ) and in the medium third of the resin tag ( selected point 4 ) , suggesting that the microfillers did n't infiltrate within collagen network nor overstep the initial portion of the resin tags .
calcium and phosphorus were predominant at the hybrid layer formed by nt ( figure 4c - selected point 2 ) .
silicon signal was detected in the adhesive layer ( selected point 1 ) and resin tag ( selected point 3 and 4 ) , suggesting the presence of nanofillers in these regions .
the selected point 2 , located at the hybrid layer formed by sb2 , was predominantly constituted of calcium , followed by phosphorus ( figure 4d ) . sodium and oxygen were minimally identified . besides the selected point 2
, silicon was detected in the other three selected points , suggesting the presence of nanofillers fillers inside the hybrid layer and resin tags .
representative tem micrographs of nondemineralized unstained sections from the bonding regions formed by the adhesive systems evaluated are illustrated ( figure 5 ) .
some round formations , which probably represent the grouping of the polialkenoic acid molecules , were observed in the adhesive layer .
microfillers could be discerned in the adhesive layer and at the initial portion of the dentinal tubules , forming particle - reinforced resin tags .
however , these particles were not present in the interfibrillar spaces of the hybrid layer , which were only infiltrated by the resin monomers of the adhesive .
the dentinal tubules are also affected by the hybridization process , forming funnel - shaped , hybridized tubular walls .
there was a tendency of the nanofillers to aggregate / agglomerate , forming electron - dense micrometric structures called clusters .
these structures could be observed within the adhesive layer and were concentrated in dentinal tubules .
some globular structures , similar to those found in sb analysis and thought to be grouping of the methacrylated - polyalkenoic acid molecules , were identified in the adhesive layer .
nanofillers were uniformly distributed throughout the entire extension of the adhesive layer , hybrid layer and in the dentinal tubules .
the concentration and distribution of nanofillers inside dentinal tubules and in the adhesive layer are similar and higher than those observed in the hybrid layer , where the nanofillers were intermixed with a collagen matrix network . at higher magnifications ( figure 6a and 6b ) , it was possible to better distinguish the nanofillers dispersed within the collagen fibrils .
some micrometric structures , probably silica particles used as thickener for the phosphoric acid etchant , were retained at the top of the hybrid layer . according to these results , the hypothesis tested is rejected .
the adhesives evaluated showed particular interaction morphology with dentin , and the bond strength of sb2 was significantly higher than that of obtained by the other materials .
the means and standard deviations ( sd ) of the microtensile bond strength ( -tbs ) test for the adhesives evaluated are presented in table 2 .
no significant differences were found between the results of sb , os and nt ( p>.05 ) .
the -tbs mean value of sb2 was significantly higher than those obtained for the other adhesives ( p<.05 ) .
the failure pattern distribution ( % ) as analyzed by sem can be observed in figure 2 .
sb2 presented the smallest percentage of the type iii failure mode and the largest incidence of the type i failure mode .
representative sem micrographs of the bonding region of the adhesives studied are illustrated in figure 3 , where authentic hybrid layers about 5 m - thick were observed .
the bonding region of sb ( figure 3a ) , os ( figure 3b ) and nt ( figure 3c ) showed similar micromorphological aspects , characterized by uniform hybrid layers and resin tags distributed along the interface .
circular formations , which probably represent a grouping of the polialkenoic acid molecules , could be identified in the adhesive layer .
another particularity of this bonding region was the presence of round grains in the medium third of some resin tags .
a qualitative and semi - quantitative elemental analysis of a point selected at the bonding region formed by the adhesive systems is shown in figure 4 .
figure 4a shows a representative elemental distribution of the selected point 2 at the bonding region formed by sb .
chlorine and oxygen were identified in reduced amounts . as was expected , other chemical elements , which could suggest the presence of inorganic filler , were not detected in any of the other selected points .
the chemical elements calcium and phosphorus were predominant in the initial portion of the resin tags of the bonding region formed by os ( figure 4b - selected point 3 ) .
the eds analyses do not provide images of high resolution , some micrometric white structures could be observed within the adhesive layer and at the initial portion of the resin tags .
the detection of silicon in these regions ( selected points 1 and 3 ) suggests that these structures are the microfillers of the adhesive .
however , silicon was not detected in the hybrid layer ( selected point 2 ) and in the medium third of the resin tag ( selected point 4 ) , suggesting that the microfillers did n't infiltrate within collagen network nor overstep the initial portion of the resin tags .
calcium and phosphorus were predominant at the hybrid layer formed by nt ( figure 4c - selected point 2 ) .
silicon signal was detected in the adhesive layer ( selected point 1 ) and resin tag ( selected point 3 and 4 ) , suggesting the presence of nanofillers in these regions .
the selected point 2 , located at the hybrid layer formed by sb2 , was predominantly constituted of calcium , followed by phosphorus ( figure 4d ) . sodium and oxygen were minimally identified . besides the selected point 2 , silicon was detected in the other three selected points , suggesting the presence of nanofillers fillers inside the hybrid layer and resin tags .
representative tem micrographs of nondemineralized unstained sections from the bonding regions formed by the adhesive systems evaluated are illustrated ( figure 5 ) .
some round formations , which probably represent the grouping of the polialkenoic acid molecules , were observed in the adhesive layer .
microfillers could be discerned in the adhesive layer and at the initial portion of the dentinal tubules , forming particle - reinforced resin tags .
however , these particles were not present in the interfibrillar spaces of the hybrid layer , which were only infiltrated by the resin monomers of the adhesive .
the dentinal tubules are also affected by the hybridization process , forming funnel - shaped , hybridized tubular walls .
there was a tendency of the nanofillers to aggregate / agglomerate , forming electron - dense micrometric structures called clusters .
these structures could be observed within the adhesive layer and were concentrated in dentinal tubules .
some globular structures , similar to those found in sb analysis and thought to be grouping of the methacrylated - polyalkenoic acid molecules , were identified in the adhesive layer .
nanofillers were uniformly distributed throughout the entire extension of the adhesive layer , hybrid layer and in the dentinal tubules .
the concentration and distribution of nanofillers inside dentinal tubules and in the adhesive layer are similar and higher than those observed in the hybrid layer , where the nanofillers were intermixed with a collagen matrix network . at higher magnifications ( figure 6a and 6b ) , it was possible to better distinguish the nanofillers dispersed within the collagen fibrils .
some micrometric structures , probably silica particles used as thickener for the phosphoric acid etchant , were retained at the top of the hybrid layer . according to these results , the hypothesis tested is rejected .
the adhesives evaluated showed particular interaction morphology with dentin , and the bond strength of sb2 was significantly higher than that of obtained by the other materials .
the bonding systems evaluated were considered effective , forming authentic hybrid layers ( figure 3 and 5 ) .
similar results were described in previous studies,1821 what contributes to fundament the clinical indication of the simplified - step adhesives evaluated .
the analysis of the -tbs results showed that os , nt and sb had no statistical differences .
these data attest that the presence of microffilers in os and nanofillers in nt did not determine superior -tbs to dentin . using similar methodology , other authors12
, 2223 also obtained similar bond strength values among adhesives with / or without filler .
however , the present study showed that sb2 , which only modification in relation to its predecessor ( sb ) was the incorporation of nanofillers ( table 1 ) , reached significantly higher bond strength compared with the others adhesives evaluated ( table 2 ) . on the other hand ,
similar influence of the nanofillers in terms of the bond strength was not verified by nunes et al13 these authors obtained statically similar mean bond strength values between nt and an experimental version without nanofillers .
they still described that the incorporation of microfillers to compose a modification of sb resulted in significant reduction of the bond strength in comparison with the original adhesive . due to these antagonistic results , it was evident that the incorporation of fillers in simplified - step adhesives can influence the bond strength to dentin only under special conditions .
furthermore , the mechanism that establishes this control is determined , probably , by the characteristics of the fillers and their interaction with dentin . to better understand the action of the inorganic fillers in this process , the chemical elements that compose the resin - dentin interfaces were assessed using sem / eds . in a general way
, the fillers of the adhesives were not clearly distinguished with sem / eds analysis that operates in low vacuum mode and carbon coated specimens , resulting in images with modest definition .
however , it is relevant to emphasize that the eds analysis using sem allows a more reliable microanalysis of the resin - dentin interface in comparison to the eds analysis performed by tem . in this case
, the detection of si could be questioned , since the elemental mapping of the polymerized sections of epoxy resin used for laboratory embedding procedures for tem examination could reveal the presence of si.11 as the epoxy resin infiltrates into spaces that were depleted following the laboratory dehydration of the specimens , the detection of si could be due to the presence of the epoxy resin and not from the structure analyzed , which would disqualify the results .
the detection of expressive amounts of calcium and phosphorus in the hybrid layers formed by the adhesives sb , nt and sb2 ( figure 4a , 4c and 4d ) , and at the initial portion of the resin tag of the adhesive os ( figure 4b ) , was expected and observed .
these chemical elements form the apatite crystals , which are the primary inorganic components of the dentin.24 they were identified due to their permanence in some regions of the dentin that were not completely devoid of the minerals , even after the phosphoric acid etching . furthermore
, an insoluble calcium phosphate complex may remain in the tissue even after the water rinsing of the etching agent . especially for os and nt ,
the detection of phosphorus in its bonding regions can also be attributed to the organic composition of these materials .
the phosphate monomers glycerol phosphate dimethacrylate ( gpdm ) and dipentaerythritolpentaacrylate monophosphate ( penta ) are present in the composition of os and nt , respectively .
the chemical elements sodium , oxygen , and chlorine , which were revealed in some of the analyses , may represent residues of the sodium hypochlorite ( naocl ) solution , used as a deproteinization agent to remove the organic phase of the specimens during its processing for eds analysis .
carbon , employed to cover the specimens and provide electronic conductance for the elemental analysis using sem , was present in each of the analyses ( figure 4 ) .
the presence of other chemical elements at the resin - dentin interfaces should not be disregarded .
probably , they were present in such low concentrations that they could not be identified in this process . to complement the study of the resin - dentin bonding interfaces , it was realized that a micro - morphologic characterization of nondemineralized unstained sections using tem was necessary . although the micromophologic aspects of the bonding regions could be better revealed with the use of stained sections , the authors preferred to process unstained sections so as to avoid the formation of nonspecific electrondense staining , located at the periphery of the collagen fibrils , as observed in a previous study using tem sections.11 this electron - dense staining could represent remnants of the proteoglycans and noncollagenous proteins that were retained within the collagen network after the acid conditioning of dentin,2526 and could be mistaken for the nanofillers of the adhesives . without the interference of these stained
collagen adjuncts,27 the study of the interaction between the adhesives and dentin was viable with the patent observation of the inorganic fillers .
when associated with the sem / eds analysis , it can be affirmed that the structures distributed in the adhesive layer and inside the dentinal tubules at the bonding interface of os and nt ( figure 5b and 5c ) are inorganic fillers from the adhesives .
likewise , the presence of nanofillers in the resin tags , adhesive layer and in the hybrid layer produced by sb2 ( figure 5d , 6a , and 6b ) , could be confirmed .
the infiltration of nanofillers within the collagen fibrils is thought to be the main and decisive factor for the -tbs values reached by sb2 .
according to this concept , after light - curing , the treated nanofillers would bond with the resin matrix of the adhesive and thereby act as a strengthening element for the severely demineralized dentin , replacing the solubilized apatite filler crystallites . as a result
, this structural condition would provide an improvement of the mechanical properties of the sb2-hybrid layer when compared with the hybrid layers produced by the other adhesives .
this can partially explain the smallest incidence of cohesive fractures in the hybrid layer ( type iii ) observed with sb2 , as demonstrated by sem analysis of the failure modes after -tbs test ( figure 2 ) .
moreover , the higher percentage of cohesive failure in the adhesive ( type i ) for sb2 ( figure 2 ) , suggests that the mechanical properties of the material was not improved and is in agreement with the results of previous study.28 so , the condition of strengthening of the hybrid layer by nanofillers was reaffirmed to explain the increased bonding effectiveness . as suggested by the interpretation of the results , the infiltration of inorganic fillers within the collagen
therefore , to obtain this interaction form , three issues must be considered , as described by tay et al11 first , the fillers should be sufficiently small ( nanofillers ) to infiltrate within the interfibrillar spaces of the demineralized dentin , which are about 12 nm in width.10 as well , the nanofillers must be physically and chemically stabilized to avoid aggregation / agglomeration during the storage and/or application of the bond solution , which creates micrometric structures called
clusters that are too large to infiltrate collagen fibrils.11 second , as the molecular weight of the nanofillers and the resin monomers of the adhesives differ markedly , the resulting diffusion rate is dissimilar .
this could preclude the complete infiltration of the nanofillers within the collagen fibrils , even if the interfibrillar spaces were maintained fully extended by the correct application of the wet - bonding technique.29 third , the infiltration of the nanofillers could be physically impeded by the presence of a hydrogel of residual ground substances , proteoglycans and noncollagenous proteins in the interfibrillar spaces of the demineralized collagen network.27 , 30 considering such premises , some considerations regarding the bonding systems analyzed in this study should be addressed . in relation to the inorganic portion of the adhesives ,
os have fillers composed predominantly of barium aluminoboro - silicate and sodium hexafluorosilicate in the order of 0.4 m .
this dimension disables them from penetrating the nanometric spaces within the collagen fibrils to compose the hybrid layer .
consequently , they remain in the adhesive layer and at the initial portion of the dentinal tubules ( figure 4b and 5b ) .
this way , the particles form an inorganic - reinforced polymer network that may serve as a shock - absorbing layer of intermediate modulus of elasticity between the hybrid layer and the resin composite , in accordance with the elastic cavity wall concept of kemp - scholte and davidson.31 however , they assume irrelevant importance in terms of bond strength , which is in agreement with the results of other studies.12,2223 the simplified - step bonding system , in spite of representing an operational simplification , needs complex chemical formulation to combine hydrophilic and hydrophobic monomers into a single bottle . therefore , characteristics regarding the chemical balance of the adhesive , the organic solvent employed,3 and operatory aspects for the bonding procedures as related to the degree of dentin moisture after acid etching , acquire larger relevance in terms of determining the bonding effectiveness.32 the nanofillers of the adhesives nt and sb2 , are constituted of synthetic silica and produced by flame pyrogenic or sol - gel techniques , respectively.33 the mean primary particle size ranged from 7 to 40 nm for nt11 and 5 nm ( manufacturer s information ) for sb2 . in the case of the fumed silica used for nt
, there is a natural tendency of the primary nanoparticles to aggregate / agglomerate forming branched and chainlike three - dimensional clusters as a result of internal interparticle van der waal s forces .
this electrostatic attraction forces are nearly 1036 more intense than the gravitational force in the nanoscale.3435 according to the derjaguim - landau - vermey - overbeek ( dlvo ) theory , the control of the aggregation / agglomeration phenomena when preparing nanoparticles may be accomplished by the inclusion of surface modifiers , which provides electrostatic repulsion between the charged surfaces.3637 however , the tendency of the nanofillers to aggregate / agglomerate can be further intensified , as a result of the surface treatment .
this can occurs due to the formation of silanol groups on the surface of the modified particles , resulting in hydrogen bridge linkages with adjacent particles38 that impede their infiltration within the collagen fibrils .
another adverse condition for the infiltration of the nanofillers to compound the hybrid layer is the possibility of a hydrogen bridge formation between silica and amino acids moieties of the collagen .
this possibility was based on the observation that similar interactions may occur among silica fillers , used as thickeners in acid conditioners , and proteins of the collagen network.11,39 in this case , the silica was retained on the top of the hybrid layer , even after thorough rinsing with water , as suggested by the figure 6 . to overcome these impediments , the manufacturers of dental materials , using the recent progresses regarding nanoscience and nanomanipulation , modify the surface of the nanofillers with a group of molecules known as silane agents .
these molecules are bifunctional , containing alkoxysilane groups at one end , which react and covalently bond with the silanol groups , and methacrylate - functinality at the other , that are available for copolymerization with the resin monomers of the adhesive.4041 thus , approximately 70% of the silanol groups on the surface of the synthesized nanoparticles were substituted with siloxane groups.42 as a consequence , the nanofillers acquire a hydrophobic character , becoming more easily dispersed in colloidal solution,40,42 and remaining un - agglomerated as observed for sb2 . besides the physical - chemical stabilization , this surface modification allows the linkage between nanofillers and the resin monomers of the adhesives , forming
nanomers , which are constituted of nanofillers and organic components.38 even after the treatment with silane , the nanofillers of the nt do not maintain the physical - chemical stability during the application of the material , forming clusters ( figure 5c ) .
as described by tay et al,11 the dispersion into individual primary particles was not observed , due in part to the incomplete chemical stabilization of the particles treated with silane , allowing the bonding with the residual hydrogen .
additionally , the destabilization of the electrostatic forces of the nanofillers may be intensified as a consequence of alterations in the ph , ionic strength and/or loss of the solvent .
all of these variations can occur during the adhesive application , contributing to the nanofillers overcoming the critical radius for repulsion from the stern s potential.36,38 especially for nt , the loss of the acetone , a highly volatile compound used as a solvent , might also contribute to destabilization of the nanofillers .
the incorporation of resin monomers in acetone is chemically complex , resulting in adhesives constituted of approximately 80% ( volume ) of solvent and only 20% ( volume ) of monomers.43 with the acetone volatilization , this proportional relationship is severely modified , which can change the ph and the chemical interaction among the molecules , contributing for the destabilization of the electrostatic forces in the surface of the nanofillers . as a consequence
, there is a reduction of the potential energy barrier of the silane and the nanofillers , which is closely approximate forming clusters .
this way , the particles of nt carry out similar functions to the micrometric fillers present in other adhesives as , for instance os , forming then an elastic cavity wall,31 but failing to strengthen the hybrid layer .
in contrast with the previous case , the nanofillers of sb2 infiltrated within collagen fibrils to form the hybrid layer ( figure 4d , 5d , and 6 ) .
special characteristics acquired for these nanofillers during manufacture allowed them to stay uniformly dispersed , even during the application of the adhesive .
the physical - chemical stability of these structures was probably obtained with an improvement of the surface treatment with silanes , after the primary particles of synthetic silica ( 5 nm ) were produced by sol - gel technique.33,42 chaimberg et al44 have reported that the amount of silane coupling agent chemisorbed on oxide supports differs drastically if changing the surface modification procedures . the type of solvents , ph , and amount of absorbed water on particles largely affected the chemisorbed content of silane.33,44 one example of improvement in the treatment of inorganic particles was recently described by iijima and kamiya,45 who modified the surface of silica nanoparticles with slight amounts of additional ph controlled water .
it was found that when 3-glycidoxypropyltrimethoxysilane was modified with the slight addition of acidic water , a relatively large steric repulsive force was measured with a colloid probe atomic force microscopy method , while the mixture modified with the slight addition of base water possessed a small steric repulsive force .
therefore , the authors of the present study consider that a similar approach could have been performed to provide larger reactivity between silane coupling agents and the surface of the nanofillers , resulting in a more efficient chemical isolation .
moreover , the water / ethanol solvent employed for sb2 is less critical regarding the chemical stability of the adhesive.46 the possible difficulty of the uniform infiltration of the nanofillers within the collagen fibrils due to its smaller diffusion rate compared with that of the resin monomers,29 was not observed for sb2 .
the chemical stability reached by this adhesive , as described above , preserved the covalent bond formed between the nanofillers and the resin monomers by the silane . with the preservation of this primary chemical bond ,
the nanofillers were carried within the collagen fibrils bonded with the organic portion of the adhesive .
the presence of the nanofillers may transpose biophysical barriers , which are formed in the course within the collagen fibrils , to reach the whole extension of the hybrid layer .
the organic matrix of the demineralized dentin as substrate for the adhesion is considered , in general , a network matrix mainly composed of collagen fibrils sustained by the water . inside this structure ,
residual substances such as proteoglycans and noncollagenous proteins were also identified.27 , 30 it was observed that after dentin demineralization , just a part of the complex of noncollagenous proteins is solubilized and extracted by the tissue for the water rinsing .
the other portion is grouped together to form a dense population of granules , with approximately 1.5 to 2 nm of diameter , along the collagen fibrils.47 in spite of these physical barriers , there is enough space for the movement of the dispersed nanofillers within the interfibrillar spaces , which are about 12 nm in width.10 as the physical - chemical stability of the nanofillers was not disordered , chemical interactions with the remaining proteoglycans , although highly loaded for polianions , was inhibited . for these reasons ,
the nanofillers of sb2 were able to infiltrate the whole extension of the hybrid layer .
the presence of nanofillers in the hybrid layer enables the concept of strengthening of the dentin that has been extensively demineralized by the acid etching .
this innovation is a result of the improvement in nanomaterials preparation that has been performed in the last years and point to a new direction for the adhesive systems .
the development of the nanoscale science is still in an early stage and it is expected that further applications will appear for dental materials in the near future .
within the limitations of this study , it can be concluded that : the infiltration of nanofillers within the interfibrillar spaces of the etched dentin strengthens the hybrid layer , improving the bonding effectiveness . | objective : to evaluate the effect of nanofillers incorporated into adhesives on the microtensile bond strength ( -tbs ) and interfacial micromorphology to dentin.methods:the occlusal enamel of 5 human molars was removed and each tooth sectioned into four quarters .
the exposed dentin was treated with one of the following adhesives : adper single bond ( sb - unfilled ) , optibond solo plus ( os - barium aluminoborosilicate , 400 nm ) , prime & bond nt ( nt - colloidal silica , 740 nm ) and adper single bond 2 ( sb2-colloidal silica , 5 nm ) .
cylinders of resin - based composite were constructed on the adhesive layers .
after 24-hour storage , the restored tooth - quadrants were sectioned to obtain stick - shaped specimens ( 0.8 mm2 , cross - sectional area ) and submitted to -tbs at a cross - speed of 0.5 mm / min .
data were analyzed using one - way anova and tukey s test ( alpha = .05 ) .
twenty - eight additional teeth were used for interfacial micro - morphologic analysis by sem ( 16-teeth ) and tem ( 12-teeth ) .
the dentin surfaces of 32 discs were treated with the adhesives ( 8 discs for adhesive ) and laminated to form disc - pairs using a flowable resin composite for sem / eds analysis . for tem ,
90nm - thick nondemineralized unstained sections were processed.results:sb2 showed significant higher bond strength than sb , os and nt .
the sem / eds and tem analysis revealed nanofillers infiltrated within the interfibrillar spaces of the sb2-hybrid layer .
fillers were concentrated around patent tubular orifices and in the adhesive layer for os and nt.conclusion:the presence of nanofillers within the interfibrillar spaces of the sb2-hybrid layer suggests its importance in the improvement of the -tbs . | INTRODUCTION
MATERIALS AND METHODS
Bonding Procedures
Microtensile Bond Testing
Scanning electron microscopy and energy-dispersive X-ray
Transmission electron microscopy
RESULTS
Bond strength results and failure analysis
Scanning electron microscopy and energy-dispersive X-ray analysis
Transmission electron microscopy analysis
DISCUSSION
CONCLUSIONS |
PMC5058570 | in recent years , concern about the impact of health care system interventions on clinical workflow processes has escalated primarily due to the implementation of electronic health records and computerized provider order entry systems [ 14 ] .
the impact and unintended consequences of these system redesign interventions have underscored the lack of knowledge about high - risk clinical processes and the concomitant patient safety risks associated with system redesign that may destabilize these processes in dynamic care delivery environments .
their dynamic nature and the complexity of healthcare environments within which they occur make them difficult to assess with current observation methods and tools .
there is a critical need for innovative methods and technologies that support a low - risk environment in which to visualize , examine , and manipulate high - risk clinical processes to assess the potential impact of redesign interventions on multilevel systems , clinician , and patient outcomes [ 5 , 6 ] .
medication errors remain a serious health care problem in the united states which result in approximately 7,000 deaths , cause harm to approximately 1.5 million people , and cost billions of dollars in hospital treatment annually [ 711 ] .
's early research into medication errors highlighted the need to examine system factors associated with medication errors .
this research used a systems analysis approach to examine all phases of the medication process , that is , physician ordering , transcription / verification , pharmacy dispensing , and nurse administration , to determine types of medication errors by stage of drug ordering and delivery . findings indicated that , of the identified medication errors ( n = 334 ) , nurse medication administration errors ( 38% ) were the second largest category of medication errors following physician medication order errors ( 39% ) .
the map is , predominantly , a nursing responsibility that has been estimated to consume approximately 40% of nursing practice time . since leape et al .
's early research on medication errors , the map has become increasingly complex due to escalating patient acuity , numerous generic and trade medication names , expanded medication delivery routes , increased use of new and diverse medication safety technologies , and an increased number of medication orders [ 13 , 14 ] . the lack of standardization of the map is also a key contributing factor in medication administration complexity . typically , the map involves a registered nurse ( rn ) performing many tasks , including , but not limited to , ( 1 ) assessing the patient to obtain pertinent data , ( 2 ) gathering medications , ( 3 ) confirming the six rights ( i.e. , right dose , patient , route , medication , time , and documentation ) , ( 4 ) administering the medications , ( 5 ) documenting administration , and ( 6 ) observing for therapeutic and untoward effects . clinical observation has confirmed a significant degree of nurse - to - nurse variability in map tasks and task sequencing that is subject to environmental interruptions that result in medication administration practices that deviate from standard practice protocols .
empirical evidence , however , is lacking about map characteristics and the nature of rn and environmental characteristics and interruptions that influence it .
this makes it very difficult to anticipate the impact of system redesign interventions undertaken to enhance medication safety , for example , introduction of medication safety technology and the potential consequences of redesign interventions . to this end , this study identified map workflow characteristics and developed a computer simulation model based on these characteristics to assess the impact of simulated map redesign interventions on selected map redesign outcomes .
it builds on our previous work where we developed a mobile application for recording live map observations and a discrete - event simulation model of the map .
the study was undertaken in a tertiary medical center in columbia , south carolina , united states .
the medical center is a 414-bed modern complex that anchors a comprehensive network of 600-plus affiliated physicians , including six strategically located community medical and urgent care centers , an occupational health center , the largest extended care facility in the carolinas , and an alzheimer 's care center .
the medical center also supports an array of health and wellness classes . the hospital employs more than 5,200 people and offers a variety of community outreach programs and education and health screenings .
a high surgical volume is supported with 29 state - of - the - art operating rooms that have cutting edge , state - of - the - art medical technology and procedures .
identification bands , proper cough etiquette , frequent hand - washing , protective wear , and public safety officers are integral parts of the medical center 's commitment to patient safety and security .
the study was conducted on two comparable medical units that served adult patients with medical , surgical , neurological , oncology , orthopedic , and renal conditions .
patients were typically admitted for chronic care management , diagnostic studies , and medical interventions .
the units were comparable in terms of size ( range = 3036 beds ) , average daily census ( range = 2631 patients ) , patient length of stay ( range = 6.57 days ) , and number of rns ( range = 2529 full - time equivalent ) , as well as numbers and types of technical and secretarial support staff .
each patient room had an individual bathroom , an inside supply cabinet , and a locked medication cabinet .
a wall - mounted locked documentation station with a drop - down writing platform was located outside each patient room .
a nurses ' station was centrally located with an adjacent dumbwaiter and various separate rooms , for example , medications , clean / soiled equipment , supplies , breaks , family consultation , and a manager office .
an integrated electronic health record ( ehr ) was used on each unit for all documentation , including medication administration and management .
two rolling computer carts were available on each hallway and rns used them during walking rounds to receive change - of - shift report , and to document all care , including medication administration .
more acutely ill patients were routinely assigned to rooms closer to the nurses ' station . since medication administration is predominantly a responsibility of rns , the study sample comprised rns .
the study did not involve patients and no patient information was collected during the course of the study .
following protocol approvals from university and medical center institutional review boards , rn volunteers were recruited from full - time rn populations for each study unit .
all full - time rns from unit 1 ( n = 16 ) and unit 2 ( n = 20 ) were invited to participate . study recruitment information was distributed at unit staff meetings by unit managers and during unit recruitment visits by the study principal investigator ( pi ) . registered nurses interested in volunteering for the study met with the pi in a unit conference room where the pi explained the study prior to them signing a consent form and completing a demographic information form .
a total of 17 rns participated in the study with 7 from unit 1 ( 44% ) and 10 from unit 2 ( 50% ) .
combined findings for all 17 rn volunteers indicated that the majority were white [ n = 15 ( 88% ) ] women [ n = 16 ( 94% ) ] who had been licensed as an rn an average of 11 years , had practiced as an rn an average of 10 years , and had worked at the medical center an average of 6 years with 60% or more of that time spent on their study unit .
the majority of rns reported that they had a totally supportive medication safety culture [ n = 11 ( 65% ) ] on their unit and that they felt totally comfortable [
most rns also indicated that they thought about medication safety frequently [ n = 15 ( 88% ) ] and that the quality of the medication safety process on their unit was extremely high
medication safety technology , such as smart intravenous infusion pumps , was readily available [ n = 13 ( 76% ) ] on the units , and the majority of the rns used it
the majority of rns had also completed 1 self - paced computer instructional program [ n = 15 ( 88% ) ] on medication safety , with 7 ( 41% ) completing this within one month prior to the study .
the majority of rns reported that they were very likely [ n = 11 ( 65% ) ] to be interrupted while engaged in the medication administration process .
their self - reports , however , did not provide any specific interruption types or patterns . to record rn medication administration functions , tasks , and interruptions in a live clinical environment and to generate baseline data for the development of our computer simulation model
imedtracker mobile application ; it is an extension of the application we developed in our previous study .
the imedtracker is a web - based application developed using the jquery mobile framework ( http://jquerymobile.com/ ) .
the team chose to develop a web - based application instead of a native application because it would allow the application to run on any smartphone or tablet .
the imedtracker represents one of the first mobile applications designed specifically to record map workflows and one that can run on multiple mobile devices .
when the item is pressed , a drop - down list is displayed for users to choose from .
there is a separate list of tasks associated with each function , and they change according to the function selected . to facilitate the recording process ,
the three menu items to the right of prepare medication allow observers to record the location of the activity ( e.g. , patient 's room , medication room , and kitchen ) , patient 's room number , if applicable , and the clinical unit number ( e.g. , 6th and 7th ) . as their names
imply , the undo menu item undoes the last recorded activity , and the
the last menu item settings takes observers to another screen where they can input their name , input the code ( instead of name ) of the rn being shadowed , and e - mail the data to project investigators .
, the left column lists the tasks , and the right column lists the interruptions . when a task or interruption is selected , the application automatically captures its start time and writes the record to the database .
the application indicates that the activity is in operation by highlighting that item , as illustrated by the
, observers would press on that item and the application would automatically capture the activity 's end time .
if a mistake is made , observers can tap on the undo button to cancel the last action .
the map observational data collection protocol was field tested in this study using expanded methods and developed in our previous studies [ 16 , 17 ] .
several steps were taken to ensure data recording consistency including ( 1 ) observer training in the use of the mobile device and imedtracker application ; ( 2 ) creation of common observation rules and data collection procedures ; and ( 3 ) assessment of observer interrater reliability ( irr ) .
three student observers participated in two 4-hour classroom training sessions that included ( 1 ) an introduction to the imedtracker application , ( 2 ) group recording practice using imedtracker and sample map videotapes from a previous study , and ( 3 ) four independent recording sessions using imedtracker and sample map videotapes .
independent observer recordings were compared and discussed by the group to assess recording variations . following classroom training , observers participated in a one - week orientation to the two study units . during this week , observers used imedtracker to record live map observations for three data collection sessions and participate in debriefing conferences with study investigators at the conclusion of each session .
debriefing feedback was used to establish observation rules to enhance observer recording consistency and clarify data collection procedures ( table 1 ) .
each assessment involved all three observers simultaneously recording a study rn 's map task sequence for each of her / his assigned patients .
edit distance ratios ( edrs ) , using the demareau - levenshtein approach , were calculated to assess irr .
edit distance is derived from information theory and is specifically used to assess irr with uneven task sequences .
it represents the difference or distance between two strings of characters in terms of the number of edit operations , that is , insertion , deletion , and/or substitution , needed to transform the first string into the second one [ 20 , 21 ] .
all edrs met the standard minimum 0.70 agreement criterion for multiobserver pairwise comparisons with time 1 edr means of 0.78 , 0.69 , and 0.69 ; time 2 means of 0.80 , 0.71 , and 0.73 ; and time 3 means of 0.86 , 0.72 , and 0.73 .
an observation was defined as an individual patient care episode that began with the first medication administration task performed and ended when the rn moved to the next patient .
trained student observers conducted a total of 54 map sessions over six weeks , during the hours of 7:00 to 11:00 am on mondays , wednesdays , and fridays in the months of june and july .
a total of 305 individual map observations were recorded during the 54 map sessions with an average of 5 map observations per rn study participant for both clinical units .
the first step of the data analysis was to develop process maps of the map .
a process map is a pictorial representation of the sequence of actions that comprise a process .
they were developed to provide baseline information on how the map was being performed and to understand its process characteristics .
analysis of the map data revealed that there were generally two distinct workflow processes : unbundled and bundled . in the unbundled workflow , an rn engaged in the map by performing only map tasks during a care episode ( figure 2 ) . in the bundled workflow , an rn completed medication administration together with other patient care responsibilities during the care episode ( figure 3 ) .
as shown in figure 2 , the unbundled map workflow typically started when the rn received and reviewed reports of patients she / he was assigned for that shift . after reviewing the reports , the rn then determined if any of her / his patients required special medications that were located in the medication room or pyxis ( a medication dispensing machine located inside the medication room ) .
if so , she / he would retrieve them . the rn then visited each patient one at a time .
this was often followed by a combination of tasks such as greet patient , clean hands or put on gloves , log in to mobile computer , and scan patient 's i d . after reviewing the patient 's medication list ,
the rn reviewed the medication box that was located in the patient 's room to determine if all of the needed medications were available .
if not , she / he needed to call the pharmacy , send a request to the pharmacy through the computer , or go to the pharmacy or medication room .
these events were considered patient - driven interruptions ; an interruption is an event that requires the rn to momentarily break away from his / her current task .
if the medications were available , then the rn would check to see if any of the medications to be given required an assessment ( i.e. , a pulse for a patient receiving digoxin ) .
once assessments were done , if applicable , the rn then proceeded with the medication administration tasks : scanning patient medications , preparing medications for administration , explaining medications to the patient , administering the medication , and documenting the medication administration .
the medication administration tasks were repeated for however many medications the patient needed . once the rn completed the medication administration tasks , she / he then closed the medication box and cleaned her / his equipment .
some rns performed this task while they were still inside the patient 's room , while others preferred to do it in the hallway . the care episode or observation ended when the rn moved to the next patient 's room .
as highlighted in figure 3 for the bundled map workflow , the key difference between the unbundled and bundled workflow was the added nonmedication assessment or other care tasks .
these included tasks such as changing a wound dressing , assessing the physical and mental capacity of the patient ( rule # 9 , table 1 ) , or providing assistance with dietary needs .
it was observed that these nonmedication tasks could take place before , during , or after medication administration .
of the 305 observations , there were almost twice as many bundled observations as there were unbundled ones ( 203 versus 102 ) .
bundled observations took nearly twice as long to complete ( 26 versus 14 minutes ) .
the contrast in standard deviations indicated that the bundled observation was highly variable , with some observations taking nearly two hours to complete .
lastly , bundled observations were more likely to be associated with interruptions ( patient - driven or time - driven ) compared to unbundled observations ( 92% versus 82% ) .
that is , the bundled map workflow had a 92% chance of having one or more interruptions .
patient - driven interruptions were defined as those that were triggered by patient - related care , whereas time - driven interruptions were triggered by other sources ( considered as random events ) ( table 3 ) .
figure 4 shows the difference in the average amount of time between unbundled and bundled map workflows for an rn performing specific map tasks , for example , prepare medication , administer medication , and document medication .
additionally , figure 4 shows the difference in the time spent on interruptions for the unbundled and bundled map workflows .
results indicated that rns spent more time preparing and documenting medication for the bundled map workflow .
the time spent administering medications was comparable between the two map workflows . while the unbundled map workflow had a lower chance of incurring interruptions , the duration of interruptions was slightly longer than those that occurred during the bundled map workflow .
this may indicate that patient - driven interruptions are more time consuming . to assess map workflow fragmentation , a timeline belt comparable to that proposed by zheng et al . was developed for both the unbundled ( figure 5(a ) ) and bundled ( figure 5(b ) ) map workflows .
twenty ( 20 ) observations were randomly selected for each workflow type . each timeline belt or row in figure 5 represents one map workflow observation . for each observation , map tasks and interruptions
were mapped into one of four functions : prepare medication ( yellow ) , administer medication ( green ) , document medication ( red ) , and interruption ( blue ) .
the length of a colored segment is proportional to the duration ( in seconds ) of the task or interruption .
thus , the longer an observation is the longer the timeline belt is , and the more fragmented an observation is , the more colored segments there are . as visually depicted in figure 5 , rns engaged in a bundled map workflow switched between map functions and tasks more frequently ( higher number of segments ) , experienced more interruptions ( more blue segments ) , and took longer to care for patients ( longer timeline belt ) .
this section presents the methodology utilized in developing the map simulation model that explicitly models the observed bundled and unbundled workflow processes .
this is accomplished via the incorporation of the statistical model discussed in section 3.1 . in section 3.2 , the design and functionality of the simulation model are explained in detail , followed by the discussion of the model input parameters and values .
the potential use of the validated simulation model is demonstrated in section 4 where it is used to assess redesign interventions .
empirically , the bundled versus unbundled map workflow processes seemed to be related to the rn 's level of experience ; that is , the more experienced the rn , the more she / he bundled care tasks to enhance organization and efficiency .
we , therefore , tested the hypothesis that nurses ' work experience would have a significant impact on the number of patient - driven interruptions in unbundled and bundled cases .
since each rn was observed multiple times during the study , a mixed poisson model was used to estimate the expected number of patient - driven interruptions during each observation .
this model incorporated the random intercept into the model to accommodate the variations of measures within and between rns . using
the mixed poisson also accounted for overdispersion ( conditional variance exceeds the conditional mean ) .
initially , we included all rn demographic variables in the model as covariates to assess the impact of moderator variables .
a stepwise model selection procedure was used to remove the nonsignificant variables based on the p values .
in addition , we also removed the variables which failed to meet the convergence criteria during the approximation procedure . in the bundled observations ( table 4 ) , the significant variance of random intercept indicated that heterogeneity existed in terms of rn patient - driven interruptions .
the significant negative effect of years of work in hospital implied that experienced rns were able to reduce the number of patient - driven interruptions .
in contrast , there was no significant variation among rns or effect of years of work in hospital in the unbundled observations .
many health care studies have utilized discrete - event simulation ( des ) to model the operation of a system as a discrete sequence of events in time .
the map workflows reflected a discrete set of events and were best modeled using des .
however , the rns were not a homogenous set of agents with identical characteristics and this had an effect on their map workflows .
in particular , the rns ' number of years worked in the hospital was correlated with the number of patient - driven interruptions for bundled workflows . for this reason , it was necessary to model rns as individual agents with their own characteristics .
pseudocode 1 provides a high - level algorithmic view of the simulation model , which was implemented using netlogo .
as illustrated in pseudocode 1 , the simulation model has three key procedures : setup , simulate , and do - task . the setup procedure performed all the preliminary steps before the workflow is simulated . in this procedure , the model initialized the variables , drew the layout of the units ( to show location of rns ) , generated a user - specified number of rns working on a unit and patients to be assigned to each rn ( the ratio of rns to patients is based on collected data ) , and assigned specific attributes to each rn . the model simulated rns ' movements according to the actual study clinical unit floor layout and patient room numbers .
this was done to track the total rn walking distance as a proxy for fatigue , which has long been considered a contributory factor in patient care errors .
the rn attributes that were explicitly modeled included the workflow type ( i.e. , bundled or unbundled ) and the years of work in hospital .
as discussed , the characteristic years of work in hospital was used to estimate the number of patient - driven interruptions each rn would encounter per observation .
time - driven interruptions were calculated per 10-minute intervals using the probability determined from data .
it maintained a queue of rns to simulate and rns were selected from this queue based on the timing of the events to be simulated .
for each rn , it determined if the rn had completed all the tasks for an observation .
if so , the rn was moved to the next room ( i.e. , observation / patient ) . if not , it assigned the next task to the rn and the time it took to complete that task .
this information was then placed in the priority queue and subsequently drawn when it got to the front of the queue .
the task processing times were determined using best - fit distributions and parameters ( determined based on sample data ) .
thus , the simulation model is a stochastic model due to the randomness in task processing times and number of interruptions .
the simulate procedure relied on the do - task function to determine the next task within the unbundled or bundled workflow .
term in the do - task function denoted a decision point in the workflow ( represented by diamond shapes in figures 2 and 3 ) .
when a conditional was encountered , the model determined the branch it followed based on the observed probabilities .
thus , while map activities were structured based on the workflows , there was significant workflow variability due to a number of conditionals ( i.e. , special medications , medication availability , medication - related assessments , and other care ) .
the graphical user interface includes the setup button that calls the setup procedure and the
additionally , the gui includes sliders that allow users to specify the number of rns to simulate , the probability that an rn follows the bundled workflow , and the probabilities that they will encounter interruptions in the unbundled and bundled workflows .
when the model is running , the gui shows which rooms the rns are in , their current activities , and relevant statistics .
the primary input is the list of tasks associated with unbundled and bundled workflows .
algorithm 1 provides a portion of the codes we implemented to model the bundled workflow .
if there is no second item in the row then the first item in the next row is the to task . if the task ends in a ?
if the conditional statement evaluates to true ( based on the observed probabilities ) then we perform the second task in the row ; otherwise , we perform the third task in the row . the task durations were drawn from the gamma distribution with parameters and , where = mean mean / variance and = mean / variance .
the durations of the commonly encountered bundled tasks are shown in table 5 ; there are over 60 tasks in each of the two workflows ; thus , not all tasks are shown .
values related to the number of patients assigned to a nurse , the number of patient - driven and time - driven interruptions , and the number of medications needed by a patient were drawn from a discrete empirical distribution constructed from the observed data .
table 6 shows the cumulative distribution function ( cdf ) values used by the simulation model to determine the values for these parameters .
that is , a random number between 0 and 1 is generated and the cdf is used to determine the value for the corresponding parameter .
model validation is the process of ensuring that the simulation model behaves in the way it was intended according to the modeling assumptions made .
the map model validation was achieved by comparing the model 's observation duration with the actual map observation duration .
netlogo 's behavior space tool was used to generate 1,000 observations ( i.e. , replications ) and each observation was run until the nurse completes map for all of her / his assigned patients .
note that the duration of each replication is dependent on a number of random factors , including how many patients are assigned to the nurse and how many medications are needed by the patients .
since the observation durations are not normally distributed , the wilcoxon rank - sum test was used to test the null hypothesis that the distribution of observation durations generated by the model was the same as that of the actual observation durations .
the wilcoxon test , using the statistical software r , yielded a p value = 0.5341 for unbundled observations and a p value = 0.7629 for bundled observations , and the null hypothesis could not be rejected .
the validated map simulation model was used to assess the impact of two hypothetical redesign interventions in a simulated setting : ( 1 ) intervention 1 increased rns ' use of an unbundled map workflow and ( 2 ) intervention 2 reduced map patient - driven interruptions . for intervention 2 , the time - driven interruptions were not considered because they are random and rns do not have control over them . to assess the impact of intervention 1 , the input to the map model was modified to have different percentages of rns follow the unbundled workflow , that is , 0% , 33.5% ( base case ) , 50% , and 100% .
the experimentation was conducted using 1,000 replications and each replication was run until the nurse completes all of her / his assignments .
table 7 provides a summary of the results for intervention 1 , and figure 7 illustrates the impact of these scenarios on the average observation duration per rn per shift .
the results indicated that as the percentage of rns following the unbundled workflow increased , the average number of interruptions and observation duration decreased . to assess the impact of intervention 2
, the input to the map model was modified to assume a reduction of 10% , 20% , and 30% in patient - driven interruptions for both unbundled and bundled workflows .
tables 8 and 9 provide a summary of the results for the unbundled and bundled workflow , respectively . as shown in table 8 , for unbundled workflow , the results indicated that as the probability of interruptions decreased , so did the interruption counts and observation duration .
this result implied that if rns could avoid situations that lead to interruptions , they would be more efficient . a similar result was observed for bundled workflow ( table 9 ) , except that the average observation duration did not follow a decreasing trend .
also , the patient - driven interruption time for bundled workflow tended to be lower than that of unbundled workflow .
thus , the fewer number of patient - driven interruptions had less of an impact on observation duration for bundled workflow than it did for unbundled workflow .
for example , the number of patient - driven interruptions was associated with an rn 's years of work in the hospital .
hence , it is possible that other rn characteristics might play a role in rn map workflow and additional research is needed in this area .
map timeline belts suggested that the bundled workflow is not desirable due to a higher incidence of map task switching ( i.e. , workflow fragmentation ) and interruptions .
previous research findings have shown that frequent task switching often increases physical activities that can result in a higher likelihood of cognitive slips and mistakes that can lead to performance error .
study qualitative findings from tape recorded comments pointed to a number of interruptions that were likely to occur from other care when rns followed a bundled map workflow pattern .
for example , rns engaged in a bundled map workflow pattern often responded to patient requests for dietary assistance and other care requests such as toileting assistance ( table 10 ) . while we classified these patient requests as interruptions
moreover , we recognize that rns can not seek to be more efficient at the expense of the quality or completeness of care .
nevertheless , these other care interruptions raised a variety of nursing practice questions about the safety impact of the bundled map workflow pattern on medication administration error outcomes and other practice considerations such as use of aseptic technique in medication administration .
this study developed a computer simulation model to assess the performance of nursing unbundled versus bundled map workflows .
it extended our previous work in several areas : ( 1 ) the imedtracker was modified to work on ipad devices instead of ipod touch to minimize scrolling and thereby facilitate the recording of live map observations , ( 2 ) data collection took place in an actual hospital setting instead of an academic simulation laboratory and thereby provided more realistic rn characteristics and map workflow observations , ( 3 ) the map agent - level des simulation model was modified to incorporate rn and map characteristics to account for patient - driven and time - driven interruptions , and ( 4 ) statistical modeling techniques were used to compliment development of the map computer simulation model . while our methods and tools were validated in this study ,
these included ( 1 ) one study hospital , ( 2 ) only 2 medical units , ( 3 ) small number of rn participants ( n = 17 ) , ( 4 ) lack of patient contextual data , ( 5 ) small number of map observations ( n = 305 ) , and ( 6 ) data collected only during morning shifts of mondays , wednesdays , and fridays .
we plan to address these shortcomings in a larger proposed field study that is currently under review .
furthermore , in future work , we plan to develop an agent - based model of map that simulates the complex behavior of rns through the use of agents .
specifically , we wish to model the autonomous behavior of the nurse agents and the interactions between the agents themselves and their environment . to accomplish this goal
, we are currently expanding the capabilities of the imedtracker to provide data for the agent - based model development and validation . | this paper presents findings of an observational study of the registered nurse ( rn ) medication administration process ( map ) conducted on two comparable medical units in a large urban tertiary care medical center in columbia , south carolina .
a total of 305 individual map observations were recorded over a 6-week period with an average of 5 map observations per rn participant for both clinical units .
a key map variation was identified in terms of unbundled versus bundled map performance . in the unbundled workflow , an rn engages in the map by performing only map tasks during a care episode . in the bundled workflow ,
an rn completes medication administration along with other patient care responsibilities during the care episode . using a discrete - event simulation model
, this paper addresses the difference between unbundled and bundled workflow and their effects on simulated redesign interventions . | 1. Introduction
2. Methods
3. Model Development and Validation
4. Assessment of MAP Redesign Interventions
5. Discussion
6. Conclusions |
PMC4768785 | adult female shiba goats maintained at the tokyo university of agriculture and technology were used in this
study .
six to eight goats were housed in a paddock with an outside area of 25 m , a sheltered area
of 15 m and a natural photoperiod .
shiba goats are annual breeders in natural daylight conditions
in japan and have been used as an experimental model of ruminants .
the goats were fed maintenance diets of
alfalfa hay cubes twice a day ( 0900 h and 1500 h ) .
all the procedures were approved by the university committee for the use and care of
animals at tokyo university of agriculture and technology ( # 27 - 18 ) .
all of the female goats were checked for
estrus once or twice daily and were considered to be in estrus when they allowed mounting by a male goat .
we
tested 16 goats ( age = 29 years , body weight = 2137 kg ) that we confirmed as being in estrus ( n = 8) or not
in estrus ( n = 8) between december 2014 and january 2015 . an observation pen ( 2.5 m 2.5 m )
was set up in the
corner of the female paddock , with one side adjacent to the male paddock .
a network camera ( dg - sf334 ,
panasonic corporation , kadoma , japan ) was fixed to the ceiling . a captured image from the video recording data
is shown in fig .
one male goat
was tied loosely with a rope to the adjacent side of the observation pen .
a bright - colored circle marker ( red
or blue , 12 cm in diameter ) was attached to the back of female goats to enable identification and tracking in
video - recordings .
the female goats were then moved gently into the observation pen by one observer ( the first
author of this paper : ne ) .
the female and male goats were allowed to partially contact each other by poking
their snouts through the bars of the adjacent side of the observation pen . after an adaptation period of
approximately 10 min , the behaviors of the goats were recorded for 10 min using a network camera recorder
( bb - hnp17 , panasonic ) connected to a personal computer .
the computer was located in a building at a distance
from the goat paddocks . during video recordings ,
the observer remained in the building to prevent disturbing
the behaviors of the goats . after finishing video recording ,
behavioral observations were performed by a well - trained observer ( ne ) using
the video recordings .
based on previous descriptions of sexual behaviors during estrus in goats , we investigated the occurrence of two behaviors as indicators of
proceptivity : approaching the male and staying near the male .
the following criteria were used to define
each behavior : approaching the male : walking or running from the other side of the observation pen toward the area adjacent
to the male paddock .
staying near the male : staying or behaving restlessly in the area adjacent to the male paddock and
occasionally contacting with the male through the bars of the pen .
the data frames were scored as 0 ( no behavior ) or 1 ( occurrence of a specific behavior ) to analyze each
behavior in terms of its frequency or duration .
the video recordings were processed using the abdigitizer software ( chinou jouhou shisutemu , kyoto , japan ) .
the positions and movements of the markers attached to the backs of the goats were tracked automatically , and
the x- and y - coordinates of the central point of the marker were outputted every 0.5 sec .
based on the results of the human observations , the following criteria were employed to identify each
behavior from the video tracking data .
the movement distance between two consecutive data points was
determined by calculating the euclidean distance , which represented the speed of movement ( cm/0.5 sec ) . standing : staying in one place and exhibiting no particular behavior .
when the distance moved between two
consecutive data points ( corresponding to 0.5 sec ) was < 3 cm , the status was considered to be
standing .
walking : when the distance moved between two consecutive data points was > 13 cm , the status was
considered to be walking .
the walking speed threshold ( 13 cm/0.5 sec ) was determined as the mean minus one
sd based on six control measurements from representative datasets .
the range between 3 and 13 cm was used as
the buffer zone . approaching the male : when the distance from the male paddock ( corresponding to x - coordinate of the marker )
decreased from > 120 cm to < 60 cm within nine consecutive data points ( corresponding to 4 sec ) ,
these criteria were based on the analysis of human
observation , in which a goat walked from the other side of the observation pen to the area adjacent to the
male paddock and the x - coordinate of the marker decreased from 120 cm to < 60 cm within nine consecutive
data points ( the mean plus sd based on six control measurements ) . staying near the male : when the x - coordinate of the marker was < 60 cm ( corresponding to the area adjacent
to the male paddock ) , the status was considered to be staying near the male .
all of the data were expressed as the mean standard error ( se ) .
parameters related to the behavioral
characteristics were analyzed using the student s t - test to compare the means between two
groups ( in estrus or not in estrus ) .
the concordance
of the results obtained by video - tracking analysis and video - recorded observation by a human observer was
examined according to a previously described method .
briefly , an
event was considered to be concordant if the behavior identified by video tracking analysis occurred within
the range of three time points before or after the time point recorded in human observations .
the concordance
rate was calculated by dividing the number of concordant events ( true positive ) by the sum of the true
positives , false positives and false negatives .
adult female shiba goats maintained at the tokyo university of agriculture and technology were used in this
study .
six to eight goats were housed in a paddock with an outside area of 25 m , a sheltered area
of 15 m and a natural photoperiod .
shiba goats are annual breeders in natural daylight conditions
in japan and have been used as an experimental model of ruminants .
the goats were fed maintenance diets of
alfalfa hay cubes twice a day ( 0900 h and 1500 h ) .
all the procedures were approved by the university committee for the use and care of
animals at tokyo university of agriculture and technology ( # 27 - 18 ) .
all of the female goats were checked for
estrus once or twice daily and were considered to be in estrus when they allowed mounting by a male goat .
we
tested 16 goats ( age = 29 years , body weight = 2137 kg ) that we confirmed as being in estrus ( n = 8) or not
in estrus ( n = 8) between december 2014 and january 2015 . an observation pen ( 2.5 m 2.5 m )
was set up in the
corner of the female paddock , with one side adjacent to the male paddock .
a network camera ( dg - sf334 ,
panasonic corporation , kadoma , japan ) was fixed to the ceiling . a captured image from the video recording data
is shown in fig .
one male goat
was tied loosely with a rope to the adjacent side of the observation pen .
a bright - colored circle marker ( red
or blue , 12 cm in diameter ) was attached to the back of female goats to enable identification and tracking in
video - recordings .
the female goats were then moved gently into the observation pen by one observer ( the first
author of this paper : ne ) .
the female and male goats were allowed to partially contact each other by poking
their snouts through the bars of the adjacent side of the observation pen . after an adaptation period of
approximately 10 min , the behaviors of the goats were recorded for 10 min using a network camera recorder
( bb - hnp17 , panasonic ) connected to a personal computer .
the computer was located in a building at a distance
from the goat paddocks . during video recordings ,
the observer remained in the building to prevent disturbing
the behaviors of the goats .
after finishing video recording , behavioral observations were performed by a well - trained observer ( ne ) using
the video recordings . based on previous descriptions of sexual behaviors during estrus in goats
, we investigated the occurrence of two behaviors as indicators of
proceptivity : approaching the male and staying near the male .
the following criteria were used to define
each behavior : approaching the male : walking or running from the other side of the observation pen toward the area adjacent
to the male paddock .
staying near the male : staying or behaving restlessly in the area adjacent to the male paddock and
occasionally contacting with the male through the bars of the pen .
the data frames were scored as 0 ( no behavior ) or 1 ( occurrence of a specific behavior ) to analyze each
behavior in terms of its frequency or duration .
the video recordings were processed using the abdigitizer software ( chinou jouhou shisutemu , kyoto , japan ) .
the positions and movements of the markers attached to the backs of the goats were tracked automatically , and
the x- and y - coordinates of the central point of the marker were outputted every 0.5 sec .
based on the results of the human observations , the following criteria were employed to identify each
behavior from the video tracking data .
the movement distance between two consecutive data points was
determined by calculating the euclidean distance , which represented the speed of movement ( cm/0.5 sec ) . standing : staying in one place and exhibiting no particular behavior .
when the distance moved between two
consecutive data points ( corresponding to 0.5 sec ) was < 3 cm , the status was considered to be
standing .
walking : when the distance moved between two consecutive data points was > 13 cm , the status was
considered to be walking .
the walking speed threshold ( 13 cm/0.5 sec ) was determined as the mean minus one
sd based on six control measurements from representative datasets .
the range between 3 and 13 cm was used as
the buffer zone . approaching the male : when the distance from the male paddock ( corresponding to x - coordinate of the marker )
decreased from > 120 cm to < 60 cm within nine consecutive data points ( corresponding to 4 sec ) ,
these criteria were based on the analysis of human
observation , in which a goat walked from the other side of the observation pen to the area adjacent to the
male paddock and the x - coordinate of the marker decreased from 120 cm to < 60 cm within nine consecutive
data points ( the mean plus sd based on six control measurements ) . staying near the male : when the x - coordinate of the marker was < 60 cm ( corresponding to the area adjacent
to the male paddock ) , the status was considered to be staying near the male .
all of the data were expressed as the mean standard error ( se ) .
parameters related to the behavioral
characteristics were analyzed using the student s t - test to compare the means between two
groups ( in estrus or not in estrus ) .
the concordance
of the results obtained by video - tracking analysis and video - recorded observation by a human observer was
examined according to a previously described method .
briefly , an
event was considered to be concordant if the behavior identified by video tracking analysis occurred within
the range of three time points before or after the time point recorded in human observations .
the concordance
rate was calculated by dividing the number of concordant events ( true positive ) by the sum of the true
positives , false positives and false negatives . | here , we report a new method for measuring behavioral patterns during estrus in goats based on video
tracking analysis .
data were collected from cycling goats , which were in estrus ( n = 8) or not in estrus ( n =
8) .
an observation pen ( 2.5 m 2.5 m ) was set up in the corner of the female paddock with one side adjacent
to a male paddock .
the positions and movements of goats were tracked every 0.5 sec for 10 min by using a video
tracking software , and the trajectory data were used for the analysis .
there were no significant differences
in the durations of standing and walking or the total length of movement .
however , the number of approaches to
a male and the duration of staying near the male were higher in goats in estrus than in goats not in estrus .
the proposed evaluation method may be suitable for detailed monitoring of behavioral changes during estrus in
goats . | Methods
Animals and housing
Video recording
Behavioral analyses by human observation
Behavioral analysis based on video tracking data
Statistical analyses |
PMC3069632 | early during an endurance training program ( e.g. , 2 wk ) there is a moderate inverse relationship between baseline aerobic fitness and improvements observed , and this physiological response is sensitive to the training modality ( 46 ) .
however , as the duration ( weeks ) of exercise training is extended to 6 wk and beyond , there is a very modest ( 9 , 10 ) or no significant relationship ( 47 , 61 , 92 ) between baseline vo2max and the improvement in vo2max observed with endurance training . in older female subjects that undertook low - intensity , low - volume training ,
the incidence of nonresponders was much higher than typically observed and did reflect baseline fitness ( 81 ) , suggesting that a minimum training stimulus is required to study the full potential of an individual 's aerobic - capacity system .
notably , the molecular markers that discriminate high responders from low responders do so regardless of whether those subjects undergo intensive interval training for 10 wk , moderately intense constant - load cycling for 6 wk , or 20 wk of incrementally load - adjusted moderately intense aerobic cycling ( 92 ) . maximal aerobic capacity is claimed to be limited by maximal delivery of oxygen to the periphery , and hence by cardiac function ( 78 ) , at least in young muscular and physically active males . however , as baseline aerobic capacity neither positively nor negatively associates with the gains in exercise training - induced maximal aerobic power , and strength training can sometimes promote an improved vo2max when endurance training does not ( 46 ) , it is more likely that vo2max in sedentary adults is a capacity determined by the integration of multiple physiological systems , including macro and micro cardiovascular function and skeletal muscle aerobic characteristics .
vo2max in vivo , insight into the molecular mechanisms determining training adaptation can still be obtained by studying skeletal muscle gene expression ( 54 , 92 ) .
the reason that this is possible reflects the fact that studying muscle gene expression provides a window into the regulation of an individual 's genome , quantifying responses to a variety of physiological stimuli , responses that will partly reflect variations in gene sequence , variations that will be equally relevant in the vascular , cardiac , and lung tissues ( where repeated tissue sampling is not possible ) .
thus i am going to discuss how muscle gene expression responses to exercise have produced insight into the molecular basis for the heterogeneous training responses in human maximal aerobic capacity .
the first examples are based on the candidate gene approach , a popular approach to link molecules to exercise - induced adaptation in aerobic function .
for example , peroxisome proliferator - activated receptor coactivator 1 ( pgc-1 ) ( 60 ) and ampk ( 51 ) are considered two major candidates for regulating skeletal muscle phenotype responses to endurance training ( 71 ) and have been extensively studied in both humans and mice .
overexpression of pgc-1 can modestly increase the percentage of slow - twitch fiber expression in mice ( 60 ) and was thus thought to influence insulin action on skeletal muscle as well as determining muscle endurance performance ( 41 , 42 ) .
pgc-1 was even thought to be specifically downregulated in the skeletal muscle of type ii diabetic patients ( 68 ) , thus explaining insulin resistance .
while this could have been due to physical inactivity ( 94 ) , it is likely the finding was a reflection of the investigators utilizing biopsy samples following , and not before , a pharmacological hyperinsulinemic clamp ( see 30 ) . indeed , pgc-1 overexpression in skeletal muscle has yielded animals that are more prone to diet - induced insulin resistance ( 21 ) while the pgc-1 knockout mouse can respond to endurance training and upregulate components of the mitochondrial proteome and improve their aerobic capacity ( 58 ) .
pgc-1 therefore provides an excellent example of genetic redundancy , where pgc-1 is able to regulate oxidative metabolism in simple cell models , yet when deleted in vivo compensation occurs by yet to be determined factors .
indeed , single - gene knockout murine models are not informative about complex polygenic in vivo human phenotypes , indeed a potentially self - evident point , and yet expenditure on manipulating single genes in mice for the benefit of understanding common human disease has never been greater , a genuinely worrying trend .
ampk has been postulated as the key energy sensor during exercise , whereby alterations in [ amp ] would activate ampk , and ampk would enact downstream transcriptional events that occur following exercise ( 51 ) .
ampk switches on lipid oxidation ( by reducing malonyl - coa inhibition of cpt1 ) in favor of the more economical fuel , carbohydrate , has been ignored and seems completely at odds with ampk 's supposed
ironically , during aerobic muscle contraction [ amp ] is stable and only during reduced blood flow ( 97 ) or high - intensity contraction ( 38 ) do amp levels change measurably , suggesting that during moderately intense aerobic exercise , amp has little to do with ampk activation .
ampk is claimed to regulate substrate uptake and oxidation during muscle contraction ; however , when genetically ablated , muscle glucose uptake during exercise is unaltered ( 64 ) .
further , neither knockout of the 2-ampk subunit or the 1-ampk subunit alters the gene expression response to endurance running in mice ( 52 ) , and the various knockout models have yielded marginal phenotypes completely inconsistent with the presentation that ampk is a master regulator of skeletal muscle phenotype ( 44 ) .
compellingly , no evidence has been presented that subject - to - subject variability in either pgc-1 or ampk activation correlates with muscle adaptation , raising the question as to why such focus has been placed on these molecules by the exercise physiology community over the last decade .
importantly , there are also no reproducible genetic associations between either pgc-1 or ampk sequence variation and gains in aerobic capacity or muscle metabolic capacity changes with endurance training ( 12 ) .
an alternative to the candidate gene approach is to use genomic screening technologies that capture expression data for a large proportion of the genome and use informatics and robust statistics to link molecules to physiological change .
microarray technologies hold many advantages over qpcr especially regarding data normalization and statistical modeling of gene networks .
for example , we recently demonstrated that the training - induced improvements in vo2max following either intensive interval training for 10 wk , moderately intense constant - load cycling for 6 wk , or 20 wk of incrementally tailored aerobic cycling could be predicted by the preexercise resting muscle expression profile of 29 genes , or a number of genetic variants in a subgroup of the same genes .
this was not a simple correlation but was a genuine blind prediction of training response across independent data sets .
it was also particularly striking that the improvement in vo2max was not at all related to the improvement in endurance performance ( 100 ) , and this was also the case for males in the study by lortie et al .
( 61 ) , while for female subjects a modest relationship was found ( 61 ) .
it is therefore likely that the molecular responses that underpin improvements in aerobic exercise performance and aerobic exercise capacity are distinct . for gains in vo2max
, do the 29 genes that make up the molecular predictor ( pretraining ) ( 92 ) shed any light on the molecular networks that determine cardiorespiratory and muscle aerobic capacity adaptation ?
the first notable point is that most of the 29 genes are not regulated ( up or down ) by exercise , while they form a network of interacting genes ( based on literature data ) that is described as being
there are some obvious members of this extended network that are known to be involved in cardiovascular adaptation .
for example , id3 is a tgf1- and superoxide - regulated gene , which interacts ( 70 ) with another predictor gene , krupple like factor 4 , and is involved with angiogenesis ( 63 ) .
these molecules provide a link between aerobic capacity and developmental processes ( 33 , 99 ) , and it is plausible that the level of expression of the 29 genes is preset before birth .
the broader network , connected to the 29 genes , was also populated by igf2 and a number of interleukins , including il-15 , il-32 , and il-8 , but not il-6 ( 92 ) , and these may represent local endocrine signals between the myocytes , extracellular matrix , and vascular cells .
the predictor genes also , based again on literature , appear to influence the expression of many of the genes more regulated by exercise in high - responder subjects ( 54 ) . however ,
to focus on the individual genes and their known biochemical functions is to partly miss the point . in genetic epidemiology
too much importance has been placed on knowing the function of an individual gene and assessing how plausible it is for that function to be associated with the physiological or disease trait being studied , to judge the validity of an association .
such thinking makes two major assumptions : first , that we have sufficient knowledge to accurately define the biochemical repertoire of a given protein or functional rna and , second , that the function of a protein or rna can be defined without consideration of the context ( e.g. , gene network ) it operates within , in a given tissue or cell .
thus further studies , looking at the simultaneous role of these 29 genes , in the various organ systems that can contribute to aerobic capacity in vivo will be required to understand in detail how they are able to predict the plasticity of the oxygen transport / consumption system in humans .
given that they are themselves not regulated ( in muscle tissue at least ) by exercise training , it may be their ability to either influence or inform about other
we have demonstrated that subjects who demonstrate a large increase in vo2max also have a unique muscle transcriptional response 24 h following their last endurance exercise training session ( 9193 ) . in this particular study ,
cycle workload was held constant during the 6 wk of aerobic training , which meant that the highest responders actually had the least relative training intensity by the final week of training . despite this , high responders were still characterized by a much larger gene expression response than low or nonresponders .
in fact , 800 mrnas were differentially expressed in the training cohort as a whole , while at least 100 of these were more regulated in the high responders than the low responders ( 92 ) .
gene ontology analysis ( 1 ) of the regulated genes indicated that modulation of extracellular matrix , mhc , and calcium signaling genes were the transducers of aerobic adaptation in humans ( 92 ) .
such genes , including integrins , appear to connect mechanical work to muscle tissue adaptation during endurance training .
analysis also indicated that calcium - regulated protein modulation , including the regulation of calcineurin , was a promising mechanism connecting endurance training and aerobic adaptation ( 19 , 20 , 72 ) .
the analysis of the human dataset is somewhat consistent therefore with the constitutively active calcineurin murine model . in that model
, there is a switch of fiber type from fast to slow , a process that can be opposed by the administration of cyclosporin ( csa ) ( 72 ) as well as the induction of mitochondrial gene expression . while the shift in the myosin heavy chain proteins is not relevant as this does not occur in humans ( 50 ) ,
the accompanying metabolic adaptations represent a common feature of human muscle remodeling following endurance training ( 34 ) . indeed , using gene - set enrichment analysis ( 84 ) we have shown that the mitochondrial related gene family ( 400 genes in this case ) is the most regulated gene family in human skeletal muscle with endurance training ( 92 ) , despite the fact that most only change by 1520% in abundance . the link between calcium signaling ( and camkii in particular ) and molecular remodeling in response to endurance exercise is further supported by other observations ( 82 ) , including human skeletal muscle studies ( 66 ) , as well as by informatic analysis in our lab ( 92 , 93 ) .
interestingly , high responders for vo2max do not demonstrate a greater mitochondrial gene expression response than low responders .
furthermore , there is no relationship between changes in muscle energy metabolism during training and the magnitude of the improvement in vo2max ( 100 ) .
these observations suggest that molecules regulated acutely by exercise are not regulating the aerobic capacity adaptive process .
rather , modulation of these molecules reflects short - term energy homeostasis within the muscle and/or deregulation of muscle molecular phenotype when the cell is exposed to an acute energy crisis .
thus , based on the available human data , aerobic capacity is an important predictor of human health ( 6 , 7 , 40 , 55 , 69 ) ; improvements in aerobic capacity can be predicted from the expression level of a group of non - exercise - responsive genes ( in muscle ) and that the molecular processes stimulated in the high responders ( for aerobic capacity ) involve calcium signaling , extracellular matrix signaling , and promotion of angiogenesis ( 91 , 92 ) .
in contrast , improvements in aerobic performance relate more to alterations in muscle energy metabolism ( 100 ) and it would be expected that the genes that control the variable training - induced improvements in performance will be distinct from those that control the health - related gains in aerobic capacity .
that is , it is a mistake to assume both of these parameters are always directly coupled .
in fact , the only credible , non - training - related strategy , for improving human performance is manipulation of muscle energy metabolism , directly ( 15 , 17 , 35 , 36 , 87 , 89 , 90 , 95 , 96 ) and through enhanced oxygen delivery in the elite athlete or patient situation ( 31 ) .
exercise performance improvements following endurance training are therefore plausibly linked to enhanced mitochondrial function , especially to modulation of energy metabolism during the rest - to - work transition period ( 95 ) . under these conditions
biomarkers of improved mitochondrial function ( 100 ) and need not causally be related to the molecular basis for fatigue .
a survey of the genetic evidence for linkage between variation in energy metabolism genes and dna sequence variation provides some interesting preliminary support for an important link ( 62 ) .
( 27 ) took the logical approach of examining a polygenic mitochondrial - related gene profile and its association with aerobic performance .
they found that a combination of polymorphisms in nrf2 , pgc-1 , and ppar , assessed by a scaled combinatorial scoring system , associated with the attainment of a better record of competitive endurance performance .
however , it is critical to emphasize that pure genetic association studies , examining the relationship between genetic variants and human performance , have been underpowered and few if any yield associations that have been universally replicated ( 12 ) while many negative studies will be missing from the literature , creating publication bias . for example , for the ace gene insertion / deletion genotype ( i / i , i / d , and d / d ) , medium - duration aerobic endurance performance is claimed to improve to a greater extent in those carrying the i / i genotype ( 16 ) . rather than figure out the connection between the ace gene i / i genotype and , for example , metabolic control , it is more productive to put this observation into context .
there have been over 60 articles examining the link between the ace gene and human fitness and performance , and currently it is not possible to draw any conclusions as to the involvement of the ace gene in human exercise capacity ( 12 ) . indeed , without numerous large ( 5,000 subject ) intervention studies and/or the application of novel genomic strategies ( 92 ) , information from genetic association studies will continue to be largely unreliable and insufficiently replicated in independent laboratories . for the time being ,
candidate molecules that demonstrate a heterogeneous response to acute exercise , rather than a consistent response , are more likely to represent genuine regulators of metabolic adaptation in human skeletal muscle and hence partly determine improvements in aerobic performance .
gains in skeletal muscle mass with resistance training are also highly variable between individuals ( 5 , 49 ) , from no change to 60% increases in muscle size .
there are a number of factors that might affect the hypertrophic response , including nutritional support and genetic variation , and a few individual genetic polymorphisms have been identified that may explain a small degree of variability in the resistance training - induced hypertrophic or strength gain phenotype ( 22 , 49 , 73 ) .
muscle hypertrophy , like the response of muscle to endurance training , is regulated by a complex series of partially redundant signaling molecules ( 23 ) , including the mtorc1 complex .
molecular responses to acute resistance exercise appear to originate from cues within the muscle tissue ( rather than in response to circulating factors ) ( 101 ) , and at some stage muscle hypertrophy is potentially limited by the availability of muscle satellite cells ( 74 ) .
what in turn determines a variable density of satellite cells , or a variable ability for them to proliferate and integrate in vivo , is unclear but could involve rna as well as protein factors .
non - protein - coding rna has emerged in recent years ( 88 ) as being of relevance to skeletal muscle biology ( 30 , 79 ) . in particular ,
micrornas ( mirnas ) are accepted regulators of mammalian cell phenotype ( 4 , 39 , 80 ) .
mirnas are 22-nucleotide posttranscriptional regulators of gene product abundance able to block the translation of protein - coding genes ( 59 ) .
mirnas regulate development and differentiation ( 26 , 76 ) , and brain and skeletal muscle tissue have the most tissue - specific mirna species ( 57 ) .
mirnas have also been implicated in the regulation of metabolism ( 25 , 26 ) , muscle disorders ( 24 , 28 ) , and recently insulin resistance in type ii diabetes ( 30 ) . in vivo in humans mirnas
may impact on protein synthesis rather than mrna stability ( 30 ) where they can be considered regulators of muscle protein expression .
mirnas are plausible candidate molecules for contributing to heterogeneous muscle hypertrophy because they have been shown to influence skeletal muscle satellite cell proliferation and differentiation .
indeed , recent data from our laboratory , in collaboration with stuart phillips , indicate that several highly expressed mirnas are selectivity regulated in subjects that represent the lowest 20% of responders in a longitudinal resistance training intervention study ( 45 ) .
clearly this represents a fruitful area for further investigations and clearly genetic variance located in noncoding dna regions will now be critical to map out .
gains in muscle size and strength also reflect the capacity to form new myofibrils . within the preexisting muscle , phosphorylation of the mtorc1 complex yields a signal transduction event resulting in ribosomal
s6 kinase 1 ( s6k1 ) and eurkaryotic initiation factor 4e ( eif4e ) binding protein phosphorylation ultimately leading to enhanced protein synthesis through reduced inhibition of eif4e .
direct inhibition of mtorc1 signaling in humans blocks the mixed - muscle protein synthesis induced by acute high - intensity fatiguing muscle contractions ( 23 ) .
this acute intervention data would imply that genes regulating translation initiation signaling determine ( or regulate ) progressive skeletal muscle hypertrophy in response to resistance training .
variation in the hypertrophy derived from supervised strength training in humans ( 5 ) allowed mayhew and colleagues ( 65 ) to explore the causal relationship between acute mtorc1 signaling and the gains in muscle mass observed after 16 wk of resistance training . both protein synthesis and mtorc1-related signaling
are elevated 24 h post acute resistance exercise and regulation of s6k1 was found to correlate with increased myofibrillar size after 16 wk of training ( 65 ) via prolonged autoinhibitory domain silencing . however
, protein synthesis measured in response to an acute bout of unaccustomed exercise does not always agree with activation of the proposed causal signaling molecules and indeed acute synthesis was not predictive of chronic hypertrophy responses in a subset of subjects ( 65 ) .
this is somewhat analogous with the observations above , whereby short - term gains in vo2max do not relate to the same parameters as longer - term changes .
likewise , muscle anabolic responses to alternative stimuli , namely infused insulin and amino acids , do not relate in a linear manner to activation of mtorc1-regulated molecules ( 37 ) demonstrating that regulation of protein synthesis ( and in this study also protein breakdown ) is distributed across further unknown pathways .
this is analogous to the observation that anabolic stimuli and hypertrophy of existing muscle fibers eventually appear limited by the further incorporation of nuclei from satellite cells ( 74 ) , i.e. , factors beyond the preexisting fibers add a new layer of regulation when one point of limitation is exceeded .
interactions between muscle protein metabolism and endurance exercise and cardiovascular conditioning exemplify additional considerations for potentially relevant genes .
for example , insulin 's ability to regulate muscle protein synthesis and mtorc1-related signaling appears dependent on an intact vascular response to insulin , and thus a bout of endurance exercise can overcome aged - related insulin resistance where insulin resistance is defined as impaired protein synthesis ( rather than impaired glucose uptake ) ( 29 ) .
it is also true that in the existing genetic association studies ( 12 ) there is some overlap between candidate genes for aerobic and strength phenotypes , suggesting that the idea that these two muscle traits are at either end of a molecular spectrum may be rather too simplistic ( 2 , 48 , 83 ) .
in fact , as will be briefly discussed below , the interaction between any two bouts of exercise may alter the molecular response observed to the initial period of contraction , and this may reflect the fact that the recovery period is also altered in nature or duration , and thus interactions are not really reflecting the differing physiological load . in support of the idea that mechanical loading of human skeletal muscle , within the diversity experienced during voluntary exercise , will activate a number of overlapping pathways ( which are not easily segregated into strength or endurance in nature ) is the recent work by the gibala laboratory ( 13 , 14 , 32 ) . here , high - intensity , short - duration sprint training yields endurance and metabolic adaptations synonymous with classic endurance training ( 3 , 100 ) .
likewise , as mentioned above , under certain circumstances strength training is more effective at improving vo2max in an individual than a similar duration of aerobic training ( 46 ) presumably because some sedentary subjects have poor muscle conditioning limiting test performance and hence the strength training protocol has a more rapid impact on improving this factor .
these data represent the only crossover - designed study that i am aware of . in a resistance training intervention study ( n = 153 )
. found that a polymorphism in the gene for interleukin 15 receptor explained a relatively large proportion of the variation in muscle hypertrophy ( 77 ) .
a similar association between il-15 and muscle hypertrophy has also been recently reported in 748 subjects ( 75 ) .
both studies are small for a genetic association study and would benefit from further replication .
intriguingly , il-15 was also connected to the gene network that predicted variation in aerobic capacity changes to endurance training ( 92 ) .
this exemplifies the idea that trainability genes could in fact be defined as tissue or organ plasticity genes , and they should probably not be defined by the physiological stimuli they are first associated with .
in the hunt for greater understanding of the molecular basis for skeletal muscle adaptation in humans , subject - to - subject variability is a very powerful aid to discovery .
second , it will become more and more apparent that the search for single gene master regulators of physiological adaptation is futile ; genes work in complex , nonlinear , redundant networks , and thus we need to take a fresh approach to molecular physiology .
third , when considering the molecular basis and signals that regulate skeletal muscle adaptation to physical activity , it is too simplistic to consider that the strength and endurance phenotypes are placed at opposing ends of a molecular spectrum , at least in the context of voluntary human muscle activation . probably the single most important philosophical question to raise at this point is why , given our apparent recent heritage as an active hunter gatherer ( 18 , 56 ) , do we have a significant number of humans unable to mount a strong physiological adaptive response to physical activity ?
is it the case that for some subjects we provide an inappropriate pattern of stimulus for their particular genotype ?
we are far away from a scientific basis for tailored exercise prescription for the general public , and recent studies examining muscle adaptation using different intervals of recovery ( 43 , 102 ) have not reproducibly demonstrated greater adaptation occurs with extended recovery , even though distinct molecular responses have been noted ( 43 , 102 ) . likewise the genes that predict aerobic capacity changes with endurance training appear independent of duration and cycling intensity ( 92 ) , at least above a certain minimum threshold .
if one considers the idea that within the range of voluntary muscle contractions possible , the gene networks that are important for both endurance and strength adaptation will demonstrate significant overlap because they are muscle plasticity networks , then there comes a point when mounting a high protein synthesis response to any type of exercise ( as we assume high responders do ) , from an energetic perspective , offsets the advantage of gaining a higher physiological capacity .
there is in essence a cost for everything in life , yet without greater insight into the actual environmental conditions that shaped our genome , or how rapid genomic and epigenomic evolution really are , providing an explanation for the clear existence of physiological nonresponders is challenging .
the studies mentioned in this review were supported by an affymetrix translational medicine grant , the swedish diabetes society , the chief scientists office , scotland , pfizer global research and development , the royal veterinary college , and the swedish national centre for research in sports .
no conflicts of interest , financial or otherwise , are declared by the author(s ) . | when human skeletal muscle is exposed to exercise training , the outcomes , in terms of physiological adaptation , are unpredictable .
the significance of this fact has long been underappreciated , and only recently has progress been made in identifying some of the molecular bases for the heterogeneous response to exercise training .
it is not only of great medical importance that some individuals do not substantially physiologically adapt to exercise training , but the study of the heterogeneity itself provides a powerful opportunity to dissect out the genetic and environmental factors that limit adaptation , directly in humans . in the following review
i will discuss new developments linking genetic and transcript abundance variability to an individual 's potential to improve their aerobic capacity or endurance performance or induce muscle hypertrophy .
i will also comment on the idea that certain gene networks may be associated with muscle
adaptability
regardless the stimulus provided . | AEROBIC AND ENDURANCE CAPACITY
MUSCLE HYPERTROPHY AND STRENGTH
CONCLUSIONS
GRANTS
DISCLOSURES |
PMC4127697 | the main aim of an endodontic obturation is to obtain a fluid tight hermetic seal of the entire root canal system ( rcs ) .
the presence of great anatomic complexity of the rcs , has advocated the evolution of different materials and techniques to achieve the desired fluid tight hermetic seal .
gutta - percha , in combination with sealer , is the most widely used solid material for endodontic root canal obturation .
cold lateral condensation as an obturation technique is widely practiced throughout because of its advantages of controlled placement of gutta - percha in the root canal and low cost .
the final filling is composed of a large number of gutta - percha cones tightly compressed together and joined by frictional grip and root canal sealer , rather than a homogeneous mass of gutta - percha .
there is usually presence of voids because of spaces between individual gutta - percha cones and the root canal walls .
this can be seen with poor root canal preparations , curved canals , inadequate lateral pressure during condensation or mismatch between gutta - percha cones and the prepared root canals .
such obturations would lack homogeneity and have to depend on sealer to fill the voids and thus would have a poor prognosis . because gutta - percha obturation techniques generally require some type of condensation ( lateral , vertical ) their final radiographic appearance may or may not be as expected due to variations in appearance such as sealer voids , spreader tracks , condenser voids and material welds ( in the case of heated techniques ) .
while various studies have attempted to relate these aberrancies with case unacceptability and failure , little correlation exist at present ; except for the standard that a dense , well - adapted root - filling is clinically and radio graphically acceptable .
since the introduction of thermoplastic obturation techniques in 1967 by schilder , new devices and systems using thermoplasticized gutta - percha as root canal filling material have been developed .
the sectional backfill technique with thermoplastic gutta - percha is one of these options , consisting of an apparatus for heating and injecting thermoplasticized gutta - percha into the root canal .
this technique has shown better adaptation to the root canal walls in comparison to lateral condensation , and also result in successful filling of simulated lateral canals .
the most recent advancement in endodontic obturating materials uses a hydrophilic polymer in the root canal , the smartseal system ( prosmart - drfp ltd . , stamford , uk ) .
the system consists of obturation points ( pro - points ) containing a polyamide core with an outer bonded hydrophilic polymer coating and an accompanying sealer which is further provided with polymer powder to be incorporated during the manipulation of the sealer .
the endodontic points are designed to expand laterally without expanding axially by absorbing residual water from the instrumented root canal space and the naturally present moisture in the dentinal tubules .
the inner core of pro - points is a mix of two proprietary nylon polymers : trogamid t and trogamid cx .
the polymer coating is a cross - linked copolymer of acrylonitrile and vinylpyrrolidone which has been polymerised and cross - linked using allyl methacrylate and a thermal initiator .
the lateral expansion of pro - points is claimed to occur nonuniformly with the expand - ability depending on the extent to which the hydrophilic polymer is prestressed ( i.e. , contact with a canal wall will reduce the rate or extent of polymer expansion ) .
different techniques and root canal materials have been used for evaluating obturation in a simulated lateral canal .
both natural and artificial teeth have been used for in order to simulate the lateral canals .
natural teeth have been more commonly used as evaluation can be done by radiographic methods , decalcification , clearing methods or a combination of both .
to the best of our knowledge , there is no reported study comparing novel smartseal system and gutta - percha system using simulated lateral canals using decalcification methods and cone beam computed tomography ( cbct ) image analysis .
the homogeneity of the materials has been evaluated using cbct with axial sections obtained at coronal , middle and apical levels . because we were evaluating a new material , a three - dimensional ( 3d ) evaluation by cbct was advised to further substantiate the results .
a total of 60 freshly extracted human single rooted teeth with fully formed apices were selected for this study .
teeth were de - coronated and roots were standardized to a working length ( wl ) of 15 mm . root canal preparation was subjected to crown down technique with rotary protaper files until file size f2 with full sequence .
after each file 1 ml of 3% sodium hypochlorite was used as an irrigating solution delivered with 27-gauge side venting needles inserted 3 mm from the apex .
ethylenediaminetetraacetic acid ( edta ) gel ( rc help ) was used as a lubricant .
this was followed by a rinse of 3 ml of 17% edta for 1 min .
after completion of root canal preparation ten specimens from each group were subjected to a tooth decalcification and clearing protocol previously described by venturi et al .
specimens were decalcified in a solution of 5% hydrochloric acid ( hexis cientfica s / a , jundia , sp , brazil ) which was changed every 24 h for 5 days .
teeth were de - mineralized until they achieved a rubber - like consistency . later , # 8 and # 10 k - files were inserted through the buccal and lingual root surfaces at 2 , 4 , and 6 mm from the wl until they reached the main root canal , creating the simulated lateral canals .
the patency and standardization of lateral canals were tested with # 8 or # 10 k - file . in order to eliminate the acid ,
teeth were washed in running tap water for 4 h and dehydrated using increasingly concentrated serial dilutions of alcohol , which reverted the specimens back to its hard consistency .
finally , specimens were immersed in ethyl salicylate ( vetec qumica fina ltda , rio de janeiro , rj , brazil ) in order to render the tissues transparent .
remaining ten specimens from each group were mounted on modeling wax for cbct analysis .
group a : smartseal systemgroup b : sectional backfill techniquegroup c : cold lateral condensation technique
group a : smartseal system group b : sectional backfill technique group c : cold lateral condensation technique group a : teeth were obturated with 6 taper master polyamide polymer cone using single - cone technique with no accessory cones being used .
the sealer used in this group was smartpaste sealer ( prosmart - drfp ltd.stamford , uk ) an epoxy amine resin - based sealer with active polymer powder .
group b : teeth were obturated using sectional backfill technique with 6 taper gutta - percha cone seared off 4 mm from the apex and the remaining canal backfilled with warmed gutta - percha .
group c : teeth were obturated using cold lateral compaction technique . a f2 0.06 taper gp cone ( diadent , vancover , bc , canada ) was placed to wl .
a fine size finger spreader was advanced to within 1 mm of the wl or to resistance , rotated and removed .
a size # 25 0.02 taper accessory gp cone ( tulsa dental ) was placed into the prepared space .
this was repeated until no more than 2 mm of the spreader could be advanced into the canal .
excess gp was removed with a # 15 scalpel blade at the coronal root surface .
sealer used in groups b and c was ah plus ( dentsply maillefer ) ; an epoxy amine resin - based sealer .
after obturation , all the specimens were stored at a temperature 37c for duration of 24 h to allow complete setting of the sealer .
proximal radiographs of each specimen ( kodak rvg 6100 ; kodak dental systems , atlanta , ga , usa ) were taken using the spectro 70 electronic x - ray unit after standardizing the positioning of the roots and the focus - film distance .
then , specimens were soaked once again in methyl salicylate , and standardized digital photographs were taken ( nikon d80 ; nikon , tokyo , japan ) .
the radiographic and photographic images showing the root canal fillings in each specimen were imported into the image tool software ( uthscsa image tool for windows version 3.0 , san antonio , tx , usa ) .
the amount of filling material observed in each lateral canal at the level of 2 , 4 and 6 mm was calculated by single calibrated examiner .
the data were expressed as linear extension ( length in mm of the lateral canal that was filled in relation to its entire length ) and of the area filled ( area in millimeter square occupied by the filling material inside the lateral canal in relation to its entire area ) .
the cbct scans were done using promax 3d mid ( planmeca oy helsinki , finland ) .
the samples were mounted on wax block for stabilizing them and were exposed to the cbct scans with 90 kv and 8 ma with field of view of 4.5 cm 4.5 cm and an isotropic resolution of 2 mm resulting in 400 - 450 slices per tooth with exposure time of 12.28 s.
a total of 60 freshly extracted human single rooted teeth with fully formed apices were selected for this study .
teeth were de - coronated and roots were standardized to a working length ( wl ) of 15 mm . root canal preparation was subjected to crown down technique with rotary protaper files until file size f2 with full sequence .
after each file 1 ml of 3% sodium hypochlorite was used as an irrigating solution delivered with 27-gauge side venting needles inserted 3 mm from the apex .
ethylenediaminetetraacetic acid ( edta ) gel ( rc help ) was used as a lubricant .
this was followed by a rinse of 3 ml of 17% edta for 1 min .
after completion of root canal preparation ten specimens from each group were subjected to a tooth decalcification and clearing protocol previously described by venturi et al .
specimens were decalcified in a solution of 5% hydrochloric acid ( hexis cientfica s / a , jundia , sp , brazil ) which was changed every 24 h for 5 days .
teeth were de - mineralized until they achieved a rubber - like consistency . later , # 8 and # 10 k - files were inserted through the buccal and lingual root surfaces at 2 , 4 , and 6 mm from the wl until they reached the main root canal , creating the simulated lateral canals .
the patency and standardization of lateral canals were tested with # 8 or # 10 k - file . in order to eliminate the acid ,
teeth were washed in running tap water for 4 h and dehydrated using increasingly concentrated serial dilutions of alcohol , which reverted the specimens back to its hard consistency .
finally , specimens were immersed in ethyl salicylate ( vetec qumica fina ltda , rio de janeiro , rj , brazil ) in order to render the tissues transparent .
group a : smartseal systemgroup b : sectional backfill techniquegroup c : cold lateral condensation technique
group a : smartseal system group b : sectional backfill technique group c : cold lateral condensation technique group a : teeth were obturated with 6 taper master polyamide polymer cone using single - cone technique with no accessory cones being used .
the sealer used in this group was smartpaste sealer ( prosmart - drfp ltd.stamford , uk ) an epoxy amine resin - based sealer with active polymer powder .
group b : teeth were obturated using sectional backfill technique with 6 taper gutta - percha cone seared off 4 mm from the apex and the remaining canal backfilled with warmed gutta - percha .
group c : teeth were obturated using cold lateral compaction technique . a f2 0.06 taper gp cone ( diadent , vancover , bc , canada ) was placed to wl .
a fine size finger spreader was advanced to within 1 mm of the wl or to resistance , rotated and removed .
a size # 25 0.02 taper accessory gp cone ( tulsa dental ) was placed into the prepared space .
this was repeated until no more than 2 mm of the spreader could be advanced into the canal .
excess gp was removed with a # 15 scalpel blade at the coronal root surface .
sealer used in groups b and c was ah plus ( dentsply maillefer ) ; an epoxy amine resin - based sealer .
after obturation , all the specimens were stored at a temperature 37c for duration of 24 h to allow complete setting of the sealer .
proximal radiographs of each specimen ( kodak rvg 6100 ; kodak dental systems , atlanta , ga , usa ) were taken using the spectro 70 electronic x - ray unit after standardizing the positioning of the roots and the focus - film distance .
then , specimens were soaked once again in methyl salicylate , and standardized digital photographs were taken ( nikon d80 ; nikon , tokyo , japan ) .
the radiographic and photographic images showing the root canal fillings in each specimen were imported into the image tool software ( uthscsa image tool for windows version 3.0 , san antonio , tx , usa ) .
the amount of filling material observed in each lateral canal at the level of 2 , 4 and 6 mm was calculated by single calibrated examiner .
the data were expressed as linear extension ( length in mm of the lateral canal that was filled in relation to its entire length ) and of the area filled ( area in millimeter square occupied by the filling material inside the lateral canal in relation to its entire area ) .
the cbct scans were done using promax 3d mid ( planmeca oy helsinki , finland ) .
the samples were mounted on wax block for stabilizing them and were exposed to the cbct scans with 90 kv and 8 ma with field of view of 4.5 cm 4.5 cm and an isotropic resolution of 2 mm resulting in 400 - 450 slices per tooth with exposure time of 12.28 s.
table 1 shows the radiographic analysis of each material of filled the simulated lateral canals ( measured in linear and area percentages ) .
smartseal system of obturation when evaluated , presented a high mean ( 89.45 16.21 ) in linear extension of root canal and high mean of ( 92.46 19.45 ) in area filled .
pro - points demonstrated a greater filling ability ( p < 0.05 ) than gutta - percha in all thirds of canal , as shown by the radiographic analysis ( extension and area ) , and as by the analysis of cleared specimens ( extension ) .
figure 2 shows the cbct analysis ( axial sections ) of all groups , in coronal ( a ) , middle ( b ) and apical ( c ) sections .
polyamide polymer reveals minimum voids in all sections indicating an obturation of greater homogeneity in comparison to gutta - percha system .
mean percentage of linear extension and area extended in coronal , middle and apical root canal photographic and radiographic analysis .
( c ) cold lateral condensation ( control ) cone beam computed tomography analysis ( axial sections ) .
( c ) apical section ( a ) polyamide polymer obturation , ( b ) sectional backfilled obturation , ( c ) cold lateral obturation ( control )
the ultimate goal of obturation is to prevent the re - infection of root canals that have been biomechanically cleaned , shaped and disinfected by instrumentation and irrigation .
successful obturation requires the use of materials and techniques capable of densely filling the entire rcs and providing a fluid tight seal from the apical segment of the canal to the canal orifice in order to prevent re - infection .
therefore , the goal of 3d obturation is to achieve an impermeable fluid tight seal within the entire rcs which will further help in preventing oral and apical microleakage .
gutta - percha along with root canal sealers is the worldwide accepted combination available till date and is considered as the gold standard . despite its many advantages and having achieved the status of a high quality material , gutta - percha still has its limitations like its inability to strengthen root as it does not bond to dentin .
although few materials have seriously challenged gutta - percha in the majority of aspects associated with gutta - percha , research continues to find alternatives that may seal better and mechanically reinforce compromised roots .
the major demerit seen with gutta - percha is its inability to reinforce endodontically treated tooth .
gutta - percha does not chemically bond to the dentin wall , that is , does not form the monoblock system . according to teixeira and trope .
gutta - percha does not from a monoblock even with the use of a resin - based sealer such as ah plus because the sealer does not bind to gutta - percha .
moreover , the sealer tends to pull away from the gutta - percha on setting .
thus , the ultimate goal of this study was to search an alternative to currently and most widely used gutta - percha system which would not only have the ideal characteristics of gutta - percha , but also further new advances installed .
smartseal system consists of polyamide polymer cones ( propoints ) and a resin sealer with additional polymer powder to be mixed during manipulation of the sealer .
the white points consist of a radiopaque core coated with a radiopaque hydrophilic polymer , which can expand laterally upon absorbing water from the tooth , adopting the shape of the canal .
the points can expand up to around 17% and will still give the same x - ray appearance as with conventional root - filling materials .
leakage studies indicate single - cone obturation methods to be inferior in their ability to achieve a fluid - tight seal . the use of expandable obturating materials to improve the seal of the root - filled canals have been reported even for gutta - percha .
gutta - percha expands in the presence of eugenol , which may reduce gaps within the filled canal space .
apart from eugenol - induced expansion , closure of microgaps in gutta - percha filled root canals by moisture present within the canal space may compensate for leakage that arises from sealer dissolution to certain extent the delayed hygroscopic expansion of pro - points when latter is coated with a hydrophobic sealer that impedes water sorption may partially compensate for the gaps arising from sealer dissolution .
farge et al . showed that lateral condensation technique results in more leakage than thermomechanical compaction technique .
brothman et al . reported that the thermoplasticized condensation technique on radiographic examination , shows nearly 2 times more lateral and accessory canals compared with lateral condensation technique .
the amount of filling achieved in the lateral canals of natural teeth has been evaluated by means of radiographs and visual analysis of decalcified and cleared specimens following a protocol described by venturi et al .
venturi et al . evaluated the diameter and shape of natural lateral canals as well as the ability of different root canal filling materials to fill these root canals using three obturation techniques . in this study ,
the lateral canal preparation was held after the demineralization of teeth until they achieved a rubber - like consistency .
the # 8 or # 10 k - files were inserted into the softened root creating standardized artificial lateral canals at 2 , 4 and 6 mm from the apex .
the clearing procedure was then completed by immersion in methyl salicylate to harden the specimens and restore their initial consistency . the radiographic analysis was performed digitally and the specimens had been previously subjected to the decalcification and clearing protocol promoting better image quality .
this study has shown results that polyamide polymer has shown a greater area and linear extension occupied in the root canal space as compared to obturation techniques using gutta - percha .
such a result can be attributed to the hygroscopic expansion property exhibited by the polyamide polymer system and the shrinkage associated with the gutta - percha system . even though , the sealers are mostly responsible for obtaining a proper seal in the lateral canals and attempt was made to show the role of different obturation techniques in filling simulated lateral canals as well as the role of a hydrophobic and a hydrophilic obturating system . an outstanding review by taylor
et al . have reported on many possible endodontic application of cbct , simon et al .
however , for this in vitro study , for the analysis of newer obturating material cross - sectional analysis using cbct was done to evaluate the homogeneity .
as group a was obturated using a single - cone technique , it can be hypothesized that it would show greater homogeneity as compared to lateral condensation and sectional backfilled methods . as seen in the images lesser number of voids
is observed in the cross - section of group a owing to its greater homogeneity ; leading to better adaptation in the root canal space . even though one can contradict the use of cbct image analysis for postobturation evaluation as various artifacts like scattering
are associated with cbct image analysis , which can be mistaken for potential voids in the obturation .
however considering the enormous benefits this cost - effect can be overlooked since the ease of the machine makes it suitable to be used also in minor diagnostic procedures .
furthermore , analysis would be required to evaluate the actual expansion shown by the polyamide polymer system ; its microleakage properties , fracture toughness , and bond strengths to the dentin .
with the introduction of novel hydrophilic smartseal system over the conventional hydrophobic gutta - percha system , has widened our range of achieving a 3d seal . this study concludes a greater efficiency in filling simulated lateral canals and a comparable homogeneity of obturation using smartseal system over gutta - percha . | aim : the aim was to evaluate and compare a novel polyamide polymer based obturating system and gutta - percha and sealer in filling simulated lateral canals and their homogeneity when used for obturating the root canals.materials and methods : a total of 60 freshly extracted human single rooted teeth with fully formed apices were selected for this study .
teeth were de - coronated , and roots were standardized to a working length of 15 mm .
root canal preparation was carried out with rotary protaper file system in all groups .
the specimens were then randomly divided into three groups a , b , and c ( n = 20 ) .
ten samples from each group were decalcified and simulated lateral canals were made at 2 , 4 , and 6 mm from the root apex .
remaining ten samples from each group were maintained calcified .
group a was obturated with smartseal system ( prosmart - drfp ltd . , stamford , uk ) .
group b was obturated with sectional backfill method .
group c was obutrated with cold lateral compaction method ( control ) .
decalcified samples from the respective groups were analyzed with digital radiography and photography and the measurement of the linear extension and area of lateral canal filling was done using uthscsa ( uthscsa image tool for windows version 3.0 , san antonio , tx , usa ) software .
calcified samples were subjected to cone beam computed tomography image analysis sectioned axially.results:group a 92.46 19.45 showed greatest extent of filling in lateral canals and denser homogeneity of oburation , followed by group b 78.43 26.45 and group c 52.12 36.67.conclusions:polyamide polymer obturation proved to have greater efficiency when compared with gutta - percha system , when used for obturation with regards to adaptation of the sealer and penetration into the simulated lateral canals . | INTRODUCTION
MATERIALS AND METHODS
Preparation of the specimens
Obturation and distribution of the specimens
Radiographic and photographic analysis of the cleared specimens
Cone beam computed tomography analysis
RESULTS
DISCUSSION
CONCLUSION |
PMC3868081 | district mental health program ( dmhp ) is an approach to decentralize mental health - care in the community using the existing public health infrastructure and additional resources .
this model has been pilot tested in bellary district of karnataka state and found to be useful in addressing the basic mental health needs of the population .
later on , this model was developed as a program , which was thought fit to be applied to the whole of india .
in most districts where the program is in operation , a team of the dmhp , consisting of a psychiatrist , psychologist , social worker , and nurse visits designated peripheral health centers .
there are no special services for special populations such as women , children , elderly , and those with substance misuse , though persons with such problems could also be approaching these clinics .
being a decentralized program , dmhp may be more accessible to older people with mental health problems and their families .
is this presumption true ? if older people tend to access dmhp more than the traditional service , then there is a strong case to suitably modify dmhp to meet the mental health needs of the rapidly growing number of older people in our country .
this would then open up an avenue for scaling up geriatric mental health services across the country .
this study aims to examine whether there is an increased attendance of elderly persons at dmhp clinics , compared to a traditional tertiary care facility ( psychiatry out - patient department of a government medical college hospital ( mch ) ) by examining records available at these two setting .
the out - patient records maintained by the department of psychiatry at a government mch , and the office of the dmhp at the nearby district and run by the same department were analyzed to find out the number and socio demographic details of out - patients seen at these two facilities during a period of 1 year .
all subjects who attended the out - patient services during the study at these two facilities period were identified from the records .
patients 60 years and older formed the geriatric group and the others formed the second group .
the sample from these two centers were compared to see if there was any significant difference in the proportion of older people seen at these two care facilities .
we also made sex - wise comparison of the older patients from both the settings to see if there was any difference between these two in terms of the proportion of women getting care .
the data in table 1 shows that elderly account for only 9.5% of out - patient attendance at the mch while they account for 16.5% in the clinics of dmhp .
comparison of patients attending a mch and community outreach clinics of dmhp ( 1 may 2007 and 30 april 2008 ) proportion of women in the geriatric age group seen at the dmhp clinics were more , compared to the sample from the mch .
older people seeking care at mch and community outreach clinics of dmhp ( 1 may 2007 and 30 april 2008 )
our results show that a significant proportion of people who receive mental health - care through the community outreach clinics of dmhp are people above the age of 60 years .
the proportion of older people among the users was dmhp clinics is 16.5% , whereas the proportion of people above the age of 60 years in the general population in the state of study is less than 11% .
it is no big surprise to note that the decentralized services provided by the dmhp are more accessible to older people .
this implies that the provision of care through community outreach clinics by the dmhp is a step in the right direction .
however , the services currently provided through the dmhp are generic , and not tailored to meet special needs of the elderly . while planning decentralized programs , planners should understand that its users are likely to include special populations and provide additional services .
there are no government funded specialized services for elderly in india and the outreach services generally do not include health - care for older people as part of their work .
we need to ensure that the services provided do meet the needs of older people who seek help .
for example , an attempt will have to be made to cater to the special needs of people with medical illness and depression .
people with disabling conditions like dementia and their caregivers may need to be provided specialized care and support .
dmhp should take up the task of supporting and guiding community based initiatives as well as other out reach services .
primary care as well as outreach services can play a crucial role in scaling up services for older people with mental health needs .
we need to develop a strategy to link the community clinics of the dmhp with primary care and outreach services .
primary care need to receive continuous support and guidance from the mental health expertise available with the dmhp .
they may be able to identify older individuals with unmet mental health needs and bring them to the notice of the services .
once this is possible , clinicians will be in a better position to identify and manage common mental health problems of late life such as depression and dementia .
they can play a huge role in providing information and education to the caregivers and could be entrusted to deliver simple caregiver interventions . support to home based care is particularly important in conditions such as dementia and various other disabling conditions affecting older people .
dmhp should reorient itself by deciding to focus on the special needs of the older people .
this need will become more evident in the future as we anticipate gradual increase in the number of older people in the population .
it is time that we reevaluate the potential for dmhp to provide better mental health - care for older people .
this will help to scale up the services for older people with mental health needs in india .
we need to convince the governments and policy makers to make such changes in the dmhp . | background : district mental health program ( dmhp ) is the flagship program of government of india to deliver mental health - care throughout the country .
being an out - reach program , it is likely that elderly people with mental health programs are accessing it more frequently .
if this is the case , there is potential for including additional components in this program so that something more than the generic service is offered to them.aim:this study aims to find out whether the elderly are accessing the dmhp services frequently.materials and methods : we compared the proportion of elderly people attending two mental health - care facilities : - a government medical college out - patient department and the clinics run by the dmhp of the same department.results:a significantly higher number of elderly people are attending the clinics of the dmhp compared to the outpatient service of a teaching hospital.conclusion and implication : it is concluded that dmhp can be further strengthened by adding additional components like service for the elderly .
this is important in view of the increasing number of older people who may need these services in future . | INTRODUCTION
AIM
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC3638265 | anterograde amnesia is a syndrome that is characterized by a sudden onset of selective amnesia during which patients can not store new memories after an event , while retrograde amnesia is a syndrome in which patients can not recall old memories after an event , and these do not involve neurological deficits3,5,29 ) .
the hallmark of transient anterograde amnesia is brief inability to form new memories and recall past memories despite otherwise normal neurological function5,29 ) . in a significant number of patients with transient anterograde amnesia
transient anterograde amnesia is occasionally observed in a number of conditions , including migraine , focal ischemia , venous flow abnormalities , and after general anesthesia5,29 ) .
transient anterograde amnesia induces impairments in memory processes involving place , time , response , and perceptual and language information9 ) .
the inhalation anesthetic , isoflurane , is halogenated ether that is used for general anesthesia .
isoflurane has many favorable effects , including analgesia , muscle relaxation , an absence of central nervous system ( cns ) excitation , and neuroprotection .
however , it occasionally induces problematic side effects , such as respiratory depression , reduced arterial blood pressure , amnesia , and unresponsiveness7,32,35 ) . of these several side effects ,
the amnesic action of isoflurane has received much interest , and many studies have been performed in order to evaluate its underlying mechanisms .
dutton et al.7 ) observed that isoflurane induced dose - dependent anterograde amnesia but not retrograde amnesia .
rau et al.26 ) suggested that -aminobutyric acid type ( gaba ) a receptor is the main causative factor of the amnestic effects of isoflurane on hippocampal - dependent declarative memory .
the frequency of oscillations was also suggested as a parameter of isoflurane - induced anterograde amnesia24 ) .
many neurotrophic factors have been suggested to be part of the underlying mechanisms associated with hippocampal - dependent memory .
these neurotrophic factors include nerve growth factor , neurotrophin-3 , neurotrophin-4/5 , and brain - derived neurotrophic factor ( bdnf ) . among them
, bdnf has been shown to have a crucial role as a modulator of long - term potentiation , synaptic plasticity , and neuronal plasticity in the adult cns4,14,18 ) .
the tyrosine kinase b ( trkb ) receptor is a specific receptor for bdnf , and it plays a key role in neuronal survival , differentiation , and synaptic plasticity6,23 ) .
lu et al.15 ) reported that the levels of bdnf in the thalamus and cortex of rats were changed by exposure to general anesthesia .
however , the effects of isoflurane on the expression of bdnf in the hippocampus in relation to anterograde amnesia are unknown . in the present study
, we investigated whether isoflurane anesthesia induces anterograde amnesia , and the underlying mechanisms of isoflurane - induced anterograde amnesia were evaluated in relation to the expressions of bdnf and trkb in the rat hippocampus .
the animals were housed under conditions of controlled temperature ( 202 ) and lighting ( 07:00 to 19:00 hour ) , and food and water were supplied ad libitum .
the experimental procedures progressed according to the animal care guidelines of the national institutes of health and the korean academy of medical sciences .
the rats were randomly divided into three groups : the control group , the 10 minutes after recovery from isoflurane anesthesia group , and the 2 hours after recovery from isoflurane anesthesia group ( n=8 in each group ) .
the rats in the isoflurane - exposed groups were anesthetized with 1.2% isoflurane in 75% nitrous oxide and 25% oxygen for 2 hours in a plexiglas anesthetizing chamber . in order to evaluate short - term memory loss
, we conducted a step - down avoidance task according to the recovery time after isoflurane anesthesia , as previously described30 ) .
the rats were positioned on a 725 cm platform with a height of 2.5 cm , and they were then allowed to rest on the platform for 2 min .
the platform faced a 4225 cm grid of parallel 0.1-cm - caliber stainless steel bars , which were spaced 1 cm apart . in the training session , the animals received a 0.5-ma scramble foot shock for 20 seconds immediately upon stepping down . in the control group ,
the training session in the other groups was conducted according to the recovery time of the respective group ( 10 minutes or 2 hours after isoflurane anesthesia ) .
the retention time in each group was assessed 10 minutes after the training session of each group .
the interval of time in which the rats stepped down and placed all four paws on the grid was defined as the latency of the step - down avoidance task .
after the determination of the retention time in the step - down avoidance task , the rats were deeply anesthetized with zoletil 50 anesthesia ( 10 mg / kg , i.p . ; virbac , carros , france ) . for immunofluorescence
, the rats were transcardially perfused with 50 mm phosphate - buffered saline ( pbs ) , which was followed by 4% paraformaldehyde in 0.5 m sodium phosphate buffer at ph 7.4 .
the brains were removed , postfixed in the same fixative overnight , and transferred to a 30% sucrose solution for cryoprotection .
serial coronal sections that were 40 m thick were cut with a freezing microtome ( leica biosystems nussloch gmbh , nussloch , germany ) .
, the hippocampus was removed from each rat brain , and the extraneous tissue was trimmed away .
the hippocampal tissues were minced and chopped in lysate buffer that contained 50 mm hepes ( ph 7.5 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm magnesium chloride hexahydrate , 1 mm ethyleneglycol - bis-(-aminoethyl ether)-n , n'-tetraacetic acid , 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 1 g / ml pepstatin , 1 mm sodium orthovanadate , and 100 mm sodium fluoride .
after homogenization , the tissues were reacted with lysate buffer for 20 min and then centrifuged at 14000 rpm for 20 minutes at 4. the supernatant was stored in a -70 deep freezer .
whole protein extracts were used to evaluate the levels of expression of the bdnf and trkb proteins .
the protein concentrations were measured using a bio - rad colorimetric protein assay kit ( bio - rad laboratories inc . , hercules , ca , usa ) .
forty g of protein was separated on sds - polyacrylamide gels and then transferred onto a nitrocellulose membrane ( schleicher & schuell bioscience gmbh , dassel , germany ) . a rabbit bdnf antibody ( 1 : 1000 ; santa cruz biotechnology inc . ,
santa cruz , ca , usa ) and a rabbit trkb antibody ( 1 : 1000 ; santa cruz biotechnology inc .
, santa cruz , ca , usa ) were used as the primary antibodies . an anti - rabbit antibody ( 1 : 2000 ;
, santa cruz , ca , usa ) was used as the secondary antibody for bdnf and trkb .
, santa cruz , ca , usa ) . for the detection of intensities of bdnf and trkb in the hippocampal ca3 region ,
after washing the free - floating sections 3 times using 50 mm pbs , we incubated the sections in 3% h2o2 for 30 minutes in order to block endogenous peroxidase activity .
next , the sections were incubated in blocking solution ( 1% bovine serum albumin and 10% goat serum in 0.05 m pbs ) for 2 hours at room temperature and then incubated overnight with rabbit bdnf antibody ( 1 : 200 ; santa cruz biotechnology inc . ) or rabbit trkb antibody ( 1 : 200 ; santa cruz biotechnology inc . ) . in order to verify the precise bdnf and trkb expression
, we performed counterstaining on the same sections using a mouse anti - neuronal nuclei antibody ( 1 : 200 ; millipore , billerica , ma , usa ) .
the sections were next incubated for 2 hours with a fitc - conjugated goat anti - rabbit secondary antibody ( vector laboratories inc . ,
burlingame , ca , usa ) and a fitc - conjugated goat anti - mouse secondary antibody ( jackson immunoresearch laboratories inc . ,
the sections were then mounted on gelatin - coated glass slides , and the coverslips were mounted using fluorescent mounting medium ( dako north america inc .
the fluorescent images were captured using confocal laser - scanning microscopy with lsm 510 meta ( carl zeiss microimaging gmbh , oberkochen , germany ) .
negative controls were performed by omitting the primary antibodies , and these sections did not show any signals . in order to compare the relative levels of expression of bdnf and trkb , detected bands for western blot and intensity for immunofluorescence
were evaluated densitometrically using an image - pro computer - assisted image analysis system ( media cybernetics inc . , silver spring , md , usa ) . the results were expressed as the meanstandard error of the mean .
all data were analyzed by one - way anova , which was followed by duncan 's posthoc test using spss ( ibm corporation , chicago , il , usa ) .
the animals were housed under conditions of controlled temperature ( 202 ) and lighting ( 07:00 to 19:00 hour ) , and food and water were supplied ad libitum .
the experimental procedures progressed according to the animal care guidelines of the national institutes of health and the korean academy of medical sciences .
the rats were randomly divided into three groups : the control group , the 10 minutes after recovery from isoflurane anesthesia group , and the 2 hours after recovery from isoflurane anesthesia group ( n=8 in each group ) .
the rats in the isoflurane - exposed groups were anesthetized with 1.2% isoflurane in 75% nitrous oxide and 25% oxygen for 2 hours in a plexiglas anesthetizing chamber .
in order to evaluate short - term memory loss , we conducted a step - down avoidance task according to the recovery time after isoflurane anesthesia , as previously described30 ) .
the rats were positioned on a 725 cm platform with a height of 2.5 cm , and they were then allowed to rest on the platform for 2 min .
the platform faced a 4225 cm grid of parallel 0.1-cm - caliber stainless steel bars , which were spaced 1 cm apart . in the training session , the animals received a 0.5-ma scramble foot shock for 20 seconds immediately upon stepping down . in the control group ,
the training session in the other groups was conducted according to the recovery time of the respective group ( 10 minutes or 2 hours after isoflurane anesthesia ) .
the retention time in each group was assessed 10 minutes after the training session of each group .
the interval of time in which the rats stepped down and placed all four paws on the grid was defined as the latency of the step - down avoidance task .
after the determination of the retention time in the step - down avoidance task , the rats were deeply anesthetized with zoletil 50 anesthesia ( 10 mg / kg , i.p . ; virbac , carros , france ) . for immunofluorescence
, the rats were transcardially perfused with 50 mm phosphate - buffered saline ( pbs ) , which was followed by 4% paraformaldehyde in 0.5 m sodium phosphate buffer at ph 7.4 .
the brains were removed , postfixed in the same fixative overnight , and transferred to a 30% sucrose solution for cryoprotection .
serial coronal sections that were 40 m thick were cut with a freezing microtome ( leica biosystems nussloch gmbh , nussloch , germany ) .
the hippocampus was removed from each rat brain , and the extraneous tissue was trimmed away .
the hippocampal tissues were minced and chopped in lysate buffer that contained 50 mm hepes ( ph 7.5 ) , 150 mm nacl , 10% glycerol , 1% triton x-100 , 1.5 mm magnesium chloride hexahydrate , 1 mm ethyleneglycol - bis-(-aminoethyl ether)-n , n'-tetraacetic acid , 1 mm phenylmethylsulfonyl fluoride , 2 g / ml leupeptin , 1 g / ml pepstatin , 1 mm sodium orthovanadate , and 100 mm sodium fluoride .
after homogenization , the tissues were reacted with lysate buffer for 20 min and then centrifuged at 14000 rpm for 20 minutes at 4. the supernatant was stored in a -70 deep freezer .
whole protein extracts were used to evaluate the levels of expression of the bdnf and trkb proteins .
the protein concentrations were measured using a bio - rad colorimetric protein assay kit ( bio - rad laboratories inc . , hercules , ca , usa ) .
forty g of protein was separated on sds - polyacrylamide gels and then transferred onto a nitrocellulose membrane ( schleicher & schuell bioscience gmbh , dassel , germany ) . a rabbit bdnf antibody ( 1 : 1000 ;
santa cruz , ca , usa ) and a rabbit trkb antibody ( 1 : 1000 ; santa cruz biotechnology inc .
, santa cruz , ca , usa ) were used as the primary antibodies . an anti - rabbit antibody ( 1 : 2000 ;
, santa cruz , ca , usa ) was used as the secondary antibody for bdnf and trkb .
for the detection of intensities of bdnf and trkb in the hippocampal ca3 region , immunofluorescence staining was performed . after washing the free - floating sections 3 times using 50 mm pbs
, we incubated the sections in 3% h2o2 for 30 minutes in order to block endogenous peroxidase activity .
next , the sections were incubated in blocking solution ( 1% bovine serum albumin and 10% goat serum in 0.05 m pbs ) for 2 hours at room temperature and then incubated overnight with rabbit bdnf antibody ( 1 : 200 ; santa cruz biotechnology inc . ) or rabbit trkb antibody ( 1 : 200 ; santa cruz biotechnology inc . ) . in order to verify the precise bdnf and trkb expression
, we performed counterstaining on the same sections using a mouse anti - neuronal nuclei antibody ( 1 : 200 ; millipore , billerica , ma , usa ) .
the sections were next incubated for 2 hours with a fitc - conjugated goat anti - rabbit secondary antibody ( vector laboratories inc . ,
burlingame , ca , usa ) and a fitc - conjugated goat anti - mouse secondary antibody ( jackson immunoresearch laboratories inc . , west grove , pa , usa ) .
the sections were then mounted on gelatin - coated glass slides , and the coverslips were mounted using fluorescent mounting medium ( dako north america inc .
the fluorescent images were captured using confocal laser - scanning microscopy with lsm 510 meta ( carl zeiss microimaging gmbh , oberkochen , germany ) .
negative controls were performed by omitting the primary antibodies , and these sections did not show any signals .
in order to compare the relative levels of expression of bdnf and trkb , detected bands for western blot and intensity for immunofluorescence were evaluated densitometrically using an image - pro computer - assisted image analysis system ( media cybernetics inc . , silver spring , md , usa ) . the results were expressed as the meanstandard error of the mean .
all data were analyzed by one - way anova , which was followed by duncan 's posthoc test using spss ( ibm corporation , chicago , il , usa ) .
the latency in the step - down avoidance task was 295.872.17 seconds in the control group , 10.252.19 seconds in the 10 minutes after recovery from isoflurane anesthesia group , and 289.255.72 seconds in the 2 hours after recovery from isoflurane anesthesia group ( fig .
the latency was significantly decreased 10 minutes after recovery from isoflurane anesthesia ( p<0.05 ) . however , latency recovered to the control level 2 hours after isoflurane anesthesia ( p<0.05 ) .
western blot analyses for the protein levels of bdnf and trkb in the hippocampus were performed in order to provide relative levels of expressions of these proteins . in the present study ,
the protein levels of bdnf and trkb in the control group were set as 1.00 .
the relative levels of expression of bdnf were 0.520.06 in the 10 minutes after recovery from isoflurane anesthesia group and 0.780.01 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the relative levels of expression of trkb was 0.590.05 in the 10 minutes after recovery from isoflurane anesthesia group and 0.850.04 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the protein levels of bdnf and trkb were significantly decreased in the 10 minutes after isoflurane anesthesia group .
however , these protein levels were increased in the 2 hours after isoflurane anesthesia group .
immunofluorescent photomicrographs of bdnf and trkb expressions in the hippocampal ca3 region are presented in fig .
the intensity levels of bdnf and trkb in the control group were set as 1.00 . the relative intensity levels of bdnf were 0.550.04 in the 10 minutes after recovery from isoflurane anesthesia group and 0.740.02 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the relative intensity levels of trkb were 0.550.03 in the 10 min after recovery from isoflurane anesthesia group and 0.760.04 in the 2 h after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the expressions of bdnf and trkb in the hippocampcal ca3 region were decreased in the 10 minutes after recovery from isoflurane anesthesia group compared to the control group
. however , the expressions of bdnf and trkb in the hippocampcal ca3 region recovered to near control values in the 2 hours after recovery from isoflurane anesthesia group .
the latency in the step - down avoidance task was 295.872.17 seconds in the control group , 10.252.19 seconds in the 10 minutes after recovery from isoflurane anesthesia group , and 289.255.72 seconds in the 2 hours after recovery from isoflurane anesthesia group ( fig .
the latency was significantly decreased 10 minutes after recovery from isoflurane anesthesia ( p<0.05 ) . however , latency recovered to the control level 2 hours after isoflurane anesthesia ( p<0.05 ) .
western blot analyses for the protein levels of bdnf and trkb in the hippocampus were performed in order to provide relative levels of expressions of these proteins . in the present study ,
the protein levels of bdnf and trkb in the control group were set as 1.00 .
the relative levels of expression of bdnf were 0.520.06 in the 10 minutes after recovery from isoflurane anesthesia group and 0.780.01 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the relative levels of expression of trkb was 0.590.05 in the 10 minutes after recovery from isoflurane anesthesia group and 0.850.04 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the protein levels of bdnf and trkb were significantly decreased in the 10 minutes after isoflurane anesthesia group .
however , these protein levels were increased in the 2 hours after isoflurane anesthesia group .
immunofluorescent photomicrographs of bdnf and trkb expressions in the hippocampal ca3 region are presented in fig .
the intensity levels of bdnf and trkb in the control group were set as 1.00 .
the relative intensity levels of bdnf were 0.550.04 in the 10 minutes after recovery from isoflurane anesthesia group and 0.740.02 in the 2 hours after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the relative intensity levels of trkb were 0.550.03 in the 10 min after recovery from isoflurane anesthesia group and 0.760.04 in the 2 h after recovery from isoflurane anesthesia group ( p<0.05 ) ( fig .
the expressions of bdnf and trkb in the hippocampcal ca3 region were decreased in the 10 minutes after recovery from isoflurane anesthesia group compared to the control group
. however , the expressions of bdnf and trkb in the hippocampcal ca3 region recovered to near control values in the 2 hours after recovery from isoflurane anesthesia group .
after surgery , the recovered patients can respond to doctors ' requests or to stimulations that evaluate neurologic symptoms or muscle relaxation reversal . however , most of the patients do not remember these checks . because the presentation of transient anterograde amnesia can be dramatic and may mimic an acute cerebral ischemic event , a thorough neurologic evaluation should be pursued .
transient anterograde amnesia is characterized by a sudden memory loss of recent and/or remote events and transient inability to acquire new knowledge .
the dramatic presentation of acute change in personality , after emergence from general anesthesia , creates a puzzling and frightening situation for both the patient 's family and the anesthesiologist5 ) .
in addition , mnte et al.22 ) demonstrated that balanced anesthesia techniques with isoflurane or propofol led to minimal implicit memory in patients undergoing lumbar disc surgery .
the amnestic effects of isoflurane have been reported in many studies24,26,34 ) . however , the exact mechanism by which the inhaled anesthetic isoflurane produces amnesia is not understood .
previous studies demonstrated that gabaa receptor is implicated in the anxiolytic , amnesic , and sedative behavioral effects of volatile anesthetics and alcohol8,26,34 ) .
perouansky et al.24 ) reported that halothane , nitrous oxide , and isoflurane slowed peak frequencies , suggesting that modulation of the rhythm contributes to the anesthetic - induced amnesia . in the present study
, we first evaluated whether isoflurane anesthesia induces transient anterograde amnesia using a step - down avoidance task .
we executed the step - down avoidance task in order to determine the latency according to the time after recovery from anesthesia ( 10 minutes or 2 hours ) . in the present results ,
the latency was shortened 10 minutes after recovery from isoflurane anesthesia , and the latency was recovered to control level 2 hours after recovery from isoflurane anesthesia .
these results indicate that temporal memory loss was induced by isoflurane anesthesia , and the memory loss disappeared 2 hours later , suggesting that isoflurane anesthesia induced transient anterograde amnesia .
transient anterograde amnesia has two types of amnesia : global type and focal ( localized ) type . in the present study ,
the rats showed transient memory loss on the electric shock 10 minutes after recovery from isoflurane anesthesia , this type of amnesia might be considered as the focal type of amnesia .
we next investigated whether the expressions of bdnf and its receptor trkb in the hippocampus were related to the transient anterograde amnesia .
bdnf enhances synaptic transmission and neuronal plasticity in the cns28 ) , resulting in increase of learning abilities and memory capabilities20 ) .
bdnf is released from synapses in an activity - dependent manner and acts on postsynaptic neurons12 ) .
this action of bdnf is involved in the representing , processing , and storing information in the complex neural networks2,16,33 ) . because trkb is a high - affinity receptor for bdnf , abnormal trkb expression impairs hippocampal - dependent memory and learning tasks19,27 ) .
overexpression of bdnf and its receptor trkb improves learning or cognitive behavior , whereas the inhibition of bdnf or trkb causes memory impairment1,13,17,19,21,25 ) .
bdnf was also suggested as the main factor in the maternal exercise - induced enhancement of memory in rat pups10 ) . in the present results ,
western blotting showed that the expressions of bdnf and trkb in the hippocampus were significantly decreased 10 minutes after recovery from isoflurane anesthesia .
however , 2 hours after recovery from isoflurane anesthesia , the expressions of bdnf and trkb in the hippocampus were recovered near to the control level .
immunofluorescence staining also revealed that the intensities of bdnf and trkb in the hippocampal ca3 region were significantly decreased 10 minutes after recovery from isoflurane anesthesia .
however , 2 hours after recovery from isoflurane anesthesia , the intensities of bdnf and trkb in the hippocampal ca3 region were recovered near to the control level .
these results indicate that isoflurane anesthesia suppressed the expressions of bdnf and trkb in the hippocampus .
however , this suppression disappeared 2 hours later , suggesting that the expressions of bdnf and trkb were closely linked with isoflurane - induced transient anterograde amnesia in rats .
in the present results , temporary memory loss was induced by isoflurane anesthesia , and this memory loss disappeared 2 hours later .
the expressions of bdnf and trkb in the hippocampus were decreased immediately after isoflurane anesthesia but were increased 2 hours after isoflurane anesthesia .
we showed that isoflurane anesthesia induced transient anterograde amnesia and that the levels of bdnf and trkb in the hippocampus might be involved in the underlying mechanism of this transient anterograde amnesia . | objectivetransient anterograde amnesia is occasionally observed in a number of conditions , including migraine , focal ischemia , venous flow abnormalities , and after general anesthesia .
the inhalation anesthetic , isoflurane , is known to induce transient anterograde amnesia .
we examined the involvement of brain - derived neurotrophic factor ( bdnf ) and its receptor tyrosine kinase b ( trkb ) in the underlying mechanisms of the isoflurane - induced transient anterograde amnesia.methodsadult male sprague - dawley rats were divided into three groups : the control group , the 10 minutes after recovery from isoflurane anesthesia group , and the 2 hours after recovery from isoflurane anesthesia group ( n=8 in each group ) .
the rats in the isoflurane - exposed groups were anesthetized with 1.2% isoflurane in 75% nitrous oxide and 25% oxygen for 2 hours in a plexiglas anesthetizing chamber .
short - term memory was determined using the step - down avoidance task .
bdnf and trkb expressions in the hippocampus were evaluated by immunofluorescence staining and western blot analysis.resultslatency in the step - down avoidance task was decreased 10 minutes after recovery from isoflurane anesthesia , whereas it recovered to the control level 2 hours after isoflurane anesthesia .
the expressions of bdnf and trkb in the hippocampus were decreased immediately after isoflurane anesthesia but were increased 2 hours after isoflurane anesthesia.conclusionin this study , isoflurane anesthesia induced transient anterograde amnesia , and the expressions of bdnf and trkb in the hippocampus might be involved in the underlying mechanisms of this transient anterograde amnesia . | INTRODUCTION
MATERIALS AND METHODS
Animals and treatments
Determination of short-term memory in the step-down avoidance task
Tissue preparation
Western blotting for the expressions of BDNF and TrkB in the hippocampus
Immunofluorescence staining for the expressions of BDNF and TrkB in the hippocampal CA3 region
Data analysis
RESULTS
Effect of isoflurane on the short-term memory
Effect of isoflurane on the BDNF and TrkB protein levels in the hippocampus
Effect of isoflurane on the BDNF and TrkB expressions in the hippocampal CA3 region
DISCUSSION
CONCLUSION |
PMC3987355 | although the first report of a live birth from a frozen oocyte dates back to 1986 ( chen , 1986 ) , oocytes have long been notoriously difficult to cryopreserve
. however , following advancements in slow freezing and especially since the introduction of vitrification , the survival rates of oocytes after thawing have risen significantly . in combination with icsi ,
good fertilization rates are achieved , making oocyte cryopreservation an efficient procedure ( rienzi et al . , 2010 ) . as safety data are also reassuring ( although long term follow - up data are not yet available ) , it is now considered both safe and efficient enough for routine clinical application by many ( cobo et al . , 2010 ; noyes , 2010 ; rienzi et al . , 2010 ; 2012 ) .
the possibility to cryopreserve oocytes to be used in ivf treatment later in life has enlarged the reproductive options of cancer patients who are faced with gonadotoxic treatments .
it also holds the promise of expanding the reproductive options of healthy women whose personal circumstances ( most often the absence of a partner ) do not allow them to reproduce in their most fertile years .
however , this latter possibility has been criticized by many and also professional bodies such as the asrm and eshre were initially reluctant to support this particular application ( asrm , 2007 ; eshre , 2004 ) .
objections that were voiced included the experimental status of vitrification , the fact that expanding the reproductive lifespan is unnatural and represents an unwarranted medicalization of reproduction and that it would lead to an unwelcome increase in the age of mothers ( jones , 2009 ; martin , 2010 ; mc cullough , 2004 ; shkedi - rafid and hashiloni - dolev , 2011 ) .
a number of ethicists have addressed these objections extensively ( dondorp and de wert , 2009 ; goold and savulescu , 2009 ; rybak and lieman , 2009 ) . in short
, it appears very difficult to maintain that it would be unacceptable to freeze eggs from a 30-year old woman and use them to establish a pregnancy when she is 40 while it is deemed acceptable to provide ivf to a similar woman of 40 suffering from age - related infertility either with her own low quality eggs or with donor eggs .
likewise , it is not clear why the innovative character of oocyte cryopreservation would plead against offering it to those women whose fertility is threatened by aging but not to those whose fertility is threatened by disease or therapy .
however , part of the debate was not based on fundamental objections , but rather on more emotionally charged arguments and prejudices , led by stereotyping of the healthy women who might request oocyte cryopreservation .
three different portrayals can be discerned in the debate about the ethics of so - called social egg freezing or
first , these women have been portrayed as selfish career - pursuing women , which leads to a position that does not support oocyte cryopreservation by healthy women .
second , healthy women who might benefit from oocyte cryopreservation have been portrayed as victims of a male - oriented society that makes it difficult for women to combine motherhood with a good education or professional responsibilities .
third , healthy women opting to cryopreserve oocytes have been portrayed as wise , proactive women who will not have to depend on oocyte donors should they suffer from age - related infertility by the time they are ready to reproduce .
although these portrayals may not lead the academic discussion , they are quite prominent in the lay media and shape public opinion .
therefore it is important to have an insight in the extent to which they are truthful and in what their shortcomings are .
when the phenomenon of egg freezing for so - called social reasons first hit the headlines , the portrayal of the women who might request this new technology was largely judgmental and negative : the popular media conjured up visions of selfish , self - absorbed career women deliberately avoiding motherhood in their 20s and 30s whilst relying on cryobiology to produce their own genetic babies for them in their 40s and 50s .
( lockwood , 2003 ) social egg freezing generally arises because a woman chooses to delay bearing children
( catt , 2009 ) professor ledger believes that is ethically questionable for women to freeze their eggs purely for
lifestyle reasons. ( fletcher , 2009 ) in contrast with women who are faced with the prospect of infertility due to cancer treatment , these women were seen as facing infertility due to their own
the reasoning goes that women who postpone motherhood to pursue a career until the point where they suffer from age - related fertility decline , are themselves accountable for this misfortune as they misplaced their priorities .
preliminary data on the profile of women requesting social egg freezing shows that most of these women are indeed highly educated , which appears to support the first narrative ( nekkebroeck et al . , 2010 ) . however , these women do not request egg freezing at a young age with the intent of putting motherhood on hold in order to pursue their careers .
rather , they present themselves when they are approaching their forties and are faced with the fact that although they want to be parents , they have not found the right partner yet ( which , granted , may be due in part to investing a lot of time in their education and careers ) .
thus , these women did not necessarily choose to delay parenthood , but their personal situation did not allow them to have children earlier in life . for these women ,
oocyte cryopreservation is a way of clinging onto the last straw of hope they have for ever establishing a family at a moment when their ovarian reserve has already diminished substantially .
the question then is : was it wrong for these women to invest in their education and career at the expense of their fertility ? would it be better if women paid more attention to childbearing than to career building at the time when they are most fertile ( between the ages of 20 and 30 ) ?
several studies have found that women find it increasingly important to first complete their education , have financial security , good housing and a stable relationship before taking on the responsibility of parenthood ( lampic et al .
. these are not selfish concerns , but considerations that are made in the best interest of their future children .
( 2008 ) have argued that the rising age of women at first childbirth is a trend that increases overall wellbeing and that should not be regretted in itself , even if it is regrettable that the peak of natural female fertility does not coincide with this age period .
they even go as far as to say that having children before the age of 23 is not to be encouraged , given the heightened risk of social deprivation .
this means that the perfect time for reproducing not too early for reasons of wellbeing , not too late for medical reasons is between the ages of 25 and 35 .
as previously argued by lockwood ( 2011 ) , both women and men , especially if they have received a tertiary - level education and have good employment prospects , are simply unprepared to cope with the consequences of the very narrow window of opportunity for parenthood that exists in the decade between realization of educational , career and economic goals and the onset of , at best , a reduced family size compared with their ideal and , at worst , involuntary childlessness .
this brings us to the second narrative : if it is in fact not the educated women s fault that they can not reproduce at the optimal age , can we then put the blame on society ?
the premise of this second narrative is that society , and most notably the way the labor market is structured , makes it difficult for women to combine motherhood with a good education or professional responsibilities .
the labor market is seen as leaving little room for family responsibilities , which was workable years ago with an all - male labor force and women as primary caregivers , but is not adapted to today s typical family with two working partners .
this second narrative can be invoked to support oocyte cryopreservation by healthy women as an intervention that women are entitled to , given the expectations of today s society : it seems unfair that society at large , which creates the economic , educational , and professional conditions that encourages deferred maternity , discourages women from using technology to bypass a biological inequity the early loss of fecundity .
( gosden and oktay , 2000 ) women face reductions in earning capacity and potentially serious financial implications that men do not . in fact , they may have very few choices at all .
( savulescu and goold , 2009 ) we are disturbed by the implied judgment that our society , having failed to sufficiently safeguard the ability of many women in their twenties and thirties to establish families without jeopardizing career advancement , can not withstand the challenges posed by elective deferral of childbearing .
alternatively , the idea that society forces women to postpone parenthood can be invoked to oppose oocyte cryopreservation . in this case
quick fix for social inequalities can heal the problem of age - onset infertility , it does not tackle the root of the problem , which ought to be remedied by taking measures that make it easier for women to have their children earlier in life : one might ask whether we actually help women [ ] by taking for granted their bad employment situation and offering them egg freezing to deal with it .
( goold and savulescu , 2008 ) technological solutions to social problems may result in a greater degree of repression rather than liberation [ ] would it not be likely [ ] that women , who already feel that they are expected by employers to postpone ( or give up ) motherhood , would now be expected to freeze their eggs if they pursue a career ? [ ] the best way to overcome society s restrictive influence on the individual s ability to act autonomously is to change the societal norms that give rise to this oppression , rather than encouraging individuals [ ] to adapt to these norms .
( shkedi - rafid and hashiloni - dolev , 2012 ) the reproductive technology of egg freezing [ ] can not escape the serious feminist worry about potentially reinforcing patriarchy and leaving the problematic social structures largely intact .
( petropanagos , 2010 ) egg freezing may leave the hard work of moving society toward greater sexual equality untouched [ ] technological solutions to social problems are inadequate and often result in the further oppression of disadvantaged groups .
( harwood , 2009 ) fertility preservation for social reasons is then a type of unnecessary medicalization of society that can be avoided by creating a better social climate for working mothers .
however , symptoms and root causes are best treated simultaneously in order to obtain the fastest results .
dondorp and de wert ( 2009 ) have pointed out that women can not afford to wait until society has been changed in a way that would allow them to have it all at the right time .
few people would argue that the individual medical treatment of obesity should be halted because the distribution of obesity over socioeconomic classes shows that social inequality or poverty is the underlying problem .
medical treatment and societal change are not mutually exclusive and thus there is no reason to abandon one to pursue the other . also , whether societal change will have any impact on the age of first - time mothers , and if such an impact is even desirable to start with , is debatable . in this context
it is both amusing and remarkable that a 1969 study from maxwell and montgomery found that at that time , there was societal pressure towards early parenthood although this is opposed to the desire of young couples for delayed parenthood .
this begs the question : has this situation reversed itself in the last 50 years and is there currently societal pressure to delay parenthood a claim that is supported by young mothers who report that
society sees them as bad mothers simply because they are young ( benzies et al . ,
2006 ) while women would prefer to have children at a younger age ? or have women s preferences stayed the same while they are now finally liberated of the societal pressure to reproduce as young as possible ?
( 2007 ) in a canadian population shows that most consider the ideal age to begin parenting to be somewhere between the ages of 25 and 35 .
this is in line with the previously mentioned findings that women prefer to complete their education , have financial security , good housing and a stable relationship before starting a family .
thus , it is very unlikely that socio - economical measures to stimulate having children before these goals have been met will have a great impact , nor that such measures are desirable .
hakim ( 2003 ) makes the same prediction that family - friendly adaptations to employment policies
although welcomed by parents ( male and female ) seeking to better combine parental and professional obligations will not automatically lead to more children and/or younger parents .
she bases herself on a preference theory with the underlying idea that such measures would benefit people who are home - centered to start with ( mainly women ) , which is the same group that would be willing to cut back on their career to have children anyhow . at the same time
, such measures are unlikely to affect people who are work - centered. this theory is also confirmed by data from van balen ( 2005 ) , indicating that a strong desire to have children overrides motivations to postpone motherhood . another way to look at
the trend to delay parenthood is to consider it in regard to an increased acceptance of voluntarily childless couples and an increased awareness that a life without children is not inferior in terms of overall wellbeing to a life with children and may indeed be more rewarding .
childfree couples remain a minority but their numbers have been growing steadily and are already estimated to be higher than involuntarily childless couples ( agrillo and nelini , 2008 ) . delaying parenthood can then be seen as a conscious decision aimed at enjoying the best of both worlds ( first without and then with children ) , rather than being some kind of second - best option . in other words
, it is too simple to claim that women are forced by a male - oriented society to delay childbearing and that changing employment policies to render them more women - friendly is all it takes to lower the age of first - time mothers .
rather , many women prefer to delay motherhood and have good reasons to do so , even in a world where having children would not have an impact on career opportunities .
this brings us to the starting point of the last narrative : neither women , nor society at large ( nor men for that matter ) should be scolded for a shift in the age of primigravid women .
societal and personal factors make it a smart choice for women and men to delay parenthood , despite the risk of remaining childless all together .
also , an abundance of dating websites can not guarantee that every woman will meet her
mister right in her early twenties , nor that he will agree to have children at that age . so does a woman in her early thirties who wants to have children but not in her current condition have other options besides storing her oocytes until the circumstances are better ?
sure , she does : she can either wait it out and risk remaining childless or having to rely on donor oocytes , or she can rush into having children without having a stable relationship or a stable financial situation .
however , these are not necessarily better options , neither for her nor her future offspring , than to store her oocytes ( even with a limited chance of success ) . reproducing as fast as possible can be a great strategy from a gynecological point of view , but it may be a very bad choice from many other points of view .
the third narrative therefore takes the fact that many women attempt to reproduce in their late thirties after their most fertile period as a given , rather than as a variable that ought to change .
according to this third narrative , the right way to present elective egg freezing is not to see it as an alternative to reproducing earlier in life as this is often simply not an option or not a good one but as an alternative to relying on donor oocytes .
it can be seen as a form of self - donation whereby the younger version of a woman donates eggs to her older version so that she is able to reproduce at an older age while keeping the genetic link between parent and child and while using younger oocytes with less risks of complications ( rybak and lieman , 2009 ; knopman et al . , 2010 ) . rather than representing unnecessary medicalization of reproduction ,
elective egg freezing then becomes a form of preventive medicine ( stoop , 2010 ; lockwood , 2011 ) : many women end up childless as a result of postponing childbearing .
as long as these women are fully informed and able to make rational decisions about their fertility , we should allow them to make their own financial decisions .
( goold and savulescu , 2009 ) what if some women do want to freeze their eggs at 30 , to
is that decision somehow less moral than using ivf at 45 with a 5% chance of a pregnancy and a 70% chance of a miscarriage , or using the precious , scarce resource of donor eggs and settling for
( lockwood , 2003 ) while this third narrative is all too easily embraced by commercial enterprises that offer egg freezing services , it is unfortunately not unproblematic .
if women would deliberately delay childbearing until their forties , they could proactively freeze their eggs around their 30th birthday and achieve good success rates .
however , this is not how things usually go in practice . as discussed earlier , women usually do not plan to have their children in their late thirties or forties , but they postpone childbearing bit by bit by lack of a partner , a demanding job , financial insecurity , etc . ( a phenomenon also known as
perpetual postponing ) and before they know it their reproductive years have passed ( lockwood , 2011 ) .
women are not inclined to undergo the demanding and costly procedure of ovarian stimulation and oocyte retrieval at the peak of their fertility .
at that moment they are either not yet thinking about starting a family or they expect to find a partner in due time .
several studies have indicated that women underestimate the speed at which female fertility declines and that the possibilities of overturning age - related infertility through ivf are highly overestimated ( hammarberg and clarke , 2005 ; lampic et al . , 2006 ; maheshwari et al . ,
moreover , cryopreserving oocytes requires a substantial financial investment that women are only willing to make when there is a substantial possibility that they will ever cash in on this investment .
it is only when their time is running out , when approaching the symbolic age of forty , that most women start to worry about remaining childless and resort to oocyte cryopreservation .
the oocytes that are preserved at that point already have a decreased potential to result in a successful pregnancy .
moreover , when lack of a partner is the problem , this problem may persist so that frozen oocytes remain unused .
thus , although egg freezing is potentially a wise and proactive measure for women in today s society , in practice it is often a desperate measure with a low utility ( mertes and pennings , 2011 ) .
one can only hope that through public education on declining fertility with age , a reduction in costs and awareness about the possibility to store oocytes at a young age , the women cryopreserving their oocytes will one day resemble the ideal of smart , proactive women rather than the image of desperate singles
the ethical debate regarding oocyte cryopreservation for healthy women has often been reduced to putting the women on trial who might benefit from it . who are these women who want to defy nature and
three different narratives can be discerned : women interested in elective egg freezing are either portrayed as selfishly prioritizing their career over motherhood , as being forced by society to postpone motherhood or as smart , proactive women who have discovered a new means to make their career compatible with motherhood .
the first narrative is probably the furthest away from reality , as the age at which healthy women currently request oocyte cryopreservation indicates that
postponement of childbearing is seldom planned at a young age and thus that freezing oocytes is rather an emergency intervention than part of a well designed life plan to have it all. however , it is argued here that also the other two narratives are misrepresentations to a certain extent . just as it is inaccurate to state that women choose to delay childbearing in order to advance their careers , it is also inaccurate to say that they have no other option but to delay childbearing .
when people have their children depends on an interplay between contextual factors and personal values and neither one will completely override the other .
finally , the image of smart , proactive women is rather an idealistic picture of who the best candidates would be than an accurate depiction of those who actually come forward . in conclusion
, it may be interesting to learn who the candidates for elective oocyte cryopreservation are , what their motives are and how they got into a situation in which they need to or want to delay childbearing until after their reproductive years .
however , a judgmental approach will not offer a clear answer to the question whether elective egg freezing is good or bad medical practice .
the central question should not be whether or not women are deserving of oocyte cryopreservation , but whether or not oocyte cryopreservation for this particular indication does more good than harm .
this evaluation will depend a lot on the utility rate , that is , on the number of women who actually return to use their frozen oocytes and on the success rates for these women . as argued elsewhere
( mertes and pennings , 2011 ) , if the only candidates for social freezing are women whose ovarian reserve is already at a critical threshold , then the utility of this procedure will be very low and women will be buying false hope at a high price .
however , if women become more aware of the effect of aging on their fertility , of the possibility to store oocytes in their fertile years ( preferably before age 35 ) and of the limits of the procedure ( especially when they are already over the age of 35 ) , oocyte cryopreservation may be a welcome intervention for women who long to preserve their fertility longer than they naturally could . | the possibility to cryopreserve oocytes to be used in ivf treatment later in life has not only enlarged the reproductive options of cancer patients who are faced with gonadotoxic treatments , but also holds the promise of enlarging the reproductive options of healthy women whose personal circumstances ( most often the absence of a partner ) do not allow them to reproduce in their most fertile years . opinions for and against this application of the cryopreservation technology are often based on different portrayals of the women who might use it .
three different portrayals can be discerned in the debate about the ethics of so - called social egg freezing or
non medical egg freezing. first , these women have been portrayed as selfish career - pursuing women .
second , healthy women who might benefit from oocyte cryopreservation have been portrayed as victims of a male - oriented society that makes it difficult for women to combine motherhood with a good education or professional responsibilities .
third , healthy women opting to cryopreserve oocytes have been portrayed as wise , proactive women who will not have to depend on oocyte donors should they suffer from age - related infertility by the time they are ready to reproduce .
each of these three portrayals has its own shortcomings that one should be wary of , as they lead to an oversimplification of the ethical debate . | Introduction
Selfish, career-pursuing women
Victims of a male-oriented society
Wise, proactive women
Conclusion |
PMC1519110 | amphibians are considered reliable indicators of environmental quality . in the western united states , a general decline of frog populations
parallels an apparent worldwide decline .
the factors thought to be contributing to declines in frog populations include habitat loss , introduction of exotic species , overexploitation , disease , climate change , and decreasing water quality .
with respect to water quality , agroecosystems use 80 - 90% of the water resources in the western united states , frequently resulting in highly eutrophic conditions .
recent investigations suggest that these eutrophic conditions ( elevated ph , water temperature , and un - ionized ammonia ) may be associated with frog embryo mortality or malformations .
however , water quality criteria for frogs and other amphibians do not currently exist . here , we briefly review data that support the need to develop water quality parameters for frogs in agroecosystems and other habitats.imagesfigure 1.figure 2.figure 3.figure 4 .
afigure 4 .
bfigure 5 . | Images |
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PMC5029213 | rna viruses have rapid replication and high error rates , leading to immense diversity within each viral replication cycle ( domingo and holland , 1997 ) . as a result
, many rna viruses are highly genetically heterogeneous and exist within infected population structures known as quasispecies .
it has been proposed that this gives these viral pathogens an increased ability to shift to a new environmental niche , such as a new host , as a suitable mutant is more likely to already exist if this opportunity arises . however ,
if the diversity within a quasispecies becomes too high , deleterious mutations can accumulate leading to loss of overall fitness ( clarke et al . , 1993 ) .
the adaptability and host range of a virus are a function of the level of diversity found within a quasispecies ( termed the quasispecies swarm size ) ( schneider and roossinck , 2001 ) . as well as a randomly generated swarm of mutants around one variant , quasispecies can also exist as a number of master variants , each with their own swarm of random mutations ( palacios et al .
, 2008 ; lauring and andino , 2010 ) . determining the extent of genetic heterogeneity in virus populations thus has important implications for predicting and preventing emerging viral diseases .
the global decline in honey bee populations over the past few decades has been attributed to the ectoparasitic mite varroa destructor and its affiliation with deformed wing virus ( dwv ) type a variant ( dainat et al . , 2012 ; martin et al . , 2012
the role of honey bees as pollinators is vital to the environment and economy , as bees are a key pollinator species for agriculture .
the economic value of honey bees is estimated to be more than 225 billion us dollars worldwide ( gallai et al . , 2009 ) .
dwv , a member of the single - stranded positive - sense rna genus iflavirus ( lanzi et al , 2006 ) , exists as a group of closely related viruses , often considered as variants of the same species complex ( ryabov et al . , 2014 ) .
the ictv ( the international committee on taxonomy of viruses ) database categorises dwv type a as two variants , dwv ( lanzi et al , 2006 ) and kakugo virus ( kv ) ( fujiyuki et al . , 2004 ) .
a second closely related virus varroa destructor virus-1 ( vdv-1 ) is also part of the genus iflavirus , which is now designated dwv type b ( martin et al . , 2012 ; mordecai et al . , 2015 ) .
dwv type b was designated a separate species based on a nucleotide identity to dwv type a of 84% that , according to demarcation criteria , is sufficient dissimilarity to warrant the creation of a new master variant ( fauquet et al . , 2005 ) .
vdv-1 was originally isolated from v. destructor but has since been reported to replicate in honey bees ( ongus et al . , 2004 ; zioni et al . , 2011 ) where it has been shown to cause wing deformities in bees ( zioni et al . , 2011 ) .
dwv type b was also recently found to dominate the dwv population in honey bees from an isolated apiary in swindon , uk ( mordecai et al . , 2015 ) ; to the apparent exclusion of any other master variants . in honey bee populations that have never been exposed to varroa mites , dwv exists in a very large variant swarm with numerous master variants ( martin et al . , 2012 )
. however , transmission of dwv by varroa reduces variant diversity to one master variant ( martin et al . , 2012 ) .
therefore , dwv exists as an endlessly mutating swarm of variants with these master variants constituting part of this quasispecies and sharing a recent common ancestor ( baker and schroeder , 2008 ; martin et al . , 2012 ) .
the dominance of one master variant over another will lead to ultimately different life histories for the colony , that is , death if dwv type a dominates ( martin et al . ,
2012 ) or health if dwv type b dominates ( mordecai et al . , 2015 ) . by existing as a diverse swarm of variants ,
certain biological traits may allow a virus to infect one cell type over another , known as cell tropism ( koyanagi et al . , 1987 ) . whether or not a virus is able to infect a susceptible cell
depends firstly on recognition of a cellular receptor on the cell surface and secondly on intracellular host factors that dictate whether the host cell is permissive to virus replication .
therefore , amino acid substitutions caused by nucleotide mutations in the structural or nonstructural region of the virus genome can affect both the host range and cell tropism of a virus . when categorising viruses based on a phylogenetic relationship it is important to note that a single amino acid change can have a substantial effect on the phenotypic traits of a virus .
therefore , when a virus exists as a collection of variants or quasispecies , although the phylogeny and ancestry of the viruses may be similar , the host range , tropism , pathogenicity and epidemiology of the variants may differ greatly ( domingo et al .
in addition , recombination between these variants is a source of further variation ( moore et al . , 2011 ) .
there are numerous biological implications of quasispecies occupying large amounts of sequence space that challenge the treatment and control of both established and emerging infectious diseases ( gomez et al . , 1999 ) .
for example , quasispecies theory has been used to describe how viruses such as hepatitis c virus and hiv are able to escape host immune responses ( pavio and lai , 2003 ; woo and reifman , 2012 ) .
in addition , effective vaccines are yet to be developed for both these viruses because of the rapid emergence of resistant mutants under vaccine selection pressure ( gaschen et al . , 2002 ; law et al , 2013 ) .
for similar reasons , the highly divergent nature of rna viruses has been implicated in the emergence of antiviral drug - resistant infections in aids ( metzner et al . , 2009 ) , hepatitis c ( halfon and locarnini , 2011 ) , hepatitis b ( nishijima et al . , 2012 ) and influenza ( boivin et al . , 2002 ) .
conventional methods to define and analyse variance within rna viruses include rna fingerprinting ' ( domingo et al . , 1978 ) and
reverse transcriptase - pcr amplification using specifically designed primers ( see , for example , highfield et al . ,
clone libraries and sanger sequencing were used to identify the dwv type a as being associated with varroa infestation and colony collapse ( martin et al . , 2012 ) .
although these techniques are valid for identifying known variants , primer - based methods are prone to missing unidentified variants and are biased towards overrepresented sequences ( gomez et al . , 1999 ) .
pcr - based methods are less appropriate to determine the extent of variation in a quasispecies that is not normally distributed that is , where multiple variants exist , each with their own spectrum of mutants ( gomez et al . , 1999 ) . in these instances
deep sequencing methods such as illumina platforms are more suited to discovering new variants as well as to diversity analysis ( wood et al . , 2014 ) .
illumina sequencing is advantageous for samples with large amounts of genetic variation because of its depth of sequencing , although significant raw read analysis is required .
reference - based assembly methods can overlook biological variants because of inaccurate read alignments and loss of data ( archer et al . , 2010 ; iqbal et al .
, 2012 ; yang et al . , 2012 ) , although former studies using high - throughput next - generation sequencing of dwv have used these methods ( moore et al . , 2011 ; ryabov et al . ,
a previous study ( highfield et al . , 2009 ) found that despite controlling varroa populations , high dwv loads were associated with overwintering colony losses ( ocl ) .
historical losses due to ocl of 10% were normal ; these have now risen to 20% since the establishment of varroa .
this suggests that a new ' non - varroa - transmitted dwv master variant may be circulating in some colonies during the winter causing ocl . to investigate this hypothesis we used a bespoke de novo assembly pipeline ( mordecai et al . , 2015 ) .
the vicuna ( broadinstitute.org/scientific-community/science/projects/viral-genomics/vicuna ) de novo assembler was used as it is designed to assemble highly heterogeneous viral populations and is well suited to the computational challenge that the dwv quasispecies present ( yang et al . , 2012 ) .
as well as assembling the dwv type a master variant , a third dwv master variant ( type c ) was assembled and confirmed to be distinct through phylogenetic inference .
illumina ( san diego , ca , usa ) hi - seq ( 2 100 ) pair - end sequencing was carried out by tgac ( the genome analysis centre ) and at the university of exeter on four samples from highfield et al .
( 2009 ) ; late june , early october and late october from gd1 , and early october from gd2 ( supplementary table s1 ) .
the samples were originally collected from devon in the southwest of england ( highfield et al . , 2009 ) .
a bioinformatics pipeline was developed to accommodate the large amount of variation found within the dwv species complex .
first , the quality of the raw reads was verified using fastqc ( babraham bioinformatics , cambridge , uk ) .
samples were then converted from fastq to fasta using the fastq_to_fasta script that is part of the fastx - toolkit ( hannon lab ) ( http://hannonlab.cshl.edu/fastx_toolkit/ ) . to isolate the dwv complex sequence reads from the host and other contaminating sequences ,
the reads were searched against a custom blast database containing the dwv , vdv-1 and kv genomes , with an e - value of 10e5 .
blast was carried out against read 1 of the illumina data . the ncbi - blast - parser perl script ( http://www.bioinformatics-made-simple.com/2012/07/ncbi-blast-parser-extract-query-and.html )
sed ' and awk ' scripts were used to delete empty lines and the reads that contained nohits ' .
the corresponding blast hits were extracted from the read 2 raw reads using qiime ( caporaso et al . , 2010 ) .
the paired reads were balanced using a custom script written in r version 3.2.0 ( r core team , 2015 ) . finally , the balanced dwv family reads were assembled using the vicuna assembler that was developed to generate consensus assemblies from genetically heterogeneous populations , specifically rna viruses .
vicuna contigs > 200 bp in length were imported into geneious ( version 7.04 , created by biomatters , auckland , new zealand ) and the map to reference tool ' was used to align the contigs with the dwv and vdv-1 reference genomes . for several of the samples the vicuna assembly yielded full - length contigs that covered the whole genome , whereas for others consensus scaffolds were created from two or more contigs .
a second novel variant was also assembled by vicuna . in order to create a consensus sequence of the novel variant ,
any contigs containing sequence other than the novel variant were removed . if a contig contained the novel variant as well as sequence belonging to the type a dwv genome because of recombination or in silico recombination , the type a dwv regions were trimmed and deleted .
a consensus genome of the novel variant was created from these sorted and trimmed contigs .
accordingly , the same steps were carried out on the type a sequence in order to assemble the type a genome consensus from devon .
the dwv scaffolds were then aligned with full - length genome sequences from the ncbi ( national center for biotechnology information ) database using the muscle alignment tool ( edgar , 2004 ) within geneious and the full - length genome was obtained .
full genome comparisons were visualised in mvista ( frazer et al . , 2004 ) .
to quantify the number of reads attributed to each dwv variant using blast ( supplementary table s1 ) , the novel type c variant genome sequence was added to the custom dwv family database and the blast for each sample was carried out a second time .
this allowed the type c variants to be categorised correctly by blast rather than attributed to the closets hit to the three other reference genomes . to validate the quantification of reads attributed to each variant ,
the balanced read 1 and 2 blast hits were aligned competitively to three reference genomes using the map to reference tool ' within geneious .
consensus genomes of type a and type c were translated using the live annotation ' tool within geneious . because of repetitive regions in the genome , assembly error took place in several sites , leading to an incomplete open reading frame .
these assembly errors were corrected by using the vicuna analysis tool to pull out individual reads covering these regions , and the correct sequence was determined .
finally , the rdp4 programme ( martin et al . , 2010 ) was used to determine whether any recombination took place between the sequences .
assembled genomes are available from the european nucleotide archive under the accession numbers ers657948 ( type a ) and ers657949 ( type c ) . to establish the phylogenetic relationship of dwv subtypes and closely related iflaviruses , we reconstructed the phylogeny of the conserved rdrp amino acid sequences for seven dwv subtypes , spanning all three types that were either sequenced and assembled from the devon hive or available from genbank ( type a : nc_005876.1 , nc_004830.2 ; type b : kc_786222.1 , nc_006494.1 , jq_413340 ) , as well as formica exsecta virus 1 ( nc_023022.1 ) and sacbrood virus ( nc_002066.1 ) .
we used a bayesian approach using mrbayes ( v. 3.1.2 ) ( huelsenbeck and ronquist , 2001 ) .
we assumed a fixed rate model of protein evolution and reconstructed the phylogeny using a model jumping method .
this method allows for different models of amino acid substitution to be used in the markov chain monte carlo ( mcmc ) procedure , with all models contributing to the final result weighted according to their respective posterior probability .
we ran two runs of four chains for 4 000 000 mcmc generations , sampling trees every 1000 generations .
all trees were drawn using figtree v.1.4.2 ( http://tree.bio.ed.ac.uk/software/figtree/ ) . to estimate the evolutionary rate of dwv and its subtypes
, we collated three independent data sets with temporal information for at least one population : ( a ) partial lp - gene ; 10 populations , n=78 , 328 nt ( 13231650 bp ) ( genbank aj489744 , ay292384 , gu109335 , hm162355 , jf346615-jf346620 , jf346624-jf346629 , jf346633-jf346639 , kf164292 , kf164293 , kj437447 , kp734726 , kp734738 , kp734747 , kp734765-kp734770 , kp734774-kp734787 , kp734817-kp734825 , kp734827-kp734846 ) ; ( b ) partial capsid - gene ; 1 population , n=167 , 1215 nt ( 26343848 bp ) ( genbank ay292384 , hq655502-hq655561 , kf314827- kf314932 ) ; ( c ) partial rdrp - gene ; 1 b. terrestris population , n=145 , 508 nt ( 80168522 bp ) ( genbank kp734326kp734470 ) . to test whether these fragments contain a molecular clock signal , we estimated the root - to - tip divergence using path - o - gen v.1.4 ( http://tree.bio.ed.ac.uk/software/pathogen/ ) to estimate how much genetic variation
we constructed maximum likelihood phylogenetic trees without the assumption of a molecular clock in phylip v. 3.695 ( evolution.genetics.washington.edu ; felsenstein , 1989 ) and tested whether the regression between root - to - tip distance in these maximum likelihood trees and the age of the samples indicated a clock - like signal .
in addition , we tested for a significant temporal signal by randomising the temporal information across each data set 100 times and compared the resulting random evolutionary rates from beast with the real data set .
a temporal signal is supported if there is a significant difference between the real data set and the randomised data sets ( ramsden et al . , 2009 ; alizon and fraser , 2013 ) .
both analyses support a temporal signal in these data sets ( supplementary figure s3 and supplementary table s4 ) .
to determine the appropriate molecular clock models for each data set , we used the path sampling maximum likelihood estimator implemented in beast 1.8 ( baele et al .
, 2012 , 2013 ) . as this method is very computationally intensive , we first used a range of simpler tests to limit the number of models to be compared by this method as suggested by alizon and fraser ( 2013 ) , and drummond and bouckaert ( 2014 ) .
for the three large temporal data sets , we used jmodeltest v.2.1.1 ( https://github.com/ddarriba/jmodeltest2 ; posada , 2008 ) to compare substitution models based on the bayesian information criterion ( alizon and fraser 2013 ) ; the resulting substitution models were the tamura - nei ( tnr ) model ( lp - fragment ) and the hasegawa , kashino and yano ( hky ) model ( vp3- and rdrp - fragments ) , both with gamma variation .
for the dwv - subtype data set , we compared the general time - reversible model and the srd06 model ( shapiro et al . , 2006 ) using path sampling .
we partitioned substitution rates between the first and second and third codon positions as , for all data sets , the third codon position had a significantly higher rate .
we tested whether a strict clock rate can be excluded by running models with a lognormal relaxed clock ; if the relaxed clock 's coefficient of variation statistic abuts the zero boundary , a strict clock can not be excluded ( gray et al . , 2011 ) .
we ran models with exponential population growth for all models ; if the exponential growth rate was significantly higher than zero , a constant population size can be excluded .
we then used path sampling to distinguish between clock models ( exponential and lognormal relaxed clock ) and demographic models ( constant population size , exponential population growth or a gaussian markov random field ( gmrf skyride ) ; drummond et al . , 2002 ; minin et al . ,
based on these analyses , we chose an exponential relaxed clock and exponential growth prior for the lp- and capsid - fragments and a lognormal relaxed clock and constant growth prior for the rdrp - fragment as well as for the dwv - subtype analyses . to generate a genome - wide estimate for the evolutionary rate in dwv , we calculated the mean of the relaxed clock means for the individual fragments ( lp - fragment : 9.097 10 ( 95% highest posterior density : 4.412 101.394 10 ) , capsid - fragment : 1.845 10 ( 1.159 102.569 10 ) , rdrp - fragment : 1.278 10 ( 4.131 102.513 10 ) , resulting in a mean evolutionary rate of 1.346 10 ( 5.41 102.627 10 ) substitutions / site / year .
we implemented this evolutionary rate as an uncorrelated lognormal clock prior for the dwv - subtype analysis with a lognormal distribution with a mean of 1.35 10 in real space and a log s.d .
we ran models with 2 runs each of 50 million mcmc generations , sampling every 5000 generations with a burn - in of 5 million generations to obtain effective sample sizes > 200 .
we examined traces for convergence using tracer v.1.6 ( http://www.tree.bio.ed.ac.uk/software/tracer/ ) and used treeannotator v.1.8 ( beast.bio.ed.ac.uk/downloads ) to produce a maximum clade credibility tree for the dwv - subtype analysis .
( 2003 ) , as implemented in dambe , to confirm that the alignment had not reached substitution saturation .
illumina ( san diego , ca , usa ) hi - seq ( 2 100 ) pair - end sequencing was carried out by tgac ( the genome analysis centre ) and at the university of exeter on four samples from highfield et al .
( 2009 ) ; late june , early october and late october from gd1 , and early october from gd2 ( supplementary table s1 ) .
the samples were originally collected from devon in the southwest of england ( highfield et al . , 2009 ) .
a bioinformatics pipeline was developed to accommodate the large amount of variation found within the dwv species complex .
first , the quality of the raw reads was verified using fastqc ( babraham bioinformatics , cambridge , uk ) .
samples were then converted from fastq to fasta using the fastq_to_fasta script that is part of the fastx - toolkit ( hannon lab ) ( http://hannonlab.cshl.edu/fastx_toolkit/ ) . to isolate the dwv complex sequence reads from the host and other contaminating sequences ,
the reads were searched against a custom blast database containing the dwv , vdv-1 and kv genomes , with an e - value of 10e5 .
blast was carried out against read 1 of the illumina data . the ncbi - blast - parser perl script ( http://www.bioinformatics-made-simple.com/2012/07/ncbi-blast-parser-extract-query-and.html )
sed ' and awk ' scripts were used to delete empty lines and the reads that contained nohits ' .
the corresponding blast hits were extracted from the read 2 raw reads using qiime ( caporaso et al . , 2010 ) .
the paired reads were balanced using a custom script written in r version 3.2.0 ( r core team , 2015 ) . finally , the balanced dwv family reads were assembled using the vicuna assembler that was developed to generate consensus assemblies from genetically heterogeneous populations , specifically rna viruses .
vicuna contigs > 200 bp in length were imported into geneious ( version 7.04 , created by biomatters , auckland , new zealand ) and the map to reference tool ' was used to align the contigs with the dwv and vdv-1 reference genomes . for several of the samples the vicuna assembly yielded full - length contigs that covered the whole genome , whereas for others consensus scaffolds were created from two or more contigs .
a second novel variant was also assembled by vicuna . in order to create a consensus sequence of the novel variant ,
any contigs containing sequence other than the novel variant were removed . if a contig contained the novel variant as well as sequence belonging to the type a dwv genome because of recombination or in silico recombination , the type a dwv regions were trimmed and deleted .
a consensus genome of the novel variant was created from these sorted and trimmed contigs .
accordingly , the same steps were carried out on the type a sequence in order to assemble the type a genome consensus from devon .
the dwv scaffolds were then aligned with full - length genome sequences from the ncbi ( national center for biotechnology information ) database using the muscle alignment tool ( edgar , 2004 ) within geneious and the full - length genome was obtained .
full genome comparisons were visualised in mvista ( frazer et al . , 2004 ) .
to quantify the number of reads attributed to each dwv variant using blast ( supplementary table s1 ) , the novel type c variant genome sequence was added to the custom dwv family database and the blast for each sample was carried out a second time .
this allowed the type c variants to be categorised correctly by blast rather than attributed to the closets hit to the three other reference genomes . to validate the quantification of reads attributed to each variant ,
the balanced read 1 and 2 blast hits were aligned competitively to three reference genomes using the map to reference tool ' within geneious .
consensus genomes of type a and type c were translated using the live annotation ' tool within geneious . because of repetitive regions in the genome , assembly error took place in several sites , leading to an incomplete open reading frame .
these assembly errors were corrected by using the vicuna analysis tool to pull out individual reads covering these regions , and the correct sequence was determined .
finally , the rdp4 programme ( martin et al . , 2010 ) was used to determine whether any recombination took place between the sequences .
assembled genomes are available from the european nucleotide archive under the accession numbers ers657948 ( type a ) and ers657949 ( type c ) .
to establish the phylogenetic relationship of dwv subtypes and closely related iflaviruses , we reconstructed the phylogeny of the conserved rdrp amino acid sequences for seven dwv subtypes , spanning all three types that were either sequenced and assembled from the devon hive or available from genbank ( type a : nc_005876.1 , nc_004830.2 ; type b : kc_786222.1 , nc_006494.1 , jq_413340 ) , as well as formica exsecta virus 1 ( nc_023022.1 ) and sacbrood virus ( nc_002066.1 ) .
we used a bayesian approach using mrbayes ( v. 3.1.2 ) ( huelsenbeck and ronquist , 2001 ) .
we assumed a fixed rate model of protein evolution and reconstructed the phylogeny using a model jumping method .
this method allows for different models of amino acid substitution to be used in the markov chain monte carlo ( mcmc ) procedure , with all models contributing to the final result weighted according to their respective posterior probability .
we ran two runs of four chains for 4 000 000 mcmc generations , sampling trees every 1000 generations .
to estimate the evolutionary rate of dwv and its subtypes , we collated three independent data sets with temporal information for at least one population : ( a ) partial lp - gene ; 10 populations , n=78 , 328 nt ( 13231650 bp ) ( genbank aj489744 , ay292384 , gu109335 , hm162355 , jf346615-jf346620 , jf346624-jf346629 , jf346633-jf346639 , kf164292 , kf164293 , kj437447 , kp734726 , kp734738 , kp734747 , kp734765-kp734770 , kp734774-kp734787 , kp734817-kp734825 , kp734827-kp734846 ) ; ( b ) partial capsid - gene ; 1 population , n=167 , 1215 nt ( 26343848 bp ) ( genbank ay292384 , hq655502-hq655561 , kf314827-
kf314932 ) ; ( c ) partial rdrp - gene ; 1 b. terrestris population , n=145 , 508 nt ( 80168522 bp ) ( genbank kp734326kp734470 ) . to test whether these fragments contain a molecular clock signal ,
we estimated the root - to - tip divergence using path - o - gen v.1.4 ( http://tree.bio.ed.ac.uk/software/pathogen/ ) to estimate how much genetic variation can be explained by the sampling date .
we constructed maximum likelihood phylogenetic trees without the assumption of a molecular clock in phylip v. 3.695 ( evolution.genetics.washington.edu ; felsenstein , 1989 ) and tested whether the regression between root - to - tip distance in these maximum likelihood trees and the age of the samples indicated a clock - like signal .
in addition , we tested for a significant temporal signal by randomising the temporal information across each data set 100 times and compared the resulting random evolutionary rates from beast with the real data set .
a temporal signal is supported if there is a significant difference between the real data set and the randomised data sets ( ramsden et al . , 2009 ; alizon and fraser , 2013 ) .
both analyses support a temporal signal in these data sets ( supplementary figure s3 and supplementary table s4 ) .
to determine the appropriate molecular clock models for each data set , we used the path sampling maximum likelihood estimator implemented in beast 1.8 ( baele et al . , 2012 , 2013 ) .
as this method is very computationally intensive , we first used a range of simpler tests to limit the number of models to be compared by this method as suggested by alizon and fraser ( 2013 ) , and drummond and bouckaert ( 2014 ) .
for the three large temporal data sets , we used jmodeltest v.2.1.1 ( https://github.com/ddarriba/jmodeltest2 ; posada , 2008 ) to compare substitution models based on the bayesian information criterion ( alizon and fraser 2013 ) ; the resulting substitution models were the tamura - nei ( tnr ) model ( lp - fragment ) and the hasegawa , kashino and yano ( hky ) model ( vp3- and rdrp - fragments ) , both with gamma variation .
for the dwv - subtype data set , we compared the general time - reversible model and the srd06 model ( shapiro et al . , 2006 ) using path sampling .
we partitioned substitution rates between the first and second and third codon positions as , for all data sets , the third codon position had a significantly higher rate .
we tested whether a strict clock rate can be excluded by running models with a lognormal relaxed clock ; if the relaxed clock 's coefficient of variation statistic abuts the zero boundary , a strict clock can not be excluded ( gray et al . , 2011 ) .
we ran models with exponential population growth for all models ; if the exponential growth rate was significantly higher than zero , a constant population size can be excluded .
we then used path sampling to distinguish between clock models ( exponential and lognormal relaxed clock ) and demographic models ( constant population size , exponential population growth or a gaussian markov random field ( gmrf skyride ) ; drummond et al .
based on these analyses , we chose an exponential relaxed clock and exponential growth prior for the lp- and capsid - fragments and a lognormal relaxed clock and constant growth prior for the rdrp - fragment as well as for the dwv - subtype analyses . to generate a genome - wide estimate for the evolutionary rate in dwv , we calculated the mean of the relaxed clock means for the individual fragments ( lp - fragment : 9.097 10 ( 95% highest posterior density : 4.412 101.394 10 ) , capsid - fragment : 1.845 10 ( 1.159 102.569 10 ) , rdrp - fragment : 1.278 10 ( 4.131 102.513 10 ) , resulting in a mean evolutionary rate of 1.346 10 ( 5.41 102.627 10 ) substitutions / site / year .
we implemented this evolutionary rate as an uncorrelated lognormal clock prior for the dwv - subtype analysis with a lognormal distribution with a mean of 1.35 10 in real space and a log s.d .
we ran models with 2 runs each of 50 million mcmc generations , sampling every 5000 generations with a burn - in of 5 million generations to obtain effective sample sizes > 200 .
we examined traces for convergence using tracer v.1.6 ( http://www.tree.bio.ed.ac.uk/software/tracer/ ) and used treeannotator v.1.8 ( beast.bio.ed.ac.uk/downloads ) to produce a maximum clade credibility tree for the dwv - subtype analysis .
( 2003 ) , as implemented in dambe , to confirm that the alignment had not reached substitution saturation .
illumina hi - seq ( 2 100 ) pair - end sequencing of asymptomatic honey bees from devon was carried out on colonies that either survived ( gd2 ) or collapsed ( gd1 ) because of ocl ( highfield et al . , 2009 ) .
to assess dwv diversity within the samples the raw reads were searched against a custom dwv family database ( including kv and vdv-1 genomes ) and reads matching to any of the dwv genomes were extracted .
the coverage for the number of reads that matched to a custom dwv blast database was estimated using the lander / waterman equation ( that is , the depth of sequencing ) .
genome coverage depth ranged from 457 to 165 927 ( average coverage was 86 838 ) ( supplementary table s1 ) .
de novo assembly of the illumina reads yielded a complete genome of a type a variant as well as of a novel dwv variant that we named type c ( supplementary table s2 )
. competitive alignment to the now three master variants revealed that out of the ~30 million dwv blast positive reads , ~27 million were assembled to one of three genomes ( supplementary table s3 and supplementary figure s1 ) . around 3 million assembled to type a , ~3.5 thousand to type b and ~24 million to type c. as expected , more reads align to the 3 region of the genome than the 5 an artefact of the reverse transcription 3 bias ( brooks et al . , 1995 ) . although reads aligned to the whole of the type c genome , the depth of coverage was lower at the 5 region ( supplementary figure s1 ) .
in addition , coverage of the 5 region of the type a variant was unusually high , indicating that this disproportionate coverage in the 5 regions of type a and c could be the evidence of recombination between the two variants . because of the low read depth and coverage for type b in our samples ,
a full genome could not be assembled ( supplementary table s3 and supplementary figure s1 ) . nonetheless ,
closer examination of 3 region where the rna - dependent rna polymerase ( rdrp ) gene is located ( baker and schroeder , 2008 ) revealed that the devon type b variant shared 100% identity to vdv-1 type b genome in this region ( supplementary figure s2 ) .
the sequence identity of our newly assembled type a and c variants was compared with other members of the dwv complex ( table 1 and figure 1 ) .
both type b and c differ from the type a nucleotide sequence in similar regions of the genome . however , type b and c share only 79% nucleotide identity in the polyprotein encoding region of the genome and 89% identity in the amino acid sequence .
phylogenetic analysis showed the relationship of the novel type a and c variants from devon to previously sequenced viruses ( figures 2 and 3 ) .
the type a genome clustered with other type a variants , whereas type c formed a distinct and separate branch , thereby confirming the originality and thus new master variant assignment of dwv type c. given that type a and c can recombine with each other , we screened for any genomes with evidence of recombination and excluded them from the analysis in order to comply with the assumptions underlying phylogenetic reconstruction .
the type c genome codes for a full - length polyprotein and translation of the genome permitted the amino acid sequence to be compared with more distant members of the genus iflavirus ( figure 2 ) .
the relationship of the dwv complex with f. exsecta virus 2 and sacbrood virus was analysed using a bayesian inference of phylogeny of a conserved region of the rdrp amino acid sequence ( figure 2 ) .
the dwv sequences were attributed to three main groups ( type a , b and c ) .
the nucleotide consensus sequences created by vicuna for type a was found to be 98.2% identical to the type a reference dwv genome .
the analysis suggested that the new type c genome is clearly related to type a and b , and forms a distinct clade to other members of the family iflaviridae ( for example , sacbrood virus ) that are all only distantly related to a dicistrovirus outgroup , f. exsecta virus .
in addition , a bayesian analysis of the polyprotein encoding region of the sequences was carried out using an mcmc model , permitting a molecular clock model to be run within beast v1.8.1 ( figure 3 ) ( drummond et al . , 2012 ) .
divergence times were calculated based on a tip - dated coalescent model , with an evolutionary rate prior based on three independent tip - dated fragments of dwv type a. the samples in the tree span 11 years ( 20002011 ) , and the samples used for estimating the evolutionary rate span 11 , 13 and 22 years for the rdrp- , capsid- and lp- fragments , respectively .
the bayesian tree of the nucleotide sequences had a similar structure to the amino acid tree , showing that the type c viral variant is distinct from type a and b. the molecular clock estimation predicts that type c diverged from the other dwv variants 319 years ago ( 571010 95% highest posterior density ) , and type a and b disassociated from each other 181 years ago ( 38497 95% highest posterior density ) .
this estimate is unlikely to be biased by substitution saturation , as there is no evidence for saturation in our data set ( xia et al . , 2003 ) .
the phylogenetic analysis concludes that the dwv quasispecies is made up of three distinct master variants .
however , the number of variants is not exhaustive and it is conceivable that more variants will be added in the future as the use of ngs becomes increasingly prolific .
the result further suggests that type c has not recently emerged , but rather is an established dwv variant .
moreover , using reverse transcriptase - pcr to amplify a region of the dwv rdrp gene , martin et al .
( 2012 ) attributed a novel variant to the type b swarm of variants , although it is now clear that this novel variant was in fact type c. the presence of type c in hawaii implies that this variant is widespread and not specific to the united kingdom .
it also confirms that it has not emerged recently as the hawaiian sample originates from kauai that has yet to be colonised by varroa ( martin et al . , 2012 ) , suggesting type c is part of the wild - type dwv quasispecies .
the high levels of heterogeneity within viral rna populations mean that viruses are able to occupy large areas of sequence space and consequently are able to exist in multiple hosts ( domingo and holland , 1997 ) .
dwv is a generalist ' known to infect bumblebees ( genersch et al , 2006 ; frst et al . , 2014 ) ,
v. destructor ( ongus et al . , 2004 ) and other insects ( reviewed in manley et al . , 2015 ) .
further work is required to ascertain whether type c causes acute wing deformities in honey bees or its presence is because of viral spill over ' from another host .
three of the four samples in this study were taken from a hive ( gd1 ) that went on to collapse from ocl . however , as type a and the type a
c 5 recombinant were also present , it remains unclear which variant was responsible for ocl .
it is worth noting however that between 85% and 98% of the reads in hive gd1 were type c that make up the type c or the a
the second hive ( gd2 ) that survived ocl contained roughly equal levels of type a and c reads ( which includes reads that make up the a
c recombinant ) ; yet , the coverage indicates that the viral load was much lower in this hive
. moreover , the low abundance of dwv type b in these devon colonies appears to confirm the observation made in the colonies from swindon ( mordecai et al . , 2015 ) ; that is , dwv type b is not present at sufficient levels to protect the honey bee from the virulent type a , or possibly c variants ( as well as any recombinants between a and c ) .
in addition , varroa mites in the devon colonies were controlled using chemical methods , thereby potentially preventing the mites from transmitting type b into the honey bees ; as observed in the swindon apiary ( mordecai et al .
, 2015 ) . a recombinant between dwv type a and b has been reported previously ( moore et al . , 2011 ) ,
and found to be hypervirulent as it was more efficient at replicating than other variants when co - injected directly into the haemolymph of honey bee larvae ( ryabov et al .
as it is presently unclear whether the type c variant or the a c recombinant lead to disease , further direct manipulation experiments such as those carried out by ryabov et al .
( 2014 could be crucial in determining whether the novel type c genome and any of its recombinants are hypervirulent . the large number of dwv type c and a
c recombinant reads in the devon samples ( supplementary table s1 and supplementary figure s1 ) suggests that these variants are able to replicate in honey bees .
the capacity of dwv to exist as a swarm of recombining variants and occupy large amounts of sequence space may be one of the factors contributing to its capability to maintain a persistent infection . defining dwv as a quasispecies with discrete master variants is comparable with the current categorisation of several other rna viruses . the genetic variability within hepatitis c virus has been classified into four hierarchical strata : genotypes , sub - genotypes , isolates and finally background variation of the quasispecies ( farci and purcell , 2000 ) .
a total of seven hepatitis c virus genotypes exist to date , differing from each other by > 15% over their complete coding region ( smith et al . , 2014 ) .
similarly , japanese encephalitis virus is currently classified into five genotypes based on sequence identity that differ in replication efficiency in a range of hosts ( han et al . , 2014 ) .
in order to further our understanding of dwv and the impact of the virus on honey bees as well as other hosts , a more progressive classification of the dwv quasispecies would be prudent , that is , the new classification of dwv as a quasispecies with at least three master variants that can recombine with each other . bioinformatics pipelines designed for eukaryotes and prokaryotes are not necessarily suited to studying viral systems .
for example , pipelines incorporating reference assemblers could hinder the discovery of novel virus variants .
the genome sequences presented here are representative of the different variants found within a sample ; however , the vicuna pipeline is not suited to analysing the further level of diversity around these variants , for which diversity models are better suited , such as those developed by wood et al .
( 2014 ) . as well as being of significance to globally important honey bee health ,
cross - species virus transmission and emergence of new epidemic diseases such as severe acute respiratory syndrome , ebola and influenza are major threats to public health ( parrish et al . ,
exploring the extent of viral diversity in rna quasispecies , of which dwv may be a suitable model , may offer insight into the mechanisms by which viruses are able to transmit between different hosts as well as how viruses are able to develop resistance to antiviral therapies ( domingo et al . , 2012 ) .
further study of the dwv quasispecies may help to explain how dwv is able to exist as multiple variants in many hosts , and may elucidate mechanisms by which it establishes a persistent infection among several hosts but only proves pathogenic in some . | treatment of emerging rna viruses is hampered by the high mutation and replication rates that enable these viruses to operate as a quasispecies . declining honey bee populations
have been attributed to the ectoparasitic mite varroa destructor and its affiliation with deformed wing virus ( dwv ) . in the current study
we use next - generation sequencing to investigate the dwv quasispecies in an apiary known to suffer from overwintering colony losses .
we show that the dwv species complex is made up of three master variants .
our results indicate that a new dwv type c variant is distinct from the previously described types a and b , but together they form a distinct clade compared with other members of the iflaviridae .
the molecular clock estimation predicts that type c diverged from the other variants 319 years ago .
the discovery of a new master variant of dwv has important implications for the positive identification of the true pathogen within global honey bee populations . | Introduction
Materials and methods
Sequencing and assembly
Amino acid-based phylogeny
Evolutionary rate
Model selection and BEAST runs
Results and discussion
Supplementary Material |
PMC3511671 | therapeutics based on rna interference hold great promise for the treatment of numerous diseases that are not currently druggable . in recent years
, both viral and nonviral methods for delivery and expression of interfering rna molecules have been explored .
examples of nonviral delivery vectors include peptides , proteins , aptamers , cationic polymers , lipoplexes , and liposomes . some of these agents are not suitable for systemic in vivo delivery due to factors such as instability in serum , undesirable pharmacokinetics , or the expense or feasibility of scaling - up to obtain therapeutic quantities .
in addition to meeting these criteria , an effective delivery agent requires targeting to disease - relevant tissue to avoid potentially toxic side effects of either the agent or the associated interfering rna .
one approach for delivering rna to specific cells is to use small proteins or polypeptides that contain a receptor - binding or cell - homing domain to direct the complex to the surface of the desired cell population .
these vectors also require rna - binding capability ; a property sometimes conferred using cationic peptide sequences that electrostatically interact with negatively charged rna backbones . as an alternative to cationic peptides , there are many examples of both prokaryotic and eukaryotic proteins that contain canonical double - stranded rna ( dsrna)-binding motifs .
these proteins can bind to rna duplexes both specifically ( i.e. , they do not bind duplexed dna ) and with high affinity . a well - studied example of these is protein kinase r ( pkr ; also known as dsrna - activated protein kinase dai ) , which contains two dsrna - binding motifs . in isolation , both motifs can bind dsrna , with affinities of kd = 3.8 10 mol / l and kd = 2
10 mol / l for the n - terminal motif 1 and c - teminal motif 2 , respectively .
together , the motifs act cooperatively to enhance the affinity of dsrna binding by 100-fold ( kd = 4 10
pkr drbd motif 1 was fused to a protein transduction domain ( ptd ) composed of three repeats of the cell - penetrating peptide ( cpp ) tat .
this fusion protein , termed ptd - drbd , and now commercially available as transductin , facilitated robust gene silencing in cultured cells and effectively reduced luciferase reporter gene expression in the nasal and tracheal passages of a transgenic mouse . while ptd - drbd is effective at small interfering rna ( sirna ) delivery in vitro or when directly administered to target tissues in vivo , it is not suitable for intravenous systemic delivery ; tat motifs interact with serum molecules such as glycosaminoglycans , which can block its internalizing activity .
further , because tat is a general cell - binding peptide and cpp , it does not target specific cells or tissue types .
we reasoned that targeting drbd with cell - homing peptides or receptor ligands in place of tat would provide a strategy for cell type - specific delivery of sirnas . in the present study , we evaluated drbds derived from pkr as a general sirna delivery vector when fused with either cell - penetrating domains or cell - homing peptides .
we found that ptd - drbd , which contains the cationic cell - binding domain tat and a single drbd , can non - specifically interact with sirna , but that stable and specific binding of sirna by fusion proteins lacking tat requires two drbds .
unexpectedly , while fusion proteins containing two drbds can mediate internalization of sirna directed by both cpp and cell - homing peptides , gene silencing is attenuated by the inclusion of the larger rna - binding domain .
in addition , regardless of the number of drbd motifs , we found that the concentration of protein required for maximal gene silencing exceeds the amount required for sirna binding .
these studies demonstrate that the structural properties of drbd , and possibly other protein delivery vectors , can significantly affect the accessibility of sirna to the rna - induced silencing complex and , ultimately , the efficacy of gene silencing .
we first tested whether drbd could complex sirna when fused to a peptide with affinity for a specific cell type .
for this , we generated a protein , pps - drbd , in which the three tandem copies of the tat peptide in ptd - drbd were replaced with the brain vascular targeting peptide dspahps ( pps ) , a motif that our lab previously identified by in vivo biopanning of mouse brain endothelium ( figure 1 ) .
for comparison we also generated drbd alone , which lacks a cell - binding domain ( figure 1 ) .
the ability of each protein to bind sirna was measured by electrophoretic mobility shift assay ( figure 2 ) .
both pps - drbd and drbd failed to induce a marked sirna mobility shift , even at a 40-fold molar excess of protein relative to sirna ( figure 2a , b ) . to address the possibility that the purification method caused a loss of rna - binding activity
, we also tested drbd purified under denaturing conditions followed by refolding ; this protein preparation yielded a comparable result ( supplementary figure s1 ) .
in contrast , incubating ptd - drbd with sirna resulted in a mobility shift in a dose - dependent manner ( figure 2c ) .
we hypothesized that the ptd domain might be mediating nonspecific interaction with sirna . to test this
, we performed an electrophoretic mobility shift assay using dsdna of the same sequence as the sirna ( figure 2c ) .
ptd - drbd induced a dsdna shift at a 4:1 molar ratio , though , in contrast to sirna , the dsdna complex failed to migrate into the gel , indicating possible aggregation ( figure 2c ) . to test if the polybasic tat motifs within the ptd domain facilitate binding , we incubated synthetic peptides with sirna and measured electrophoretic mobility
. sirna migration was shifted when incubated with tat , but not control peptides , including the polybasic tlh and b2 peptides ( figure 2d ) .
these data indicate that drbd alone or fused to the pps - targeting domain is not sufficient to stably bind sirna . however , the tat motifs in ptd - drbd enable nonspecific nucleic acid binding .
it was previously reported that proteins containing the complete pkr rna - binding motif ( 2 drbd ) can complex with small dsrna ( ~2025 base pairs ) and sirna .
given this , we next tested whether the pps domain ( pps-2 drbd ) fused to 2 drbd could interact with sirna .
both 2 drbd alone and pps-2 drbd yielded near complete sirna binding at a 2:1 molar ratio ( figure 3a , b ) . an additional mobility shift was observed at 4:1 molar ratios , which is consistent with previous studies indicating that the length of a small dsrna is sufficient to allow binding of two molecules of 2 drbd in a concentration - dependent manner . increasing the concentration of protein did not result in a further mobility shift or aggregation as evidenced by the same band migration pattern at ratios as high at 16:1 ( figure 3c ) . unlike ptd - drbd ,
ptd fused to 2 drbd also did not bind to dsdna , and it displayed a sirna - binding profile nearly identical to 2 drbd ( figure 3d ) . in a previous report ,
ptd - drbd was complexed with sirna at relatively high molar ratios ( 20 - 60:1 ) for in vitro gene silencing . at these concentrations , complexes formed with ptd-2
drbd migrate into the gel but those with ptd - drbd are retained in the gel well , indicative of aggregation ( figure 3e ) .
interestingly , at ratios of 40 and 60:1 , the complex undergoes a third mobility shift compared to that at 4:1 ( figure 3d versus figure 3e ) .
this suggests that at high concentrations , a third protein molecule may be able to associate with the sirna .
collectively , these results indicate that 2 drbd fused to either ptd or cell - targeting peptides can specifically and stably bind sirna , but not dsdna .
having established that our 2 drbd fusion proteins could bind to sirna , we next evaluated their ability to deliver sirna and mediate gene silencing in vitro .
we hypothesized that the greater specificity and affinity of the sirna interaction with ptd-2 drbd would confer more potent knockdown relative to ptd - drbd . to test this
, we measured knockdown of the housekeeping gene hypoxanthine - guanine phosphoribosyltransferase ( hprt ) in hela cells treated with sirna complexes formed with each protein ( figure 4 ) .
pps-2 drbd served as a negative control as pps peptide does not bind to hela cells ( supplementary figure s2 ) .
as previously mentioned , at least a 2060-fold molar excess of ptd - drbd is typically complexed with sirna for in vitro gene silencing , despite our observation that much less is required for sirna binding . in order to directly compare silencing efficacy of ptd - drbd to ptd-2 drbd , we chose to form complexes with a 60-fold excess of protein over sirna .
after 24 hours , hprt message was reduced ~82% in cells treated with ptd - drbd , and no knockdown was observed from treatment with pps-2 drbd ( figure 4 ) .
unexpectedly , treatment with ptd-2 drbd only resulted in ~19% knockdown ( figure 4 ) . to test
if the size of the transduction domain might somehow be inhibitory when fused to 2 drbd , we also tested a truncated form of ptd that only contained a single tat motif ( 1 tat-2 drbd ) .
however , this protein did not mediate any knockdown of hprt ( figure 4 ) . to determine whether a cpp other than tat might yield knockdown , we tested 2 drbd fused to the cpp , penetratin ; however this construct also failed to mediate knockdown ( supplementary figure s3 ) .
together , these findings suggest that 2 drbd , but not drbd , inhibits robust gene silencing in vitro .
a possible explanation for lack of gene silencing is that the interaction between the ptd and the larger rna - binding domain prevents efficient binding and/or internalization into cells . to address this , we incubated hela cells with complexes containing protein and fluorescently labeled sirna ( figure 5 ) .
after incubation , cells were acid washed to remove extracellular complexes and sirna localization was monitored by fluorescence microscopy .
cells treated with ptd - drbd showed punctate , vesicular signal , localized near the nucleus ( figure 5a ) .
treatment with ptd-2 drbd resulted in a similar pattern but the puncta were larger and there was a higher overall level of internal cell fluorescence ( figure 5a , b ) .
treatment using pps-2 drbd or sirna alone did not result in appreciable intracellular sirna localization ( figure 5a ) .
since sirna needs to escape into the cytoplasm to interact with the silencing machinery , we also evaluated whether the vesicular localization of sirna changed over time when treated with the different proteins .
after 24 hours of incubation , sirna delivered with both ptd - drbd and ptd-2 drbd remained primarily punctate with perinuclear staining ( figure 5c ) .
these results show that sirna complexed to both ptd - drbd and ptd-2 drbd can bind to and be internalized into cells .
we hypothesized that minimal gene silencing resulted from limited dissociation of the sirna from the high affinity rna - binding domain and/or that the sirna that did dissociate remained trapped in endocytic compartments . to test these possibilities , we treated cells with sirna complexes in the presence or absence of chloroquine , an endosomolytic agent used to enhance gene delivery ( figure 6a ) .
in contrast to treatment without chloroquine , hprt message was reduced 92% in cells treated with either ptd - drbd or ptd-2 drbd ( figure 6a ) .
hprt levels also decreased after treatment with 1 tat-2 drbd , though the silencing was more modest ( 59% ) ( figure 6a ) . a comparable level of knockdown ( ~60% )
was achieved with penetratin-2 drbd , indicating that sirna delivery can be achieved with a distinct cpp when fused to 2 drbd ( supplementary figure s3 ) . minimal knockdown ( 12% )
was observed using pps-2 drbd ( figure 6a ) . as our initial hypothesis was that ptd-2 drbd 's higher affinity for sirna would enhance silencing activity ( i.e. , less protein
is required to deliver the same amount of sirna ) , we tested whether lower concentrations of protein ( 0.4 versus 3.0
mol / l in figure 6a ) could mediate knockdown ( figure 6b ) . in the absence of chloroquine , no protein constructs , including ptd - drbd , reduced hprt mrna levels ( figure 6b ) . however , in the presence of chloroquine , cells treated with ptd-2 drbd but not ptd - drbd had significantly lower levels of hprt message ( 63 versus 4% , respectively ) ( figure 6b ) .
together , these results suggest that lack of silencing with ptd-2 drbd in the absence of chloroquine is due to restricted endosomal escape of sirna rather than a lack of dissociation from the rna - binding domain .
further , on a molar basis , ptd-2 drbd has a greater ability to deliver sirna into cells as compared with ptd - drbd .
it is possible that targeting 2 drbd fusion proteins to specific receptors , rather than using nonspecific cpps , may result in an altered trafficking pathway that yields greater localization of cargo to the cytoplasm versus the endosome . to test this and to evaluate sirna delivery with a receptor - targeted fusion protein
, we generated a 2 drbd fusion protein containing three repeats of the b2 peptide sequence ( ghkvkrpkg ) in place of the tat ptd domains ( figure 1a ) .
we previously identified the b2 peptide by phage display panning against recombinant transferrin receptor ( tfr ) and showed that adenovirus capsids engineered to present b2 display enhanced transduction of cells in a tfr - mediated manner .
coincidentally , the b2 peptide also shares a high degree of similarity with the mature prion protein ( prp ) n - terminal polybasic domain ( kkrpkp ) , which has been shown to have membrane interacting and cell - internalizing properties ( figure 7b ) .
we first tested delivery of sirna and hprt knockdown in hela cells , which express tfr and have been used to study transferrin trafficking .
interestingly , in the absence of chloroquine , treatment with b2 - 2 drbd sirna complexes resulted in a 30% reduction in hprt message , which is 12% greater knockdown than that obtained with ptd-2 drbd ( figure 7a versus figure 4 ) . in the presence of chloroquine , hprt knockdown
was increased to 65% ( figure 7a ) . to test whether sirna delivery and knockdown
occurs through the tfr , we competed binding by pre - incubating cells with transferrin or bovine serum albumin control protein before adding the sirna complex .
while there was a decrease in hprt knockdown with transferrin pre - treatment relative to the control ( 67 versus 49% ) , this difference was variable and not statistically significant ( figure 7a ) .
these results indicate that b2 - 2 drbd can mediate gene silencing but that internalization might be occurring through multiple pathways which include binding tfr and a cell - penetrating mechanism involving the prp - like internalization motif present within the b2 sequence .
we first tested whether drbd could complex sirna when fused to a peptide with affinity for a specific cell type .
for this , we generated a protein , pps - drbd , in which the three tandem copies of the tat peptide in ptd - drbd were replaced with the brain vascular targeting peptide dspahps ( pps ) , a motif that our lab previously identified by in vivo biopanning of mouse brain endothelium ( figure 1 ) .
for comparison we also generated drbd alone , which lacks a cell - binding domain ( figure 1 ) .
the ability of each protein to bind sirna was measured by electrophoretic mobility shift assay ( figure 2 ) .
both pps - drbd and drbd failed to induce a marked sirna mobility shift , even at a 40-fold molar excess of protein relative to sirna ( figure 2a , b ) . to address the possibility that the purification method caused a loss of rna - binding activity
, we also tested drbd purified under denaturing conditions followed by refolding ; this protein preparation yielded a comparable result ( supplementary figure s1 ) .
in contrast , incubating ptd - drbd with sirna resulted in a mobility shift in a dose - dependent manner ( figure 2c ) .
we hypothesized that the ptd domain might be mediating nonspecific interaction with sirna . to test this
, we performed an electrophoretic mobility shift assay using dsdna of the same sequence as the sirna ( figure 2c ) .
ptd - drbd induced a dsdna shift at a 4:1 molar ratio , though , in contrast to sirna , the dsdna complex failed to migrate into the gel , indicating possible aggregation ( figure 2c ) . to test if the polybasic tat motifs within the ptd domain facilitate binding , we incubated synthetic peptides with sirna and measured electrophoretic mobility
. sirna migration was shifted when incubated with tat , but not control peptides , including the polybasic tlh and b2 peptides ( figure 2d ) .
these data indicate that drbd alone or fused to the pps - targeting domain is not sufficient to stably bind sirna . however , the tat motifs in ptd - drbd enable nonspecific nucleic acid binding .
it was previously reported that proteins containing the complete pkr rna - binding motif ( 2 drbd ) can complex with small dsrna ( ~2025 base pairs ) and sirna .
given this , we next tested whether the pps domain ( pps-2 drbd ) fused to 2 drbd could interact with sirna .
both 2 drbd alone and pps-2 drbd yielded near complete sirna binding at a 2:1 molar ratio ( figure 3a , b ) . an additional mobility shift was observed at 4:1 molar ratios , which is consistent with previous studies indicating that the length of a small dsrna is sufficient to allow binding of two molecules of 2 drbd in a concentration - dependent manner . increasing the concentration of protein did not result in a further mobility shift or aggregation as evidenced by the same band migration pattern at ratios as high at 16:1 ( figure 3c ) . unlike ptd - drbd ,
ptd fused to 2 drbd also did not bind to dsdna , and it displayed a sirna - binding profile nearly identical to 2 drbd ( figure 3d ) . in a previous report ,
ptd - drbd was complexed with sirna at relatively high molar ratios ( 20 - 60:1 ) for in vitro gene silencing . at these concentrations , complexes formed with ptd-2
drbd migrate into the gel but those with ptd - drbd are retained in the gel well , indicative of aggregation ( figure 3e ) .
interestingly , at ratios of 40 and 60:1 , the complex undergoes a third mobility shift compared to that at 4:1 ( figure 3d versus figure 3e ) .
this suggests that at high concentrations , a third protein molecule may be able to associate with the sirna .
collectively , these results indicate that 2 drbd fused to either ptd or cell - targeting peptides can specifically and stably bind sirna , but not dsdna .
having established that our 2 drbd fusion proteins could bind to sirna , we next evaluated their ability to deliver sirna and mediate gene silencing in vitro .
we hypothesized that the greater specificity and affinity of the sirna interaction with ptd-2 drbd would confer more potent knockdown relative to ptd - drbd . to test this , we measured knockdown of the housekeeping gene hypoxanthine - guanine phosphoribosyltransferase ( hprt ) in hela cells treated with sirna complexes formed with each protein ( figure 4 ) .
pps-2 drbd served as a negative control as pps peptide does not bind to hela cells ( supplementary figure s2 ) .
as previously mentioned , at least a 2060-fold molar excess of ptd - drbd is typically complexed with sirna for in vitro gene silencing , despite our observation that much less is required for sirna binding . in order to directly compare silencing efficacy of ptd - drbd to ptd-2 drbd , we chose to form complexes with a 60-fold excess of protein over sirna .
after 24 hours , hprt message was reduced ~82% in cells treated with ptd - drbd , and no knockdown was observed from treatment with pps-2 drbd ( figure 4 ) .
unexpectedly , treatment with ptd-2 drbd only resulted in ~19% knockdown ( figure 4 ) . to test
if the size of the transduction domain might somehow be inhibitory when fused to 2 drbd , we also tested a truncated form of ptd that only contained a single tat motif ( 1 tat-2 drbd ) .
however , this protein did not mediate any knockdown of hprt ( figure 4 ) . to determine whether a cpp other than tat might yield knockdown , we tested 2 drbd fused to the cpp , penetratin ; however this construct also failed to mediate knockdown ( supplementary figure s3 ) .
together , these findings suggest that 2 drbd , but not drbd , inhibits robust gene silencing in vitro .
a possible explanation for lack of gene silencing is that the interaction between the ptd and the larger rna - binding domain prevents efficient binding and/or internalization into cells . to address this
, we incubated hela cells with complexes containing protein and fluorescently labeled sirna ( figure 5 ) .
after incubation , cells were acid washed to remove extracellular complexes and sirna localization was monitored by fluorescence microscopy .
cells treated with ptd - drbd showed punctate , vesicular signal , localized near the nucleus ( figure 5a ) .
treatment with ptd-2 drbd resulted in a similar pattern but the puncta were larger and there was a higher overall level of internal cell fluorescence ( figure 5a , b ) .
treatment using pps-2 drbd or sirna alone did not result in appreciable intracellular sirna localization ( figure 5a ) .
since sirna needs to escape into the cytoplasm to interact with the silencing machinery , we also evaluated whether the vesicular localization of sirna changed over time when treated with the different proteins .
after 24 hours of incubation , sirna delivered with both ptd - drbd and ptd-2 drbd remained primarily punctate with perinuclear staining ( figure 5c ) .
these results show that sirna complexed to both ptd - drbd and ptd-2 drbd can bind to and be internalized into cells .
we hypothesized that minimal gene silencing resulted from limited dissociation of the sirna from the high affinity rna - binding domain and/or that the sirna that did dissociate remained trapped in endocytic compartments . to test these possibilities , we treated cells with sirna complexes in the presence or absence of chloroquine , an endosomolytic agent used to enhance gene delivery ( figure 6a ) .
in contrast to treatment without chloroquine , hprt message was reduced 92% in cells treated with either ptd - drbd or ptd-2 drbd ( figure 6a ) .
hprt levels also decreased after treatment with 1 tat-2 drbd , though the silencing was more modest ( 59% ) ( figure 6a ) . a comparable level of knockdown ( ~60% )
was achieved with penetratin-2 drbd , indicating that sirna delivery can be achieved with a distinct cpp when fused to 2 drbd ( supplementary figure s3 ) . minimal knockdown ( 12% )
as our initial hypothesis was that ptd-2 drbd 's higher affinity for sirna would enhance silencing activity ( i.e. , less protein is required to deliver the same amount of sirna ) , we tested whether lower concentrations of protein ( 0.4 versus 3.0
in the absence of chloroquine , no protein constructs , including ptd - drbd , reduced hprt mrna levels ( figure 6b ) . however , in the presence of chloroquine , cells treated with ptd-2 drbd but not ptd - drbd had significantly lower levels of hprt message ( 63 versus 4% , respectively ) ( figure 6b ) .
together , these results suggest that lack of silencing with ptd-2 drbd in the absence of chloroquine is due to restricted endosomal escape of sirna rather than a lack of dissociation from the rna - binding domain .
further , on a molar basis , ptd-2 drbd has a greater ability to deliver sirna into cells as compared with ptd - drbd .
it is possible that targeting 2 drbd fusion proteins to specific receptors , rather than using nonspecific cpps , may result in an altered trafficking pathway that yields greater localization of cargo to the cytoplasm versus the endosome . to test this and to evaluate sirna delivery with a receptor - targeted fusion protein
, we generated a 2 drbd fusion protein containing three repeats of the b2 peptide sequence ( ghkvkrpkg ) in place of the tat ptd domains ( figure 1a ) .
we previously identified the b2 peptide by phage display panning against recombinant transferrin receptor ( tfr ) and showed that adenovirus capsids engineered to present b2 display enhanced transduction of cells in a tfr - mediated manner .
coincidentally , the b2 peptide also shares a high degree of similarity with the mature prion protein ( prp ) n - terminal polybasic domain ( kkrpkp ) , which has been shown to have membrane interacting and cell - internalizing properties ( figure 7b ) .
we first tested delivery of sirna and hprt knockdown in hela cells , which express tfr and have been used to study transferrin trafficking .
interestingly , in the absence of chloroquine , treatment with b2 - 2 drbd sirna complexes resulted in a 30% reduction in hprt message , which is 12% greater knockdown than that obtained with ptd-2 drbd ( figure 7a versus figure 4 ) . in the presence of chloroquine , hprt knockdown
was increased to 65% ( figure 7a ) . to test whether sirna delivery and knockdown occurs through the tfr , we competed binding by pre - incubating cells with transferrin or bovine serum albumin control protein before adding the sirna complex .
while there was a decrease in hprt knockdown with transferrin pre - treatment relative to the control ( 67 versus 49% ) , this difference was variable and not statistically significant ( figure 7a ) .
these results indicate that b2 - 2 drbd can mediate gene silencing but that internalization might be occurring through multiple pathways which include binding tfr and a cell - penetrating mechanism involving the prp - like internalization motif present within the b2 sequence .
in this study , we sought to determine whether pkr - derived drbd could serve as a protein platform for cell type - specific targeted delivery of sirna . for this , we compared knockdown mediated by cell - binding peptides fused to either a single drbd or the complete pkr rna - binding motif that contains two drbds .
our evaluation revealed that ptd - drbd , which can mediate robust gene silencing as shown in earlier work , does not stably bind sirna in the absence of ptd .
this result is consistent with previous studies of the rna - binding activity of pkr demonstrating that although the n - terminal drbd motif 1 can bind short rnas ( 2025 bp ) , the binding is of low affinity and unstable .
this suggests that the tat peptides in ptd - drbd are acting in tandem with the drbd to enable stable sirna binding through electrostatic interaction , which is further supported by the observation that dsdna also binds to ptd - drbd when in molar excess .
the requirement for ptd raises the question as to what degree the drbd contributes to the sirna - binding activity of ptd - drbd .
it could be that the drbd is not absolutely required and that a polypeptide composed of only the ptd or several tandem repeats of tat may also have sirna complexation properties that could confer silencing activity . in the contrast to ptd - drbd
, we showed that the inclusion of both pkr - derived drbds results in stable and dsrna - specific binding , even when the drbds are fused to the tat - containing ptd .
this means that 2 drbd can be fused to various cell - binding peptides , including highly positively charged sequences , and still maintain binding of the sirna restricted to the drbd .
this property is important as it prevents the cell - binding peptide from interacting with the sirna , which could interfere with its ability to associate with cell surface moieties .
both ptd - drbd and ptd-2 drbd can complex with sirna and be internalized , however , ptd-2 drbd complexes are more readily internalized at lower concentrations compared with ptd - drbd , as inferred from a greater degree of gene silencing .
this may occur because at lower molar ratios of protein to sirna , the sirna binds to the ptd domain of ptd - drbd and this impedes uptake of the complex into the cell .
in contrast , at the same molar ratios , sirna binding to ptd-2 drbd is restricted to the drbds , allowing the unencumbered ptd to mediate more effective cell surface binding and internalization . despite the improved sirna binding and internalization properties of 2 drbd fusion proteins , we found that these complexes are not nearly as robust at mediating gene silencing as ptd - drbd .
this was true for both tat and penetratin fusions , which supports the hypothesis that it is not the specific cpp that is restricting silencing but rather a property of the 2 drbd domain .
penetratin has been used to mediate rapid internalization of sirna when directly conjugated to modified sirna via a disulfide bond .
the resulting molecule is smaller than the complexes tested in our studies , which may explain its greater silencing activity . interestingly , despite the observation that sirna can bind to 2 drbd proteins at a 24 molar excess , maximal silencing requires a greater than eightfold molar excess .
it may be that excess , unbound protein facilitates knockdown in a manner that does n't necessarily require direct interaction with the sirna .
one possibility is that at high concentrations , free cpp stimulates cargo uptake as reported for tat - dna complexes .
alternatively , the high protein concentration may prevent sirna dissociation from drbd proteins after dilution into the transfection media .
treatment of cells with chloroquine resulted in silencing comparable with ptd - drbd , which indicates that the 2 drbd complexes are sequestered to endosomal / lysosomal compartments and do not efficiently escape into the cytoplasm .
this is consistent with earlier studies showing that sirna complexed with a protein containing the pkr drbds could facilitate gene silencing , but only when admixed with the fusogenic peptide kala .
one explanation for this observation is that both ptd - drbd and 2 drbd fusion proteins traffic through the same compartments but that the larger relative size and/or structural features of the 2 drbd fusion protein complex hinder endosomal escape .
alternatively , the 2 drbd constructs might enter the cell through a pathway distinct from ptd - drbd .
it has been reported that ptd - drbd and other tat fusion proteins utilize a macropinocytic uptake mechanism that promotes cytoplasmic cargo delivery .
however , there are differing reports on the exact mechanism of tat uptake and recently it was shown that tat peptide fused to relatively small cargo could directly translocate across the plasma membrane whereas fusion to larger cargo results in endocytic internalization . when considering the relationship between complex size and bioactivity , it is of note that at the molar concentrations of protein used for in vitro gene silencing ( 20 - 60:1 ) , ptd - drbd complexes are aggregated .
counter - intuitively , aggregates are more efficient at mediating gene silencing than soluble or lower - order complexes .
thus , 2 drbd complexes might preferentially traffic through endocytic compartments that lead to entrapment while ptd - drbd trafficking allows for enhanced cytoplasmic localization of bound sirna . in support of the concept that utilizing different internalization mechanisms may affect gene - silencing efficacy ,
our transferrin receptor - targeted protein provided enhanced knockdown relative to ptd-2 drbd in the absence of chloroquine . however , because transferrin did not compete with b2 - 2 drbd - mediated gene knockdown , the b2 peptide may be utilizing both the transferrin receptor as well as other cell surface moieties , via interaction with the b2 polybasic motif , for internalization .
one possibility is that the net positive charge of the b2 domain facilitates nonspecific electrostatic interaction with negative charges on the cell surface ( e.g. , from glycosaminoglycans ) . in support of this ,
internalization of the prp cell - penetrating motif is inhibited by exogenous heparin and chondroitin sulfate .
this property may , in part , explain the incomplete inhibition of silencing by transferrin receptor competition
. it would be interesting to evaluate whether the b2 peptide , in addition to binding tfr , is a novel cpp .
overall , our study demonstrates that protein - based sirna delivery vectors utilizing canonical drbds display several properties that could be used for cell type - specific targeting .
however , the effect of the drbd structure on the bioavailability of sirna delivered with the 2 drbd proteins evaluated in this study poses a significant limitation to their utility . it would be interesting to test whether further vector engineering could overcome these shortcomings .
for example , a membrane - destabilizing fusogenic / endosomolytic peptide could be introduced into the 2 drbd fusion protein .
alternatively , chemical modification of the proteins with endosome - disrupting groups might enhance cytoplasmic cargo delivery .
future studies will show whether these , or other strategies , will yield more potent 2 drbd - based vectors for delivery of sirna targeted to specific cell types .
hela cells were cultured in complete medium ( dulbecco 's modified eagle 's medium containing 10% fetal bovine serum ) at 37 c , 5% co2 .
unlike the other constructs , penetratin-2 drbd contains a flexible linker previously used to generate a penetratin - mcherry protein for cellular delivery .
oligonucleotides ( idt , coralville , ia ) were annealed , extended , and pcr amplified .
digested pcr products were ligated into pet11d expression vector ( stratagene , santa clara , ca ) backbone generated by digesting with ncoi and bamhi restriction enzymes ( new england biolabs , ipswich , ma ) .
the sequence of each expression plasmid was confirmed by sequencing performed at the university of iowa dna core facility .
all proteins were expressed and purified except for ptd - drbd , which was obtained from idt ( commercially sold as transductin ) .
pet11d expression plasmids were transformed into bl21 star e. coli ( life technologies , carlsbad , ca ) .
a 25 ml starter culture grown using a freshly transformed colony was inoculated into 500 ml of lb containing 100 g / ml ampicillin and grown at 37 c , 220 rpm shaking , until the od600 reached between 0.6 and 0.8 .
protein expression was induced with 1 mmol / l iptg and cultures were grown an additional 4 hours at 37 c , 220 rpm shaking .
cultures were centrifuged at 5,000 rpm at 4 c for 15 minutes to pellet bacteria .
protein constructs containing 2 drbd required denaturing purification and on - column refolding in order to remove bacterial rna bound to the protein . for purification under denaturing conditions ,
the pellet was resuspended in 5 ml of buffer a ( 50 mmol / l sodium phosphate , ph 7.4 ) containing complete mini protease inhibitors ( roche , basel , switzerland ) ; 30k units of rlysozyme ( novagen , madison , wi ) , 150 units of benzonase ( sigma - aldrich , st .
louis , mo ) , and mgcl2 to a final concentration of 2 mmol / l and the suspension was incubated on ice for 20 minutes .
ten milliliter of 1.1 buffer b ( 50 mmol / l sodium phosphate , 555.5 mmol / l nacl , 22.2 mmol / l imidazole , 6.66 mol / l guanidine hcl , 2.2 mmol / l -mercaptoethanol ; ph 7.4 ) was added to the suspension that was then sonicated on ice seven times for 30 seconds with the amplitude set at 50 using a model ge50 ultrasonic processor ( sonics & materials , newton , ct ) ; 35 ml of buffer b was added for a final volume of 50 ml and the lysate was allowed to incubate on ice for 25 minutes to solubilize proteins .
the lysate was clarified by centrifugation for 40 minutes at 16,000 g , 4 c .
the supernatant was then filtered through a 0.4 m pvdf filter ( millipore , billerica , ma ) . using a biorad biologic lp system ( bio - rad , hercules , ca ) ,
denatured proteins were bound to a hitrap imac column ( ge healthcare , waukesha , wi ) charged with cobalt chloride .
the column was washed with 10 ml of buffer b diluted to 1 with buffer a. bound protein was refolded on - column by applying a 0100% linear gradient over 80 minutes at 0.5 ml / minute flow rate starting with 100% buffer b/0% buffer c ( 50 mmol / l sodium phosphate , 500 mmol / l nacl , 20 mmol / l imidazole ; ph 7.4 ) and ending with 0% bufferb/100% buffer c. purified , refolded proteins were eluted with buffer d ( 50 mmol / l sodium phosphate , 300 mmol / l nacl , 150 mmol / l imidazole ; ph 7.4 ) and concentrated using amicon ultra 10k mwco centrifugal filters ( millipore ) .
concentrated eluates were buffer exchanged into phosphate - buffered saline + 10% glycerol using zeba desalting columns ( thermo fisher scientific , waltham , ma ) .
for non - denaturing purification of the drbd and pps - drbd proteins used in figure 2a , b , benzonase- and lysozyme - treated lysates were clarified by centrifugation and then combined with 1 buffer b containing 750 mmol / l nacl .
extracts were applied to the imac cobalt column and washed with buffer b containing 1 mol / l nacl .
after elution , extracts were treated again with benzonase to remove co - purifying bacterial nucleic acid .
electrophoretic mobility shift assays . the sirna used was ambion silencer rest sirna ( catalog no .
dsdna was prepared using dna oligonucleotides ( idt ) of the same sequence as the sirna .
dna oligonucleotides were annealed by heating to 95 c for 4 minutes in annealing buffer ( 100 mmol / l potassium acetate , 2 mmol / l magnesium acetate , 30 mmol / l hepes , ph 7.4 ) and then allowed to cool to room temperature . in all gel shifts except for in figure 3e , 8 pmol of sirna or dsdna were combined with varying amounts of each protein in a final volume of 8 l , yielding concentrations of 1 mol / l sirna or dsdna . in figure 3e , 1 pmol of sirna was complexed with proteins in order to achieve the higher molar protein ratios in a small volume .
mixtures were incubated at room temperature for 15 minutes and then combined with 2 l of 5 tbe loading buffer ( life technologies ) .
the entire reaction volumes were electrophoresed on a 0.5 tbe , 6% acrylamide gel at 90 v for 45 minutes in 0.5 tbe running buffer .
the gel was stained with 0.5 g / ml ethidium bromide ( bio - rad ) and visualized using a versadoc 5000 mp ( bio - rad ) . for gel shifts with synthetic peptides , n - terminally biotinylated tat ( rkkrrqrrr ) ,
pps ( dspahps ) , tlh ( gwtlhnk ) , or b2 ( ghkvkrpkg ) peptides ( american peptide company , sunnyvale , ca ) were incubated at a final concentration of either 20 or 40 mol / l with 1 mol / l sirna and analyzed as described above .
hela cells were plated in 48-well dishes at 23 10 cells / well in regular media ( dulbecco 's modified eagle 's medium with 10% fetal bovine serum ) 1416 hours before sirna treatment .
complexes were formed by incubating 6 pmol of hprt s1 dicer - substrate sirna ( idt ) or universal negative control ( nc1 ) dicer - substrate sirna ( idt ) with either 48 or 360 pmol of protein on ice for 30 minutes .
dulbecco 's modified eagle 's medium containing 10% q - serum ( q - media ) prepared as previously described was added to complexes to yield a final concentrations of 50 nmol / l sirna and 0.4 or 3 mol / l protein .
media and sirna / protein mixtures were added to cells that were previously washed one time with q - media and then preincubated in q - media for 30 minutes at 37 c .
complexes were incubated with cells for 4 hours and then removed and replaced with fresh complete medium .
for chloroquine experiments , chloroquine was added during both the sirna incubation step and during the 24-hour incubation period . for transferrin receptor blocking experiments , hela cells were pretreated for 30 minutes with 5
mol / l holo - transferrin ( sigma - aldrich ) or bovine serum albumin ( new england biolabs ) in q - media before knockdown .
trizol reagent ( life technologies ) was used to extract and purify total rna from treated cells .
cdna was synthesized from rna using the multiscribe high capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's protocol .
cycling was performed on a 7900ht fast real time pcr system ( applied biosystems ) .
gapdh was used to calculate the ct for hprt in hela cells , knockdown with hprt sirna was calculated as the average relative quantity from three independent experiments and variation is represented as the sd of the three relative quantities .
the variation of hprt in cells treatment with nc1 sirna was calculated as the population sd of the ct s for each treatment condition in the three independent experiments .
hela cells were incubated with complexes containing 50 nmol / l scrambled sequence cy3-sirna ( idt ) and 2 mol / l protein prepared as described above for hprt gene - silencing experiments .
after 3 hours of incubation , the medium containing complexes was removed and the cell nuclei were stained for 10 minutes at 37 c with hoechst 3342 dye ( life technologies ) diluted into complete media .
cells were then washed three times with complete media followed by washing with low ph acid buffer ( 0.2 mol / l glycine , ph 2.8 ) .
after the acid wash , cells were washed one additional time with dulbecco 's pbs ( life technologies ) before examination by fluorescence microscopy .
the brightness and contrast of the primary images were adjusted equivalently using image j software ( nih , bethesda , md ) .
the fluorescence intensity ( integrated density ) was measured for 16 cells per analyzed image .
the integrated density was calculated and corrected by subtracting background signal from regions proximal to the measured cells .
mean fluorescence intensity and sd was calculated from two independent experiments . peptide cell binding and recombinant protein characterization .
electrophoretic mobility shift assay of sirna binding to drbd prepared under denaturing purification conditions .
silencing of hprt mediated by penetratin-2 drbd fusion protein in the presence or absence of chloroquine .
silencing of hprt mediated by penetratin-2 drbd fusion protein in the presence or absence of chloroquine . | delivery of small interfering rna ( sirna ) targeted to specific cell types is a significant challenge for the development of rna interference - based therapeutics .
recently , ptd - drbd , a double - stranded rna binding domain ( drbd ) fused to the tat protein transduction domain ( ptd ) , was shown to be effective at delivering sirna in a non - cell type - specific manner . here
, we evaluated the potential of drbd as a general protein platform for targeted small interfering rna ( sirna ) delivery .
we found that a single drbd was insufficient to stably complex sirna when fused to targeting peptides other than ptd , which facilitated nonspecific nucleic acid binding .
in contrast to ptd - drbd , fusion proteins containing two drbds ( 2 drbd ) yielded specific and stable sirna binding .
these proteins could mediate the cellular uptake of sirna in vitro , though compared with ptd - drbd gene silencing was attenuated by endosomal entrapment .
our findings suggest that unlike a single drbd , 2 drbd inhibits sirna escape into the cytoplasm and/or induces an internalization pathway distinct from that of ptd - drbd .
collectively , these data indicate that while 2 drbd retains sirna - binding activity when fused to different cell surface - interacting peptides , the utility of 2 drbd for cell - specific rna interference is limited without further protein engineering to enhance the bioavailability of the delivered sirnas . | Introduction
Results
Complex formation with siRNA and DRBD fusion proteins
siRNA delivery and gene silencing with DRBD fusion proteins
Transferrin receptor-targeted delivery of siRNA with a peptide 2 DRBD fusion protein
Discussion
Materials and Methods
Supplementary Material |
PMC4616739 | adequate vascular access is of critical importance for patients who require extracorporeal renal replacement therapy .
although an arteriovenous fistula ( avf ) is the golden standard access for hemodialysis , temporary dialysis catheters are frequently required . the national kidney foundation kidney disease outcomes quality initiative ( k / doqi ) suggests that catheter access is usually achieved through cannulation of 1 of the 3 easily accessible central veins , namely the femoral , internal jugular , or subclavian veins .
the internal jugular veins are preferable locations for insertion . however , the femoral vein is often selected as well , depending on the patient 's condition .
several complications are associated with the insertion of these catheters , including infection , bleeding , deep venous thrombosis , and injuries to arteries .
thrombosis and infections are the common complications during catheter insertion as well as in the already inserted catheters .
an avf is a rare but severe complication following catheterization for patients who require hemodialysis .
we describe a case of an iatrogenic avf after cannulation of the femoral vein for temporary hd leading to leg swelling 6 months after removal of a hemodialysis catheter .
a 42-year - old asian man with a history of diabetic ketoacidosis and acute renal failure ( arf ) was first admitted to a satellite hospital 6 months ago .
a temporary double - lumen hd catheter was smoothly inserted into the right femoral vein .
blood samples were collected through the catheter to confirm its venous origin , and hd was performed through the catheter .
bleeding from the catheter insertion site was observed 5 days after admission , when he walked to the restroom . at that time , the platelet count was normal and international normalized ratio ( inr ) was 2.45 .
six days later , the serum creatinine level become normal and urine volume was much more than 1500 ml / day after the comprehensive treatment .
the catheter was removed to stop the continuous bleeding ; as a result , the patient was able to walk without bleeding and subsequently discharged .
the patients received insulin therapy and stayed in good condition in the follow - up .
six months after the catheter removal , he was admitted to our institution because of right lower limb swelling and calf pain . on physical examination ,
the temperature was 36.9c and blood pressure was 140/80 mmhg , the jugular venous pressure was normal , and the pulse was regular at 75 beats / min . body mass index ( bmi , weight / height ) was 21.5 .
a bruit was heard on auscultation and a thrill was felt at the inguinal area .
serum levels of creatinine , blood urea nitrogen ( bun ) , and hemoglobin were 98 mol / l , 5.6 mmol / l , and 10.8 g / dl , respectively .
his serum potassium and sodium were 4.9 and 138 meq / l , respectively .
an avf was detected by color doppler ultrasound between the right superficial femoral artery ( sfa ) and the right femoral vein and subsequently it was confirmed by 3-dimensional reconstruction with ct and angiography ( figures 1 and 2 ) .
three - dimensional ct revealing an arteriovenous fistula between the right superficial femoral artery and the right femoral vein ( arrow ) .
angiography revealing the arteriovenous communication ( between the superficial femoral artery and superficial femoral vein ) .
initial treatment involved dilation of the stenotic lesion immediately beneath the bifurcation of the right common iliac artery using a balloon and stent insertion . during operation , the surgeon found a 3-mm diameter fistula between the right femoral vein and the right sfa .
the right femoral vein and sfa were separated carefully . then , vascular repairing was carried out with running nonabsorbable sutures .
a 42-year - old asian man with a history of diabetic ketoacidosis and acute renal failure ( arf ) was first admitted to a satellite hospital 6 months ago .
a temporary double - lumen hd catheter was smoothly inserted into the right femoral vein .
blood samples were collected through the catheter to confirm its venous origin , and hd was performed through the catheter .
bleeding from the catheter insertion site was observed 5 days after admission , when he walked to the restroom . at that time
, the platelet count was normal and international normalized ratio ( inr ) was 2.45 .
six days later , the serum creatinine level become normal and urine volume was much more than 1500 ml / day after the comprehensive treatment .
the catheter was removed to stop the continuous bleeding ; as a result , the patient was able to walk without bleeding and subsequently discharged .
the patients received insulin therapy and stayed in good condition in the follow - up .
six months after the catheter removal , he was admitted to our institution because of right lower limb swelling and calf pain . on physical examination ,
the temperature was 36.9c and blood pressure was 140/80 mmhg , the jugular venous pressure was normal , and the pulse was regular at 75 beats / min . body mass index ( bmi , weight / height ) was 21.5 .
a bruit was heard on auscultation and a thrill was felt at the inguinal area .
serum levels of creatinine , blood urea nitrogen ( bun ) , and hemoglobin were 98 mol / l , 5.6 mmol / l , and 10.8 g / dl , respectively .
his serum potassium and sodium were 4.9 and 138 meq / l , respectively .
an avf was detected by color doppler ultrasound between the right superficial femoral artery ( sfa ) and the right femoral vein and subsequently it was confirmed by 3-dimensional reconstruction with ct and angiography ( figures 1 and 2 ) .
three - dimensional ct revealing an arteriovenous fistula between the right superficial femoral artery and the right femoral vein ( arrow ) .
angiography revealing the arteriovenous communication ( between the superficial femoral artery and superficial femoral vein ) .
initial treatment involved dilation of the stenotic lesion immediately beneath the bifurcation of the right common iliac artery using a balloon and stent insertion . during operation , the surgeon found a 3-mm diameter fistula between the right femoral vein and the right sfa .
it is reported that traumatic avf , a complication of femoral vein catheter insertion for hd , is an exceptional disease .
however , when it occurs , it is often caused by damage to an artery while inserting a catheter into a vein .
chronic limb edema , false aneurysms degeneration , and high output cardiac failure due to fistula are common complications . to prevent this ,
several studies have demonstrated that real - time ultrasound guidance is superior to traditional landmark insertion techniques for the insertion of dialysis catheters because real - time ultrasound guidance is of particular value in increasing
first - pass rates and reducing the risk of hematoma formation and other complications related to catheter insertion .
moreover , the use of a fine - gauge needle to locate the vein before introduction of the guide wire using the seldinger needle is suggested as well .
avf as a complication of dual - lumen hd catheter insertion has been described in the medical literature .
kuramochi et al described a case of right superficial femoral avf with a symptom of arterial bleeding .
chiba et al reported a case of an avf associated with calf pain 2 months after the removal of the catheter . according to the literature
, the most common initial symptoms were swelling , hemorrhage , limb pain , hematoma , thromboembolism , and cardiac failure .
limb swelling along with calf pain as the initial symptom was rare in any of the cases .
but in this case , the patient was initial with a symptom of limb swelling and calf pain .
chiba et al reported a case of femoral avf observed 2 months after catheter removal . in the present case ,
it seems that femoral avf might be observed later than cervical avf , probably because of inaccessibility to the groin area .
several explanations were possible for the formation of femoral avf : ( 1 ) the femoral head provides posterior bony support , and it might be difficult to achieve hemostasis for the lack of bony support during compression following catheter removal .
( 2 ) avf once closed by a hematoma might be reopened by movement or anticoagulation with heparin during hd .
( 3 ) presumably , femoral avf formation is caused by relatively larger caliber of the catheter and occult arterial puncture . in the present case
, the anticoagulation with heparin during hd might be a reason for femoral avf formation .
the risks factors that increase the likelihood of developing avfs included distal groin punctures , arterial hypertension , antiplatelet therapy , and peri - procedural anti - coagulation in this case , the low puncture might increase the likelihood of developing avfs .
moreover , there was no real - time ultrasound - guided that had been used for catheter insertion .
the traditional landmark insertion techniques are commonly performed using anatomic landmarks to guide vessel cannulation .
these traditional techniques can not direct visualization of the target vein during catheter placement and are associated with an increased complication rate .
so , it is recommended that real - time ultrasound is performed to direct guidance of the needle insertion to avoid repeated blind punctures .
femoral venous catheterization is frequently selected to achieve temporary access to circulation in patients requiring acute hemodialysis .
thus , it is important for nephrologists and nursing staff to be aware of this potential complication and to perform thorough clinical and medical examinations at the insertion and removal of temporary hd catheters .
it is recommended that real - time ultrasound guidance should be used for catheters insertion to avoid inadvertent arterial puncture . | abstractdouble - lumen catheters have been used widely to obtain temporary access in patients who are in need of acute hemodialysis ( hd ) because of acute renal failure .
several complications are associated with the insertion of these catheters , including bleeding , infection , injuries to arteries , and deep venous thrombosis .
an arteriovenous fistula ( avf ) is a rare but significant complication following catheterization for temporary hd .
herein , we present a case of avf associated with leg swelling 6 months after the removal ofa double - lumen hd catheter.we describe a special case of a 42-year - old man who experienced acute renal failure secondary to diabetic ketoacidosis ( dka ) .
a 12-fr dialysis catheter was inserted in the right femoral vein .
six months after catheter removal , the patient was admittedfor pain and swelling in the right leg .
color doppler ultrasound and three - dimensional computed tomography ( ct ) revealed an avf between the right femoral vein and the right femoral superficial artery .
the fistula was repaired successfully by vascular surgeons.this case highlights that an avf is a rare but significant complication after catheterization for temporary hd .
the nephrologist should be wary of the potential of this complication and perform clinical and medical examinations at the insertion and removal of temporary hd catheters . | INTRODUCTION
CASE PRESENTATION
DISCUSSION
CONCLUSION |
PMC5094612 | pancreatic cancer ( pc ) is the fourth most common cause of cancer death in the usa , whereas in europe , it is the fourth for women and fifth for men.1,2 pc is the third most common cancer in the gastrointestinal tract after colorectal and esophagogastric cancer.2 the danish pancreatic cancer database ( dpcd ) aims to prospectively describe the epidemiology , diagnostic workup , histological or cytological diagnosis , treatment , and outcome of patients with pc in denmark at an institutional and national level . furthermore , dpcd collects data for continuous quality control of diagnostic workup and treatment .
dpcd also provides baseline data for epidemiological research in pc . to ensure a high quality of the registered data ,
dpcd is organized as part of the danish pancreatic cancer group ( dpcg ) , a multidisciplinary group established for the publication of national guidelines and to promote research in the field of pc in denmark .
the dpcd has included patients diagnosed with pancreatic ductal adenocarcinoma in denmark from may 1 , 2011 onward . only patients with a histological or cytological diagnosis are included .
the total number of patients registered as of june 30 , 2014 , was 2,217 , corresponding to an average monthly inclusion rate of 58 patients .
patients with pancreatic neoplasms other than ductal adenocarcinoma , eg , neuroendocrine tumors , are excluded .
the data in the dpcd have been cross - referenced with the danish pathology registry and the danish national patient registry to ensure the completeness of registrations .
lists of missing patients are distributed annually to the participating institutions in order to enter missing data .
the completeness of the registry has gradually improved from 76% the first year ( 2011 ) to 83% in 2014 . from 2012 ,
patient age at diagnosis , tissue specimen for diagnosis ( ie , core needle biopsy , fine - needle aspirate , resection specimen ) , precise histological diagnosis , localization of the tumor , sex , and world health organization performance status at the time of diagnosis and/or start of treatment are registered .
also the precise modalities used for the diagnostic workup , including imaging techniques , and the pretreatment tumor node metastasis stage , usually determined at the multidisciplinary team conference , are registered .
the charlson comorbidity index can be calculated based on data from the national patient registry .
the following treatment variables are included : type of operation , first adjuvant or neoadjuvant therapy , and first palliative chemo- or chemoradiation therapy . for patients treated by surgery ,
pathological staging and margin status are registered . the type of vascular resection , if any , has been registered since 2013 .
baseline data of selected variables are shown in table 1 . in order to track and monitor the clinical pathway ,
several key data are registered , including the following dates : referral , end of diagnostic workup , multidisciplinary team conference , patients acceptance of treatment proposal , and first surgical and/or oncological treatment .
table 2 shows the currently registered indicators that , due to dpcg , are the measurable key indicators of quality and desired timeline of workup and treatment initiation and fulfill the criteria setup by the danish government by law .
the main end point is death , and the date of recurrence is not registered directly in the database .
instead , dpcd relies on data from the danish civil registry to monitor the survival status of the study population , and this registration is virtually complete .
the results with respect to baseline characteristics , key indicators , and survival ( table 2 ) are audited by all participating institutions and published annually .
the data from dpcd are readily available to researchers , and all administrative regions in denmark , danske regioner
[ danish regions ] have established standardized application forms for usage of data from all danish national health databases .
data obtained from different national registries are coupled using the unique danish civil personal registration number .
this coupling enables data from all registries , including dpcd , to be readily available for research studies.36
the treatment of pc in denmark has been centralized at four surgical centers and seven oncological departments , all of which contribute with data for the dpcd .
the members of both dpcd and dpcg represent surgeons , oncologists , radiologists , and pathologists from the participating institutions .
dpcd receives statistical , epidemiological , and data management support free of charge from danske regioner .
most of the development and some of the registrations are based on good will from the involved clinicians .
dpcd is currently undergoing a major revision , aiming at direct transfer of data from the national patient registry and the danish pathology registry , avoiding the need for manual registration by surgeons and oncologists .
afterward , the study population also includes patients from whom it was not possible to obtain a cytological or histological diagnosis , in many cases due to early death .
these patients , with a clinical pc diagnosis only , constitute 20% of all patients with pc in denmark and have a median overall survival as low as 1 month.79 hence , the overall survival of patients registered in dpcd is therefore expected to decrease .
the four departments of pathology handling pancreatic resection specimens in denmark have from 2015 adopted a national , standardized protocol for pancreaticoduodenectomy specimen examination , involving multicolor inking of resection margins and specimen surfaces , axial slicing , and extensive tissue sampling.10 using this protocol , the rate of pancreaticoduodenectomy specimens with microscopic tumor - free margins ( r0 resections ) is expected to decrease .
in the dpcd , data from patients with a histological diagnosis of ductal adenocarcinoma of the pancreas are included since may 1 , 2011 .
moreover , data from patients with a histological diagnosis of periampullary and duodenal adenocarcinoma are included since 2012 .
the total number of patients with pc registered as of june 30 , 2014 , was 2,217 , and the completeness of registration was 82% .
dpcd supports continuous quality control assessment of diagnostic workup , treatment , and outcome and provides baseline data for basic , epidemiological , and clinical research projects in an unselected , geographically well - defined western population of patients with pc . | aim of databasethe danish pancreatic cancer database aims to prospectively register the epidemiology , diagnostic workup , diagnosis , treatment , and outcome of patients with pancreatic cancer in denmark at an institutional and national level.study populationsince may 1 , 2011 , all patients with microscopically verified ductal adenocarcinoma of the pancreas have been registered in the database . as of june 30 , 2014 ,
the total number of patients registered was 2,217 .
all data are cross - referenced with the danish pathology registry and the danish patient registry to ensure the completeness of registrations.main variablesthe main registered variables are patient demographics , performance status , diagnostic workup , histological and/or cytological diagnosis , and clinical tumor stage .
the following data on treatment are registered : type of operation , date of first adjuvant , neoadjuvant , and first palliative chemo- or chemoradiation therapy , and dates for milestones in referrals , diagnostic workup , treatment decisions , and treatment . for patients undergoing resection , data on operative
evaluation of tumor stage , histological diagnosis , and duration of hospital stay are registered.descriptive datadeath is monitored using data from the danish civil registry .
this registry monitors the survival status of the danish population , and the registration is virtually complete .
all data in the database are audited by all participating institutions , with respect to baseline characteristics , key indicators , and survival .
the results are published annually.conclusionthe danish pancreatic cancer database has registered data on 2,217 patients with microscopically verified ductal adenocarcinoma of the pancreas .
the data have been obtained nationwide over a period of 4 years and 2 months .
the completeness of registration was 82% .
the observed overall 3-year survival after diagnosis was 6% . | Aim of database
Study population
Main variables
Follow-up
Examples of research
Administrative issues and funding
Future development
Conclusion |
PMC4363713 | diabetes mellitus ( dm ) is a widespread disorder with a prevalence of about 285 million in 2010 and predicated increase to 439 million by 2030 .
dr is one of the most common complications of dm and affects about 93 million people worldwide .
clinically , dr is divided into two forms : nonproliferative dr ( npdr ) and proliferative dr ( pdr ) .
diabetic macular edema ( dme ) and retinal neovascularization are the two main causes of visual impairment and blindness in patients with dr .
its pathological features include increased vascular permeability or breakdown of brb , neovascularization ( nv ) , capillary nonperfusion , endothelial cell damage , and apoptotic cell death of retinal neurons , endothelial cells , and pericytes . the early events , such as endothelial cell - leukocyte adhesion ( or retinal leukostasis ) and oxidative stress , contribute to these clinical and pathological characteristics in dr .
vegfr1 has been reported to play various roles in the vascular development , angiogenesis , cell survival , and inflammation .
first of all , as a vegf - a trap or sink , vegfr1 ( mainly soluble vegfr1 or flt1 ) , has been characterized as a negative regulator in both embryonic and postnatal vascular development [ 4 , 5 ] .
secondly , vegfr1 has been shown to be a positive mediator of pathological angiogenesis in the experimental models of some primary tumors and wet age - related macular degeneration ( amd ) .
for instance , in the oxygen - induced retinopathy ( oir ) model , vegfr1 activation by placental growth factor ( plgf ) could prevent vessel obliteration or degeneration during the hyperoxia phase , thereby preventing the subsequent vessel proliferation during the hypoxia phase .
in addition , vegfr1 signaling plays a role in regulating the chemotaxis of inflammatory cells [ 810 ] .
the functions of vegfr1 vary depending on the pathophysiological microenvironment , the type of ligand that binds ( plgf , vegf - a , or vegf - b ) , and the formation of vegfr1-vegfr2 heterodimers . whether the vegfr1 plays a role in the pathogenesis of dr remains unknown . in the present study ,
this vegfr1-specific antibody has been previously reported by us and other investigators [ 8 , 10 , 11 ] .
we found that vegfr1 blockade prevented vascular leakage and retinal leukostasis , degeneration , and disorganization of the tight junction protein zonula occludens- ( zo- ) 1 and the adhesion molecule vascular endothelial ( ve ) cadherin in dr .
all animals were used in accordance with the approved protocols by the institutional animal care and use committee of johns hopkins university school of medicine and the guidelines of the arvo statement for the use of animals in ophthalmic and vision research .
two mouse models of diabetes were used : one was streptozotocin- ( stz- ) induced method and the other was an akita diabetic mouse , both of which were described by our previous paper .
50 mg antibody per 1 kg mouse body mass was intraperitoneally ( ip ) injected three times per week as we performed previously ; rat igg was used as the treatment control .
this dose was used because it showed high efficacy in inhibiting pathological angiogenesis and infiltration of inflammatory cells in the mouse models of laser - induced cnv and oxygen - induced retinopathy ( oir ) [ 6 , 11 ] . in the present study ,
the preventative approach was implemented : the treatments started shortly after the onset of hyperglycemia and long before the occurrence of diabetic complications .
retinas were homogenized in an ice - cold lysis buffer [ 150 mm nacl , 20 mm tris ( ph 7.4 ) , 2 mm ethylenediaminetetraacetic acid ( edta ) , 1% triton x-100 , and complete mini edta - free protease inhibitor ] by sonication for 35 seconds , were incubated for 30 minutes , and were centrifuged at 14,000 g at 4c for 10 minutes .
supernatant was collected and protein concentrations were measured by the bicinchoninic acid ( bca ) method .
twenty - to - thirty micrograms of protein were electrophoresed on 4%15% gradient sds page gels and then transferred to nitrocellulose membranes . after blocking with 5% bovine serum albumin ( bsa ) or 5% nonfat milk for 1 hr
, protein blots were incubated with primary antibodies in the cold room overnight , followed by horseradish peroxidase- ( hrp- ) conjugated secondary antibodies ( 1 : 2,0005,000 ) and developed with chemiluminescence reagents ( pierce technology co. , holmdel , nj ) . the optical density ( od ) of each protein band was determined with image j software according to the user 's instructions .
the od of vegfr1 protein was normalized by dividing with that of -actin of the same protein sample .
the normalized od ratio of the diabetic sample to the nondiabetic sample was designated as fold changes .
10 m cryosections were blocked / permeabilized with pbs buffer containing 0.25% triton x-100 and 10% goat serum for one hour and incubated at 37c for two hours or overnight at 4c with primary antibodies : rat anti - vegfr1 ( imclone , somerville , nj ) , mouse anti - icam-1 ( dshb , iowa city , ia ) , rabbit anti - albumin ( nordic , capistrano beach , ca ) , rat anti - cd31 ( novus biologics , littleton , co ) , mouse anti -
ve cadherin ( dshb ) , and rat anti - zo-1 ( dshb ) . after
a rinse was repeated 3 times with pbs buffer , specimens were incubated with appropriate secondary antibodies at 1000 dilution . for double - labeling if staining of albumin and cd31/pecam1 , the cocktail of two primary antibodies from distinct species was applied , and the appropriate secondary antibodies conjugated with alex fluo 594 or 488 were used to visualize the staining with fluorescence microscopy .
dapi was used for counter - staining . in the case of mouse primary antibodies , the anti - mouse secondary antibody was preincubated with 0.03 mg / ml normal mouse igg ( invitrogen , cat no : 10400c ) to prevent its association with the endogenous mouse igg , thus minimizing background staining . for the quantitative comparison of if images ,
the specimens and images were prepared to eliminate the errors caused by variations with particular care , as described previously .
briefly , the eye samples of control and experimental groups ( n = 4 each group ) were cryopreserved in the same module with the superior quarter up . the cryosections including optical nerves were collected for immunostaining .
a zeiss axioplan2 fluorescent microscopy was used to acquire if images with axion 4 software .
the if - stained specimens were used for quantification within 1 week . the fluorescence intensity and area of if images were quantified by image j software .
the if - positive areas were first identified as a region of interest ( roi ) by running image > adjust > threshold . the mean intensity and area of roi
the results were averaged from the 4 cryosections and then expressed as mean sd per section ( 10 m ) .
total rna was prepared from the retinas of 5~6-month - old akita diabetic male mice ( with 4~5 months ' diabetic duration ) and the nondiabetic littermate male mice by using trizol agents based on the manufacturer 's manual .
the mouse inflammatory cytokine pcr array ( cat no : 330231 pamm-011a , sabiosciences ) , containing 84 important cytokine , chemokine , or receptor genes , was used to screen the novel genes whose expression was affected by diabetes .
real - time qpcr was used to quantify the gene expression alterations of cytokine and chemokine due to vegfr1 blockade .
primers were designed as follows : the mrna nucleotide sequences were obtained from the unigene database by searching their gene symbols .
the achieved sequences were aligned with the mouse genome by using an online blat program to identify the locations of exons .
the primers were designed with the primer3 software . to circumvent the contaminations from genomic dna with intron sequences , the forward and reverse primers
were located in the two or more distinct exons ( see supplementary table 1 for sequence information available online at http://dx.doi.org/10.1155/2015/605946 ) .
the quantitative brb assay was performed according to a previously described technique with some modifications .
mice were sedated as above and given an ip injection of 1 ci / gram body weight of [ h]-mannitol .
one hour after injection , the mice were sedated and retinas from the experimental and control eyes were rapidly removed . the posterior portion of the globe was firmly grasped with forceps and a razor blade was used to cut across the cornea and extrude the lens , vitreous , and retina .
retinas were dissected free from the lens , vitreous , and any rpe that was extruded and were placed within preweighed scintillation vials within 30 seconds of sacrifice . the thoracic cavity was opened and the left superior lobe of the lung was removed , blotted free of excess blood , and placed in another preweighed scintillation vial .
a left dorsal incision was made and the retroperitoneal space was entered without entering the peritoneal cavity .
the renal vessels were clamped with forceps and the left kidney was removed , cleaned of fat , blotted , and placed into a preweighed scintillation vial .
the vials containing the tissue were weighed and tissue weights were calculated and recorded . 1 ml of ncsii solubilizing solution was added to each vial and the vials were incubated overnight in a 50c water bath .
solubilized tissue was brought to room temperature ( rt ) and decolorized with 20% benzoyl peroxide in toluene in a 50c water bath .
the vials were brought to rt and 5 ml of cytoscint es and 30 l of glacial acetic acid were added .
radioactivity was counted with a ls 6500 liquid scintillation counter ( beckman , brea , ca ) .
the cpm / mg tissue was measured for the lung , kidney , and experimental and control retinas .
mice were first anesthetized with excess carbon dioxide , the descending aorta was clamped , and the right atrium was cut .
the mice were perfused with 5 ml pbs to remove erythrocytes and nonadherent leukocytes , followed by perfusion of fluorescein - conjugated con - a to label the adherent leukocytes .
the eyes were harvested and fixed for more than 1 hour with phosphate - buffered formalin .
the cornea and lens were removed and , under a stereomicroscope ( stemi 2000c ; carl zeiss meditec , inc . ,
thornwood , ny ) , the entire retina was carefully dissected from the eye cup and rapidly cut from the edge to the equator in all four quadrants and was flat - mounted with the photoreceptors facing upward .
leukocytes adherent to the vessel walls were labeled with fluorescein , and leukocytes within the vessels of each retina were counted under an epifluorescence microscope ( axio - pan2 ; carl zeiss meditec , inc . ) by an investigator masked to the nature of the specimen .
the vessel at the 12-o'clock position was first examined from the optical disk to the edge of vasculature , and the focus was adjusted as necessary to include all the arteries , veins , and capillaries in the field .
this process was repeated in a clockwise direction for each vessel radiating from the optic disc , so the total number of adherent leukocytes in all of the vessels of the retina was counted .
the mann - whitney test was used for the statistical analyses of comparisons between groups : diabetic retina versus nondiabetic retina and antibody treatment versus rat igg treatment .
increased expression of vegfr1 has been previously shown in the retinal vasculatures of diabetic rat and human [ 1416 ] , but whether its expression is also increased in diabetic mice remains unknown .
the wb results showed that vegfr1 expression was increased in stz - induced diabetic mice ( 3-month diabetes duration ) compared with nondiabetic controls ; and if staining further showed its vascular localization ( figure 1 ) . in akita
diabetic mice ( both 6- and 8-month diabetes duration ) , a robust and specific protein band of approximately 180 kda was detected by anti - vegfr1 antibody , but no or weak protein band was detected in nondiabetic mice ( supplementary figures 1(a ) and 1(b ) ) .
similarly , if staining showed that there was no or barely detectable immunoreactivity for vegfr1 in nondiabetic mice ( supplementary figure 1(c ) ) ; whereas if intensity was evidently elevated in diabetic mice with an apparent vascular localization ( supplementary figure 1(d ) ) .
quantification showed that vegfr1 protein expression was significantly upregulated by diabetes ( supplementary figures 1(e ) and 1(f ) ) .
six - week - old stz - induced diabetic mice ( about 2-week diabetes duration ) and nondiabetic mice of the same age were used for this analysis .
similar to our previous results , 2-week diabetes duration caused a significant increase of leukocytes in comparison with the nondiabetic mice ( figure 2(a ) ) .
we observed that the leukocytes presented in capillary as well as arteries and veins in the diabetic retina ( figures 2(b)2(d ) ) .
it was obvious that the cells in the capillary vessels transverse the whole lumen space , thus becoming an obstacle for blood flow . on some occasions , the cells aggregated at the root of branches from the larger vessels to the smaller capillaries ( figure 2(c ) ) , preventing the blood from circulating into the capillaries .
in nondiabetic mice , icam-1 immunoreactivity was generally weak in the majority of blood vessels ; very few blood vessels showed strong immunoreactivity ( figure 3(a ) ) .
icam-1 immunoreactivity was strong in all icam-1(+ ) blood vessels in diabetic mice ( figure 3(b ) ) .
icam-1 immunoreactivity was weaker in the mf1-treated mice than in rat igg - treated ones .
the signal intensity of icam-1 immunoreactivity was faint in the majority of blood vessels but strong only in very few blood vessels ( figure 3(c ) ) .
mf1 treatment apparently resulted in a reduction of icam-1 immunostaining intensity compared with the mice treated with rat igg in the vasculature .
quantification further showed the significant differences among the three treatment groups ( figure 3(d ) ) .
albumin staining was used to examine the sites of vascular leakage and the effects of vegfr1 blockade .
double - labeling immunofluorescence staining showed that the albumin protein colocalized well with the endothelial cell marker cd31/pecam-1 in nondiabetic mice , suggesting its localization in the blood vessels without leakage ( figures 4(a)4(c ) ) .
by contrast , albumin immunoreactivity was not restrained to the cd31 ( + ) vessel areas in the diabetic mice treated with rat igg ( figures 4(d)4(f ) ) . in the diabetic mice treated with mf1
, the staining results showed that albumin protein colocalized largely with cd31 ( figures 4(g)4(i ) ) , which was very similar to those from the nondiabetic mice .
quantification showed the intensity ratio of albumin and cd31 immunoreactivity was significantly different among the three groups ( figure 4(j ) ) .
in addition , the degree of vascular leakage was further measured by a quantitative brb assay .
the results showed that retina to lung leakage ratio ( rllr ) and retina to renal leakage ratio ( rrlr ) were significantly reduced by vegfr1 blockade ( figure 5 ) .
we investigated the effects of vegfr1 blockade on the expression and localization of zonula occludens- ( zo- ) 1 and vascular endothelial ( ve ) cadherin , which are the tight junction and adhesion proteins of brb .
ve cadherin immunoreactivity was very strong and showed a very sharp and blood vessel - like staining pattern in nondiabetic mice , whereas the staining looked foggy and cloudy in diabetic mice . in mf1 , the staining showed clear and vessel - like patterns similar to those observed in the nondiabetic retinas ( figures 6(a)6(c ) ) .
the quantification showed that the fluorescence intensity of ve cadherin was significantly lower in diabetic mice than nondiabetic mice and mf1 treatment significantly increased its expression compared with the rat igg control treatment .
in nondiabetic mice , if staining of zo-1 showed a ring and string pattern , which may reflect that zo-1 proteins are well localized in the walls of both large retina arteries and veins as well as in the small capillaries ( figure 7(a ) ) ; in diabetic mice , the staining looked like short rod and stick , indicating that zo-1 proteins were displaced in the large vessels and were diminished in the capillaries ( figure 7(b ) ) . in the antibody
quantification showed that the mean size of the zo-1(+ ) vessels was significantly different ( figure 7(d ) ) .
we investigated the influences of vegfr1 blockade on the expression of 84 important cytokine and chemokine genes that were contained in an array plate . in total , the expression of 67 genes was found in the retina .
the expression of 9 genes was detected only in the diabetic retinas but not in the nondiabetic retinas : cxcr2 , cxcl9 , itb , ccl8 , il1f6 , ccl4 , cxcl10 , cxcl11 , and ccl20 .
expressions of 11 genes were upregulated by diabetes : cxcl13 , ccl5 , il18 , pf4 , il13 , abcf1 , cxcr5 , il10 , c3 , il16 , and il15 ( figure 8(a ) ) , and expressions of 31 genes were downregulated by diabetes : such as tnfrsf1a , casp1 , ccr7 , and cxcl10 ( figure 8(b ) ) .
furthermore , the genes whose expressions were altered by diabetes were selected to investigate the effects by vegfr1 blockade .
expressions of 10 genes were found to be altered by vegfr1 blockade compared with rat igg control : cxcl10 , il10 , ccl8 , il1f6 , cxcl15 , ccl4 , il13 , ccl6 , casp1 , and ccr5 ( figures 8(c ) and 8(d ) ) .
in this study , we first found that vegfr1 protein expression was increased more than 10-fold in the retinas of diabetic mice compared with nondiabetic mice .
this result is consistent with previous reports that showed upregulation of vegfr1 expression in several other species , such as rat and human [ 1416 ] .
we then found that vegfr1 blockade prevented vascular leakage and retinal leukostasis , degeneration , and disorganization of the tight junction protein zo-1 and the adhesion molecule ve cadherin in dr .
we started treating the mice shortly after the onset of hyperglycemia and long before the occurrence of any complications of dr .
therefore , the results of this study display the preventative effects of vegfr1 blockade on vascular complications dr . in order to know whether vegfr1 blockade can also be therapeutically effective or reverse the disease progression
, it would be necessary to treat mice at a later stage when complications of diabetes have already taken place because dr is a chronic ischemic and inflammatory disorder and many diabetes - associated complications start appearing at relatively later stages . finally , we found that vegfr1 blockade inhibited gene expression of inflammatory marker icam-1 and other cytokine and chemokine genes , providing further evidence that vegfr1 signaling is involved in the regulation of inflammation in dr .
our findings suggest that targeting vegfr1 may be a complementary strategy to anti - vegf therapy , which is widely used to treat vascular disorders , including , age - related macular degeneration ( amd ) , dr , and dme .
for instance , vegfr1 signaling has been suggested to be associated with the diseased cells and tissues more than the normal ones , so inhibiting its activity likely results in less side effects than anti - vegf therapy .
the potency of blocking vegfr1 appears comparable to that of anti - vegf antagonists in the experimental models of cancer and ocular angiogenesis [ 6 , 18 ] .
vegfr1 has been shown to inhibit infiltration of inflammatory cells or orchestrate interplay of vegf - a , plgf , and vegfr2 [ 11 , 19 ] .
in addition , another possible alternative strategy could target the upstream regulators of vegf , such as prolyl hydroxylase ( phd ) and hypoxia inducible factor ( hif ) .
because they regulate a broad spectrum of gene expressions , inhibition of their activities may lead to a normalization of vasculature instead of impairment and degeneration .
in addition to vegf and its receptor signaling , a number of other molecules and signaling pathways are implicated in the pathogenesis of dr , such as protein kinase c- ( pkc- ) /- [ 21 , 22 ] , transforming growth factor- ( tgf- ) , tumor necrosis factor- ( tnf- ) , -catenin ( a key regulator of wnt pathway ) , cyclooxygenase- ( cox- ) 2 , nadph oxidase ( nox)2 , arginase-1/-2 , aldose / aldehyde reductase ( ar ) , nuclear factor kappa - light - chain - enhancer of activated b ( nf-b ) cells , forkhead box protein o1 ( foxo1 ) , inducible nitric oxide synthase ( inos ) , and altered o - glcnac signaling .
understanding these underlying molecular cascades helps develop therapeutic strategies and ushers in clinical trials or applications , especially targeted therapy .
the most successful example is vegf , which was originally discovered as a vascular permeability and mitogen factor [ 33 , 34 ] .
the two vegf antagonists lucentis ( ranibizumab ) and eylea ( aflibercept ) have been approved by us food and drug administration to treat the patients with dme . despite the success , special attention
must be paid to the safety and efficacy of treatments in patients , because they do not provide a cure and not all patients respond ; there are potential side effects since vegf is a survival factor for choriocapillary , retinal neuron , and rpe [ 3537 ] .
therefore , search for more optimal therapeutic strategies is necessary for the improvement of anti - vegf therapy . in summary
, the results from this study suggest that vegfr1 is involved in the pathogenesis of dr and a potential target for this disease .
the development of vegfr1 inhibitors is clinically relevant because they could be potentially used for alternative and/or combinatory treatments to anti - vegf therapy . | diabetic retinopathy ( dr ) is a leading cause of blindness in working age adults .
the objective of this study is to investigate the effects of vascular endothelial growth factor receptor 1 ( vegfr1 ) blockade on the complications of dr .
experimental models of diabetes were induced with streptozotocin ( stz ) treatment or insulin2 gene mutation ( akita ) in mice . protein expression and localization
were examined by western blots ( wb ) and immunofluorescence ( if ) .
mrna expression was quantified by pcr array and real - time pcr .
the activity of vegfr1 signaling was blocked by a neutralizing antibody called mf1 .
vascular leakage was evaluated by measuring the leakage of [ 3h]-mannitol tracer into the retina and the if staining of albumin .
vegfr1 blockade significantly inhibited diabetes - related vascular leakage , leukocytes - endothelial cell ( ec ) adhesion ( or retinal leukostasis ) , expression of intercellular adhesion molecule- ( icam- ) 1 protein , abnormal localization and degeneration of the tight junction protein zonula occludens- ( zo- ) 1 , and the cell adhesion protein vascular endothelial ( ve ) cadherin .
in addition , vegfr1 blockade interfered with the gene expression of 10 new cytokines and chemokines : cxcl10 , il10 , ccl8 , il1f6 , cxcl15 , ccl4 , il13 , ccl6 , casp1 , and ccr5 .
these results suggest that vegfr1 mediates complications of dr and targeting this signaling pathway represents a potential therapeutic strategy for the prevention and treatment of dr . | 1. Introduction
2. Methods
3. Results
4. Discussion |
PMC5407697 | in response to a rise in blood glucose level , the pancreas secretes insulin into the bloodstream where it is quickly distributed to its target tissues such as skeletal muscle , via resistance arteries and capillaries .
the delivery of insulin to the skeletal muscle interstitium has been shown to be a rate limiting step for the metabolic actions of insulin that promote glucose disposal234 . within 10 - 15 min , insulin increases the capillary blood volume ( microvascular recruitment ) , an effect that occurs before total blood flow increases56 .
microvascular recruitment expands the endothelial surface area available for exchange of nutrients ( and insulin)78 .
insulin - mediated microvascular recruitment precedes and is independently associated with changes in skeletal muscle glucose uptake89 .
the effect of insulin on the vasculature has been termed ' vascular insulin sensitivity ' .
it has been shown that insulin - mediated microvascular recruitment and insulin - induced vasodilation are impaired in obese zucker rats1011 .
moreover , lean mice with reduced capillary density display muscle insulin resistance12 . in their influential work , kubota et al . showed that impaired insulin signaling in endothelial cells caused reduction in insulin - induced microvascular recruitment , which reduced glucose uptake in skeletal muscle by approximately 40%13 .
these abnormalities in microvascular function do not only occur in muscle , but also in multiple other tissues and organs such as the heart , retina and kidney141516 .
these examples and other studies17181920 suggest that vascular effects of insulin are an important mechanism in the ( patho)physiology of insulin resistance and its complications . while there is substantial evidence that insulin increases microvascular blood volume ( mbv ) in skeletal muscle56 , the mechanisms by which this happens are not completely understood9 .
endothelium - dependent vasodilation is essential in many aspects of vascular insulin sensitivity212223 at different levels of the vasculature .
vascular insulin sensitivity can manifest itself by insulin - induced relaxation of resistance arteries and by relaxation of pre - capillary arterioles to increase the perfused microvascular exchange surface area72425 .
intravital microscopy ( ivm ) has been used in a variety of tissue preparations including skinfold chambers of the mouse dorsum26 , the mesentery of the mouse and rat27 , models of limb ischemia in the mouse28 and the hamster cheek pouch29 .
contrast - enhanced ultrasonography ( ceus ) is another imaging technique that allows assessment of the microcirculation in cardiac30 as well as skeletal muscle31 .
it utilizes inert gas - filled microbubbles which behave rheologically as red blood cells and remain entirely within the vascular lumen .
these microbubbles are infused intravenously at a constant rate to attain a steady state . a high energy ultrasound wave , then
the microbubbles ' replenishment speed in the region of interest ( roi ) represents flow velocity ( mfv ) .
ceus can be performed repeatedly ( also in humans ) and it has advanced the understanding of vascular dysfunction that occurs in insulin - resistant states ( discussed in barrett et al.32 ) . in the current study we describe a new technique for studying regulation of muscle perfusion , through simultaneous use of both ivm and ceus . here
we focus on insulin 's vascular actions in the adductor compartment of the mouse hindlimb .
this compartment is one of the largest skeletal muscle groups in the mouse , enabling studies of local glucose uptake in a representative muscle .
this compartment is ideal for ivm as the preparation and visualization of the arteries are readily accessible by a standardized surgical procedure28 .
moreover , our own group and others have shown that ceus can be used in this compartment3334 . an advantage to the combined ivm and ceus technique is the possibility to assess insulin 's effect at the level of the larger arterioles ( feed or resistance arteries ) and the microcirculation ( capillary beds ) in the same muscle group .
in addition , the simultaneous application of the two methods provides insight into the temporal action of insulin at the levels of the resistance arteries and microcirculation .
this combined ivm and ceus technique can also be implemented in other vascular biology fields .
for example , the role of various proteins and certain pathophysiological conditions affecting the endothelium can be studied using knockout models . moreover ,
both techniques can be used in one mouse at multiple time points reducing the time and cost of research .
all animal experiments have been approved by the local animal care and ethical committee . the entire protocol from induction of anesthesia in the mouse until the end of the hyperinsulinemic - euglycemic
clamp takes approximately 2 h. induce anesthesia along with analgesia in a male mouse weighing 20 - 25 g after a 14 h or overnight fasting with an intraperitoneal injection of a fentanyl ( 0.31 mg / kg ) , midazolam ( 6.25 mg / kg ) and acepromazine ( 6.25 mg / kg ) ( fma anesthesia ) and place it on a rectal - temperature - controlled homeothermic heating pad which maintains body temperature at 37 c . disinfect the operation table and the equipment several times using an alcohol - based solution . attach a 27 g needle to a 10 cm long polyethylene tube ( pe-20 ) and attach the tube to a 4-way connector .
this cannula will be used for infusion of microbubbles , insulin , glucose and anesthesia .
note : adding heparin ( 5 u / ml ) in sterile saline during the cannulation process to flush the tail vein ( approximately 10 l ) reduces the possibility of clogged cannulas . throughout the surgical procedures and experimental protocols ,
maintain anesthesia by a continuous intravenous infusion of fma anesthesia via the tail vein cannula at rate of 33.75 l / kg / min .
place the mouse with the ventral side up and fix the feet using a thermostable tape to expose the upper thigh area .
use a slight exorotation of the hip joint ( hindlimb paws facing up ) and a 40 - 60 angle at the knee joint to standardize the stretch of the muscle at the adductor compartment of the thigh . remove the hair at the groin and upper thigh areas bilaterally using a depilatory cream .
place the mouse under a stereomicroscope and perform the following surgical steps using 10x to 16x magnification .
make a 2 cm incision using skin scissors running parallel to the inguinal ligament , just lateral to the abdominal curvature ( figure 1 ) .
apply traction on the distal side of the incision distally using a bulldog clamp ( figure 1d ) .
this will help to adjust the window as needed and help hold the paraffin oil ( described in 1.12 ) .
gently separate the fat pad from the wall instead of dissecting directly through the pad .
gentle traction at the fat pad in the distal direction will facilitate the process ( figure 1c ) . identify the femoral artery and follow it down to the first major branches ( the epigastric artery and the gracilis artery ) ( figure 2 ) .
the gracilis artery is the first major branch of the femoral artery running on the adductor magnus muscle and then runs deep to the gracilis muscle .
the gracilis artery will be used for ivm . identify the transparent deep fascia covering the muscles and the vessels . using sharp forceps , pull the fascia upwards and cut it using a microscissor . cover the exposed muscle with a drop ( 200 l ) of medicinal liquid paraffin ( room temperature , or pre - warmed to 37 c ) to prevent the prepared tissue from drying out .
adjust the skin folds of the incision using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels .
place the mouse under a previously calibrated microscope ( 16x optical magnification ) in such a way that the gracilis artery is vertical on the computer screen .
attach the microscope to a camera and computer based analysis system that can extract the vessels ' diameter from the image data set .
place the light source at a sufficient distance ( minimum 20 cm ) from the hindlimb to reduce head conduction from the light .
carefully adjust the angle and direction of the ultrasound probe to get a cross - sectional view of the adductor muscle group .
take care to keep the position of the ultrasound probe in relation to the mouse stable to keep the same imaging plane for baseline and hyperinsulinemic measurements .
the diameter of the gracilis artery should be stable for 10 min before documenting the baseline diameter .
make sure that the baseline gracilis artery diameter is saved by the computer program used for the ivm .
prepare the microbubbles in advance as described35 and count with coulter counter to a concentration of 2.5 x 10 bubbles / ml just before the experiment .
as the microbubbles can only be viewed in the contrast - mode of the ultrasound machine , control the parameters which affect the image and the contrast data collected ( described in 2.3.1 ) and use consistently during acquisition .
use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest .
use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest .
start infusing the microbubbles using the tail vein cannula at a 5 l / min rate .
place the infusion tube on a vibrating vortex ( 200x per min ) to maintain a uniform suspension of microbubbles .
allow 5 min of continuous infusion of microbubbles so that a steady - state level is reached .
proceed with obtaining the time - intensity curves of the bubbles using the microbubble destruct function ( mbd ) on the ultrasound machine at 5 min and at 10 min after the start of microbubbles infusion ( figure 3a ) .
take the average signal of these two measurements to get the baseline perfusion data .
after obtaining the baseline data , start the hyperinsulinemic - euglycemic clamp as described34 . use the tail cannula ( placed in 1.3 ) to administer the insulin and glucose ( and anesthesia ) .
in short ,
induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion
use a variable infusion of 20% d - glucose to maintain euglycemia.assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate .
determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min .
in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion
assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate .
determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min . make sure that the diameter of the gracilis artery is documented at the desired time periods ( for example at 10 , 30 and 60 min ) of the start of the hyperinsulinemic - euglycemic clamp with the computer program .
after 25 min and/or 55 min of the insulin clamp , start the second ( hyperinsulinemic ) ceus measurement to document the mbv at 30 and/or 60 min , respectively .
after completion of ivm and the ceus measurements 60 min after the start of insulin infusion , withdraw blood from the mouse by a heart puncture procedure for later analysis .
this will also euthanize the mouse . carefully dissect the gracilis and femoral arteries and store them for further desired experiments ( for e.g. , western blots , immunohistochemistry , ex vivo pressure myography experiments363738 ) .
note : the analyses of ivm and ceus measurements should be performed offline by a blinded investigator .
ceus offers the possibility to distinguish the microcirculation from larger vessels by temporarily destructing the microbubbles by high intensity ultrasound waves using the mbd function .
the signal ( measured in arbitrary units ( a.u ) ) in larger vessels is restored quicker than those in the microcirculation because of the microbubbles speed in the corresponding vessels .
use an offline workstation or the software on the ultrasound machine to do the analyses . draw a region of interests ( roi ) to include the microcirculation . draw a separate roi to include the larger femoral vessels ( figure 3a ) .
duplicate the microcirculation 's and larger vessels ' rois for the background , baseline and hyperinsulinemic measurements by using the roi copy function built in the software .
subtract the intensity signal of the background measurement from the baseline and the hyperinsulinemic measurements .
divide the intensity signal of the microcirculation by the intensity signal of the femoral vessels .
induce anesthesia along with analgesia in a male mouse weighing 20 - 25 g after a 14 h or overnight fasting with an intraperitoneal injection of a fentanyl ( 0.31 mg / kg ) , midazolam ( 6.25 mg / kg ) and acepromazine ( 6.25 mg / kg ) ( fma anesthesia ) and place it on a rectal - temperature - controlled homeothermic heating pad which maintains body temperature at 37 c . disinfect the operation table and the equipment several times using an alcohol - based solution . attach a 27 g needle to a 10 cm long polyethylene tube ( pe-20 ) and attach the tube to a 4-way connector .
this cannula will be used for infusion of microbubbles , insulin , glucose and anesthesia .
note : adding heparin ( 5 u / ml ) in sterile saline during the cannulation process to flush the tail vein ( approximately 10 l ) reduces the possibility of clogged cannulas . throughout the surgical procedures and experimental protocols ,
maintain anesthesia by a continuous intravenous infusion of fma anesthesia via the tail vein cannula at rate of 33.75 l / kg / min .
place the mouse with the ventral side up and fix the feet using a thermostable tape to expose the upper thigh area .
use a slight exorotation of the hip joint ( hindlimb paws facing up ) and a 40 - 60 angle at the knee joint to standardize the stretch of the muscle at the adductor compartment of the thigh . remove the hair at the groin and upper thigh areas bilaterally using a depilatory cream .
place the mouse under a stereomicroscope and perform the following surgical steps using 10x to 16x magnification .
make a 2 cm incision using skin scissors running parallel to the inguinal ligament , just lateral to the abdominal curvature ( figure 1 ) .
apply traction on the distal side of the incision distally using a bulldog clamp ( figure 1d ) .
this will help to adjust the window as needed and help hold the paraffin oil ( described in 1.12 ) .
dissect the adipose tissue away from the abdominal wall . to avoid bleeding , gently separate the fat pad from the wall instead of dissecting directly through the pad .
gentle traction at the fat pad in the distal direction will facilitate the process ( figure 1c ) . identify the femoral artery and follow it down to the first major branches ( the epigastric artery and the gracilis artery ) ( figure 2 ) .
the gracilis artery is the first major branch of the femoral artery running on the adductor magnus muscle and then runs deep to the gracilis muscle .
the gracilis artery will be used for ivm . identify the transparent deep fascia covering the muscles and the vessels . using sharp forceps , pull the fascia upwards and cut it using a microscissor . cover the exposed muscle with a drop ( 200 l ) of medicinal liquid paraffin ( room temperature , or pre - warmed to 37 c ) to prevent the prepared tissue from drying out .
adjust the skin folds of the incision using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels .
place the mouse under a previously calibrated microscope ( 16x optical magnification ) in such a way that the gracilis artery is vertical on the computer screen .
attach the microscope to a camera and computer based analysis system that can extract the vessels ' diameter from the image data set .
place the light source at a sufficient distance ( minimum 20 cm ) from the hindlimb to reduce head conduction from the light .
carefully adjust the angle and direction of the ultrasound probe to get a cross - sectional view of the adductor muscle group .
take care to keep the position of the ultrasound probe in relation to the mouse stable to keep the same imaging plane for baseline and hyperinsulinemic measurements .
the diameter of the gracilis artery should be stable for 10 min before documenting the baseline diameter .
make sure that the baseline gracilis artery diameter is saved by the computer program used for the ivm .
prepare the microbubbles in advance as described35 and count with coulter counter to a concentration of 2.5 x 10 bubbles / ml just before the experiment .
as the microbubbles can only be viewed in the contrast - mode of the ultrasound machine , control the parameters which affect the image and the contrast data collected ( described in 2.3.1 ) and use consistently during acquisition .
use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high ; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off . level the location of focal zones to the center of the region of interest .
use the following settings on the ultrasound machine : contrast gain at 35 decibels ; the time gain compensation to off ; line density to high
; number of focal zones to wide ; transmit power to 4% ; transmit beam width to standard ; sv gate to 4 ; sensitivity to 1 ; persistence to off .
start infusing the microbubbles using the tail vein cannula at a 5 l / min rate .
place the infusion tube on a vibrating vortex ( 200x per min ) to maintain a uniform suspension of microbubbles .
allow 5 min of continuous infusion of microbubbles so that a steady - state level is reached .
proceed with obtaining the time - intensity curves of the bubbles using the microbubble destruct function ( mbd ) on the ultrasound machine at 5 min and at 10 min after the start of microbubbles infusion ( figure 3a ) .
take the average signal of these two measurements to get the baseline perfusion data .
after obtaining the baseline data , start the hyperinsulinemic - euglycemic clamp as described34 . use the tail cannula ( placed in 1.3 ) to administer the insulin and glucose ( and anesthesia ) .
in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion ( 7.5 mu / kg / min ) for 60 min . use a variable infusion of 20% d - glucose to maintain euglycemia.assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate .
determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min .
in short , induce a hyperinsulinemic state by introducing a 200 mu / kg insulin bolus followed by continuous insulin infusion
assess blood glucose from the tail vein every 5 min with a glucose monitoring device , and maintain at 5 mm by adjusting the variable glucose infusion rate .
determine insulin sensitivity by averaging the mean glucose infusion rate during the last 30 min .
make sure that the diameter of the gracilis artery is documented at the desired time periods ( for example at 10 , 30 and 60 min ) of the start of the hyperinsulinemic - euglycemic clamp with the computer program .
after 25 min and/or 55 min of the insulin clamp , start the second ( hyperinsulinemic ) ceus measurement to document the mbv at 30 and/or 60 min , respectively .
after completion of ivm and the ceus measurements 60 min after the start of insulin infusion , withdraw blood from the mouse by a heart puncture procedure for later analysis .
this will also euthanize the mouse . carefully dissect the gracilis and femoral arteries and store them for further desired experiments ( for e.g. , western blots , immunohistochemistry , ex vivo pressure myography experiments363738 ) .
note : the analyses of ivm and ceus measurements should be performed offline by a blinded investigator .
ceus offers the possibility to distinguish the microcirculation from larger vessels by temporarily destructing the microbubbles by high intensity ultrasound waves using the mbd function .
the signal ( measured in arbitrary units ( a.u ) ) in larger vessels is restored quicker than those in the microcirculation because of the microbubbles speed in the corresponding vessels .
use an offline workstation or the software on the ultrasound machine to do the analyses . draw a region of interests ( roi ) to include the microcirculation . draw a separate roi to include the larger femoral vessels ( figure 3a ) .
duplicate the microcirculation 's and larger vessels ' rois for the background , baseline and hyperinsulinemic measurements by using the roi copy function built in the software .
subtract the intensity signal of the background measurement from the baseline and the hyperinsulinemic measurements .
divide the intensity signal of the microcirculation by the intensity signal of the femoral vessels .
glucose infusion rate during the hyperinsulinemic - euglycemic clamp ( insulin sensitivity ) was 180.21 19.81 mol / kg / min . local application of paraffin oil on the adductor muscle compartment to stabilize the vessel did not change the average baseline diameter of the arteries ( 73.6 29.0 m vs. 68.8 17.9 m ; p = 0.58 ) but helped reduce the variation the animals tested ( figure 4a ) .
insulin consistently increased the gracilis artery diameter ( by 14.58 6.2% at 60 min ; n = 9 ) which was significantly different ( p < 0.0001 ) from the diameter change caused by saline infusion ( -6.3 4.9% ; n = 6 ) .
insulin - induced vasodilation was appreciable after 10 min ( 10.09 5.1% ; p = 0.002 ) and reached approximately 95% of its maximum dilatory capacity after 30 min .
using ceus , insulin consistently increased muscle mbv ( figure 5a ) by 33.5% ( 31.04% ,
n = 7 ; p = 0.0009 ) when compared to saline infusion ( -10.63 27.87% , n = 6 ) ( figure 5b ) .
the data presented are the signal intensities of the muscle mbv divided by that in the femoral vessels .
this reduces experimental variation between different measurements and between different mice ( data not shown ) .
the signal intensity in the femoral vessels corresponds linearly with the concentration of microbubbles in the circulation ( figure 3c ) .
correcting for the femoral vessels ' signal theoretically corrects for differences in concentrations of microbubbles used ( figure 3d ) .
( a ) an incision is made at the groin , parallel to the direction of the inguinal ligament .
( b ) gentle traction at the fat pad in the distal directions ( black arrows ) will present the connective tissue ( * ) between the fat pad and the abdominal wall .
( c ) the skin folds of the incision can be adjusted using the bulldog clamp to create a small cavity to hold the paraffin oil that bathes the vessels .
( d ) the ultrasound probe is positioned on the contralateral upper hindlimb after the prepared gracilis artery is viewed using a calibrated microscope .
the femoral artery ( a ) gives rise to the epigastric artery ( b ) and the gracilis artery ( c ) which runs over the adductor muscle group ( d ) .
figure 3:signal intensity of contrast - enhanced ultrasonography in muscle microvascular blood volume and femoral vessels .
( a ) view of the nonlinear contrast imaging mode of the digital imaging platform during microbubbles constant infusion in a male mouse upper hindlimb .
right panel : two rois are drawn to represent the muscle mbv and the femoral vessels . only the superficial part of the adductor muscle compartment is included in the roi as the signal intensity decreases with depth . left panel : time - intensity curve from the muscle mbv roi .
vertical lines represent the destruction of the microbubbles ( mbd ) with high energy waves . immediately after the mbd , no contrast agent is present in the imaging plane which starts to fill with microbubbles gradually .
after 10 - 15 s , the peak of the contrast enhancement has been reached .
( b - d ) after a steady - state signal was reached , the infusion rate of 2.5 x 10 bubbles / ml was doubled ( 5 , 10 , 20 l / min ) . signal intensity from muscle mbv ( b ) and
femoral vessels ( c ) paralleled the doubling of the microbubble concentration in the circulation .
( d ) correcting muscle mbv for the femoral vessels signal removes the variability in signal intensity caused by different microbubble concentrations ( n = 9 ; error - bars represent standard deviation ) .
( a ) paraffin oil reduces the variation of the gracilis arteries of different animals ( which is 29.0 m without paraffin vs 17.9 m after applying the oil ) while keeping the average baseline diameter stable ( 73.6 m vs. 68.8 m ; p = 0.58 ) .
( b ) arterial diameters at baseline and after 60 min of insulin or saline infusions .
( c ) insulin - induced vasodilation occurs at 10 min after the start of infusion ( p = 0.002 ) and reaches 95% of the maximum at 30 min .
error - bars represent standard deviation ; unpaired student 's t - test is used for statistics .
figure 5:microvascular blood volume measurements using contrast - enhanced ultrasonography of the adductor muscle compartment of the mouse hindlimb .
( a ) insulin resulted in a consistent increase in mbv 30 min after the start of insulin infusion .
( b ) the difference between the hyperinsulinemic and the baseline measurements ( mbv change ) is denoted as the insulin - mediated microvascular recruitment .
insulin induced a 33.5% ( 31.04% , p = 0.016 ; n = 7 ) microvascular recruitment compared to saline infusion ( -10.63 27.87% , n = 6 ) .
error - bars represent standard deviation ; unpaired student 's t - test is used for statistics .
we have developed a technique to simultaneously estimate insulin 's vascular actions on the larger arteries ( using ivm ) and the skeletal muscle microcirculation ( using ceus ) .
the critical steps for successful and reliable measurements are : 1 ) correctly exposing the gracilis artery without bleeding ; 2 ) preventing leakage of the paraffin oil bathing the artery ; 3 ) having a patent venous access ( tail vein cannula ) for infusion of vasoactive compounds ( insulin ) and contrast agent ( microbubbles ) .
the study of microvascular dysfunction in muscle has been gaining attention in the context of obesity and insulin resistance14253940 .
the negative impact of obesity and insulin resistance on vascular function is manifested at different levels of the vascular tree .
the combined use of the ivm and ceus techniques in the same mouse provides a powerful tool to quantify insulin 's effects at different levels of the vasculature .
ivm allows direct visualization and quantitative analysis of the resistance artery and ceus allows for assessment of insulin - induced changes in muscle perfusion .
the arteries are readily accessible and the superficial nature of the incision makes it possible to close the skin incision with a 5.0 absorbable sterile suture after the experiment is finished .
the animals were injected subcutaneously with buprenorphine after the experiments as an analgesic at a dose of 0.1 mg / kg and allowed to recover in a warm environment .
the mice tolerated the procedure very well and we experienced no loss of animals nor infections of the hindlimb in more than 35 animals studied .
this makes it possible to follow - up or study the animals in a longitudinal fashion .
the animals used in these experiments , however , were anesthetized using 1.8% inhaled isoflurane balanced with oxygen flowing at 0.4 l / min though an anesthesia mask . in contrast to isoflurane anesthesia4142 , fma anesthesia does not disturb peripheral insulin sensitivity . a future plan is to study how well the mice recover from fma anesthesia .
the adductor muscle compartment is also useful since various vasoactive compounds mediating local and downstream vascular effects can be evaluated .
for example , topical application of these compounds to the target tissue is feasible using superfusion techniques28 or surgical manipulation and implantation of drug - eluting cuffs surrounding the vessels43 .
our group and others have gathered substantial experimental evidence using the pressure myograph to document the effects of insulin and other vasoactive compounds on this artery ex vivo363738 .
a limitation inherent to the use of the ivm technique is the surgical exposition of the muscle and application of paraffin oil to stabilize the vessels .
however , figure 3a shows that the baseline diameter of the gracilis artery bathed in paraffin oil does not change considerably .
it has been also shown that mineral oil successfully inhibits the diffusion of oxygen , protecting the tissue from hyperoxic conditions44 .
moreover , the oil helps to reduce the variation in the baseline diameter of the arteries .
this is why we advocate to use paraffin oil and let the preparation rest for at least 30 min .
of note , the use of buffered saline instead of oil or no oil at all resulted in highly variable diameters and constriction of the vessel ( data not shown ) . moreover , at the end of the experiments , we isolated the gracilis arteries - bathed in paraffin oil - and tested their reactivity in the pressure myograph ex vivo .
the paraffin oil - bathed arteries reacted similarly to control arteries when stimulated with insulin and acetylcholine ( a vasodilator ) ( data not shown ) .
the consistent insulin - induced vasodilation clearly shows that the ivm protocol described in this study produces reliable results .
the advantage of applying both techniques in the same mouse overcomes some of the intrinsic limitations of one technique by the other : ceus estimates mbv in the undisturbed muscle in vivo , but individual vessels can not be seen ; ivm makes it possible to see individual vessels , albeit it can not estimate mbv .
a future plan is to utilize ivm microscopy of the cremaster muscle in combination with ceus of the adductor muscle on the contralateral side .
this modification can provide an estimate of the mbv ( using ceus ) and a direct optical access to the capillaries ( using ivm ) .
the protocol can be further modified ; the 4-way connector used for the tail cannula can be switched to a 5-way connector . by this
, we can avoid detaching the anesthesia tube while performing the second ceus measurement ( described in point 2.9 ) .
another modification that can be made to this protocol is the insulin clamp rate used .
we used 7.5 mu / kg / min clamp rate which is considered supra - physiological . depending on the study ,
a lower insulin clamp rate ( for example 3 mu / kg / min ) can be used . while we have found the described protocol reliable , there are specific limitations that need attention .
it is crucial that the paraffin oil does not leak from the vessel environment as supplementing it with new oil will disturb the vessel and change the diameter , making it necessary to let the artery rest for another 30 min .
in addition , the reflection of the light ( described in step 1.14 of the protocol ) on the surface of the paraffin oil was sometimes too bright , making it difficult to view the artery .
this can be counteracted by directing the light source so that the light falls at an angle to the paraffin oil surface and parallel to the artery . in conclusion
, the combination of ivm and ceus techniques described in this study makes it possible to quantify different effects of insulin at different levels of the vasculature .
ivm of the gracilis artery provides insight into the upstream vascular changes contributing to downstream microvascular perfusion measured using ceus .
we advocate the combination of several experimental techniques in the same mouse to better assess the vascular function .
visual sonics inc . covered the open access fees , whereas the structure and content of the article remained the full responsibility of the authors . | it has been demonstrated that insulin 's vascular actions contribute to regulation of insulin sensitivity .
insulin 's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin - sensitive tissues .
we here describe a technique for combining intravital microscopy ( ivm ) and contrast - enhanced ultrasonography ( ceus ) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo .
simultaneously assessing insulin 's effect at multiple levels of the vascular tree is important to study relationships between insulin 's multiple vasoactive effects and muscle perfusion .
experiments in this study were performed in mice .
first , the tail vein cannula is inserted for the infusion of anesthesia , vasoactive compounds and ultrasound contrast agent ( lipid - encapsulated microbubbles ) .
second , a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment .
the ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross - section . to assess baseline parameters , the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume ( mbv ) . when applied before and during a hyperinsulinemic - euglycemic clamp , combined ivm and ceus allow assessment of insulin - induced changes of arterial diameter , microvascular muscle perfusion and whole - body insulin sensitivity . moreover , the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified .
it is also possible to follow - up the mice longitudinally in time , making it a valuable tool to study changes in vascular and whole - body insulin sensitivity . | Introduction
Protocol
1. Microsurgical Preparation
2. Baseline and Hyperinsulinemic Measurements
3. Offline Analysis
Representative Results
Discussion
Disclosures |
PMC3426986 | in 1889 , when charles lloyd tuckey produced the first modern british medical work on hypnotism , many physicians eagerly anticipated enormous changes in medical thought and practice .
following on the heels of numerous physiological and pathological discoveries , to many physicians hypnotic therapy appeared to be the next stage in an all - encompassing scientific revolution in medicine . taking tremendous care to distance modern scientific hypnotism from the dubious work of mesmer and his followers , british medical supporters of hypnotism situated their experimental investigations in a scientific tradition that began more directly with their compatriot james braid the manchester surgeon who coined the term in 1843 .
unlike practitioners of mesmerism in the first half of the nineteenth century , braid had viewed the patient s trance state as deriving from mental , rather than physical
, the trance state was induced by the patient him / herself because it was based upon a willed decision to become entranced a state which he likened to sleep through the rigorous focus of the individual s full attention . in therapeutic practice
, he held that the physician acted only as a guide for the hypnotised patient in helping to focus attention on producing relief from a wide variety of ailments .
looking back to braid s important discoveries in the 1840s ,
hypnotism s supporters criticised the british medical profession for having failed to pursue hypnotic therapy after braid s death in 1860 .
following many decades of scientific resistance to the study of phenomena deemed psychological as metaphysical and
thus beyond the domain of physical laws in the early 1890s , medical experimenters in hypnotism believed that they were living at a moment of far - reaching transformation in both therapeutic and psychological understanding .
it was against this backdrop of expectant possibility that , from the mid-1880s , scientific interest in hypnotic research exploded across europe . as the hypnotic work of the obscure privately practising french physician ambroise libault was brought to public attention following the discovery of his therapeutic successes by the chief physician at nancy hospital , dr hippolyte bernheim , in 1882
numerous physicians flocked to his practice in nancy to witness the near - miraculous cures for themselves .
although braid was credited with disputing the fluid materiality of the mesmeric trance , physicians agreed with libault and bernheim s assessment that he had overlooked a very significant descriptive and explanatory detail the effects of the physician s suggestions on the hypnotised patient .
bernheim provided the theoretical elaboration for this new understanding of hypnotism in suggestive therapeutics in 1884 , in which he defined hypnosis as nothing more than a heightened state of normal waking suggestibility .
in his view
, the hypnotic state arose through the physician s suggestion of hypnotic sleep , and enabled him more effectively to make therapeutic suggestions to the sick patient to cure him / herself .
he held that hypnosis acted only as a means to make patients more receptive to their physicians health - restorative suggestions .
although braid was frequently proclaimed by british physicians as the original pioneer in the development of
modern hypnotism , bernheim was in fact most responsible for the explosion of medical interest in the subject in britain in the 1890s his theory of universal suggestibility provided the theoretical basis for british therapeutic hypnotism throughout the 1890s and 1900s .
following bernheim , supporters of hypnotism held that it was the physician s suggestions , rather than the hypnotic state of
although medical interest in hypnotism in the early 1890s had been generated through the enthusiastic support of physicians in contact with medical research being done in france , interest in hypnotism was by no means exclusive to medical practitioners .
myers were the first individuals to engage in a systematic study of hypnotism in britain ( in the early 1880s ) .
while gurney and myers had observed and were impressed by the therapeutic uses of hypnotism on a visit to nancy in 1885 , they did not limit their own experimental research to hypnotism s therapeutic potential .
for spr investigators , hypnotism provided an invaluable point of entry into the innermost recesses of the human mind , and opened up the possibility for exploring its true nature and full capacities : there is no reason why experiments should be confined to the hospital , or even to the psycho - physical laboratory it would be a grave retardation of science were it assumed that this strange metapsychosis was a medical curiosity alone .
it is the key which seems likeliest to unlock the mysteries of attention and memory ; of sleep , dreams , and hallucination ; of
thus , perhaps , may [ we ] with most reason hope to lay the corner - stone of a valid experimental psychology , and to open up our deepest inlet to the inner man .
there is no reason why experiments should be confined to the hospital , or even to the
psycho - physical laboratory it would be a grave retardation of science were it assumed that this strange metapsychosis was a medical curiosity alone .
it is the key which seems likeliest to unlock the mysteries of attention and memory ; of sleep , dreams , and hallucination ; of
double consciousness and of religious ecstasy thus , perhaps , may [ we ] with most reason hope to lay the corner - stone of a valid experimental psychology , and to open up our deepest inlet to the inner man .
gurney and myers held that the hypnotised individual exhibited an even stronger will and greater mental power than the waking self as was seen , they argued , in the curative effects of hypnotic suggestions .
they theorised that a secondary consciousness was active and dominant in the hypnotised subject while the primary ( or waking ) consciousness lay dormant .
while the task of understanding this secondary consciousness was to preoccupy psychical researchers for the next fifty years , spr investigations only infrequently directly intersected with british physicians hypnotic investigations as their experimental work was more directly concerned with the underlying psychological explanation for hypnotic mental phenomena .
when hypnotism was introduced into british medical discussion in the 1890s , it was firmly embedded within a nexus of discussion and debate over the nature of the mind and its relationship to the body , and was not attached to one coherent set of ideas and practices .
there were several interpretations of the hypnotic state and hypnotic susceptibility and many methods for employing hypnotism in medical practice .
suggestion , to which british supporters of hypnotism appealed in explaining hypnotism s therapeutic effects , bore no consistent meaning in medical discussions .
it was variously associated with the long - acknowledged contagious effects of a physician s attitude ,
while it also gestured towards a host of possible medical and more broadly social abuses . the absence of a definitive explanation for how suggestibility functioned , and uncertainty as to the precise nature of its role in everyday social life , brought a sense of urgency to medical discussions . although concern over the potentially dangerous implications of individual suggestibility quickly became a central feature of discussions of hypnotism in the 1890s , the dominant tone of the lively discussions published in both the british medical journal and the lancet in the months following bramwell s demonstration was an optimistic openness to the possibility of hypnotism s value for medical practice .
even the staunchest opponents to hypnotism supported the bma s decision to pursue the matter more thoroughly . although dr norman kerr proclaimed that hypnotism created a potentially disastrous affective bond between the hypnotist and the hypnotised which he described in vivid terms as a jelly - fish slavery without mental or moral backbone
he cast his vote in favour of further investigation . as a result , at the conclusion of the annual meeting of the bma s psychological section in 1890 , when the bma s president , sir willoughby francis wade , put to the vote the formation of a committee of medical men with the object of endeavouring to ascertain the true nature of its phenomena , and the propriety , or otherwise , and the value of its use in the treatment of disease, it was carried unanimously.
witnessing physicians enthusiasm in 1890 at the prospect of a new therapeutic revolution in medicine , few present at this gathering would have predicted hypnotism s status of disrepute by 1898 .
suggestive therapeutics appeared for many to be the logical outcome of another revolution in scientific discovery .
the question of who was susceptible to hypnosis was a crucial preoccupation from the start . in light of critics claims that only men and women of a low nervous and intellectual type
could be hypnotised , determining whether the capacity to be hypnotised was symptomatic of health or illness was uppermost on the agendas of both physicians and spr investigators .
british physicians accounts of trips to nancy and paris emphasised that the choice of subjects for hypnotic experimentation both reflected and helped shape the scientist s beliefs about hypnotic susceptibility .
moreover , the choice of experimental subjects also had a significant impact on the phenomena witnessed and then associated with the hypnotic state .
british physicians never failed to mention their pleasant surprise at seeing how remarkably differently hypnotised patients appeared and behaved in nancy in comparison to the hysterical patients in paris at the salptrire hospital for women .
medical psychologist george robertson noted that in contrast to the carefully orchestrated theatrics of jean martin charcot s hysteric hypnotic subjects who , guided by his commands , expressed from one moment to the next trance - like lethargy and effusive emotion
the effects of hypnosis were so subtle at the hospital in nancy that it was a very difficult thing to tell by any external sign when hypnosis had occurred as the patients look[ed ] so natural and [ were ] so conscious of all that goes on around.
the differences between the phenomena exhibited by charcot s and bernheim s subjects were vast , and medical visitors unfailingly noted the quiet , ordinary , everyday tone of libault s medical practice in marked contrast to the picture drawn by [ salptrire physicians ] binet and fr of the morbid excitement shown at the salptrire :
the patients told to go to sleep apparently fell at once into a quiet slumber , then received their dose of curative suggestions , and when told to awake , either walked quietly away or sat for a little to chat with their friends libault took especial pains to explain to his patients that he neither exercised nor possessed any mysterious power , and that all he did was simple and capable of scientific explanation .
the patients told to go to sleep apparently fell at once into a quiet slumber , then received their dose of curative suggestions , and when told to awake , either walked quietly away or sat for a little to chat with their friends libault took especial pains to explain to his patients that he neither exercised nor possessed any mysterious power , and that all he did was simple and capable of scientific explanation .
the calm
everyday tone of nancy experiments with hypnosis was held up by british supporters of hypnotism as proof that when induced by a trustworthy adherent of the nancy school , the hypnotic state was simply a heightened and , at worst , innocently comical version of conscious waking life .
hypnotism s most ardent supporters constructed their arguments attesting to its therapeutic benefits around their beliefs about the non - pathological nature of hypnotic susceptibility .
determining who was susceptible to hypnosis and , perhaps more importantly , who was not was crucial to persuading their scientific audiences that it was a necessary addition to physicians therapeutic canon .
neurotic people were the most easily hypnotised : private soldiers and agricultural labourers are good subjects for hypnotism .
strong muscular and intelligent men and women are , in my experience , the best subjects for hypnotism.
bramwell similarly noted that many of [ his ] most successful cases were strong healthy males , who were easily hypnotised for operative and experimental purposes.
explicitly challenging the assumptions prevalent among hypnotism s detractors , bramwell argued that hysterical women were among the most difficult to hypnotise while healthy young men were almost uniformly susceptible : the intelligent man with the well - balanced brain is more or less easily influenced.
the picture which hypnotism s supporters generally presented of the hypnotisable subject was male , intelligent , and in good physical and mental health . in adopting bernheim s theory of universal suggestibility as the explanatory basis for the healthy nature of hypnotic susceptibility , british supporters of hypnotism were confronted with a new conception of social life . describing hypnotism as a
normal state of heightened suggestibility , and thereby revealing human beings to be highly receptive to the suggestive power of the outside world , bernheim radically altered how the human being s relationship to the external world was conceived .
formulated in the immediate wake of universal enfranchisement in france , bernheim s theory of suggestibility posed a serious challenge to the notion that individuals could make autonomous rational and moral decisions .
hypnotic treatment via suggestion implied that the patient suspended their independence of thought and believed everything that the physician communicated without question . noting how extreme was the deeply hypnotised patient s level of suggestibility , one medical hypnotist , dr george kingsbury , described how the subject will imitate every gesture of the operator in the manner of a puppet .
bmj editor ernest hart pointed to the dangers of hypnotism in maintaining that the hypnotised subject became a
marvellous machine , astonishingly , blindly , actively obedient to your wildest orders or most bizarre suggestions.
both critics and supporters of hypnotism invoked the vivid language of automatons ,
instruments and machines in their descriptions of the profoundly suggestible hypnotised subject . resolving the dilemma of whether physicians should be allowed to exert overt control over their patients was of central importance in considerations of whether hypnotism should be made an acceptable medical practice . with the potentially immoral , and even dangerous , consequences of heightened suggestibility and hypnotic
the possibility of illicit sexual relations between physicians and their female patients pervaded discussions of hypnotism in the 1890s .
both supporters and detractors of hypnotism regularly invoked lurid criminal scenes involving hypnotised women and morally depraved hypnotists to support their calls for strict legislation limiting hypnotic practice to qualified medical practitioners .
popular warnings in the medical literature involved hypnotised women impregnated against their will , as well as otherwise moral women forced through suggestion to commit crimes on behalf of their hypnotist . not only did the prospect of hypnotic therapy present new possibilities for criminal abuse , the theory of hypnotic suggestibility also called into question the much - prized notion of innate moral consciousness .
hypnotism s supporters lacked a systematic explanation of the hypnotic state that preserved both the hypnotized subject s autonomy and moral conscience .
if universal suggestibility were to be accepted as a fact of human life , then moral values would lose their cherished status as
natural , and be reduced to illusory , and eminently vulnerable , socially prescribed concepts .
john milne bramwell criticised bernheim s challenge to innate morality , claiming that it was not only dangerous , but illogical : when one considers that bernheim
boldly asserts that there is nothing in hypnotism but the name ; that it does not create a new condition , and that hypnotic acts are only exaggerated normal ones
bramwell condemned hypnotism s supporters in britain for failing to resolve the troubling contradiction in bernheim s view of the hypnotised subject s incapacity for moral awareness .
when one considers that bernheim boldly asserts that there is nothing in hypnotism but the name ; that it does not create a new condition , and that hypnotic acts are only exaggerated normal ones
although there was much enthusiasm for hypnotic research in the 1890s , the porousness of mental boundaries implied in bernheim s theory of universal suggestibility opened up a host of frightening possibilities .
as hypnotism in late nineteenth - century medical practice intrinsically involved relationships of potential influence between individuals of differing social backgrounds , the complex set of tensions at the heart of modern social relationships exploded onto the pages of medical journals .
doctors committed to promoting hypnotism s remarkable effects in restoring health were faced with the difficult task of convincing their audiences that its therapeutic benefits outweighed its potentially damaging costs to an increasingly tenuous social order .
amidst debate over the potentially dangerous and amoral consequences of the hypnotic heightening of individual suggestibility , physicians unanimously agreed on one issue : public demonstrations of hypnotism needed to be stopped . medical journals throughout the 1890s
were replete with warnings about the dangers of hypnotic demonstrations before unscientific audiences . in identifying public demonstrations of hypnotism as a danger ,
both medical supporters and critics of hypnotism commonly established clear distinctions between scientific and unscientific audiences . at the bma s annual meeting in 1891
, the physicians present unanimously agreed that the public exhibition before unscientific and miscellaneous audiences , and for the purposes of gain or amusement , of the phenomena of hypnotism should be banned , as they were antagonistic to public morality.
in emphasising the unscientific and miscellaneous status of the audiences in addition to the unethical agenda of stage hypnotists who were motivated by a desire for personal profit physicians were particularly concerned about audience members suggestibility as manifested in an inability to detect the fiction involved in hypnotic performance , and confuse entertainment with reality .
physicians worried that the public s suggestibility in combination with the deceptions of stage performers would lead to a general degradation of people s minds , resulting not only from witnessing a fiction masquerading as truth , but in actively desiring this kind of performance and deriving pleasure from the possibility of its imagined reality . stressing the dangers of public performers gross misrepresentation of scientific facts in their opposition to stage hypnotism , medical supporters of hypnotism emphasised that their own exhibitions were concerned purely with the disinterested demonstration of genuine phenomena . as the dissemination of medical knowledge about hypnotism was greatly dependent upon hypnotic demonstrations , supporters needed to assert their ability to detect deceit successfully . in a brief description of a series of medical demonstrations given in the 1880s , scottish physician r.w .
felkin was careful to note that scientific audiences were sufficiently sceptical to recognise the difference between genuine and fraudulent phenomena : when hypnotic phenomena are witnessed in a scientific spirit by competent observers , fraud , or even unconscious deception , is not likely to pass muster.
felkin s insistence that scientific audiences possessed the observational skills necessary to avoid being fooled by
trained hypnotic subjects was premised upon the view that non - scientific ( and uncritical ) audiences lacked the necessary intellectual training to be so discerning .
participants in the hypnotism debate thus distanced themselves from the natural suggestibility of uneducated audiences which they elided with concerns about public morality through an emphasis upon the mental rigour of a scientific education .
the explicitly articulated assumption that university education countered the potential for succumbing to false suggestions enabled physicians to establish clear distinctions in levels of suggestibility between educated and uneducated social classes .
physicians deep reservations about the potential consequences of the suggestible nature of unscientific and miscellaneous audiences was rooted in a general mistrust of those large sections of the british population that had only in the last few decades become increasingly literate , educated at a primary school level , and enfranchised .
bramwell singled out the negative effects of a mass press that thrived on the propagation of sensational , and often inaccurate , information as an obstacle to physicians hypnotic research since nearly every one ha[d ] read , and been more or less influenced by , various unfounded newspaper stories regarding the dangers of hypnotism.
as the public generally ha[d ] accepted the misleading statement that hypnosis is characterised by unconscious and suspended volition , patients under the influence of these ideas could not be hypnotised .
in order to make this point clear
, he described his successes with hypnotism in the late 1880s and early 1890s on patients in goole , in east yorkshire , where hypnotism had not been discussed in newspaper and magazine articles.
in bramwell s view , the effect of the media on suggestible minds was so remarkable that it interfered with patients ability to be hypnotised .
public was strongly shaped by their interests as members of the expanding middle class . as defenders of middle - class values ,
physicians vision of a healthy society coincided with bourgeois assumptions about the immorality of the lower classes .
the unintended consequences of modern progress , and some of its most frequently discussed threats in the late nineteenth century , were perceived increases in urban criminality , insanity , alcoholism and venereal disease all understood as originating and proliferating among the lower classes , and increasingly explained in the medical language of degeneration .
as a key marker of this medicalisation of social problems , physicians conflated moral anxieties about the lifestyle of the less educated
public morality. in a similar spirit , participants in the hypnotism debate elided concerns about the implications of individual suggestibility for doctor
patient relations with the potential influence which public performers , political orators ,
and sensational writers had on their presumably uneducated lower - class audiences . throughout the 1890s , suggestibility was aligned with the more troubling signs of britain s seemingly inexorable democratisation . although universal suggestibility signified a threat to social order , and the possible need for tighter legal controls over what the public saw , read and heard , it was also interpreted by a number of physicians as having potentially beneficial uses , not simply in curing unhealthy patients , but in reforming deviant behaviour .
the possibility that the public , or at least its most undesirable members , could be molded through hypnosis to conform to late nineteenth - century behavioral norms was explored by many physicians alongside therapeutic research . in 1901 , american physician r. osgood mason produced a work devoted entirely to the subject of hypnotism as a mode of
educational field of hypnotic investigation was beginning to be opened up alongside the therapeutic and the psychological fields .
the influence which may be exerted by hypnotism upon the development and improvement of the mind.
he was optimistic that this new educational field , may yet prove of greater interest and utility than either of the others when one views the number of children brought into the world with imperfect mental organizations and vicious tendencies , and sees how little impression in general is made upon them by the ordinary and even the special processes of education .
may yet prove of greater interest and utility than either of the others when one views the number of children brought into the world with imperfect mental organizations and vicious tendencies , and sees how little impression in general is made upon them by the ordinary and even the special processes of education .
although mason claimed that this new area of investigation was only in its early phases of development , british physicians had been using hypnotism to cure a variety of moral defects
kleptomania , perverted sex , nail biting and even lies for a decade .
the promises which individual suggestibility held out as a locus of moral persuasion and site of social reform were significant , however the absence of clear understanding concerning its precise limits made any firm distinction between hypnotist and subject , doctor and patient , scientific audience and the public difficult to establish .
the near - impossibility of definitively determining the direction of influence and the precise nature of suggestions mysterious operations were subtle , but crucial , issues that hypnotism s medical detractors brought to the discussion .
if suggestibility was universal , as hypnotism s supporters claimed , then where did it stop ?
how could physicians be certain that they were not on the receiving end of suggestions when they experimented with hypnotised patients , and that the hypnotic phenomena witnessed were not being deliberately , or even unconsciously , performed ?
in response to the problem of experimental subjects deception , described by psychical researchers as the potential for mal - observation ,
physicians could do nothing more than appeal to their scientific training as a guarantee against mistaking a subject s conscious ( or unconscious ) performance for reality . in his condemnation of therapeutic hypnotism ,
ernest hart appealed to the unknown limits of suggestibility in arguing against physicians ability to perceive cases of fraud . in a sensational expos of british medical researchers work with a trained hypnotic subject , hart described how he had paid a professional hypnotic subject to perform for a group of prominent medical men including bramwell . believing the trained subject referred to only as
l. to be genuine , hart reported that the medical men were duped because of their desire to see real hypnotic phenomena . noting the particularly
histrionic quality of l.s over - acted performance , hart questioned the integrity of the judgement of the medical men , and speculated that if such well - respected men of science could be fooled , then very little could be expected of the lay public : if such things be possible in the green wood of an intellect originally trained to scientific observation , what is likely to happen in the dry sticks and shakings of half educated , wholly uncritical , and superstitious minds ready to take fire at the slightest spark of the mysterious ?
challenging distinctions presumed to separate scientific demonstrations from public entertainment , hart argued that medical demonstrations of hypnotism held no scientific worth other than their
foolometric value.
if such things be possible in the green wood of an intellect originally trained to scientific observation , what is likely to happen in the dry sticks and shakings of half educated , wholly uncritical , and superstitious minds ready to take fire at the slightest spark of the mysterious ?
in his exposure of the physicians credulity ,
hart inadvertently presented a critique that could have been levelled at a wide variety of forms of scientific inquiry : men are easily induced to see what they are anxious to see , and even the dry light of science does not always keep its votaries out of this pitfall .
suggestion often acts more powerfully on the operator than on the subject.
invoking the power of suggestion to explain how medical hypnotists could fall prey to illusion during
scientific investigations of hypnotism , hart turned the problem of suggestibility onto professional medical men .
he also further complicated the already complex relationship between hypnotist and hypnotised by concluding that hypnotic subjects were not only born , but
made.
arguing that the phenomena of hypnotism were only at best partially genuine , he claimed that physicians unwittingly trained their hypnotic subjects , moulding them to perform the symptoms that they were anxious to see. in support of his claim , he pointed to jean - martin charcot s peculiar hysterical hypnotic subjects , whose highly theatrical symptoms exhibited under hypnosis could not be found anywhere outside the sphere of the french neurologist s direct influence .
hart s mistrust of hypnotic subjects , in combination with audiences expectations of a specific kind of trance performance , was in line with relatively common concerns about the problematic nature of normal waking suggestibility .
his emphasis on medical investigators unreliability due to their suggestible natures was consistent with the interpretation taken up by crowd psychologists at the turn of the century . in his highly influential 1895 work on crowd psychology ,
gustave le bon argued that the even the most rational scientists could fall prey to an unconsciously derived suggestibility when working in a group . premised upon his foundational theory that individuals in groups lose their critical abilities as a direct result of an affective and contagious suggestibility ,
le bon argued that during scientific studies of phenomena associated with spiritualism , scientists became blinded by a collective suggestibility generated through group association . as a result
, they often witnessed things that had not occurred in any real , or objective , sense : the faculty of observation and the critical spirit possessed by each of them individually at once disappears . here ,
as always , we have the power of the hypnotiser over the hypnotised.
le bon s theory was widely known in britain by the end of the nineteenth century , and , in 1907 , wilfred trotter , an english surgeon and budding social psychologist , acknowledged le bon s interpretation of suggestibility as a predecessor to his own , more up - to - date biological version .
appealing to the concept of suggestibility to explain not simply crowd behaviour but all human behaviour ,
trotter argued that suggestibility was a primary attribute of the herd instinct , and a foundation of all human social life : if the biological explanation of suggestibility here set forth be accepted , the latter must necessarily be a normal quality of the human mind .
man is not , therefore , suggestible by fits and starts , not merely in panics and in mobs , under hypnosis , and so forth , but always , everywhere , and under any circumstances variations [ in suggestibility ] are due to the differing extent to which suggestions are identified with the voice of the herd .
if the biological explanation of suggestibility here set forth be accepted , the latter must necessarily be a normal quality of the human mind .
man is not , therefore , suggestible by fits and starts , not merely in panics and in mobs , under hypnosis , and so forth , but always , everywhere , and under any circumstances variations [ in suggestibility ] are due to the differing extent to which suggestions are identified with the voice of the herd .
as a result , the suggestions of the individual s social group ( or herd ) determined how they behaved . following from this initial premise
, trotter argued that men of science were not naturally compelled to approach the world rationally , but were guided through the suggestions of their community their teachers and colleagues to adhere as much as possible to the rigorous strictures of reason in their work .
by the first decade of the twentieth century
the concept of suggestibility had been appealed to in explanations of a wide variety of social phenomena that involved individuals at every level of british society .
fitting scientists into a grand social psychological theory of suggestibility was in many ways to take bernheim s initial concept as an explanation for the effects of hypnotism to its full and logical conclusion . from initially signifying the slightly increased influence of the physician over the patient in the late 1880s ,
its theoretical consequences expanded over the course of the 1890s and early 1900s as it was increasingly invoked as a means of explaining a wide variety of aspects of social life . by the early twentieth century , suggestibility had tremendous explanatory power for understanding social behaviour from the effects of newspapers on readers and public entertainment on audiences , to politicians hold over crowds , to the discoveries made by small groups of scientists .
the appeal of suggestibility within the newly developing field of social psychology at the turn of the century centered upon the view that traditional hierarchical society was quickly dissolving into modern
while suggestibility was a cause for concern , and signified an obstacle to both patients and physicians capacity for rational responsible behaviour , trotter attempted to resolve its potentially negative consequences through practical solutions provided by his biologically derived social psychology .
herd could work in favour of truth : as the suggestibility of man does not necessarily or always act against the advancement of knowledge , traditional ideals of rationality and progress did not need to be abandoned .
trotter urged that it was the task of scientists to make scientific modes of thought and behaviour socially desirable , and so seemingly powerful that everyone would be compelled to follow . by 1907 , when trotter formulated his optimistic solution to the apparent chaos of human suggestibility , discussions of hypnotism in medical journals had been in retreat for several years .
when tuckey summed up the recent developments in hypnotic research in the preface to the fifth edition of his textbook on hypnotism in 1907 , he required only one sentence : the seven years which have elapsed since the publication of the fourth edition of this work have not been marked by any striking development of hypnotic theory or practice.
by 1900 , neither the dangerous implications of suggestibility nor the precise nature of hypnotism s social effects had been resolved by hypnotism s supporters .
physicians could not even establish consensus over the exact meaning and implications of suggestive therapeutics. riven by internal disagreement , supporters of hypnotism increasingly turned their focus away from public medical debate to the safety and seclusion of private investigation .
although the theory of suggestibility was riddled with ambiguities from the moment that it was conceived , hypnotism s retreat from medical discussion was not a foregone conclusion in the 1890s .
the nancy conception of universal suggestibility was not the only explanation for hypnotism available to british physicians .
subliminal self , which explained hypnotic susceptibility without espousing suggestibility , entered into public discussion in the 1890s and 1900s .
despite the fact that myers was not a physician , nor an academically trained scientist , he was invited to the bma s annual meeting in the autumn of 1898 to present a paper outlining his theory of hypnotic consciousness .
the months following his psychologically complex address were marked by a noticeable silence in response to his approach to the potential problem of suggestibility .
as we shall see , the absence of medical interest in myers revolutionary theoretical paradigm for understanding mental life signalled the end of the hypnotism debate .
the medical receptiveness to hypnotism s open - ended possibilities that marked the early 1890s had been closed down by 1898 .
the problems raised by the theory of universal suggestibility could not be resolved within late - victorian physicians traditional epistemological framework , and as a result hypnotism was pushed off the public medical agenda . as myers explained in his 1898 paper , the hypnotist s suggestions did not act in the place of the subject s will , or contrary to the subject s desires , but set going some intelligent organic faculty in the man which laid dormant till that moment , and which proves more effectual for healing than the man s
conscious will.
shifting the focal point of theoretical explanation from the influence of the hypnotist s suggestions to the hypnotised individual s self - suggestion , myers argued that the hypnotist served to unlock some fountain of energy which was latent within the man s
own being.
myers explained this crucial , but psychologically complex , point to the physicians present using the metaphor of a factory to describe the mind , and the factory proprietor to describe the waking self : in waking consciousness i am like the proprietor of a factory whose machinery i do not understand .
my foreman my subliminal self weaves for me so many yards of broadcloth per diem ( my ordinary vital processes ) as a matter of course
. if i want any pattern more complex , i have to shout orders in the din of the factory , where only two or three inferior workmen hear me , and shift their looms in a small and scattered way
. such are the confined and capricious results of the first , the more familiar stages of hypnotic suggestion . at certain intervals , indeed , the foreman stops most of the looms , and uses the freed power to stoke the engine and to oil the machinery .
this , in my metaphor , is sleep , and it will be an effective hypnotic trance if i can get the foreman to stop still more of the looms , come out of his private room , and attend to my orders my self - suggestions for their repair and rearrangement .
in waking consciousness i am like the proprietor of a factory whose machinery i do not understand . my foreman my subliminal self
weaves for me so many yards of broadcloth per diem ( my ordinary vital processes ) as a matter of course .
if i want any pattern more complex , i have to shout orders in the din of the factory , where only two or three inferior workmen hear me , and shift their looms in a small and scattered way
. such are the confined and capricious results of the first , the more familiar stages of hypnotic suggestion . at certain intervals , indeed , the foreman stops most of the looms , and uses the freed power to stoke the engine and to oil the machinery .
this , in my metaphor , is sleep , and it will be an effective hypnotic trance if i can get the foreman to stop still more of the looms , come out of his private room , and attend to my orders
my self - suggestions for their repair and rearrangement .
stemming from his belief that the hypnotised individual remained under the control of their secondary consciousness
further , myers argued that hypnotised subjects could not be forced to commit crimes that they would not otherwise commit because hypnotised individuals always acted in accordance with their own self - suggestions and moral conscience .
myers approach to hypnotic consciousness provided the basis for a very different , and more hopeful , view of the hypnotised subject . opposing the image of the automaton
a popular metaphor to describe hypnosis for participants on both sides of the debate adherents of myers theory of the
subliminal self refuted the notion that there was any lapse in the hypnotised individual s sense of social and moral propriety .
describing his experiences with hypnotism in 1896 , bramwell related how three young female patients lost neither the ability to reason , moral sensibility , nor sense of social propriety while in a hypnotic state .
miss c. , aged 19 , an uneducated girl
would not , while hypnotised , allow him to extract a decaying tooth until he had carefully and rationally explained the medical reasons for his intervention .
miss f. , aged 19 , in good health , intelligent , and well - educated , would not carry out the post - hypnotic suggestion to go to the sideboard in [ bramwell s ] room , pour out a glass of water , and drink it.
when her mother was later asked why she had refused , she explained that she did not know [ bramwell ] well enough to help herself to a glass of water in [ his ] house without being asked.
when he asked
miss e.maid to one of [ his ] patients , while hypnotised to reveal the private details of an event involving her employer , she absolutely refused to say a word on the subject.
bramwell , following myers , hypothesised that the hypnotised individual was animated by a secondary , and more powerful , consciousness : if i were asked in one word to describe the difference between the hypnotised and the non - hypnotised subject , i should say that it consisted in the superiority of the former over the latter in his having reached a far - reaching power over his own organism , which the other does not share .
this view of the hypnotic state is not a novel one , and has already been ably described by mr frederic myers .
if i were asked in one word to describe the difference between the hypnotised and the non - hypnotised subject , i should say that it consisted in the superiority of the former over the latter in his having reached a far - reaching power over his own organism , which the other does not share .
this view of the hypnotic state is not a novel one , and has already been ably described by mr frederic myers .
not only did the hypnotised individual remain in full control of her surroundings but also self - control was more resolute while she was hypnotised . in following myers approach to hypnotism , bramwell was a notable and vocal exception among british physicians .
medical professionals responses to myers paper in 1898 were generally unsympathetic , and ranged from dismissive to explicitly hostile .
one contributor to the bmj rejected myers theory as unsubstantiated and ultimately unhelpful in understanding hypnotism : it is not clear that anything has been gained by the introduction of the term subliminal consciousness .
we are not persuaded that mr myers has given us proof that hypnotism has the power to arouse faculties which are dormant in the normal condition .
it is not clear that anything has been gained by the introduction of the term subliminal consciousness .
we are not persuaded that mr myers has given us proof that hypnotism has the power to arouse faculties which are dormant in the normal condition .
according to this anonymous contributor , myers was inconsistent in claiming that the hypnotised individual responded to suggestions made for curative purposes , but did not respond to criminal orders : the exponents of the hypnotic faith can not have it both ways ; they can not urge that hypnotism is effective and yet not effective as may suit the argument of the moment .
there is no hypnosis , in the sense indicated by dr bramwell , without suggestion ; and we deprecate its use while it holds out any inducement to a man , however feeble , to resign his responsibility and become the passive agent of another however well - intentioned .
the exponents of the hypnotic faith can not have it both ways ; they can not urge that hypnotism is effective and yet not effective as may suit the argument of the moment .
there is no hypnosis , in the sense indicated by dr bramwell , without suggestion ; and we deprecate its use while it holds out any inducement to a man , however feeble , to resign his responsibility and become the passive agent of another however well - intentioned .
myers theory , which proclaimed all suggestions as self - suggestions , was swiftly dismissed as another ( and incorrect ) version of mental pathology : it would appear that , using the example cited by mr myers we might more correctly and more suggestively speak of the fringes of consciousness.
hysteria and mania were then cited as exemplary conditions stemming from the fringes of consciousness. although many physicians were troubled by the possible criminal and amoral implications of the suggestible hypnotised subject , almost none rejected this version of the self in favour of myers subliminal self , despite the fact that it preserved innate individual morality and maintained hypnotism s association with health . for most physicians ,
myers hopeful contemplation of humanity s spiritual evolution conflicted with widely perceived social problems in britain in the late nineteenth century .
subliminal self , suggestibility provided a language for understanding the perceived unruliness and irrationality of the lower classes , while it also provided an explanation for why such individuals posed a threat to middle - class social order . at the same time as suggestibility helped to explain the nature of britain s social problems
, it opened up a potential practical method for reforming and controlling the socially undesirable through still unexplored forms of education. in the end , the suggestible subject more aptly corresponded to the disruptive influences in british society that numerous physicians were already engaged in medicalising .
as a practice underwritten by the concept of suggestibility , it played into concerns about democratisation , the emergence of mass culture , and the difficulty of establishing a basis for morality in an increasingly secular age .
rapport exposed the seeming ambiguity of personal boundaries , questioned the inviolability of individual autonomy , and ultimately presented a threat to the stability of the victorian social order . furthermore , because the majority of hypnotism s medical supporters with the notable exception of john milne bramwell accepted bernheim s doctrine of suggestibility and all it implied , even advocates of hypnotism were unable to mount a convincing case for the practice .
no amount of medical evidence demonstrating that hypnotism could be used to cure a variety of illnesses could persuade the majority of physicians that the hypnotic
ironically , the rejection of hypnotism as a medical practice was a testament to its persuasiveness as a theory for understanding the problems of modern social life . given its problematic implications ,
the theory of universal suggestibility provided a compelling framework for imagining social existence at a moment when a far - reaching re - orientation of social and political life appeared both inevitable and necessary .
it made sense to physicians at the turn of the century , however committed they might be to an ideal of social progress rooted in individual rational autonomy .
although ernest hart asserted in 1893 that the hypnotic subject was not only born
but made , he retained the concept of suggestibility as a means to explaining how it was made .
at once constructed and contested during the hypnotism debates , the suggestible hypnotic subject and its accompanying consequences for social life demonstrated an enormous intractability in refusing to be unmade , or remade into myers more profoundly moral subliminal self .
as a symptom of tensions deeply rooted in the fabric of middle - class responses to social change at the turn of the century , physicians could not force the suggestible subject out of existence . beyond the context of medical therapeutics , universal suggestibility had tremendous appeal within the newly developing field of social psychology as the most fruitful basis for understanding
the theory was widely taken up by leading crowd and social psychologists at the turn of the century to explain a wide range of irrational social phenomena , from fashion fads to religious revivals , financial panics , and the violence of mobs . by 1907 ,
as wilfred trotter noted , suggestibility had replaced irrationality as the despair of the social reformer : now the trouble is not irrationality , not a definite preference for unreason , but suggestibility that is , a capacity for accepting reason or unreason if it comes from the proper source.
as the capacity for independence of thought was being questioned in many quarters , and individual autonomy was increasingly seen as illusory , universal suggestibility signified an ever - present potential for social chaos .
an apparently unavoidable fact of social life at the turn of the century , suggestibility could at best be only carefully managed .
medical participants in the hypnotism debate were keenly aware of these larger social implications . for most physicians
suggestibility was resistant to regulation , and the networks of hypnotic influence too diffuse to be patrolled .
in the potential medical adoption of therapeutic hypnotism , not only were the trust and authority of physicians at stake , but also the social structure in which they were profoundly implicated .
hypnotism was in retreat from public medical discussion soon after its rediscovery not because it was perceived as therapeutically ineffective , or because its practice was considered too difficult to regulate professionally .
hypnotism was judged an impossible practice because it was seen as potentially destabilising and affirmed educated middle - class fears concerning the threat to lasting social stability in a modern democratic world .
moreover , it was rooted in an acknowledgement that , despite claims of progress , modern medicine was unable to provide a definitive solution to the inescapable problems which universal suggestibility implied . solving the problem of the suggestible subject meant solving the many social problems that underwrote progress in the late nineteenth century , and this was beyond the capacity of professional medicine . as a result , the medical debate over the adoption of hypnotism as a therapeutic practice foundered and effectively fell into a protracted silence by the end of the nineteenth century . | during the late nineteenth century , many british physicians rigorously experimented with hypnosis as a therapeutic practice . despite mounting evidence attesting to its wide - ranging therapeutic uses publicised in the 1880s and 1890s , medical hypnosis remained highly controversial .
after a decade and a half of extensive medical discussion and debate surrounding the adoption of hypnosis by mainstream medical professionals including a thorough inquiry organised by the british medical association it was decisively excluded from serious medical consideration by 1900 .
this essay examines the complex question of why hypnosis was excluded from professional medical practice by the end of the nineteenth century .
objections to its medical adoption rarely took issue with its supposed effectiveness in producing genuine therapeutic and anaesthetic results .
instead , critics objections were centred upon a host of social and moral concerns regarding the patient s state of suggestibility and weakened will - power while under the physician s hypnotic spell. the problematic question of precisely how far hypnotic
rapport and suggestibility might depart from the victorian liberal ideal of rational individual autonomy lay at the heart of these concerns .
as this essay demonstrates , the hypnotism debate was characterised by a tension between physicians attempts to balance their commitment to restore patients to health and pervasive middle - class concerns about the rapid and ongoing changes transforming british society at the turn of the century . | Hypnotism Returns to Britain: The Context for the Medical Debate
Defining the Hypnotically Susceptible: The Suggestible Subject and the Problems of Influence
Determining the Limits of Suggestibility: Medicine, the Public, and the Challenge to Social Order
The Medical Rejection of Hypnotism and the Triumph of Suggestible Subjectivity
Conclusion |
PMC4616094 | strength and endurance training can lead to many physiological changes within the human
body and it aims to improve overall physical performance .
training improves performance , in
part , due to muscular adaptations in morphology , such as conversion between fiber types , and
altered biomechanical properties , tendon stiffness , and increased elasticity of muscles1 .
such intrinsic muscular adaptations of
mechanical properties are thought to enhance , or impact , muscle performance during muscle
contraction2 , 3 .
therefore , training is performed in an attempt to enhance the
ability of a muscle to generate force , or to control the length change of its fibers in
relation to the stretch of its tendon , which depends on the architecture and the
physiological properties of its fibers4 .
these morphological and biomechanical changes may impose an increased risk of injury on the
muscle itself , due to the development of the stiffer muscle tissue necessary to generate
increased force . in a study performed to determine the relationship between running economy
and muscle stiffness
it was postulated that there was a positive relation between muscle
stiffness and power production5 .
as a
muscle increases in strength , it loses flexibility in order to maintain the elasticity to
create a forceful contraction .
injuries , such as a hamstring strain , are commonly
associated with exercise that involves fast movements and sudden acceleration .
the
hamstrings are recognized as being the most frequently injured structure , and are estimated
to account for between 12% and 16% of injuries in professional soccer leagues8 , 9 .
hamstring injuries are injuries that often require a long recovery period and are especially
prone to recurrent injury10 .
the physical fitness component of police cadet training often emphasizes muscle power and
endurance training .
it
is our understanding that there is no literature on the changes in muscle properties
elicited by police cadet training .
therefore , our study aimed to investigate the effects of
this physical exercise on lower extremity muscle properties . by observing the effects of
physical exercise on lowell police academy cadets , in lowell , ma in the united states , this
study aimed to describe the relationship between overall performance in physical activity ,
muscle property , and police cadet training
the study s hypothesis was that over the 20-week
training period , the cadets would show changes in muscle properties and a decline in the
flexibility of the hamstring and calf compound .
fifty - nine ( 10 females and 49 males ) police cadets from the lowell police academy , class of
2013 , were initially recruited for the study .
eight males dropped out from the study due to
schedule conflicts preventing them from attending all assessment sessions .
all subjects
prior to the study reported no known neuromuscular disorders , no recent ( within a year )
lower extremity injury or surgery , and no prosthetic joint .
subjects were excluded if their
range of a straight leg raise ( slr ) was less than 30 , greater than 100 , or the stabilized
leg during slr could not maintain at 0 of hip flexion .
this study was approved by the institutional review board of university
of massachusetts lowell , ma , usa .
table 1table 1descriptive characteristics of subjects at the time of recruitmentmale ( n=41)female ( n=10)overall ( n=51)age ( yrs)27 ( 3.8)24.9 ( 3.2)26.6 ( 3.7)height ( cm)177.4 ( 9.3)166.8 ( 7.2)175 ( 9.9)weight ( kg)85.2 ( 15.6)64.5 ( 37.7)81.2 ( 17.2)body fat ( % ) 15.6 ( 6.1)22.8 ( 13.3)17.0 ( 6.7)values are mean ( sd ) shows the demographics of the 51 subjects at the time they were recruited
before undergoing the standard cadet training .
standard fitness tests and quantitative assessments of muscle properties were conducted
pre , during ( 1st and 2nd evaluations ) and at the end ( 3rd evaluation ) of cadet training at
five to six weeks interval during the cadet training .
standard fitness tests included upper
extremity muscle strength and endurance ( bench - press , push - up and pull - up ) , trunk muscle
strength and endurance ( sit - up ) , the flexibility of back and leg muscles ( sit and reach
test ) , and general power ( timed 1.5 miles run ) . the body size and the body composition
were
recorded at each of the four sessions . to quantify the biomechanical properties of the
hamstrings and calf compound , ajjolos
( seign , llc , north reading , ma , usa ) shown in fig .
1fig .
1.experimental setup for evaluating the biomechanical properties of the hamstring and
the calf compound .
a device containing a pressure plate and an inflatable ball
connected to an electric pump was connected to a computer to record the tension
changes during passive stretching .
dead
man switch so that the experimenter could stop ball inflation by taking a finger off
the switch . was used to evaluate subjects within the same week of the standard fitness test .
subject assumed the pre - stretch position , and he / she was positioned supine on the mat .
a
coin flip determined which lower extremity would be measured first for each subject .
once
the subject was in proper alignment with the device and self - reportedly comfortable , the
subject was instructed to raise the randomly selected leg and place it ( ankle or calf ) upon
the inflatable ball . in most cases ,
the subject s initial positioning resulted in the raised
leg having a slr of approximately 70 or more depending on each individual s flexibility
( fig .
once the test position was reached ,
the subject was requested to relax and to allow the device to passively apply a gentle
stretch to the lower extremity .
the device s passive applicator was gradually inflated to
0.3 psi , over approximately 5 seconds , applying a gentle stretch to the subject s hamstring
musculature . at the end of the inflation , the pressure placed on the ball over a 60 second
after 60 seconds , the device gradually released the applied passive
stretch by deflating the ball .
once the subject was back in the initial ( pre - stretch )
position , he / she was asked to assume a similar position with the opposite lower extremity ,
and the procedure was repeated . experimental setup for evaluating the biomechanical properties of the hamstring and
the calf compound .
a device containing a pressure plate and an inflatable ball
connected to an electric pump was connected to a computer to record the tension
changes during passive stretching .
dead
man switch so that the experimenter could stop ball inflation by taking a finger off
the switch .
the recorded pressure data were plotted against time over the 60-second period for each
lower extremity ( fig .
2.an example of the pressure data measured by the device during the test in two
consecutive sessions ( before and after the police cadet training ) .
the decay curve
indicates the stretched muscle became more relaxed over the one minute of stretching
resulting in less pressure being applied to the inflated ball .
the initial stretch
force indicates the resistance caused by the stretched leg . ) .
the slope
of the fitted curve was defined as the relaxation coefficient ( relx ) in this study . a
smaller relx ( more negative number )
implies a more compliant , i.e. less stiff , passive
element of the soft tissues .
the highest point of the stretching curve was defined as the
initial stretch force ( isf ) .
an example of the pressure data measured by the device during the test in two
consecutive sessions ( before and after the police cadet training ) .
the decay curve
indicates the stretched muscle became more relaxed over the one minute of stretching
resulting in less pressure being applied to the inflated ball .
repeated measures anova was used in this study to compare measures ( the relaxation
coefficient ( relx ) , initial stretched force ( isf ) , sit - and - reach ( sr ) distance , sit - up and
1.5 miles run time ) and body size / composition changes among the four longitudinal sessions .
bonferroni correction was then used as post - hoc test to examine the main
effects .
all analyses were performed using ibm spss statistics 20 ( ibm , chicago , il , usa )
with a significance level of 0.0125 .
table 2table 2.body size and compositionheight ( cm)weight ( kg)body fat ( % ) mean ( sd)mean ( sd)mean ( sd)pre175.4 ( 9.4)82.1 ( 16.2)17.0 ( 6.3)1st eval175.8 ( 8.9)82.0 ( 16.1)16.0 ( 5.8)*2nd eval175.8 ( 8.9)80.1 ( 15.2)*^16.3 ( 5.6)3rd eval175.9 ( 9.5)79.8 ( 14.4)*^16.5 ( 5.6)values are mean ( sd ) . * : p<0.0125 compared to pre session , ^ :
p<0.0125 compared to second session demonstrates the body size and the body composition of all subjects over the
time course of the study .
no significant height changes were seen while statistically
significant weight losses were observed at the second and third evaluations compared to the
pre and first evaluation sessions ( p<0.001 ) .
body fat decreased significantly after the
first five weeks of the cadet training ( p<0.001 ) but increased at the second ( p=0.009 )
evaluation session .
values are mean ( sd ) . * : p<0.0125 compared to pre session , ^ :
p<0.0125 compared to second session table 3table 3.time course changes in general fitness of the police cadetssit- up1.5 mile runsit - and - reachpre36.1 ( 8.0)710.6 ( 67.3)17.3 ( 2.4)1st eval41.0 ( 7.4)*653.8 ( 63.9)*18.4 ( 2.2)*2nd eval43.5 ( 7.1)*^641.5 ( 81.7)*19.1 ( 2.3)*^3rd eval45.0 ( 6.0)*^#641.2 ( 84.2)*19.9 ( 2.1)*^#values are mean ( sd ) . * p<0.0125 compared to pre session , ^ :
p<0.0125 compared to post session , # : p<0.05 compared to the
previous session shows the changes of general fitness after the cadet training . the general
fitness improved as expected .
the time spent in running 1.5 miles significantly reduced at
about the five weeks into the cadet training ( p<0.001 ) and the effect was maintained to
the end of training .
group data demonstrated that sr and sit - ups improved significantly at
all three evaluation sessions ( p<0.001 ) .
* p<0.0125 compared to pre session , ^ :
p<0.0125 compared to post session , # : p<0.05 compared to the
previous session table 4table 4.biomechanical properties of the hamstring and calf compoundisf ( dominant)isf ( non - dominant)relx ( dominant)relx ( non - dominant)pre10.73 ( 2.94)10.03 ( 2.46)0.0079 ( 0.0118)0.0075 ( 0.0135)1st eval9.63 ( 2.76)9.66 ( 2.67)0.0061 ( 0.01054)0.0055 ( 0.01322)2nd eval9.43 ( 2.55)*9.53 ( 2.62)0.0079 ( 0.01162)0.0101 ( 0.0119)3rd eval9.23 ( 2.61)*8.96 ( 2.31)*0.0035 ( 0.02329)0.0039 ( 0.1314)#values are mean ( sd ) . * : p<0.0125 compared to pre session , # :
p<0.0125 compared to the previous session shows the biomechanical properties of back leg soft tissues on each side .
the
isf of the dominant leg decreased after the training ( p=0.03 ) and was maintained at the
follow - ups while there was no significant change of relx .
the isf of the non - dominant side
did not change after the training , but there was a significant difference between pre and
2nd follow - up ( p=0.007 ) .
a significant difference was found in relx of the non - dominant side
between the first and 2nd follow - up ( p=0.025 ) .
values are mean ( sd ) . * : p<0.0125 compared to pre session , # :
p<0.0125 compared to the previous session
as expected , an improvement in general fitness was observed during and at the end of the
cadet training .
there was no significantly negative change in the flexibility - related
measures ( sit - and - reach , isf , relx ) of the subjects between the pre and first evaluation
sessions .
the isf decreases observed at the 2nd and 3rd evaluation sessions indicate that
back leg musculature became less stiff and required less force to be moved to the extreme
position .
however the changes in isf were not symmetrical which indicates the importance of
being able to recognize the differences between two legs .
although the changes in the sr
test and isf were contrary to our hypothesis , the relx of the both legs showed an increasing
trend ( less compliant ) but it was not statistically significant except at the end of the
cadet training .
the usage of sr is controversial , but it was recommended for estimating the extensibility
of the hamstring when other forms of flexibility tests are impractical11 .
the criterion - related validity of the sr test might be
impacted by the hamstring extensibility specifically among subjects with reduced hamstring
extensibility12 . moreover
, the sr test
alone does not discriminate the potential differences between two legs ( and there were
asymmetrical changes in isf ) or the contributors to muscle properties .
for example , a stress relaxation curve was
used to estimate muscle extensibility post - exercise in rabbits13 : a second order polynomial was fitted to the relaxation
curve over the period of 300 seconds after the stress was applied . in our study
we a chose 60-seconds
recording window and a linear regression model , which is appropriate because most tension
decay occurs in the first 60 seconds after the application of a stretching force13 . sr and isf demonstrated changes opposite
to relx , but it is possible that the changes in relx were confounded by back muscle
extensibility , abdominal muscle strength , body and head positions , etc .
our data shows there
were increases in both sit - up numbers and sr distance .
therefore , the possibility of an
increase in sr distance as a result of an enhanced abdominal muscle performance can not be
ruled out .
measuring the straight leg raise with more sensitive sensors would allow us to
better study the compliance of hamstrings . though the odds of developing a hamstring injury may seem intimidating , animal evidence
shows that techniques such as passive stretching tend to reduce skeletal muscle
injuries14 .
this might be one of the
reasons that although the standard police cadet training emphasizes muscle power and
endurance training , with warm - up and cool - down performed in the training , it seemed to not
increase the risk of hamstring injuries . in our data , larger variances in relx of both legs
that might be due to the different activities they performed or reduced
physical exercise changes in muscle properties .
however , it is one of the limitations of
this study , that since the activities during the cadet training could not be monitored or
restricted , it is not possible to draw a causal conclusion .
the non - dominant legs became less compliant at the end of the cadet training ;
however no injury was reported at the follow - up , it might be due to the activities that
police cadets conduct daily are not the sudden and fast motions made by athletes such as
soccer players . nonetheless our data show that muscle compliance changes due to the training
involving and that the lower extremities should be taken into account when designing a
training protocol .
a future study of a longer training period ( more than 20 weeks ) is needed
to see if the muscles involved become less compliant . | [ purpose ] this study was performed to examine the relationship between physical
performance and muscle properties during police cadet training .
the study s hypothesis was
that improved physical performance brought about by training , would in turn cause a
reduction in muscle flexibility .
[ subjects and methods ] fifty - nine police cadets were
included in this study .
standard fitness tests and quantitative assessments of muscular
biomechanical properties were conducted before , during and after the 20-week cadet
training . [ results ]
general fitness had improved at the end of the police cadet training .
there was no significant decrease in muscle flexibility as measured by the sit - and - reach
test .
however , muscle compliance of the non - dominant leg measured by the relaxation
coefficient had decreased at the end of the police cadet training .
[ conclusion ] the
increased sit - and - reach distance could be due in part to strengthening of the abdominal
muscles . on the other hand , the biomechanical test , which was specific to muscle
extensibility , showed a reduction in the relaxation coefficient of the non - dominant leg .
our data suggests that changes in muscle compliance as a result of lower extremity
training should be considered .
this data may be useful in the design of a training
protocol that prevents the potential injuries caused by reduced muscle flexibility . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC4166433 | we approach the countdown for the mdg5 for improved maternal health , and how to deliver quality antenatal care ( anc ) in low - income countries becomes an important issue to address . with the mdgs ,
maternal health has received increasing political attention ; however , little attention has been given to anc , even though the guidelines for focused anc ( fanc ) were launched concurrently .
the risk of maternal death is by and large not predictable during pregnancy , and it is argued that anc has limited effects on maternal death [ 3 , 4 ]
. the most important strategy for reduced maternal mortality is to ensure that women give birth by skilled attendance [ 57 ] .
consequently , there is an ongoing debate about the importance of anc for maternal health .
nevertheless , pregnant women are embracing the idea of care during pregnancy ; in sub - saharan africa , three in four pregnant women receive at least one anc visit .
generally , guidelines for fanc have not been fully implemented in low - income settings , and the quality of anc is often low [ 912 ] .
the anc is increasingly focused on and acknowledged for providing health education , instructions on danger signs during pregnancy , and birth preparedness , as this is considered to be lifesaving and to reduce delay in seeking help .
the opportunities of the anc program to counsel women in the need for skilled birth attendance have not been sufficiently investigated .
thus , while the importance of a highly utilized health program is under debate , the priority of the area ( hence the quality of the actual care provided ) is low , and research on how to strengthen the anc is needed . in ethiopia ,
maternal mortality rates have been among the highest in the world , although currently there are indications of a marked decline . according to estimates ,
the maternal mortality ratio was 990 per 100,000 live births in 1990 but was estimated to 676 in 2010 . at national level , most recent data show that only 10% give birth under the care of a skilled provider , 51% in urban and 4% in rural settings . from 2005 to 2011 , the proportion attending anc increased from 28 to 34% .
local studies characterise the anc service by unclear guidelines , lack of training of providers [ 18 , 19 ] , and poor health system registration , but the preventing mother - to - child transmission ( pmtct ) program seems to receive higher priority than the anc program .
one study mentions that in 2005 fanc was not implemented , but in other studies [ 16 , 18 , 20 ] coverage of four visits is reported .
fanc guidelines suggest that four visits per pregnancy will suffice , if the woman does not experience complications .
how anc services are prioritised by the ethiopian health authorities might be reflected in the national reproductive health strategy 20062015 .
overall , anc is not central to the document , and the priority areas are social and cultural determinants of women 's health , fertility and family planning , maternal and newborn health , hiv / aids , reproductive health of young people , and reproductive organ cancers .
thus , ethiopia seems a relevant case for the study of how to strengthen the implementation of quality anc for improved maternal health .
the aims of this study were to analyse if the health system priorities matched the user expectation and to assess the needs for anc strengthening for improved maternal health in the jimma area of ethiopia .
the specific objectives of the quantitative component were to analyse the content of anc and to identify the predictors of low anc satisfaction , while the specific objectives of the qualitative component were to understand perceptions , practices , and policies of anc .
the study was a needs assessment aimed at informing the development of a complex intervention study that would test the feasibility of anc strengthening in ethiopia .
we applied the who guidelines for fanc as the best practice for efficient care , and the present paper provides an in - depth understanding that can guide adaption of fanc to the jimma context . using
the terminology from the british medical research council ( mrc ) guidance on complex interventions , this needs assessment is contributing to the initial phases of trial development , where the program theory is developed and modelled .
implementation of policies for reduction of maternal mortality without involvement of local stakeholders and contextual understanding has previously been found to be ineffective . our choice of conducting an in - depth analysis of anc in the local setting is in line with the theories for the planning of health promotion programs , where need for user - involvement and application of qualitative research is highlighted [ 24 , 25 ] .
the study was conducted in jimma city , which has a population of 121,000 , and in the surrounding urban communities of serbo and agaro .
the anc program of all public health facilities in the area was studied , four health centres ( jimma town , higher 2 , serbo , and agaro ) and one hospital ( jimma university specialized hospital ) .
the hospital serves as a referral site and provides specialized care for southwestern ethiopia , with a catchment population of about 15 million , and anc service is provided for women with complicated as well as uncomplicated pregnancies . at the health centres , pregnant women from the nearby urban and rural communities are seen , and primary health care services ( including anc , delivery services , postpartum care , and family planning ) to all population groups are provided . the registration system is poor , but an estimated 6,500 pregnant women are seen at the facilities per year .
field work was conducted in the study area from august 2008 to august 2009 by sfv , with linguistic , cultural , and logistical support from at .
all women residing in the study area who gave birth in the year preceding the interview ( date of birth from april 28 , 2008 , to june 20 , 2009 ) were invited to participate .
we collected information about the use of and experience with health facilities during the last pregnancy , and women were asked to recall the content of care received during anc .
the questions were based on the components of care recommended in the who fanc model and were inspired by the ethiopian demographic health survey .
the questions on laboratory testing were divided into ( 1 ) urine analysis , ( 2 ) hiv test , and ( 3 ) other blood analyses ( haemoglobin , blood group , rh status , and syphilis test ) .
the women were asked to rate how much they were satisfied with the service received during last pregnancy .
user satisfaction was originally coded as follows : very satisfied , acceptable , and not satisfied , but for the regression model very satisfied and acceptable were merged .
the questionnaire was in amharic and the translation was conducted by a group of local nurses and corrected by dn and agm independently , and the questionnaire was pilot tested .
local guides from the kebeles ( smallest administrative unit ) helped to identify eligible women by walking from door to door .
the qualitative component included observations at anc facilities , in - depth interviews with recent mothers and health system leaders , focus group discussions ( male spouses and tbas ( traditional birth attendants ) ) , and workshops with health professionals .
observations took place regularly at all anc facilities , adding up to 2040 hours per site , and were focused on the infrastructure , availability of technology , types of health staff , and interaction between user and provider .
interviews were conducted with 13 recent mothers ( 20 days to 5 months after delivery ) to understand their perceptions and experiences regarding pregnancy and utilization of anc .
we used purposeful sampling to capture the variation in anc service delivered at different levels of care , women who followed anc at hospital ( 3 ) or health centre level ( 6 ) or did not have any anc at all ( 4 ) .
the anc attendants were selected from the anc archive and nonattendants from the archive of childhood immunizations .
the tracing of women was difficult and time consuming due to incomplete registration at facility level and an informal address structure in the community .
interview guidelines were developed in the local setting together with health professionals , pilot tested , and translated into the local language .
a focus group discussion with eight male spouses visiting jimma town health centre was conducted to gain information about their perceptions of pregnancy and to understand decision - making processes at the family level .
these men attended the facility for different purposes on the day of the focus group .
further , a focus group discussion with 12 tbas , who were recruited through a local nongovernmental organization ( family guidance association ) , was conducted to explore their roles regarding pregnancy and delivery at the community level .
the discussions were conducted in the local language and facilitated by agm . all interviews and discussions
workshops with clinical staff members were conducted at serbo , jimma town , and higher 2 health centre and at the hospital ( no physicians participated ) .
the workshops consisted of two hours of group - based discussions on strengths , weaknesses , and solutions in anc provision .
the groups subsequently presented posters with their main perspectives for the rest of the group .
key informant interviews were performed with health authorities from the town health bureau and managers from the obstetric department at the hospital in order to understand their perceptions of the organisation of the service as well as the regional and national priorities .
the survey data were analysed using mixed effect logistic regression to identify predictors of being dissatisfied with the service .
health facility was analysed as the random effect parameter to adjust for the potential clustering of women using each health facility .
further adjustment was made for maternal age , socioeconomic status , and number of anc visits .
all analyses were performed in stata 11.0 ( statcorp , texas , usa ) . the initial analysis of the qualitative interview data and
the categories were grouped in more superior domains , and patterns and relations between these were explored .
posters from the workshops with health professionals were analysed , and the context of the workshops and the interpersonal dynamics among the health professionals were kept in mind .
perspectives evolving from the field observations were discussed with colleagues and local health professionals for validation and contextualisation .
this indicates that the research questions of the two approaches were not initially integrated and the two approaches were designed to apply with the standards of the two disciplines separately . in the discussion of this study , however , the qualitative and quantitative findings were triangulated and the conclusions on the research question were based on whether the findings were converging , diverging , or complementary .
ethical permission was obtained from the jimma university ethical review committee of the college of public health and medical sciences , and permission to observe the practice at health facilities was obtained from the relevant town and zonal health bureaus as well as the hospital administration .
all informants were ensured anonymity and confidentiality , and they gave their informed consent after appropriate explanation of the study objectives and content .
the mean ( range ) age was 24.5 ( 1546 ) years ( table 1 ) .
almost 20% had never been to school and 42% were primiparous . during the previous pregnancy , 83% attended anc and 67% gave birth in a health institution .
few women had only one visit ( 3% ) ; 25% had four , whereas 44% had more than four visits .
measurements of blood pressure , weight , tetanus toxoid ( tt ) immunization , and hiv - testing were performed for more than 90% of attendants ( table 3 ) .
abdominal examinations as well as blood tests were performed less frequently , and the numbers differed between the facilities .
about 25% experienced discomfort due to many students or waiting for more than one hour .
overall satisfaction with the service was high ( 31% very satisfied and 59% acceptable ) , but 10% reported being not satisfied .
lack of blood pressure testing , abdominal examination , hiv - testing , and tt immunization was not associated with satisfaction ( table 4 ) .
women who did not have other laboratory tests than the hiv test were more likely to report being not satisfied with anc .
moreover , health education , long wait time , poor cleanliness of the health facility , and discomfort due to many students were related to the dissatisfaction .
participants in in - depth interviews were women aged 18 to 32 years , most of whom were primiparous , but also women with three and four children were interviewed .
nine attended anc service ; two did not attend at all ; and another two had contact with the service but did not classify themselves as attendants : one because she never reached the awareness that anc is a special program for follow - up care for pregnant women .
a central aspect shared by both women and men was that women were considered vulnerable while pregnant and hence needed to be taken care of by their husbands and relatives . for some women , pregnancy added to the worry associated with poor living conditions and concerns about how to take care of their family .
relatives , especially the mothers of the pregnant women , also played a significant supervising role during pregnancy .
seeking health care was seen as a way to cope with the increased vulnerability of being pregnant .
the only references to guidelines were a ministry of health poster and a training manual for health professionals found at health centres , both based on the fanc guidelines ( with four visits per pregnancy ) .
the booking of anc visits followed the fanc guidelines at health centres , where , in contrast , the hospital recommended monthly appointments until 28 weeks , biweekly appointments until 36 weeks , and then weekly appointments until the birth . at all facilities , health professionals and consultation rooms were dedicated to anc provision . at some facilities ,
the anc duty would shift among all staff ; at others , one person was responsible . at the health centres ,
the permanent staff members providing anc were all at the nurse or midwife level ; however , occasionally , the service would be provided by students , alone or under supervision . at the hospital
, the anc facility was staffed by nurses who did the measurement of blood pressure and body weight , whereas all other physical examinations and consultations were provided by medical students , who cycled through the department every 14 days , which resulted in a lack of continuity of care .
the students were providing service with reference to obstetricians , but the level of supervision was low , as the obstetricians were busy attending a high number of complicated deliveries and other teaching assignments .
further , at the hospital , collaboration problems between nurses and medical students were frequent .
the nurses and midwives indicated that the medical students received more attention and feedback from the senior medical staff than they did , and this made them feel less appreciated .
the nurses would not have authority over medical students , so nurses refrained from taking responsibility in the presence of medical students . during observations we noted that there was a lack of alignment of procedures in the facilities where the staff rotated or students were in charge of the anc service .
this was visible in the registration procedure , which changed from provider to provider , as well as in the placement of the equipment . during visits to the clinics , it was difficult to find a person who took the lead of providing the anc services .
in contrast , at serbo and higher 2 health centres , one person was in charge of anc , and this created continuity and a sense of leadership .
though always present , the registration procedures were faulty with no basis for reflection or adjustment of procedures .
it was observed that supervision of the anc staff and practice was minimal , which seemed to lead to apathy in the service provision .
the observed lack of continuity , thorough registration procedures , and supervision were not reported a concern by the health staff during the workshops . however
, they did note that preventive services were not prioritized as much as curative services .
the interviews with the senior health staff indicated that these issues were part of the clinical culture and that solving them was not a priority . at the workshops
, the health professionals themselves requested training on fanc , especially in - service training that could improve their skills during their normal working hours .
they felt that it was challenging to provide good - quality service due to the lack of equipment and the low standards of the physical environment .
serbo , higher 2 , and agaro health centres only had laboratory facilities for hiv - testing and not for analysis of urine , haemoglobin , and syphilis and determination of blood group .
therefore , the staff referred anc attendants to private clinics , where testing could be done based on user fees . at the hospital and jimma town health centre ,
the laboratory facilities were available , but , at the health centre , a minimum fee was charged : haemoglobin 3 etb ( 0.17 usd ) , syphilis 3 etb , blood group and rh status 4 etb ( 0.23 usd ) , and urine analysis 3 etb .
women expected anc service to be free , and therefore the costs of laboratory tests caused frustration .
this affected their satisfaction with the health centres : unlike the hospital , the health centres are short of laboratory equipment and chemicals . if it is well equipped like the hospital it will be acceptable ( female participant ) .
a woman , at her first anc visit , was asked to take laboratory tests at a private clinic with user fees . at the second anc visit ,
she was asked to take more tests at the private clinic and was refused tt immunization before she had the laboratory results .
she did not have money for more laboratory services and refrained from further check - ups .
the women associated pregnancy and the need for health care with hiv , but with no other specific diseases .
the health system in jimma had previously received funding for hiv / aids activities , for example , training of staff .
our observations revealed that the staff members were more careful to provide good - quality service and to make thorough registrations for the pmtct service than they were for the basic anc service .
the increased attention might be due to close follow - up and guidance by senior doctors , health authorities , and donor agencies .
some women mentioned that hiv - counselling was provided , whereas the majority said that no health education was given .
this inconsistency was supported by our observations , and the staff expressed a wish to improve the health education .
the interactions between users and providers were dominated by the provider , and conversations were often formal and very limited .
further , the women indicated that the service sometimes occurred without the expected content of anc and that the staff sometimes delayed services , even though they were not busy .
both women and men reported that some health professionals had a reputation of purposely insulting the women .
a particularly important issue for the women and their partners was that anc was not conducted in privacy , due to the presence of a high number of students .
we observed that it was common to have five health providers in the room conducting anc services for one woman . a woman
, it is horrible , when you exhibit an undressed body . there was no time where i was examined in privacy .
this lack of privacy made women feel embarrassed , and several refrained from further anc visits .
the women sometimes felt unsafe in their hands : i was not examined by trained health providers .
it is based on her advice that they gave you help . since they pushed down my abdomen without care ,
i did not build any confidence in them ( female participant ) . in adjacent rooms , to which the doors could not close ,
a father recommended that if many health providers had to be present , the supervision and discipline should be improved : if nobody enters the room during examination , it is better .
if somebody enters the room during examination the mother might feel shy and will not answer the questions the woman will be forgotten on the examination couch and the staff will start to discuss with each other .
poor conduct of staff was identified as a problem by the staff themselves , but they did not reflect on the issues of lack of privacy and the negative effect on the well - being of their clients .
the need for anc strengthening was assessed using a mixed - method approach . there were no national guidelines for anc in ethiopia , and the providers in jimma did not have support to give high priority to anc . within the health system , teaching of health professional students
was given high priority , and that contributed to a lack of continuity and privacy for women .
poor user - provider interaction was a serious concern for the women , and contributed to a lack of trust in the providers .
the interaction was deemed poor because of a combination of a poor privacy culture , a high number of students present , and a lack of dialogue and attention to the women 's individual needs .
therefore , we conclude that the women and the health system have different priorities , and , from the women 's point of view , the agenda of the health system compromises the care provided , and that hinders trust and mutual respect .
further , the care provision was compromised by inadequacies in leadership , supervision , skills of professionals , and routine registrations as well as a shortage of laboratory facilities .
however , in the pmtct services integrated in the anc program , priority and funding made it possible to overcome these issues .
the measurement of satisfaction with care in the survey was relevant as an overall assessment of the women 's evaluations of the care received ; however , satisfaction has previously been shown to be dependent on the expectations towards care [ 30 , 31 ] , and , for example , primigravidas might have difficulties knowing what to expect from anc . in this study ,
a relatively small proportion of women reported being not satisfied with anc , and this might be due to underreporting dissatisfaction , as respondents tend to report favourably on questions of perceived quality of care or satisfaction . by triangulating the findings of the qualitative data against those of the qualitative study , we found that the analysis of the predictors of dissatisfaction offers some guidance towards what women do not want from care .
the qualitative results give a deeper understanding of why laboratory tests , privacy issues , and conduct of health staff had strong statistical association with satisfaction .
lack of health education was also significantly associated with dissatisfaction but was not a strong theme in the qualitative interviews .
however , limited verbal interaction between provider and user was , and it is , interpreted as a barrier for counselling during anc .
limited verbal interaction between user and provider has been described in other sub - saharan settings [ 33 , 34 ] , and it has been ascribed in part to the fact that existing social hierarchies in the surrounding society are reflected inside the facility ; poor and less - educated patients are often treated less politely and are given less attention than middle - class or wealthy patients [ 35 , 36 ] . in this study ,
67% of the women gave birth in a health facility , which is higher than the national urban average ( 51% ) , but still lower than the anc coverage ( 83% ) .
some studies show that the use of anc predicts the use of health institution delivery , whereas others do not [ 7 , 38 ] .
provider attitudes have been described as barriers for use of health institution delivery [ 34 , 39 ] .
we hypothesize that increased attention to what women want from anc could increase both the number of follow - up visits during pregnancy and the number of health facility deliveries .
this proposition is supported by a recent multicountry study which found that the relation between the use of anc and the place of delivery in previous studies has been underestimated and that governments and ngos should place more importance on the role of anc in efforts to promote skilled birth attendance .
further , it could be speculated that the ability of the health professionals to improve the health of the pregnant women might increase , if the women are involved as more active partners in discussing their health status and in the screening for health problems .
this study revealed that both the health system and the women had high awareness of hiv , which may reflect how a vertical program can be well implemented in a less functional horizontal program .
both the high priority of hiv and the general health system falling behind have been described in other settings [ 40 , 41 ] .
today the efforts to improve public health in low - income countries are partly funded by global health initiatives , and these activities lead to new inequalities in health care provision unless better integrated in the overall health system .
we believe that the success of the implementation of pmtct services should be considered a model for how health system strengthening can be accomplished for improved maternal health .
although the importance of anc for maternal health is under debate , the guidelines for fanc are based on best available evidence , anc is implemented globally , and coverage is high .
therefore , it is surprising how poorly the fanc guidelines are implemented in low - income countries , including ethiopia .
langley and denis suggest that quality improvement initiatives in general seem to neglect that innovations ( although scientifically sound ) imply a distribution of costs and benefits to several groups of stakeholders who have more or less interest in and power to support changes .
further , they argue that different stakeholders hold different values about what is good and right , and that power relations in health care systems are diffused because of the need for both professional and managerial expertise . in the present study ,
it was clear that quality assurance of anc / fanc was not a shared interest ; an example is that for the medical students it might be beneficial to keep the traditional model for booking of anc visits ( with many visits per woman ) as they would gain more experience .
they might have had relatively good chances for upholding this practice , as they seemed to be close collaborators of the senior medical doctors .
further , the collaborative challenges seen between the nurses , medical students , and senior doctors might reflect differences in values about what good care is .
clearly , the differences in the priorities of the women and the health system are an example of differences in what good care is considered to be .
finally , the diffuse leadership of anc and hence the lack of supervision and guidelines were noted , and those were obviously due to the low priority and funding of anc .
thus , if dedicated implementation of fanc was a goal in ethiopia , it is clear that in the implementation process the micropolitics of power , interests , and values would be important to address .
the survey is based on a relatively large study population , with a very high participation rate .
we can not preclude selection bias ; socioeconomically disadvantaged women , women with stillbirths , and mothers who experienced infant deaths might be underrepresented .
nevertheless , we did everything feasible to ensure inclusion of all women : local female data collectors were trained and kebele guides assisted in identifying the women .
the qualitative interviews with women were conducted in their own homes because it was assumed that they would be more comfortable there .
this was an important step to reduce the effects it could have when relatively powerful people from the local community together with a foreigner ask questions about the health system
. in general , the participants appeared happy to meet us and to reflect on the theme introduced . at the workshops with the health professionals ,
it was known by all that this study was part of a needs assessment for a future intervention .
further , the health centre leaders were present , and it might have been difficult to express critical perspectives on the local leadership , including perspectives on supervision , continuity , and job responsibilities , and this is one limitation that could possibly have influenced our data . however , the extensive field stay ( sfv ) and the participation of local researchers ( dn , agm , and at ) in this study alleviated this risk of bias and provided a strong background for developing interview guides and for the contextualisation and interpretation of the data .
the findings were relatively consistent , and this consistency enhanced the validity and relevance of our conclusions .
data in this study stem from a single area in ethiopia , and a discussion of transferability is relevant [ 45 , 46 ] . in jimma ,
the coverage of laboratory testing during anc was higher than the national coverage ; thus , the issue of poor - quality laboratory facilities seems national . from other low - income countries
, it is reported that the quality of anc is compromised by shortages of laboratory reagents and drugs , low coverage of health education , and low compliance with the fanc guidelines and also that service varies between facilities and countries [ 912 , 41 , 4750 ] . in the present study , we found the same challenges and thus conclude that they seem to apply generally .
however , the quality of this program depends on the health system it is nested in . on a large scale , funding and leadership are found to be cornerstones for health system strengthening in low - income countries , and therefore it is likely that the themes raised in this study are of particular interest for ethiopia , but also of general interest for health system strengthening in low - income settings .
it seems essential that anc services should be given increased attention and that stronger leadership should be implemented with more supervision and monitoring of the service providers .
based on our findings , one suggestion for improved continuity of care could be to avoid job rotations .
further , if many health professional students have to be present during service provision , a different culture for how to approach the women could be encouraged . in other settings
, it has been suggested that improved quality of care can be achieved by in - service training and improved supportive structures at managing levels [ 10 , 41 , 47 , 52 ] .
based on this needs assessment , an anc strengthening intervention ( the maternity study ) is to be developed . according to the mrc guidelines for complex interventions
, the intervention will be considered an exploratory trial , where the feasibility of implementing the intervention and its acceptability to providers and users will be tested .
the focus of the intervention should be on adaption and implementation of anc guidelines , availability of laboratory facilities and supply , development and implementation of health education materials , in - service training of health professionals in anc services , guidelines for ensuring privacy during consultations , and regular and supportive supervisions of anc professionals .
improvements are needed on guidelines development and implementation , upgrading of laboratory facilities , health education , and user - provider interaction as well as training of health professionals .
national and international decision makers need to be aware of the consequences of having one of the most utilized health care services running without guidelines and structured supervision , for example , inadequate quality of care and the loss of health professionals ' motivation and user 's trust in the health care system
. more importance on the role of anc in efforts to promote skilled birth attendance and maternal health is needed .
however , it is clear that dedicated implementation of fanc takes strong leadership and we suggest supportive supervision of health care providers geared towards building trust and mutual respect to protect maternal and infant health . |
objective . we assessed how health system priorities matched user expectations and what the needs for antenatal care ( anc ) strengthening were for improved maternal health in jimma , ethiopia .
methods . a questionnaire survey among all recent mothers in the study area
was conducted to study the content of anc and to identify the predictors of low anc satisfaction .
further , a qualitative approach was applied to understand perceptions , practices , and policies of anc . results .
there were no national guidelines for anc in ethiopia . within the health system ,
the teaching of health professional students was given high priority , and that contributed to a lack of continuity and privacy .
to the women , poor user - provider interaction was a serious concern hindering the trust in the health care providers .
further , the care provision was compromised by the inadequate laboratory facilities , unstructured health education , and lack of training of health professionals .
conclusions .
health system trials are needed to study the feasibility of anc strengthening in the study area . nationally and internationally
, the leadership needs to be strengthened with supportive supervision geared towards building trust and mutual respect to protect maternal and infant health . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions and Perspectives |
PMC2795155 | fluorescence resonance energy transfer ( fret ) is becoming increasingly popular as a tool to study membrane protein structure and to monitor conformational changes in membrane proteins ( corry et al .
2006 ) . unlike nuclear magnetic resonance ( nmr ) spectroscopy and x - ray crystallography
, fret can not provide high - resolution structural information on membrane proteins . on the other hand ,
the technique has obvious advantages in that it is applicable over a wide range of experimental conditions and has superior sensitivity .
however , to obtain distance constraints from fret experiments , it is in general necessary to use fluorescent labels that are foreign to the protein .
the use of probes introduces uncertainties in the interpretation of the fret data , and in the extrapolation to the native structure of the protein . clearly , to fully exploit fret as a structural technique ,
it is vital that we understand the factors that govern the conformation and dynamics of fluorescent labels used in structural studies .
only recently have fluorescent labels been taken into account explicitly in molecular modeling studies ( corry and jayatilaka 2008 ; gustiananda et al .
similar approaches have been carried out for the simulation of electron spin resonance ( esr ) spectra of spin - labeled phospholipids ( hkansson et al .
2001 ) and proteins ( desensi et al . 2008 ; steinhoff et al . 2000 ) .
the fluorescent probe studies demonstrate that it is crucial to take into account the orientation , dynamics , and conformational space of the fluorescent labels for the calculation of accurate energy transfer efficiencies . for this reason ,
it is important to learn what factors affect the orientation and conformation of the fluorescent labels used in fret experiments .
this is especially true in the case of fluorescent labeled membrane proteins , where specific interactions of the fluorescent label with the phospholipid bilayer are an additional factor complicating the interpretation of fluorescence experiments . moreover , due to the low dielectric environment inside the lipid bilayer , the effect of neighboring residues on the behavior of a fluorescent label could be more pronounced in the case of membrane proteins . several molecular dynamics simulation studies have been performed to date that attempt to understand the relationship between the conformational behavior of a fluorescent label and the conformational features of a polymer on an atomic level ( see , for instance , kosovan et al .
even though a number of modeling studies have taken fluorescent labels into account explicitly ( sparr et al . 2005 ) , no systematic study on the behavior of fluorescent labels covalently attached to membrane proteins has been carried out to date . here , we perform molecular dynamics simulations of a number of aedans - labeled single cysteine mutants of a small reference membrane protein , m13 major coat protein , covering 60% of its primary sequence .
m13 major coat protein is a single membrane - spanning , -helical membrane protein with a relatively large water - exposed region in the n - terminus ( vos et al .
we analyze the behavior of the aedans label and the native tryptophan , which were used as acceptor and donor in our previous fret experiments ( nazarov et al .
the effect of lipids on the tryptophan and aedans conformational space is quantified and discussed .
the effect of neighboring side - chains in a low - dielectric membrane environment is studied by comparison with simulations of a series of aedans - labeled polyalanine peptides .
our data show that the effect of both lipids and of neighboring side - chains on the aedans conformational space is limited .
thus aedans is an excellent fluorescent label to probe the direct chemical environment of membrane proteins , which is quite unhindered by neighboring amino acid side - chains , lipid or water molecules . as such
, our approach could help to develop a general strategy to study membrane protein structure and function in the future using aedans as a probe in fret or other fluorescence experiments .
a straight -helical conformation of m13 major coat protein , as proposed by vos et al .
the -helix was constructed using the computer program swiss pdbviewer ( guex and peitsch 1997 ) .
the aedans label was incorporated into the molecular model in an extended configuration with all chain dihedral angles at 180 using the computer program molmol ( koradi et al .
proteins were inserted into dopc bilayers using the method of blowing up the bilayer on a grid and shrinking it again as described in kandt et al .
the proteins were positioned with a tilt angle of 23 relative to the bilayer normal , according to the orientation as determined in the literature with the valine residue at position 29 positioned at the center of the bilayer with its side - chain pointing downwards ( koehorst et al .
simulated systems contained the labeled protein , 126 dopc molecules ( 63 per leaflet ) , and approximately 8,840 simple point charge ( spc ) water molecules ( berendsen et al .
1981 ) . as a control for interactions between the protein side - chains and the aedans label , polyalanine helices ( 25 residues ) with the labeled cysteine at various positions ( 2 , 5 , 10 , 13 , 17 , 20 , 23 ) were incorporated parallel to the z - axis in additional simulations with the same dopc membrane .
the rest of the parameters for the simulations of the polyalanine simulations were identical to the parameters used in the case of the coat protein .
for all mutants a 10-ns molecular dynamics simulation was performed after energy minimization and a short protein - restrained run .
all simulations were performed with gromacs 3.3.1 ( berendsen et al . 1995 ; lindahl et al .
2001 ) and the optimized potential for liquid simulations ( opls ) all - atom protein force field ( tieleman et al .
( 1997 ) , combined with the spc water model ( berendsen et al .
the force field parameters for the aedans label were calculated using the jaguar software ( schrdinger 2000 ) and can be obtained from the authors on request .
water , lipids , and protein were coupled separately to a berendsen thermostat at 1 atm with a coupling constant of 0.1 ps ( berendsen et al .
the pressure was coupled semi - isotropically to a berendsen thermostat using a coupling constant of 1 ps and a compressibility of 4.5 10 atm in the xy plane and normal to the membrane .
long - range electrostatic interactions were calculated using the particle - mesh ewald ( pme ) algorithm ( darden et al .
1997 ) , allowing for a 2-fs time step . to avoid biasing due to the starting configuration of the dihedral angles the first 2 ns of simulation time were discarded . the orientation factor and energy transfer efficiency e
were calculated for each frame of the remaining 8 ns of the trajectory and averaged .
the rest of the analyses was performed with vmd ( humphrey et al . 1996 ) .
the energy transfer efficiency is related to the distance between the donor and the acceptor and to the orientation of the two chromophores via ( dale and eisinger 1976):1\documentclass[12pt]{minimal }
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\begin{document}$$ < e_{i } > = \left\langle { { \frac{{\kappa_{i}^ { 2 } } } { { c^ { - 1 } r_{i}^{6 } + \kappa_{i}^{2 } } } } } \right\rangle .
$ $ \end{document } for simplicity , neglecting effects of boltzmann weighting , i runs over all possible conformations of the donor and the acceptor .
parameter e is the energy transfer efficiency , and is the orientation factor as defined in eq . 2 .
parameter c describes the refractive index of the medium , the spectral overlap integral characteristic for this donor
acceptor pair , and the quantum yield of the donor in the absence of acceptors .
it was determined to be 2.9 10 in our previous work ( vos et al .
the distance between the donor and the acceptor , r , is taken as the distance between the middle of the central bond of the indole and middle of the central bond of the dansyl chromophore .
the orientation factor is related to the orientation of the donor transition dipole moment and acceptor absorption dipole moment ( gustiananda et al .
2004):2\documentclass[12pt]{minimal }
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\begin{document}$$ \kappa^{2 } = ( \cos \theta_{\text{t } } - 3\cos \theta_{\text{d } } \cos \theta_{\text{a } } ) ^{2 } .
$ $ \end{document } here , is the angle between the donor transition dipole moment and the acceptor absorption dipole moment , d is the angle between the donor transition dipole moment and donor acceptor interconnecting vector , and a is the angle between the absorption dipole moment and the donor acceptor interconnecting vector .
the indole chromophore has two emitting states that are dependent on solvent polarity . in apolar solvents ,
the chromophore emits from the lb state , whereas in polar solvents , the chromophore emits from the la state ( albinsson and norden 1992 ; sobolewski and domcke 1999 ) .
since in our case , tryptophan is buried inside the phospholipid membrane , being a hydrophobic environment , we will assume that the indole chromophore emits exclusively from the lb state .
chemically , the tryptophan side - chain is very similar to 5-methylindole , having a carbon atom connected to the indole ring at the same position .
therefore , we expect the electronic configuration in tryptophan to be similar to that in 5-methylindole , enabling us to use the transition dipole moment of the lb state of 5-methylindole as reported in the literature ( albinsson and norden 1992 ) in our calculations of . this results in vector d shown in fig . 1 .
the value and orientation of the absorption dipole moment of the aedans dansyl chromophore ( vector a in fig .
1 ) was taken from the literature , using the absorption dipole moment of 1,5-accys - aedans at 330 nm ( van der heide et al .
the calculation of the angles , d , and as well as the calculation of the distance between the donor and acceptor was done using a perl script.fig .
1vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor
a straight -helical conformation of m13 major coat protein , as proposed by vos et al .
the -helix was constructed using the computer program swiss pdbviewer ( guex and peitsch 1997 ) .
the aedans label was incorporated into the molecular model in an extended configuration with all chain dihedral angles at 180 using the computer program molmol ( koradi et al .
proteins were inserted into dopc bilayers using the method of blowing up the bilayer on a grid and shrinking it again as described in kandt et al .
the proteins were positioned with a tilt angle of 23 relative to the bilayer normal , according to the orientation as determined in the literature with the valine residue at position 29 positioned at the center of the bilayer with its side - chain pointing downwards ( koehorst et al .
simulated systems contained the labeled protein , 126 dopc molecules ( 63 per leaflet ) , and approximately 8,840 simple point charge ( spc ) water molecules ( berendsen et al .
1981 ) . as a control for interactions between the protein side - chains and the aedans label , polyalanine helices ( 25 residues ) with the labeled cysteine at various positions ( 2 , 5 , 10 , 13 , 17 , 20 , 23 ) were incorporated parallel to the z - axis in additional simulations with the same dopc membrane .
the rest of the parameters for the simulations of the polyalanine simulations were identical to the parameters used in the case of the coat protein .
for all mutants a 10-ns molecular dynamics simulation was performed after energy minimization and a short protein - restrained run .
all simulations were performed with gromacs 3.3.1 ( berendsen et al . 1995 ; lindahl et al .
2001 ) and the optimized potential for liquid simulations ( opls ) all - atom protein force field ( tieleman et al .
( 1997 ) , combined with the spc water model ( berendsen et al .
the force field parameters for the aedans label were calculated using the jaguar software ( schrdinger 2000 ) and can be obtained from the authors on request .
water , lipids , and protein were coupled separately to a berendsen thermostat at 1 atm with a coupling constant of 0.1 ps ( berendsen et al .
the pressure was coupled semi - isotropically to a berendsen thermostat using a coupling constant of 1 ps and a compressibility of 4.5 10 atm in the xy plane and normal to the membrane .
long - range electrostatic interactions were calculated using the particle - mesh ewald ( pme ) algorithm ( darden et al .
1997 ) , allowing for a 2-fs time step . to avoid biasing due to the starting configuration of the dihedral angles the first 2 ns of simulation time were discarded . the orientation factor and energy transfer efficiency e
were calculated for each frame of the remaining 8 ns of the trajectory and averaged .
the rest of the analyses was performed with vmd ( humphrey et al . 1996 ) .
the energy transfer efficiency is related to the distance between the donor and the acceptor and to the orientation of the two chromophores via ( dale and eisinger 1976):1\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$ < e_{i } > = \left\langle { { \frac{{\kappa_{i}^ { 2 } } } { { c^ { - 1 } r_{i}^{6 } + \kappa_{i}^{2 } } } } } \right\rangle .
$ $ \end{document } for simplicity , neglecting effects of boltzmann weighting , i runs over all possible conformations of the donor and the acceptor .
parameter e is the energy transfer efficiency , and is the orientation factor as defined in eq . 2 .
parameter c describes the refractive index of the medium , the spectral overlap integral characteristic for this donor
acceptor pair , and the quantum yield of the donor in the absence of acceptors .
it was determined to be 2.9 10 in our previous work ( vos et al .
the distance between the donor and the acceptor , r , is taken as the distance between the middle of the central bond of the indole and middle of the central bond of the dansyl chromophore .
the orientation factor is related to the orientation of the donor transition dipole moment and acceptor absorption dipole moment ( gustiananda et al .
2004):2\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
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\begin{document}$$ \kappa^{2 } = ( \cos \theta_{\text{t } } - 3\cos \theta_{\text{d } } \cos \theta_{\text{a } } ) ^{2 } .
$ $ \end{document } here , is the angle between the donor transition dipole moment and the acceptor absorption dipole moment , d is the angle between the donor transition dipole moment and donor acceptor interconnecting vector , and a is the angle between the absorption dipole moment and the donor acceptor interconnecting vector .
the indole chromophore has two emitting states that are dependent on solvent polarity . in apolar solvents ,
the chromophore emits from the lb state , whereas in polar solvents , the chromophore emits from the la state ( albinsson and norden 1992 ; sobolewski and domcke 1999 ) .
since in our case , tryptophan is buried inside the phospholipid membrane , being a hydrophobic environment , we will assume that the indole chromophore emits exclusively from the lb state .
chemically , the tryptophan side - chain is very similar to 5-methylindole , having a carbon atom connected to the indole ring at the same position .
therefore , we expect the electronic configuration in tryptophan to be similar to that in 5-methylindole , enabling us to use the transition dipole moment of the lb state of 5-methylindole as reported in the literature ( albinsson and norden 1992 ) in our calculations of . this results in vector d shown in fig . 1 .
the value and orientation of the absorption dipole moment of the aedans dansyl chromophore ( vector a in fig .
1 ) was taken from the literature , using the absorption dipole moment of 1,5-accys - aedans at 330 nm ( van der heide et al .
1992 ) . the calculation of the angles , d , and as well as the calculation of the distance between the donor and acceptor was done using a perl script.fig .
1vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor vectors of the tryptophan emission dipole ( d ) , the aedans absorption dipole ( a ) , and the interconnecting vector ( t ) used for the calculation of the orientation factor
depending on the initial configuration of a molecular dynamics simulation the phospholipid bilayer can take considerable time to equilibrate . for this reason we took a pre - equilibrated phospholipid bilayer as a starting configuration in all our simulations , and we used the inflategro script ( kandt et al .
2007 ) to insert the proteins , thereby minimizing disturbances of the lipids . to evaluate equilibration of the phospholipids , the area per lipid was analyzed over an extended simulation time of 30 ns ( data not shown ) for the aedans - labeled a18c and a27c mutant . during the first 2 ns of the simulation , the area per lipid decreased from ~0.75 to ~0.65 nm for both mutants , in good agreement with values reported in the literature ( tieleman et al .
this indicates that after ~2 ns the bilayer is sufficiently equilibrated for our purpose of evaluating the behavior of the aedans probe and the tryptophan side - chain .
therefore , the rest of our analyses were performed over the timeframe 210 ns . both the tryptophan side - chain and the aedans label adopt different conformations depending on the position of the aedans - labeled cysteine residue for membrane - embedded m13 coat protein mutants .
this is illustrated in fig . 2 , which shows a surface plot of the tryptophan side - chain ( blue ) and the aedans label ( red ) over the course of the entire simulation for three representative aedans label positions : a mutant in the n - terminus ( a16c ) , in the transmembrane segment ( v29c ) , and in the c - terminus ( s46c ) .
in the case of the mutants a16c and s46c , the tryptophan surface plot appears more disc shaped , whereas the tryptophan surface plot for the v29c mutant is more spherical , indicative of different rotameric states during the course of the simulation .
the aedans conformational space has a confined , disc - like shape in the case of the v29c mutant .
also for the a16c mutant , the aedans conformational space is relatively confined , whereas the conformational space of the s46c mutant has a bilobal shape , indicative of two major rotameric states.fig .
2space - filling representation of the conformational space of the tryptophan side - chain ( blue ) and of the aedans - labeled cysteine residue ( red ) over the course of 8-ns simulation time for membrane - embedded aedans - labeled a16c , v29c , and s46c m13 coat protein mutants , respectively .
the protein is represented as a grey ribbon space - filling representation of the conformational space of the tryptophan side - chain ( blue ) and of the aedans - labeled cysteine residue ( red ) over the course of 8-ns simulation time for membrane - embedded aedans - labeled a16c , v29c , and s46c m13 coat protein mutants , respectively .
the protein is represented as a grey ribbon during the simulations , membrane - embedded m13 coat protein remains close to an -helix , enabling the use of the long axis of the helix as a reference to describe the orientation of the tryptophan and aedans side - chains .
thus , to quantify the conformational space occupied by the tryptophan side - chain , the angle of the tryptophan emission dipole moment with the long axis of the helix , w , is depicted in fig . 3 for membrane - embedded wild - type m13 coat protein and the mutants a16c , v29c , and s46c .
in the case of the wild - type protein , the angular distribution of the tryptophan dipole moment with helical axis is symmetrical , centered on a value of 70. the angular distributions of the mutants a16c and s46c are also symmetrical , centered on 65 and 70 , respectively .
however , in the case of the v29c mutant , the angular distribution resembles two peaks , centered on values of 70 and 95 , respectively.fig .
3distribution of the angle between the tryptophan emission dipole moment and the helical axis for membrane - embedded wild - type m13 coat protein ( wt ) , and the mutants a16c , v29c , and s46c distribution of the angle between the tryptophan emission dipole moment and the helical axis for membrane - embedded wild - type m13 coat protein ( wt ) , and the mutants a16c , v29c , and s46c an overview of values of the center of the distributions describing the tryptophan conformational space for all mutants is given in fig .
one peak is observed centered at a value of w around 60. some mutants show a second peak at a higher value of w , mostly centered on 85. an exception is observed for mutants a7c and a9c , where the value of w of the second peak is 115 and 120 , respectively . only in the case of mutant a18c
are three peaks observed , giving a third value for w of 118. all values of w fall within the range from 50 to 120 , indicating that the tryptophan emission dipole has a tendency to adopt a perpendicular orientation with respect to the helical axis . throughout the aedans label position ,
furthermore , no large deviations from the value of the wild - type protein ( w = 70 ) are observed .
these observations indicate that the effect of the aedans label on the tryptophan conformational space is limited.fig .
4center of the distribution of angles between the tryptophan dipole and helical axis of the m13 coat protein , w , max , for all different mutants as a function of aedans label position .
the filled circle represents the case of three peaks ( mutant a18c ) . for well - resolved peaks ,
error margins were estimated based on the width of the peak at half height center of the distribution of angles between the tryptophan dipole and helical axis of the m13 coat protein , w , max , for all different mutants as a function of aedans label position .
the filled circle represents the case of three peaks ( mutant a18c ) . for well - resolved peaks ,
error margins were estimated based on the width of the peak at half height in fig . 5 ,
the angle between the aedans absorption dipole and long axis of the protein helix , a , is depicted for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c . in the case of mutant a16c
, the angular distribution of the aedans absorption dipole with respect to the helical axis is centered on a value of 80. for mutant v29c , two peaks are observed in the angular distribution : one large peak centered on 50 and a smaller peak centered on 110. the angular distribution of mutant s46c shows two peaks , centered at 20 and 65.fig .
5distribution of the angle between the aedans excitation dipole moment and the helical axis for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c distribution of the angle between the aedans excitation dipole moment and the helical axis for membrane - embedded m13 coat protein mutants a16c , v29c , and s46c in fig . 6 , the values of the center of the distributions describing the aedans conformational space are depicted .
the values of a fall within a range from 25 to 125 , indicating a wider distribution of conformational space of the aedans label as compared with the tryptophan side - chain , probably due to the longer aedans linker . since the protein makes a tilt of 23 with respect to the membrane normal ( koehorst et al .
2004 ) , the smallest values of a imply that for certain mutants the aedans label is almost aligned with the lipid tails .
similar as in the case of tryptophan ( fig . 4 ) , no clear trend can be observed in the values of a , although it is obvious that the aedans conformational space varies strongly among different positions of the cysteine point mutation.fig .
6center of the distribution of the angles between the aedans absorption dipole and the helical axis of the m13 coat protein , a , max , for all different mutants as a function of aedans label position .
the filled circle represents the case of three peaks ( mutant 18 ) . for well - resolved peaks ,
error margins were estimated based on the width of the peak at half height center of the distribution of the angles between the aedans absorption dipole and the helical axis of the m13 coat protein , a , max , for all different mutants as a function of aedans label position .
the filled circle represents the case of three peaks ( mutant 18 ) . for well - resolved peaks ,
error margins were estimated based on the width of the peak at half height for the membrane - embedded m13 coat protein , the behavior of the aedans label is expected to be determined by three main factors : ( 1 ) the local rotations of the aedans side - chain attached to the protein backbone , ( 2 ) the restrictions of the rotamers by the backbone and side - chains of the neighboring amino acid residues , and ( 3 ) the restrictions imposed by the surrounding lipids . to investigate these possibilities , an additional series of molecular dynamics simulations was carried out , using a 25-residue membrane - spanning -helical polyalanine with a single aedans - labeled cysteine point mutation at different positions in the helix . even though polyalanines do not form a transmembrane helix in vitro ( lewis et al .
2001 ) , polyalanines are frequently used in molecular dynamics simulations as model transmembrane -helices ( choma et al .
since the alanine side - chain is relatively small , specific interactions between the polyalanine helix and the aedans label are expected to be minimal , making it an ideal model transmembrane helix for our purpose .
therefore , in case of the polyalanine helix the conformational space of the aedans label is expected to be determined mainly by restrictions imposed by the surrounding lipids .
the aedans conformational space varies strongly among different positions of the cysteine point mutation , as shown by the values of a in fig .
all values of a fall within a range from 20 to 100 , indicating a slightly narrower distribution of conformational space of the aedans labels in polyalanine as compared with aedans labels attached to m13 coat protein ( fig .
this might be related to the more homogeneous structure of the polyalanine - membrane system.fig .
7center of the distribution of the angles between the aedans absorption dipole and the helical axis , a , max , for all different mutants in the case of a membrane - embedded polyalanine helix .
the open triangles represent the angles in case a second peak is observed . for well - resolved peaks , error margins were estimated based on the width of the peak at half height center of the distribution of the angles between the aedans absorption dipole and the helical axis , a , max , for all different mutants in the case of a membrane - embedded polyalanine helix .
the open triangles represent the angles in case a second peak is observed . for well - resolved peaks , error margins were estimated based on the width of the peak at half height
the goal of this work is to advance fluorescent techniques , in particular fret , as tools to study membrane protein structure through studying the effect of the lipids and neighboring side - chains on the conformational space of a fluorescent labels attached to a model membrane protein . to evaluate the implications of our findings with respect to the energy transfer efficiencies measured in our previous work ( vos et al .
8 . when the donor acceptor pair in the experimental ensemble has the same fixed orientation or the same extent of dynamic averaging it is valid to substitute a single or average value for in eq . 2 ( dale and eisinger 1979 ) .
qualitatively , the energy transfer efficiencies calculated here are in good agreement with the experimental values for mutant positions 139 , which could suggest that sampling might be sufficient , even though it is not complete for all individual mutants . however , the quality of the fit is not improved much compared with the values calculated in our previous work based on the < > = 0.67 approximation . for mutant positions 4050 ,
8 is therefore that the energy transfer efficiencies in the c - terminus of the protein ( mutants 4050 ) show a systematic deviation towards lower values .
interestingly , recent fret studies by our group on the same protein reconstituted in bilayers of hydrophobic mismatch indicate that residues 3850 have a high propensity for helical deformation ( vos et al .
it is therefore likely that the discrepancy between the theoretical efficiencies calculated in the work presented here and the previously measured experimental efficiencies are due to the -helical starting structure that was used in silico , whereas the helix would be more distorted in vitro .
however , since the protein backbone is effectively rigid during the time span of the simulations , this will most likely not have an effect on the aedans or the tryptophan conformational space , which is the focus of the current work.fig .
8experimental ( filled diamonds ) and theoretical ( open triangles ) energy transfer efficiencies for the aedans - labeled mutants used in the molecular dynamics simulations experimental ( filled diamonds ) and theoretical ( open triangles ) energy transfer efficiencies for the aedans - labeled mutants used in the molecular dynamics simulations the surface plots in fig .
2 of the tryptophan side - chain and of the aedans label of mutant v29c suggest a direct interaction between the tryptophan side - chain and the aedans label .
the tryptophan and the aedans chromophores appear to be oriented in parallel planes , suggesting that the two chromophores are strongly interacting .
the tryptophan conformation is best described as a disc - like conformation , which is also visible in the case of the a16c and s46c mutants . in this disc - like conformation , which is observed in the simulation of all mutants , the tryptophan emission dipole makes an angle of ~70 with the helical axis and the tryptophan emission and aedans absorption dipoles are more likely to align .
4 shows that this conformation of w = 60 is present for most mutants . for a large number of mutants
, a second conformation is observed , represented by a peak centered on a value w = 90. a third conformation , with w = 115 , is visible in the case of the a7c and a9c mutants .
the first conformation , with a value w = 70 , could be the conformation with the lowest energy .
the other two conformations are not always probed , although the second conformation , with a value w = 90 , shows up in ~50% of all mutants .
the third conformation is the rarest , only showing up in 14% of all mutants . only in the case of the a18c mutant
the fact that there is no apparent correlation between the position of the aedans label and the conformational space of the tryptophan suggests that the differences between the different mutants and simulations are caused by incomplete probing of the tryptophan conformational space rather than by specific interactions affecting the energy of the different tryptophan rotameric states .
even though 10 ns of simulation time represents a considerable amount of computational time , such a simulation might not be sufficient for exhaustive probing of the tryptophan conformational space . to address this issue , the angle of tryptophan dipole with the local helix axis over the simulation time
was evaluated for a selection of mutants : 7 , 15 , 23 , 31 , 36 , 41 , and 46 ( data not shown ) .
the main implications of this analysis are summarized in table 1 . except for the mutant s46c
, tryptophan shows many transitions between the different conformations , indicating that sampling is significant .
the tryptophan conformational space is thus well described by three different rotameric states , even though not all conformational states appear in each simulation.table 1sampling analysis of the angles of the tryptophan and aedans dipoles with the local helix axis over the simulation time ( 10 ns ) for the aedans - labeled coat protein .
the analysis was carried out for a selection of mutantsmutant positiontryptophanaedans720160 , many transitions0180 , many transitions1520160 , many transitions0180 , few transitions230180 , many transitions20150 , many transitions3110180 , many transitions50120 , many transitions360180 , many transitions0160 , many transitions4120120 , many transitions50120 , many transitions46few transitionsfew transitionsabout 100 transitions per nsabout 1 transition per ns sampling analysis of the angles of the tryptophan and aedans dipoles with the local helix axis over the simulation time ( 10 ns ) for the aedans - labeled coat protein .
the analysis was carried out for a selection of mutants about 100 transitions per ns about 1 transition per ns compared with the tryptophan emission dipole moment , the values of the angular distribution of the aedans absorption dipole moment is wider .
the maxima of the a values can not be categorized into groups with almost discrete values , possibly because the conformational space of the aedans group is much larger than that of the tryptophan side - chain due to the long linker between the aedans chromophore and the protein backbone . for comparison ,
the aedans group is linked to the protein backbone by seven bonds about which the chromophore can rotate freely , compared with two bonds with free rotation in the case of tryptophan , making the aedans conformational space significantly larger . however , even though the values for the maxima of the aedans angular values do not fall into neatly defined categories , it is worthwhile to analyze the angular distribution of the aedans dipole with the helical axis further
. this is done by categorizing the values of the maxima depicted in fig . 5 into different groups : groups a , b , c , d , e , and f , corresponding to values of a of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 , respectively .
groups a , e , and f , representing the most extreme values of a , are least densely populated .
possibly , this reflects a purely statistical effect , indicating that aedans behaves like a relatively unperturbed vector , sweeping a conical surface . throughout all the molecular dynamics simulations , a ranges from 30 to 50 ( group
b ) . even though the values of a throughout the different simulations do not form a peak themselves
, the results presented here do suggest that values 30 a
90 ( groups b , c , and d ) have a higher probability , regardless of the mutant position , and that extreme a values have a lower probability . to evaluate whether sampling of the aedans conformational space is significant , the angle of the aedans dipole with the local helix axis over the simulation time was evaluated for a selection of mutants : 7 , 15 , 23 , 31 , 36 , 41 , and 46 ( data not shown ) .
mutants 15 and 46 show only a few transitions , indicating that sampling is limited , while for the other mutants the angles sample a significant range with many transitions , indicating that sampling is significant in most cases.fig .
9probability of finding the aedans labels in various conformations averaged over all mutants for aedans - labeled polyalanine ( black bars ) and for aedans - labeled coat protein ( white bars ) .
groups a , b , c , d , e , and f , correspond to values of a , max of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 ,
respectively probability of finding the aedans labels in various conformations averaged over all mutants for aedans - labeled polyalanine ( black bars ) and for aedans - labeled coat protein ( white bars ) .
groups a , b , c , d , e , and f , correspond to values of a , max of 1030 , 3050 , 5070 , 7090 , 90110 , and 110130 , respectively to elucidate to what extent specific interactions with the side - chains or with the neighboring lipid and water molecules affect the aedans conformational space , an additional set of simulations of aedans - labeled polyalanine was analyzed in the same way .
the angular distribution diagrams for both the aedans - labeled coat protein and the aedans - labeled polyalanine show a considerable spread , depending on the position of the mutation . since specific interactions between the aedans label and the amino acid side - chains
are not very important in the case of the polyalanine helix , the spread in the maxima of the angular distribution functions which is observed in the simulations of aedans - labeled polyalanine is not caused by specific , position - dependent interactions with neighboring side - chains .
this raises the question of what determines the spread in a values observed in fig . 7 , i.e. , whether specific interactions between the label and lipid and water molecules are responsible or whether the different simulations probe different parts of the aedans conformational space , which is , in fact , very similar for the various mutant positions .
if the interactions between the label and the lipids change the aedans conformational space , a certain degree of symmetry would be expected in fig . 7 .
for instance , the distance between the mutant positions 2 and 5 and the lipid head group region is roughly the same as the distance between mutant positions 23 and 20 and the opposite lipid head group region .
this implies that the aedans groups are exposed to approximately the same lipid environment for mutants 2 and 5 as for mutants 23 and 20 , respectively .
however , the a values for positions 2 and 23 are different , as are the a values for positions 5 and 20 . also for the other mutant positions , where the aedans label is buried deeper into the bilayer , no symmetry can be inferred from the a values .
therefore , it is not likely that the interaction with the lipid and water molecules is the decisive factor that determines the spread in a values as observed in the polyalanine simulations .
more likely , the spread in a values observed here is caused by the fact that different simulations probe different parts of the aedans conformational space , as is the case even for the much smaller tryptophan side - chain . in fact , in the case of aedans , the spread in a values is expected to be even bigger than the spread in w values in the case of the tryptophan side - chain , due to the larger mass of the aedans label and the increased magnitude of the aedans conformational space .
the comparison between the a values in the case of aedans - labeled coat protein and aedans - labeled polyalanine indicates that specific interactions between the aedans label and neighboring side - chains do not have a large effect on the aedans conformational space .
secondly , the fact that the distribution in a values in the case of the aedans - labeled polyalanine is essentially random suggests that the position spread in the aedans conformational space is not strongly affected by interactions with neighboring lipid and water molecules .
more likely , the spread in a values between the different mutant positions is caused by incomplete probing of the conformational space in each individual simulation . to test the latter idea ,
it is worthwhile to make a comparison between the distribution of the a values in the case of the aedans - labeled coat protein and the aedans - labeled polyalanine , averaged over all mutants .
if position - dependent specific interactions between the label and neighboring side - chains , and between the label and surrounding lipid and water molecules , do not have a large effect on the aedans conformational space , the conformational space as observed in the simulations of the polyalanine simulations would be similar to the conformational space as observed in the simulations of the coat protein simulations across all different mutants .
the a values found across the different simulations of m13 coat protein and polyalanine are depicted in fig .
clearly there are differences between the behavior of aedans when attached to a polyalanine helix and to m13 major coat protein .
for instance , when attached to a polyalanine single helix the label is not found in group b , whereas the label is frequently present in group b when it is attached to coat protein .
possibly , this difference is because the number of mutants for the polyalanine is only limited as compared with those for coat protein , which means that sampling is less exhaustive for the label attached to the polyalanine helix then for the label attached to coat protein . to illustrate this ,
we compare combined probabilities for groups a and b , c and d , and e and f , respectively , for the aedans - labeled polyalanine and aedans - labeled coat protein .
the combined probabilities for a / b , c / d , and e / f are 0.3 , 0.6 , and 0.1 in the case of polyalanine . for coat protein ,
hence , for both the aedans - labeled polyalanine helix and the coat and for the aedans - labeled coat protein , the label is most likely to be found in groups c / d , although in the case of aedans - labeled polyalanine the label is equally likely to be found in groups a / b . for both aedans - labeled polyalanine and coat protein ,
this result suggests that the average conformational space for aedans is similar in the case of the polyalanine simulations as in the case of the m13 simulations , indicating that specific interactions between the label and neighboring side - chains do not affect the aedans conformational space significantly . in summary ,
our results indicate that neither specific interactions between the aedans label and the lipid or water molecules , nor interactions between the aedans label and neighboring side - chains , have a large effect on the aedans conformational space .
interestingly , this finding advocates the use of aedans as a relatively inert environmental probe that can move relatively unhindered through the lipid membrane .
an important factor when using fret as a spectroscopic tool is the mutual orientation of the donor and acceptor during energy transfer . for this reason
, we evaluate the average value of the orientation factor for all different aedans - labeled major coat protein mutants over the course of the simulation , < > , depicted in fig .
we note that this value represents a static average , and therefore the extrapolation to energy transfer efficiencies as measured in a fluorescence experiment is not straightforward . however
, for our purpose of evaluating potential systematic trends in the mutual orientation of donor and acceptor dipoles , evaluation of < > suffices . for most mutants , the value of < > is lower than 0.67 ( the isotropic dynamic average that is frequently used to calculate distances from energy transfer experiments ) . in certain cases ,
< > is almost zero , indicating a perpendicular orientation between the tryptophan emission dipole and the aedans absorption dipole .
notably , for mutant positions 2332 , < > is strongly increased to as high as 2.5 , approaching the theoretical maximum of 4 , indicating that the dipoles are almost aligned .
for this reason it is worthwhile to evaluate the average value of the orientation factor for all different aedans - labeled major coat protein mutants over the course of the simulation , < > , as depicted in fig .
the value < > is lower than 2/3 , which is the isotropic dynamic average that is frequently used to calculate distances from energy transfer experiments . in certain cases ,
< > is almost zero , indicating a perpendicular orientation between the tryptophan emission dipole and the aedans absorption dipole .
notably , for mutant positions 2332 , the energy transfer for most mutants is strongly increased to as high as 2.5 , approaching the theoretical maximum of 4 , indicating that the tryptophan emission dipole and the aedans absorption dipole are almost aligned , at least through part of the simulation.fig .
10implications for the average orientation factor < > implications for the average orientation factor < > even though the effect of neighboring side - chains on the aedans and tryptophan conformational space is limited , the data presented in fig .
9 suggest that there is some interaction between the aedans and the tryptophan group , resulting in an increased < > value for mutants in the transmembrane region .
this could suggest a small degree of stacking between the aedans and tryptophan chromophores , as illustrated in fig . 2 , increasing the probability of dipole alignment .
6 gives no indications of a systematic effect on the overall conformational space , suggesting that the increase in < > could be due to an indirect effect , with both tryptophan and aedans aligning along the lipid acyl chains .
our computer simulations suggest that the conformational space of the aedans label is only slightly affected by the presence of amino acid side - chains or lipids .
this finding advocates the use of aedans as a relatively inert environmental probe that is structurally unhindered by the membrane lipids . as such
, aedans fluorescence spectroscopy and fret could contribute to the development of a general strategy to study membrane protein structure and function in the future . | computer simulations were carried out of a number of aedans - labeled single cysteine mutants of a small reference membrane protein , m13 major coat protein , covering 60% of its primary sequence .
m13 major coat protein is a single membrane - spanning , -helical membrane protein with a relatively large water - exposed region in the n - terminus . in 10-ns molecular dynamics simulations
, we analyze the behavior of the aedans label and the native tryptophan , which were used as acceptor and donor in previous fret experiments .
the results indicate that aedans is a relatively inert environmental probe that can move unhindered through the lipid membrane when attached to a membrane protein . | Introduction
Methodology
Details of the computer simulations
Calculation of the energy transfer efficiency from the molecular dynamics simulations
Results
Discussion
Concluding remarks |
PMC4864009 | more than 2.1 billion people close to 30% of the global population today are overweight or obese.1 obesity can therefore be seen as a global epidemic and thus as a major public health problem .
both overweight and obesity are characterized by the accumulation of excessive levels of body fat , and this creates an increased risk for cardiovascular diseases , some types of cancer , and overall mortality.2 recent evidence has shown similar adverse effects of obesity on the brain.3 in particular , abdominal obesity is associated with significant metabolic changes that impinge upon the central nervous system in even younger , nonclinical (= general ) populations at midlife.4 furthermore , obesity has been implicated to play a role in cognitive deficits in patients with dementia5 and mental disorders.6 the underlying reasons for becoming obese are not well understood ; however , it is likely that both genes and environmental factors , especially an age - related decrease in physical activity , contribute to this problem.7 obesity is caused by perturbations of the balance between food intake and energy expenditure .
enlarged adipocytes and activated macrophages secrete proteins and lipids , known as adipokines , that influence inflammation and overall carbohydrate and lipid metabolism.8 moreover , an upregulation of indoleamine 2,3-dioxygenase-1 ( ido1 ) activity , caused by immune - mediated inflammation , which catalyzes the formation of kynurenine ( kyn ) and limits the availability of tryptophan ( trp ) , could be a key component in the initiation of obesity.9 thereby , the kyn pathway is induced,10 and the elevation of the ratio of kyn to trp concentrations that estimates the trp breakdown rate is often linked with conditions of inflammation11 and neuropsychiatric symptoms.12 the accelerated trp breakdown during inflammation is mainly fed by endogenously formed interferon- and is also influenced by other cytokines , such as tumor necrosis factor- , interleukin ( il)-6 , and lipopolysaccharide.13,14 as a precursor for 5-hydroxytryptamine ( serotonin ) , the essential amino acid trp is also a key player in caloric intake regulation by predominantly inhibiting carbohydrate intake.15 in the epiphysis , the sleep - regulating hormone melatonin is biosynthesized out from serotonin .
lifestyle modification , specifically changes in diet , physical activity , and exercise are considered the cornerstone of obesity management.16 however , the adherence of victims to diet and exercise regimens is often limited and they may quit after the first six months before reaching their goals.17 recently , we observed that low - calorie weight loss diet lowered not only levels of leptin but also levels of trp significantly.18 the disturbed metabolism of trp might affect the biosynthesis of serotonin and could thereby increase the susceptibility for mood disturbances and carbohydrate craving , increasing the cessation probability of weight reduction programs ( fig .
1 ) . on the other side , moderate physical exercise a potent stimulus to modulate proinflammatory cytokines , which may affect trp levels could be helpful in improving mood status and preventing uncontrolled weight gain.19 the purpose of this review is to provide a brief overview on the effects of diet and exercise on weight control and to describe the important role of trp metabolism during dietetic interventions combined or not with physical exercise .
a review of literature was conducted through pubmed database to identify relevant studies , especially published in the past 10 years .
the following keywords were used alone or in various combinations : diet , exercise , mood , tryptophan , weight loss , and maintenance .
reference lists from original and review articles were also reviewed in order to identify additional relevant studies .
mechanistic influences on energy imbalance arise from changes in resting metabolic rate , nonexercise activity thermogenesis , fat - free mass , and energy intake .
the impact of these changes on energy imbalance translates to changes in body weight.20 dietary restriction and exercise are useful methods to create a negative energy balance . however , most of the exercise studies suggest that exercise activity alone has only a minor influence on body weight reduction21 and that mainly the combination of both diet and exercise training leads to significant reduction of body mass.22 the classic explanation for the secondary role of exercise is that exercise alone can not generate enough energy expenditure to create a negative energy balance to the extent possible with caloric restriction .
the frequency and intensity of exercise at public health recommendation levels may explain why exercise does not improve weight loss above that achieved with caloric restriction .
ross et al.23,24 showed that the energy expenditure of exercise has to be between 500 and 700 kcal / d to provide a body mass loss of approximately 6 kg in women and 8 kg in men within 12 weeks .
this is true , but it is also true that , independent of the method of weight loss , a negative energy balance of 500700 kcal / d always causes a fat loss of 68 kg within three months . when the energy deficit imposed by diet only and diet plus exercise interventions is similar , weight loss and/or percentage change in body weight are similar.25 nevertheless , to be successful in losing weight , it takes a considerable amount of time and effort .
several studies have examined weight compensation after aerobic exercise training and concluded that the major factors limiting the expected weight loss from aerobic exercise were dietary compensation and a low exercise dose.20,26 currently , fewer than 30% of european adults meet exercise guidelines with a lack of time , the major barrier to regular exercise participation .
accordingly , in contrast to traditional high - volume endurance training ( at 35.9 metabolic equivalent tasks ) , vigorous intensity aerobic endurance training ( at 6 metabolic equivalent tasks ) or resistance training ( muscular strength exercises using a resistance equivalent to 60%80% of the individual s one repetition maximal effort , including free weights , machines with stacked weights or pneumatic resistance , and resistance bands ) can be a time - efficient strategy to reduce body fat and improve metabolic health.27 it has been shown that in terms of weight change , when different intensities of aerobic exercise are matched for caloric expenditure or dose , both vigorous and moderate intensity aerobic trainings result in similar amounts of weight loss.28 furthermore , vigorous intensity aerobic endurance training can serve as an effective alternate to traditional endurance - based training , inducing even superior health benefits for important risk factors , including visceral fat,29 measures of glucose / insulin metabolism,30 and cardiorespiratory fitness.31 although the addition of resistance training to dietary restriction has been shown to have limited effectiveness in reducing body weight increases in muscle mass with resistance training may off - set loss of adipose tissue
the combination of diet with resistance training seems to be more effective than diet or diet with endurance training in the reduction of body mass and fat mass.32 resistance training significantly increases resting metabolic rate after a training session33 and has the power to reduce low - grade inflammation independently from weight loss in overweight / obese adults.34 muscle contractions lead to the production and release into the circulation of il-6 , which appears to have numerous biological effects , including effects on glucose and fat metabolism .
acute elevations in il-6 produced by contracting the skeletal muscle downregulate the production of tumor necrosis factor- by monocytes and stimulate the release of il-1 receptor antagonist from monocytes and macrophages , thus increasing the circulating concentrations of anti - inflammatory cytokines ( such as adiponectin and il-10 ) from adipose tissue.35 in addition to exercise interventions , many studies have manipulated the macronutrient content of energy - restricted diets and have reported favorable changes in body composition with many dietary approaches.36,37 thus , reducing the calorie intake below expenditure results in a predictable initial rate of weight loss that is related to the energy deficit.38 based on recent research and meta - analytic evidence , weight loss is achieved by adherence to any diet that successfully reduces calorie intake , but adherence rates are low with most diets.39 although a low - carbohydrate diet may be associated with greater short - term weight loss , superior weight loss in the long - term has not been established.40 furthermore , very - low carbohydrate weight loss diets may be associated with more frequent side effects , such as mood swings.41 carbohydrates are responsible for helping drive trp across the blood brain barrier and having an effect on the brain serotonin levels.42 thus , by eating a low carbohydrate meal , the amount of trp entering the brain will be substantially lower compared with the normal situation , contributing to symptoms of depression , such as mental fatigue and low mood .
in addition , trp competes with the other large neutral amino acids ( lnaa ) for transport across the blood brain barrier . since plasma amino acids change in obese persons on hypocaloric diet , a decrease in trp
lnaa ratio may further influence serotonin synthesis.43 previous work reported a decrease in plasma trp and trp / branched - chain amino acids ( bcaas ) ratio in men and women by dieting.44 as a common consequence , caloric restriction weight loss diets , especially if based upon macronutrient content that is low in carbohydrate and high in protein , lead many dieters to revert to their old eating habits , eating more and using carbohydrate - rich foods , to feel better , which is a frequent cause of weight gain , the so - called yo - yo effect.45 in a recent study,18 concentrations of essential amino acid trp decreased significantly with a caloric restriction weight loss diet , and lowest trp concentrations were observed in the group of individuals with the lowest calorie intake .
the decline of trp levels can be referred to its reduced intake during caloric restriction diet as it was unrelated to the immune activation status of individuals , which remained unchanged .
however , there may be gender differences in the response of plasma trp to dieting .
for example , in women , but not in men , dieting significantly lowered the plasma total and free trp , indicating that dieting alters brain serotonin function in women , perhaps as a consequence of reducing the availability of plasma trp.46 calorie restriction decreases resting and total energy expenditure.47 declines in energy expenditure favoring the regain of lost weight persist well beyond the period of dynamic weight loss.48 findings from the pounds lost study confirmed a decrease in total energy expenditure , mainly contributed by a decrease in resting energy expenditure , in both men and women.49 the authors found , further , a decrease in energy expenditure from physical activity as dietary carbohydrate decreases followed by a decrease in lean body mass with a high fat diet .
thus , weight loss leads to both physiological and psychological changes that promote subsequent weight regain .
the path to overcome this tendency for weight regain may involve exercise and dietary strategies that improve adherence , counter the physiological and behavioral adaptations , and reestablish the balance between intake and expenditure.50
although many individuals have success in losing weight with diet , weight maintenance is a challenge , regardless of the initial modality used for weight loss .
long - term adherence to restrictive diets is made difficult because of the reduction in energy expenditure that is induced by weight loss but also because of changes in the peripheral hormone signals that increase appetite.51 hence , most dieters subsequently regain much or all of the lost weight . exercise and behavioral interventions may help individuals maintain weight loss since exercise programs modify responses in a direction expected to enhance satiety and permit weight loss and/or maintenance.50 weight loss leads to compensatory changes in the homeostatic processes , including alterations in energy expenditure , substrate metabolism , and hormone pathways involved in appetite regulation that result in increased hunger and energy storage , favoring weight regain.51 in a recent review , maclean et al.52 summarized the adaptations to energy - restricted weight loss that are thought to promote weight regain . during weight loss ,
metabolic requirements decline as a function of ( i ) lost body mass , ( ii ) reduced consumption of food , and ( iii ) increased metabolic efficiency of peripheral tissues .
neuroendocrine signals from the periphery transfer a message of energy depletion and low nutrient availability favoring signals of hunger to the hypothalamus and hindbrain that serve as the primary control centers for energy balance regulation .
the response is that appetite increases and the expenditure of energy declines , named as the energy gap.52 to maintain the reduced weight , food intake must be restricted to the level that expended energy is suppressed or adherence to exercise must be enhanced in order to reduce the gap between appetite and expenditure .
another reason why it is so hard to maintain weight loss is the evidence that weight loss may be associated with increased depressive symptoms.53 mood improvements often occur early in treatment , prior to achieving significant weight loss ; however , in the long term , personal costs of losing weight exceed the benefits .
lowered trp availability during weight loss may limit the production of neurotransmitter serotonin , and this may result in mood disturbances and can , further , diminish serotonin functions ultimately leading to satiety dysregulation and increased food intake.54 it is also apparent that disturbed mood can affect the self - rewarding mechanisms of food consumption , which is likely to involve foods that are high , both in carbohydrates and saturated fats , and which in turn may promote weight gain , which could further depress mood and self - esteem over the long term.55 notably , calorie restriction diet is not only associated with a decline in the amino acid trp , the precursor of the neurotransmitter serotonin , but also with a reduction in phenylalanine , the precursor of the tyrosine
dopamine pathway.18 furthermore , in obese individuals , increased activity of the immunomodulatory enzyme ido1 during immune activation results in trp depletion , and this persists in spite of significant weight reduction,56 which may slow - down the production of serotonin in the brain and further lower mood.57 following weight loss , compensatory changes include changes in the levels of circulating appetite - related hormones , such as increases in orexigenic hormones ( eg , ghrelin ) , which stimulate appetite , and decreases in anorexigenic hormones ( eg , leptin ) , which inhibit appetite , with the net result that appetite is increased.51 currently , there is a disparity in the literature concerning the influence of exercise on appetite .
some researchers suggest that chronic exercise training does not induce substantial weight loss due to compensatory responses , ie , increase in appetite and food intake,58,59 while some others consistently document that exercise has no influence on appetite or ad libitum food intake.60,61 based on a recent meta - analysis , acute exercise has a trivial effect on subsequent energy intake.62 differences in lean body mass , fat mass loss , volume and duration of exercise interventions , and inclusion of different genders are likely to be factors contributing to the discrepancy .
staten noted a gender difference in compensatory food intake after exercise , with an increased caloric intake in men but not in women.63 one possible reason for this is that men exhibit greater amounts of fat - free mass.64 indeed , resting metabolic rate ( largely determined by fat - free mass ) is associated with hunger , self - determined meal size , and daily energy intake and may represent a marker for appetite.65 on the contrary , the appetite and energy intake response to exercise did not differ between men and women in a recent study.66 however , women exhibit compensatory appetite , gut hormone , and food intake responses to acute food restriction - induced energy deficit but not in response to an acute bout of exercise .
furthermore , there may be variability in weight - reduced subjects according to individual physiological characteristics , with clear responders and nonresponders.67 since serotonergic mechanisms may reduce body weight by accelerating the onset of satiety68 besides suppressing excessive snacking of carbohydrate - rich foods , nonpharmacological methods of raising brain serotonin during weight loss and/or maintenance could be highly useful in preventing uncontrolled weight gain .
it has been shown that physical exercise ( mild stress ) normalizes levels of proinflammatory cytokines and may counteract the activation of inflammation / ido1 pathways , which may decrease the susceptibility for mood disturbances and carbohydrate craving.69 animal and human studies have shown that aerobic exercise can stimulate brain serotonin activity and trigger parallel elevations in plasma - free trp and brain trp.7072 in the primary study by chaouloff et al.70 , nonesterified fatty acid elevation increased free trp , hence its entry in the brain for serotonin synthesis and also in the liver .
acute exercise seems to elevate the activity of trp 5-monooxygenase , the enzyme involved in the rate - limiting step in the synthesis of serotonin , and so leads to an increase in the concentration of serotonin in some areas of the brain , ie , the brain stem and hypothalamus.73 chronic exercise ( 30 min / d , six days per week for four weeks ) causes neural adaptations by activating not only the synthesis but also the metabolism of serotonin in the cerebral cortex.74 furthermore , salivary and serum cortisol levels in humans and corticoster - one in rats are increased by exercise,75,76 and this could induce liver trp 2,3-dioxygenase , as demonstrated in rats .
the combined effects of increased liver trp and trp 2,3-dioxygenase induction can elevate serum kyn in humans77 and in rats.78 in both of these latter studies , increases in serum kyn after exercise have been shown . although exercise increased the ido1 activity of macrophages in rats,78 until now ,
there is no direct information on the effect of exercise on ido1 activity in humans .
furthermore , the effects of elevations in pro- and anti - inflammatory cytokines by exercise on ido1 activity still remain to be examined .
however , a recent study in healthy human volunteers showed a significant induction of trp breakdown as indicated by an increase in kyn / trp , which could be referred to an enhanced activity of ido1 because a significant association of kyn / trp with the immune activation marker neopterin was observed.79 trp is not only a precursor of the serotonin biochemical pathway but also the key element for the formation of nicotinamide adenine dinucleotides , nad and nadh , via the kyn pathway.80 potential signals during exercise include increased nad / nadh . with increasing exercise intensity , the cytosolic nad / nadh ratio declines as lactate accumulates.81 nad regulates the expression of sirtuins ( sirt ) and peroxisome proliferator - activated receptor- coactivator ( pgc-1 ) .
enhanced sirt1 and pgc-1 activities are associated with improved mitochondrial function and exercise performance82 and protection against obesogenic feeding.83 recently , a mechanism was discovered by which overexpression of pgc-11 in muscle mimics antidepressant effects of exercise by promoting kyn aminotransferase expression . by this way ,
crossing of kyn through the blood brain barrier and the disruption of neural plasticity are prevented.84 during exercise , the entry of trp into the brain through the blood
brain barrier is favored by increased muscle use of bcaas that inhibit trp transport into the brain and elevated plasma fatty acids , as this elevates the ratio of unbound trp to bcaa .
because of the increase in plasma trp and decrease in bcaa , there is a substantial increase in trp availability to the brain , consequently leading to higher serotonin concentrations in some areas of the brain.85 serotonin plays a key role in signal transduction between neurons , and an exercise - induced increase in the concentrations of serotonin has been linked to central fatigue.86,87 although there is some evidence that bcaa supplementation may increase exercise intensity at lactate threshold,88 upper body muscle power,89 and the rate of perceived recovery after strenuous exercise,90 the association between the ergogenic effect of the bcaa intake and the reduction in serotonin production in the brain has not been tested in humans . in a recent study,91 during a half - ironman triathlon , serum amino acids concentrations were reduced by > 20% .
however , neither the changes in serum - free amino acids nor the trp / bcaa ratio was related to muscle fatigue or muscle damage during the race .
paradoxically , trp supplementation can decrease fatigue perception during an aerobic exercise and likewise may improve physical performance , possibly by acting via endogenous opioids.92,93 taken together , exercise increases trp availability to the brain , and these findings provide support to the hypothesis that increases in serotonin synthesis and activity might be involved in the antidepressant effect of exercise.94 however , too high serotonin levels may impair mood and increase the sensitivity to fatigue , a situation similar to the development of a transient serotonin syndrome.95 given that there is a reciprocal link in mood disorders and obesity,48 physical exercise could mitigate the biological changes that occur with weight loss ( ie , decreased metabolic mass , increased metabolic efficiency , and increased hunger and depression ) via both an increase in energy expenditure ( with the potential to generate an energy deficit ) and the induction of an anti - inflammatory environment ( with a subsequent increased release of serotonin ) .
however , one has to distinguish recreational physical activity ( activity that people engage in during their free time , that people enjoy , and that people recognize as having socially redeeming values ) from repeated high level training sessions or intense endurance sports .
although the recreational physical activity performed every other day maximum will exert the above - mentioned positive impact on physiological pathways and parameters , repeated high - level activities could easily turn toward a negative net effect .
exhaustive physical activity significantly impacts on inflammation cascades that involve several proinflammatory cytokines , such as interferon- and down - stream biochemical pathways.96,97 this includes also alterations of amino acid profiles , eg , during and after a half iron man triathlon.91 such data fit well with the observation that intense training was associated with an accelerated trp breakdown and an increased kyn / trp.79 interestingly , intense versus moderate physical activities might also exert contrasting effects on neuropsychiatric circuits ( fig .
acute and moderate physical exercises , such as noncompetitive physical exercise such as jogging , increase the activity of gtp - cyclohydrolase-1 ( gch1 ) and contribute to increased productions of pteridine derivatives.98 by contrast , in our recent study , a drop in phenylalanine and phe / tyr concentrations by exhaustive exercise was noted,79 which indicates a decreased pah activity that could be due to a decline in tetrahydrobiopterin ( bh4).99 in the same population , nitrite levels dropped ( unpublished data ) that could represent another consequence of lowered no production related to diminished bh4 . unfortunately , the direct measurements of bh4 are almost impossible because only very stringent preanalytical measures can limit the oxidative loss of this compound and practically limit its monitoring to laboratory animal or in vitro studies .
still , data fit well with existing literature , which shows that acute endurance exercise may increase serotonin availability as was reflected in the periphery by increased concentration of free trp.100 the pathogenesis of negative mood is certainly multifactorial , trp and serotonin metabolism can be of some greater relevance , and implicating reactive oxygen species ( ros ) and bh4 is currently still rather speculative , although successful therapy of depression with bh4 has been reported already several years earlier.101 one may conclude that moderate physical exercise enhances the production of neurotransmitters , such as serotonin , noradrenaline , adrenaline , and dopamine , and can thereby contribute to a heightening of mood.102 however , only sports performed occasionally with two or three days interval when performed as recreational activity seems to exert this beneficial effect on general well - being , whereas intense training will rather adversely affect both mood103 and immune system function.104 other factors such as psychological stress , lack of sleep , and malnutrition can increase the risk of infection when immunity becomes suppressed .
this relationship will become especially important when sports activity is performed together with a reduction in food intake as a part of a weight - loss program .
after short time , calorie restriction diet was associated with a decline in essential amino acid trp in serum / plasma followed by a significant drop in phenylalanine.18 trp would further decline when the weight loss diet is combined with heavy physical exercise that induces trp degradation .
it could result in deficiency of the essential amino acids that because of their relevance for neurotransmitter biosynthesis
may affect the adherence to weight loss programs . to avoid this , one might consider supplementation with trp or 5-hydroxy - trp during such periods to improve weight loss maintenance .
indeed , it has been shown that acute trp supplementation while dieting could be helpful in improving mood status and preventing uncontrolled weight gain or neuropsychiatric symptoms.13 however , in healthy subjects , only large increases in brain trp levels are able to improve mood significantly , whereas relatively small increases in brain trp result in an improved mood in vulnerable subjects . so eating foods with relatively high trp content , such as turkey meat , cacao , nuts , and other seeds ,
is considered to increase trp availability to the brain , albeit slightly , and contributes to some mood enhancement.105 this conclusion is , however , weakened by the fact that such food usually also contains high concentrations of competing lnaa that reduces the trp : lnaa ratio and thus again limits the transport of trp to brain via the leucine - preferring l1 system.106 by contrast , in vitro observations show that antioxidant compounds are able to suppress ido1 , which supports the conclusion that healthy food rich in antioxidants could exert some additional positive effect on trp availability when trp breakdown is slowed down.107
weight loss leads to compensatory changes in the homeostatic processes , including alterations in energy expenditure , substrate metabolism , and hormone pathways involved in appetite regulation that result in increased hunger and energy storage , favoring weight regain.51 in a recent review , maclean et al.52 summarized the adaptations to energy - restricted weight loss that are thought to promote weight regain . during weight loss ,
metabolic requirements decline as a function of ( i ) lost body mass , ( ii ) reduced consumption of food , and ( iii ) increased metabolic efficiency of peripheral tissues .
neuroendocrine signals from the periphery transfer a message of energy depletion and low nutrient availability favoring signals of hunger to the hypothalamus and hindbrain that serve as the primary control centers for energy balance regulation .
the response is that appetite increases and the expenditure of energy declines , named as the energy gap.52 to maintain the reduced weight , food intake must be restricted to the level that expended energy is suppressed or adherence to exercise must be enhanced in order to reduce the gap between appetite and expenditure .
another reason why it is so hard to maintain weight loss is the evidence that weight loss may be associated with increased depressive symptoms.53 mood improvements often occur early in treatment , prior to achieving significant weight loss ; however , in the long term , personal costs of losing weight exceed the benefits . lowered trp availability during weight loss may limit the production of neurotransmitter serotonin , and this may result in mood disturbances and can , further , diminish serotonin functions ultimately leading to satiety dysregulation and increased food intake.54 it is also apparent that disturbed mood can affect the self - rewarding mechanisms of food consumption , which is likely to involve foods that are high , both in carbohydrates and saturated fats , and which in turn may promote weight gain , which could further depress mood and self - esteem over the long term.55 notably , calorie restriction diet is not only associated with a decline in the amino acid trp , the precursor of the neurotransmitter serotonin , but also with a reduction in phenylalanine , the precursor of the tyrosine
dopamine pathway.18 furthermore , in obese individuals , increased activity of the immunomodulatory enzyme ido1 during immune activation results in trp depletion , and this persists in spite of significant weight reduction,56 which may slow - down the production of serotonin in the brain and further lower mood.57
following weight loss , compensatory changes include changes in the levels of circulating appetite - related hormones , such as increases in orexigenic hormones ( eg , ghrelin ) , which stimulate appetite , and decreases in anorexigenic hormones ( eg , leptin ) , which inhibit appetite , with the net result that appetite is increased.51 currently , there is a disparity in the literature concerning the influence of exercise on appetite .
some researchers suggest that chronic exercise training does not induce substantial weight loss due to compensatory responses , ie , increase in appetite and food intake,58,59 while some others consistently document that exercise has no influence on appetite or ad libitum food intake.60,61 based on a recent meta - analysis , acute exercise has a trivial effect on subsequent energy intake.62 differences in lean body mass , fat mass loss , volume and duration of exercise interventions , and inclusion of different genders are likely to be factors contributing to the discrepancy .
staten noted a gender difference in compensatory food intake after exercise , with an increased caloric intake in men but not in women.63 one possible reason for this is that men exhibit greater amounts of fat - free mass.64 indeed , resting metabolic rate ( largely determined by fat - free mass ) is associated with hunger , self - determined meal size , and daily energy intake and may represent a marker for appetite.65 on the contrary , the appetite and energy intake response to exercise did not differ between men and women in a recent study.66 however , women exhibit compensatory appetite , gut hormone , and food intake responses to acute food restriction - induced energy deficit but not in response to an acute bout of exercise . furthermore , there may be variability in weight - reduced subjects according to individual physiological characteristics , with clear responders and nonresponders.67 since serotonergic mechanisms may reduce body weight by accelerating the onset of satiety68 besides suppressing excessive snacking of carbohydrate - rich foods , nonpharmacological methods of raising brain serotonin during weight loss and/or maintenance could be highly useful in preventing uncontrolled weight gain .
it has been shown that physical exercise ( mild stress ) normalizes levels of proinflammatory cytokines and may counteract the activation of inflammation / ido1 pathways , which may decrease the susceptibility for mood disturbances and carbohydrate craving.69 animal and human studies have shown that aerobic exercise can stimulate brain serotonin activity and trigger parallel elevations in plasma - free trp and brain trp.7072 in the primary study by chaouloff et al.70 , nonesterified fatty acid elevation increased free trp , hence its entry in the brain for serotonin synthesis and also in the liver .
acute exercise seems to elevate the activity of trp 5-monooxygenase , the enzyme involved in the rate - limiting step in the synthesis of serotonin , and so leads to an increase in the concentration of serotonin in some areas of the brain , ie , the brain stem and hypothalamus.73 chronic exercise ( 30 min / d , six days per week for four weeks ) causes neural adaptations by activating not only the synthesis but also the metabolism of serotonin in the cerebral cortex.74 furthermore , salivary and serum cortisol levels in humans and corticoster - one in rats are increased by exercise,75,76 and this could induce liver trp 2,3-dioxygenase , as demonstrated in rats .
the combined effects of increased liver trp and trp 2,3-dioxygenase induction can elevate serum kyn in humans77 and in rats.78 in both of these latter studies , increases in serum kyn after exercise have been shown . although exercise increased the ido1 activity of macrophages in rats,78 until now ,
there is no direct information on the effect of exercise on ido1 activity in humans .
furthermore , the effects of elevations in pro- and anti - inflammatory cytokines by exercise on ido1 activity still remain to be examined .
however , a recent study in healthy human volunteers showed a significant induction of trp breakdown as indicated by an increase in kyn / trp , which could be referred to an enhanced activity of ido1 because a significant association of kyn / trp with the immune activation marker neopterin was observed.79 trp is not only a precursor of the serotonin biochemical pathway but also the key element for the formation of nicotinamide adenine dinucleotides , nad and nadh , via the kyn pathway.80 potential signals during exercise include increased nad / nadh . with increasing exercise intensity
, the cytosolic nad / nadh ratio declines as lactate accumulates.81 nad regulates the expression of sirtuins ( sirt ) and peroxisome proliferator - activated receptor- coactivator ( pgc-1 ) . enhanced sirt1 and pgc-1 activities are associated with improved mitochondrial function and exercise performance82 and protection against obesogenic feeding.83 recently , a mechanism was discovered by which overexpression of pgc-11 in muscle mimics antidepressant effects of exercise by promoting kyn aminotransferase expression . by this way , crossing of kyn through the blood brain barrier and the disruption of neural plasticity are prevented.84 during exercise , the entry of trp into the brain through the blood brain barrier is favored by increased muscle use of bcaas that inhibit trp transport into the brain and elevated plasma fatty acids , as this elevates the ratio of unbound trp to bcaa . because of the increase in plasma trp and decrease in bcaa , there is a substantial increase in trp availability to the brain , consequently leading to higher serotonin concentrations in some areas of the brain.85 serotonin plays a key role in signal transduction between neurons , and an exercise - induced increase in the concentrations of serotonin has been linked to central fatigue.86,87 although there is some evidence that bcaa supplementation may increase exercise intensity at lactate threshold,88 upper body muscle power,89 and the rate of perceived recovery after strenuous exercise,90 the association between the ergogenic effect of the bcaa intake and the reduction in serotonin production in the brain has not been tested in humans . in a recent study,91 during a half - ironman triathlon , serum amino acids concentrations were reduced by > 20% . however ,
neither the changes in serum - free amino acids nor the trp / bcaa ratio was related to muscle fatigue or muscle damage during the race .
paradoxically , trp supplementation can decrease fatigue perception during an aerobic exercise and likewise may improve physical performance , possibly by acting via endogenous opioids.92,93 taken together , exercise increases trp availability to the brain , and these findings provide support to the hypothesis that increases in serotonin synthesis and activity might be involved in the antidepressant effect of exercise.94 however , too high serotonin levels may impair mood and increase the sensitivity to fatigue , a situation similar to the development of a transient serotonin syndrome.95 given that there is a reciprocal link in mood disorders and obesity,48 physical exercise could mitigate the biological changes that occur with weight loss ( ie , decreased metabolic mass , increased metabolic efficiency , and increased hunger and depression ) via both an increase in energy expenditure ( with the potential to generate an energy deficit ) and the induction of an anti - inflammatory environment ( with a subsequent increased release of serotonin ) .
however , one has to distinguish recreational physical activity ( activity that people engage in during their free time , that people enjoy , and that people recognize as having socially redeeming values ) from repeated high level training sessions or intense endurance sports .
although the recreational physical activity performed every other day maximum will exert the above - mentioned positive impact on physiological pathways and parameters , repeated high - level activities could easily turn toward a negative net effect .
exhaustive physical activity significantly impacts on inflammation cascades that involve several proinflammatory cytokines , such as interferon- and down - stream biochemical pathways.96,97 this includes also alterations of amino acid profiles , eg , during and after a half iron man triathlon.91 such data fit well with the observation that intense training was associated with an accelerated trp breakdown and an increased kyn / trp.79 interestingly , intense versus moderate physical activities might also exert contrasting effects on neuropsychiatric circuits ( fig .
acute and moderate physical exercises , such as noncompetitive physical exercise such as jogging , increase the activity of gtp - cyclohydrolase-1 ( gch1 ) and contribute to increased productions of pteridine derivatives.98 by contrast , in our recent study , a drop in phenylalanine and phe / tyr concentrations by exhaustive exercise was noted,79 which indicates a decreased pah activity that could be due to a decline in tetrahydrobiopterin ( bh4).99 in the same population , nitrite levels dropped ( unpublished data ) that could represent another consequence of lowered no production related to diminished bh4 . unfortunately , the direct measurements of bh4 are almost impossible because only very stringent preanalytical measures can limit the oxidative loss of this compound and practically limit its monitoring to laboratory animal or in vitro studies .
still , data fit well with existing literature , which shows that acute endurance exercise may increase serotonin availability as was reflected in the periphery by increased concentration of free trp.100 the pathogenesis of negative mood is certainly multifactorial , trp and serotonin metabolism can be of some greater relevance , and implicating reactive oxygen species ( ros ) and bh4 is currently still rather speculative , although successful therapy of depression with bh4 has been reported already several years earlier.101 one may conclude that moderate physical exercise enhances the production of neurotransmitters , such as serotonin , noradrenaline , adrenaline , and dopamine , and can thereby contribute to a heightening of mood.102 however , only sports performed occasionally with two or three days interval when performed as recreational activity seems to exert this beneficial effect on general well - being , whereas intense training will rather adversely affect both mood103 and immune system function.104 other factors such as psychological stress , lack of sleep , and malnutrition can increase the risk of infection when immunity becomes suppressed
. this relationship will become especially important when sports activity is performed together with a reduction in food intake as a part of a weight - loss program .
after short time , calorie restriction diet was associated with a decline in essential amino acid trp in serum / plasma followed by a significant drop in phenylalanine.18 trp would further decline when the weight loss diet is combined with heavy physical exercise that induces trp degradation .
it could result in deficiency of the essential amino acids that because of their relevance for neurotransmitter biosynthesis
, one might consider supplementation with trp or 5-hydroxy - trp during such periods to improve weight loss maintenance .
indeed , it has been shown that acute trp supplementation while dieting could be helpful in improving mood status and preventing uncontrolled weight gain or neuropsychiatric symptoms.13 however , in healthy subjects , only large increases in brain trp levels are able to improve mood significantly , whereas relatively small increases in brain trp result in an improved mood in vulnerable subjects . so eating foods with relatively high trp content , such as turkey meat , cacao , nuts , and other seeds ,
is considered to increase trp availability to the brain , albeit slightly , and contributes to some mood enhancement.105 this conclusion is , however , weakened by the fact that such food usually also contains high concentrations of competing lnaa that reduces the trp : lnaa ratio and thus again limits the transport of trp to brain via the leucine - preferring l1 system.106 by contrast , in vitro observations show that antioxidant compounds are able to suppress ido1 , which supports the conclusion that healthy food rich in antioxidants could exert some additional positive effect on trp availability when trp breakdown is slowed down.107
the obesity epidemic that we are facing today may relate to various aspects , such as increased general availability of energy - rich foods , increased sedentary activity , and decreased occupational physical activity ( traveling to or from work or school by a means involving physical activity , such as walking and riding a bicycle).108 in theory , eating less and increasing physical activity will be able to counteract this development .
however , obesity is often associated with disturbed mood , which is closely linked with the inability to lose or to stop gaining weight , and a craving for carbohydrates is manifested by many people who are overweight or are becoming so . the disturbed metabolism of trp in overweight victims might affect the biosynthesis of serotonin and could thereby increase the susceptibility for mood disturbances and carbohydrate craving , increasing the cessation probability of weight reduction programs .
however , breakdown of trp triggered by proinflammatory cascades that are elicited during excessive physical exercise may again relate to the development of neuropsychiatric symptoms , such as fatigue and low mood .
thus , a balance between eating habits and the optimal dose of physical activity needs to be reached . | an association between mood disturbance , the inability to lose or to stop gaining weight , and a craving for carbohydrates is manifested by many people who are overweight or are becoming so . in a recent study , we observed that low - calorie weight loss diet lowered not only levels of leptin but also levels of essential amino acid tryptophan ( trp ) significantly .
the disturbed metabolism of trp might affect biosynthesis of serotonin and could thereby increase the susceptibility for mood disturbances and carbohydrate craving , increasing the cessation probability of weight reduction programs .
alternatively , moderate physical exercise a potent stimulus to modulate ( reduce / normalize ) proinflammatory cytokines , which may affect trp levels could be helpful in improving mood status and preventing uncontrolled weight gain .
in contrast , excessive physical exercise may induce breakdown of trp when proinflammatory cascades together with trp - degrading enzyme indoleamine 2,3-dioxygenase-1 are stimulated , which may lead to neuropsychiatric symptoms such as fatigue and low mood . | Introduction
Diet- Versus Exercise-Induced Weight Loss
Improving Weight Loss Maintenance
Mechanisms of weight regain after weight loss
Countering biology with physical exercise
Conclusion |
PMC3461785 | female genital mutilation ( fgm ) , previously inappropiately called female circumcision is the partial or total removal of the female external genitalia or other deliberate injury to the female genital organs , either for cultural or non - therapeutic reasons .
the world health organization ( who ) estimates that between 100 and 140 million girls and women worldwide are presently living with fgm , and every year about three million girls are at risk .
the practice is most prevalent in african countries such as nigeria , ethiopia , sudan , egypt , and some areas of the middle east .
the practice of fgm has been perpetuated in large part by cultural and religious beliefs .
for example , fgm is believed to attenuate sexual desire and thus ensures chastity and fidelity in marriage .
female genital mutilation has been classified into four types ( 14 ) by the who , depending on the degree of scarification and distortion of the female external genitalia .
type 1 involves excision of the prepuce , with or without total or partial excision of the clitoris .
type 2 involves excision of the clitoris , with partial or total excision of the labia minora .
type 3 involves total or partial excision of the external genitalia , and stitching and narrowing of the vaginal opening ( infibulation ) .
type 4 unclassified , includes several other procedures such as pricking , piercing , or incising the clitoris and / or the labia , and also cauterization of the clitoris , cutting the vagina , and any other procedure that can be included in the definition of fgm .
fgm type 1 and 2 are the most prevalent 80% , while 15% of the fgm is type 3 .
female genital mutilation may result in immediate complications such as severe pain and bleeding and long - term complications such as : psychological , psychosexual , trauma , infertility , susceptibility to bacterial vaginosis , and genital herpes and obstetric complications including perinatal death.[1310 ] all types of fgm have been shown to be associated with complications . in this report , we present the case of a 23-year - old nigerian girl , with obstructed micturition and genital tract obstruction , as a complication of type 3 fgm , with a brief literature review .
a 23-year - old undergraduate presented to our urology service with a history of obstructed micturition dating to infancy .
she specifically complained of impaired urinary stream and splashing of the urine on her upper thighs during micturition .
more recently she noticed she had difficulty with coitus ; inability to admit the partners erect penis because of a partially obstructed introitus .
she had a normal 28-day menstrual cycle . on examination she was in apparent good health .
examination of the external genitalia revealed a partially amputated labia majora and minora , with the fused labia minora remnant that completely covered the vestibule , except for a small opening at the posterior fourchette that could barely admit the tip of the index finger .
( the external urethral meatus was completely covered and the introitus was partially covered ) [ figure 1 ] .
. clinical photograph of a 23-year - old , nigerian girl , with type 3 fgm , showing post mutilation fusion of the labia minora ; the external urethral meatus is completely covered and the introitus is partially covered laboratory investigations consisting of urinalysis , complete blood count , and hiv screening were entirely normal .
she had elective defibulation , which consisted of splitting the fused labia minora in the midline superiorly , till the remnant of the clitoris was exposed and freed from the tethering fibrous tissue .
the cut edges of the labia minora were sutured together with running sutures of 2 - 0 chromic catgut and seperated by means of gauze dressings , during the healing period , to prevent cross adhesions .
she was discharged home on the same postoperative day and reviewed in the outpatient clinic on the eighth postoperative day and two weeks later .
at the six - month follow - up she was happily married and had no further complaints .
she had elective defibulation , which consisted of splitting the fused labia minora in the midline superiorly , till the remnant of the clitoris was exposed and freed from the tethering fibrous tissue .
the cut edges of the labia minora were sutured together with running sutures of 2 - 0 chromic catgut and seperated by means of gauze dressings , during the healing period , to prevent cross adhesions .
she was discharged home on the same postoperative day and reviewed in the outpatient clinic on the eighth postoperative day and two weeks later .
at the six - month follow - up she was happily married and had no further complaints .
although recognized as a form of violation of the rights of young girls and discrimination against women , and despite several international and regional human rights treaties and consensus documents condeming the practice of fgm , the practice is still ongoing in several parts of rural africa , including nigeria .
for example , fgm is erroneously believed to attenuate sexual desire , and thus . ensures chastity and fidelity in marriage .
a survey from gambia shows that 72.9% of gambian women will like their daughters to undergo fgm .
another reason is the lack of knowledge regarding the complications of fgm , especially among the healthcare professionals , like nurses and midwifes .
immediate health complications include hemorrhage , shock , infectious and psychological consequencies , pelvic and femoral fracture , and severe pain.[36 ] an anesthetic death has been reported in a young girl undergoing fgm in egypt , warranting the banning of the procedure in that country . in the long term , chronic pain , infections , keloid formation , sexual dysfunction , primary infertility , birth complications ,
danger to the new born , and psychological consequences can occur.[39 ] urological complications have also been reported to occur following fgm . in a study by dirie and lindmark , urinary retention , urinary tract infection , and urinary problems
were documented . in that study , urinary retention occurred in the first three days after fgm and was attributed to any of the following factors ; postoperative pain , irritation of the raw areas by urine , and obstruction of the external urethral meatus by skin flaps or blood clots . in some instances
we highlight a late urological complication of fgm ; the obstruction of the urinary stream by the fused labia minora , following type 3 fgm . during micturition ,
the urinary stream first impinges on the fused labia minora and then exits through the narrowed opening in the vulva causing urinary splashing around the perineum and upper thighs .
dirie and lindmark also documented recurrent urinary tract infections , and according to them , these occurred because the meatus was covered by the infundibulum , causing the vaginal discharge to accumulate and favor the growth of bacteria .
our index patient did not have urinary tract infection at the time of presentation , as evidenced by the negative urine cultures .
our index patient presented primarily because of difficulty in penetration , at the point of getting married .
this was due to the post fgm fusion of the labia minora , leaving only a narrow opening in the vulva , which could not admit the erect penis of an adult [ figure 1 ] .
abdelhady found that circumcised women had significantly higher rates of psychological problems than women who were not circumcised .
others have also found a higher prevalence of posttraumatic stress disorder and sexual health problems among circumcised women .
she appeared , however , to be mentally stable , although she did not have an expert psychological evaluation , because the facilities for this were not readily available .
after defibulation , she was found to have a normal vagina , and has since been happily married .
this has been attributed to several factors such as the use of nonsterile instruments , an increased number of blood transfusions , due to blood loss during surgery , increased anal intercourse due to difficult or painful vaginal intercourse , and tearing of the vagina during intercourse . however , some other studies have found no statistically significant associations .
the so - called gabbar 's defibulation surgery can be done under regional or general anesthesia .
it involves incising the fused labia minora to restore the introital opening , with excision of clitoral or epidermoid inclusion cysts , if present .
more extensive plastic reconstruction of the external genitalia may be warranted in some instances , but was not necessary in our index patient . where the clitoris has been amputated , little
can be done to restore it , but the remnant of it could be freed from its tethering by fibrous tissue , as was done in the index case .
gordon , who has a lot of experience in these surgeries , reports that in 60% of the cases , all natural functions are restored .
female genital mutilation is a dehumanizing and dangerous practice that needs to be stopped . however
there is need for a massive educational campaign on the harmful effects of fgm at all levels , such as ; the community level , women 's groups , traditional birth attendants , and healthcare professionals .
also adequate psychological , social , and medical support needs to be provided for those who are already victims of this condition . | female genital mutilation ( fgm ) is the partial or total removal of the female external genitalia or other deliberate injury to the female genital organs , either for cultural or non - therapeutic reasons .
this barbaric act is accompanied by a variety of complications ranging from hemorrhage , fracture , infective complications , gynetresia , with its attendant sexual and obstetric difficulties , and death .
a 23-year - old girl , with urinary and genital tract obstruction following female genital mutilation(infibulation ) is presented .
she was managed by elective defibulation , with a satisfactory outcome .
robust health education strategies are needed for the eradication of fgm . | INTRODUCTION
CASE REPORT
Operative technique
DISCUSSION
CONCLUSION |
PMC3733283 | clinical education is a critical part of nursing curriculum , as more than half of the school time is spent on it , and both the clinical competencies and professional identity of the future nurses are formed in this period.[13 ] many factors may affect clinical learning , which may be related to the learner , teacher , or the environment .
one of the important and frequently reported factors that may influence students learning , especially in clinical settings , is stress .
the pattern of specific and nonspecific responses an organism makes to stimulus events that disturb its equilibrium and tax or exceed its ability to cope .
learning and personal change is considered as a stressor per se , but nursing education could be more stressful to learners , as it is blended with technical tasks , professional skills , human relationships , and sympathy feelings . therefore , identifying additional stressors in the clinical context is very crucial for providing measures to minimize students stress to a tolerable level and helping them to cope better .
there is considerable number of reports on stressors in clinical nursing education ; naming student assessments , instructor s teaching skills , not cooperating staff , inappropriate communication between instructors and students , as main stressors.[41015 ] but it seems that perceived stress and related stressors may be different in different cultural context and organizational settings . in taiwan ,
tsai reported that educational protocols , especial situation of nursing , evaluation , and time management were the most important stressors .
all of these themes are context - bond , and may differ by changing in environment and local rules .
however , another study performed in five countries , emphasized on the common stress perception among nursing students , in spite of the influence of their different cultural backgrounds .
it seemed most probable that the stressors in clinical nursing education in iranian nursing schools to be of different order from other contexts .
investigating the published literature , we found that there may be considerable reports of original researches exploring these stressors .
so , we decided to perform a systematic review to gather all published reports and provide an evidence base about stressors in clinical nursing education in iran .
the main review question was : what are the factors related to stress in clinical nursing education in iranian nursing schools ?
the prepared evidence base about stress sources in clinical education could help nursing schools to have better management of stressors , and recognizing coping strategies as an integral part of teaching -learning process .
the main review question was what factors have been recognized to be related to stress in the settings of clinical nursing education in iran ? inclusion and exclusion criteria : all papers about clinical nursing education , which were published from 1989 to 2009 , which resulted from original research ( irrespective of the research design ) , were entered in the study . in this step a much broader area of clinical education
then , studies which were not related to stress factors and those whose full text was not available were excluded [ figure 1 ] .
the process of screening and selection of documents for review both electronic and hand searching was performed :
electronic searching : all iranian research databases ( including iranmedex , magiran , irandoc and sid ) and the most popular international databases ( pubmed , google scholar and eric ) were searched using appropriate key words [ table 1 ] .
also , the whole electronic archive of 14 persian peer reviewed scientific journals about nursing , as well as 3 english- language iranian journals about either nursing or medical education was searched according to the mentioned key words.hand searching : since there were peer reviewed nursing or medical sciences journals in iran which have not been completely indexed in e - data bases , their back issues was searched manually , too .
electronic searching : all iranian research databases ( including iranmedex , magiran , irandoc and sid ) and the most popular international databases ( pubmed , google scholar and eric ) were searched using appropriate key words [ table 1 ] . also , the whole electronic archive of 14 persian peer reviewed scientific journals about nursing , as well as 3 english- language iranian journals about either nursing or medical education was searched according to the mentioned key words .
hand searching : since there were peer reviewed nursing or medical sciences journals in iran which have not been completely indexed in e - data bases , their back issues was searched manually , too . therefore , the archives of 39 journals were searched .
search strategy for extraction of research reports on clinical nursing education in iran , as the broader concept all retrieved records that entered the step 2 of the screening process [ figure 1 ] , were entered to an excel data sheet .
the next processing of documents ( including examination of their relevance to the research question , the quality of the report , and the extracted codes from each report ) was recorded in that sheet . to evaluate the quality of reports ,
each document was scored by two independent researchers , and in controversial cases , the third rater came in .
the scale used for evaluation of the quality of documents content analysis of selected documents was done by one of the researchers in team , through determining main codes in each paper , finding themes , and classification of themes .
the data rigor was achieved via double checking of the process by two other researchers in the team .
both electronic and hand searching was performed :
electronic searching : all iranian research databases ( including iranmedex , magiran , irandoc and sid ) and the most popular international databases ( pubmed , google scholar and eric ) were searched using appropriate key words [ table 1 ] .
also , the whole electronic archive of 14 persian peer reviewed scientific journals about nursing , as well as 3 english- language iranian journals about either nursing or medical education was searched according to the mentioned key words.hand searching : since there were peer reviewed nursing or medical sciences journals in iran which have not been completely indexed in e - data bases , their back issues was searched manually , too .
electronic searching : all iranian research databases ( including iranmedex , magiran , irandoc and sid ) and the most popular international databases ( pubmed , google scholar and eric ) were searched using appropriate key words [ table 1 ] . also , the whole electronic archive of 14 persian peer reviewed scientific journals about nursing , as well as 3 english- language iranian journals about either nursing or medical education was searched according to the mentioned key words .
hand searching : since there were peer reviewed nursing or medical sciences journals in iran which have not been completely indexed in e - data bases , their back issues was searched manually , too .
therefore , the archives of 39 journals were searched . search strategy for extraction of research reports on clinical nursing education in iran , as the broader concept
all retrieved records that entered the step 2 of the screening process [ figure 1 ] , were entered to an excel data sheet .
the next processing of documents ( including examination of their relevance to the research question , the quality of the report , and the extracted codes from each report ) was recorded in that sheet . to evaluate the quality of reports ,
each document was scored by two independent researchers , and in controversial cases , the third rater came in .
the scale used for evaluation of the quality of documents content analysis of selected documents was done by one of the researchers in team , through determining main codes in each paper , finding themes , and classification of themes .
the data rigor was achieved via double checking of the process by two other researchers in the team .
at the first step of electronic searching , 1066 records were retrieved related to the general subject of clinical nursing education . also , 38 more records found by hand searching .
then , the title and authors of retrieved records were sorted and checked for duplications twice , in excel sheet . after omitting the repetitive items , 424 records remained . after going through the second and third steps of screening [ figure 1 ] , 15 documents remained which were relevant to the research question ( specific subject of stress and clinical nursing education ) , and
unfortunately , the titles of grey documents ( dissertations and unpublished research reports ) although retrieved in the searching phase , were not accessible in spite of great effort from researchers .
conference proceedings in iran only included abstracts , which were not so informative to be analyzed .
the specifications of the 15 analyzed papers , published between 1998 - 2010 , are presented in [ table 3 ] .
general specifications of papers finally analyzed to find factors related to stress in clinical nursing education the frequency distribution of papers according to the place of research is depicted in figure 2 .
as observed , there is an acceptable distribution of researches from different places throughout the country , so that they give a good picture of the whole .
distribution of number of papers according to the place of study ( province ) evaluation of papers , according to the indices mentioned in table 2 , showed that all papers had passed the minimum required score ( 5 out of 10 ) .
as the figure shows , the quality score of papers has been improved in the recent years .
evaluation scores of 15 papers entered in content analysis in the review two papers had reported stressors from the instructors point of view . summarizing the items ,
4 main themes were found as the stress roots : 1 ) factors related to educational programming ; 2 ) factors related to educational environment ; 3 ) instructor s competencies and responsibilities ; 4 ) student - related factors . description of factors under each theme is given in [ table 4 ] .
stressors in clinical nursing education , according to the instructors since more papers were dealing with stressors according to students view points , all mentioned items in these papers were extracted and discussed in the review team , trying to classify items according to their nature to build more abstract and understandable themes .
for example , if items working with critically ill patients and observing critically ill patients suffering and pain were mentioned in a paper as two items , a more general item giving care to critically ill patients and observing their suffer was formed .
detail of themes and the frequency of items under each theme are represented in [ table 5 ] .
stressors in clinical nursing education according to the nursing students : detail of themes and the frequency of items
under each theme summarizing the frequency of reported factors in table 5 , the most frequently reported stressors in clinical nursing education are as follows : factors related to interpersonal relationships and interactions ( as 41% of all reported issues ) and factors related to the nature of nursing profession ( as 24% of all reported issues ) , then , main domain of education ( 15% ) , clinical environment ( 11% ) , and finally , factors related to the students clinical competence and ability to accomplish their roles ( 9% ) .
relative frequency of themes ( sources of stress ) according to students opinions in the reviewed documents
at the first step of electronic searching , 1066 records were retrieved related to the general subject of clinical nursing education . also , 38 more records found by hand searching .
then , the title and authors of retrieved records were sorted and checked for duplications twice , in excel sheet . after omitting the repetitive items , 424 records remained . after going through the second and third steps of screening [ figure 1 ] , 15 documents remained which were relevant to the research question ( specific subject of stress and clinical nursing education ) , and
unfortunately , the titles of grey documents ( dissertations and unpublished research reports ) although retrieved in the searching phase , were not accessible in spite of great effort from researchers .
conference proceedings in iran only included abstracts , which were not so informative to be analyzed .
the specifications of the 15 analyzed papers , published between 1998 - 2010 , are presented in [ table 3 ] .
general specifications of papers finally analyzed to find factors related to stress in clinical nursing education the frequency distribution of papers according to the place of research is depicted in figure 2 .
as observed , there is an acceptable distribution of researches from different places throughout the country , so that they give a good picture of the whole .
distribution of number of papers according to the place of study ( province ) evaluation of papers , according to the indices mentioned in table 2 , showed that all papers had passed the minimum required score ( 5 out of 10 ) .
as the figure shows , the quality score of papers has been improved in the recent years .
two papers had reported stressors from the instructors point of view . summarizing the items , 4 main themes were found as the stress roots : 1 ) factors related to educational programming ; 2 ) factors related to educational environment ; 3 ) instructor s competencies and responsibilities ; 4 ) student - related factors . description of factors under each theme is given in [ table 4 ] .
since more papers were dealing with stressors according to students view points , all mentioned items in these papers were extracted and discussed in the review team , trying to classify items according to their nature to build more abstract and understandable themes .
for example , if items working with critically ill patients and observing critically ill patients suffering and pain were mentioned in a paper as two items , a more general item giving care to critically ill patients and observing their suffer was formed .
detail of themes and the frequency of items under each theme are represented in [ table 5 ] .
stressors in clinical nursing education according to the nursing students : detail of themes and the frequency of items
under each theme summarizing the frequency of reported factors in table 5 , the most frequently reported stressors in clinical nursing education are as follows : factors related to interpersonal relationships and interactions ( as 41% of all reported issues ) and factors related to the nature of nursing profession ( as 24% of all reported issues ) , then , main domain of education ( 15% ) , clinical environment ( 11% ) , and finally , factors related to the students clinical competence and ability to accomplish their roles ( 9% ) .
relative frequency of themes ( sources of stress ) according to students opinions in the reviewed documents
there is great emphasis on the influence of stressor on clinical nursing education , and the number of published original researches in this field in english language literature seems to be considerable .
however , we could not find any systematic review on this topic , so the present work could be considered as the first one at least in iran .
we followed the thematic analysis , as well as quantitative summarizing of the stressors in clinical nursing education , hoping to provide a practical summary to be used as an evidence for prioritization of corrective interventions . also , depicted figure of previous research could help investigators locate the important gaps of knowledge for further research .
it is found that most published reports ( 13 out of 15 ) , are based on descriptive research design .
the importance of descriptive studies in providing background information on the present situation could not be denied .
however , researchers are expected to use the background knowledge to synthesize related hypotheses and test them through interventional studies .
the results of the present work may help them to find suitable points for intervention .
the geographical distribution of studies reviewed in this paper includes samples from north , center , and south of iran [ figure 2 ] . according to the cultural and organizational diversity in different parts of iran
instructors play an important role in stressor recognition and control , both for themselves and students , so that without their effective partnership in stress reduction , students will not attain a good situation for effective clinical experience and learning .
two studies in the review showed that according to instructors , stressors related to educational programming have the highest importance in imposing stress in clinical education . the next important stressor was factors related to the instructors competencies and responsibilities .
therefore , correcting the present programming processes , especially with instructors partnership , as well as empowering them , may attenuate their stress .
also , mentorship programs employing more expert instructors to help juniors , and running workshops on stress management have been reported to be effective interventions for instructors that may also result in better situation for students , too .
the most frequently reported stressors in the reviewed literature belong to the interpersonal relationships and interactions , according to students .
this finding brings an important message for curriculum planners and educational managers in nursing schools .
these measures could include providing training courses or workshops on communication skills for both instructors and students as a curricular or extracurricular activity .
this solution has been addressed in many studies,[8152123 ] and its effectiveness should be further tested .
also , better supervision of educational management on clinical environment , to find problems and improve human relationships between students , instructors , and non - academic staff is essential . providing a supportive counseling system for students and instructors
all of these interventions may be tested in experimental or at least action research studies , to clarify their real value in stress reduction and students development .
the second important domain of stressors was the nature of nursing profession as a hard profession responsible for giving critical and diverse health services to critical clients . it may be suggested that objectives regarding professional issues ( values , rights and responsibilities , ethics , ) be included in the curriculum of nursing . attaining to these objectives
should be ensured so that students clearly know their future career and get ready to cope with this sort of stressors . on the other hand , efforts to make society better acknowledge and respect nursing will improve students attitude toward their future career and hence , decrease their stress .
as table 5 shows , a considerable source of stress comes from inappropriate educational planning by nursing schools . here , the important role of instructors has been emphasized . besides the previously mentioned strategies , having clear and effective program for selecting instructor from knowledgeable and ethically committed nursing experts
along this , nursing schools should explore the causes of theory - practice gap in their context , clearly conceptualize clinical student assessments , and define their tasks and responsibilities in advance.[132426 ] problems in clinical environment have been addressed as sources of stress in 11% of the reviewed studies . according to the institutional diversity in the studies context , special shortcomings in some of them , including having enough teaching hospitals , student admission policies , and financial resources for clinical education could bring some pressure to students .
although , this source is not consistent in all studies , but there should be attention to decrease its effect if it is found to be influential in a given context . there were inconclusive items in the studies which are explained briefly here : although in foreign studies , giving care to patient with the other sex is considered as a source of stress , only one study in iran has paid attention to this item . according to the iranian culture and islamic regulations , many students and patients prefer to have close contact with the same sex .
therefore , it would be expected that this issue be explored more extensively to examine its real effect on the psychological stress among students , and possibly , patients . in some reviewed studies ,
stress perception was more in female students compared to male , which is similar to other studies in non - iranian students .
if this finding is supported in further investigations , providing special support to female students and instructors will be essential .
in some studies , students with higher grades had more stress , while the opposite situation was found in other studies .
probably the research in portugal compared the effects of different stressors in different school years may clarify the observed discrepancies . as they found that the relative influence of different stressors are different according to the students grade .
for instance , perception of special situations in nursing as a stressor was more frequent among second year students , compared to the third and fourth years . on the other hand , first year students perceived clinical assessment as stressor less than the students in higher grade .
therefore , studying the relative importance of different stressors according to the students grade may help recognize the points of intervention in each school grade .
it is noticeable that in most foreign studies such as , surveys are accompanied by investigations on the students coping strategies and their effectiveness , the issue has not been attended in iranian reviewed studies , except for one case which was written in english and was beyond clinical training .
first , limited access to grey literature , most of them were dissertations that were not accessible to us .
the second one was the diversity of questionnaires and scoring system that were used in studies .
therefore , we had to just count the frequency of reporting each stressor , and could not give reliable quantitative summary of their findings . in three studies ,
of course , since most nurses and nursing students in iran are females , the bias was not expected to be great enough to distort the results .
based on the study findings , there may be some suggestions for investigators , as well as suggestions to managers : compiling a comprehensive tool , based on the findings of this review and good practices abroad , and validating it through participation of students and instructors ( for instance by delphi technique ) seems to be essential .
thereafter , doing a multicenter research using such a tool may help having a comparative and clear figure about the situation of stressors in iranian nursing schools , and a good baseline data to be used in further comparisons after planned interventions .
it may be recommended to investigators in the field to deal more with interventions to improve the present status , and measure the effect and value of each intervention objectively .
some of interventions that seems to be effective are :
to pay more attention to communication skills and stress management in nursing curriculumto have special programs for clinical orientation of nursing studentsto provide counseling and support services for students and instructorsto revise clinical curriculum , so that clinical rotations become more meaningful to students , and they can benefit from more support ( for instance having peer mentors from higher grades)to improve the school s supervision and governance in clinical environment , to recognize problems , as soon as possible and react to them appropriately .
to pay more attention to communication skills and
stress management in nursing curriculum to have special programs for clinical orientation of nursing students to provide counseling and support services for students and instructors to revise clinical curriculum , so that clinical rotations become more meaningful to students , and they can benefit from more support ( for instance having peer mentors from higher grades ) to improve the school s supervision and governance in clinical environment , to recognize problems , as soon as possible and react to them appropriately .
the paper scores according to the indices represented in table 2 are shown in the vertical axis .
the horizontal axis labels show the code of each paper ( the same as the reference number in the references section of this article ) and its publication year .
first , limited access to grey literature , most of them were dissertations that were not accessible to us .
the second one was the diversity of questionnaires and scoring system that were used in studies .
therefore , we had to just count the frequency of reporting each stressor , and could not give reliable quantitative summary of their findings . in three studies ,
of course , since most nurses and nursing students in iran are females , the bias was not expected to be great enough to distort the results . based on the study findings
, there may be some suggestions for investigators , as well as suggestions to managers : compiling a comprehensive tool , based on the findings of this review and good practices abroad , and validating it through participation of students and instructors ( for instance by delphi technique ) seems to be essential .
thereafter , doing a multicenter research using such a tool may help having a comparative and clear figure about the situation of stressors in iranian nursing schools , and a good baseline data to be used in further comparisons after planned interventions .
it may be recommended to investigators in the field to deal more with interventions to improve the present status , and measure the effect and value of each intervention objectively .
some of interventions that seems to be effective are :
to pay more attention to communication skills and stress management in nursing curriculumto have special programs for clinical orientation of nursing studentsto provide counseling and support services for students and instructorsto revise clinical curriculum , so that clinical rotations become more meaningful to students , and they can benefit from more support ( for instance having peer mentors from higher grades)to improve the school s supervision and governance in clinical environment , to recognize problems , as soon as possible and react to them appropriately .
to pay more attention to communication skills and
stress management in nursing curriculum to have special programs for clinical orientation of nursing students to provide counseling and support services for students and instructors to revise clinical curriculum , so that clinical rotations become more meaningful to students , and they can benefit from more support ( for instance having peer mentors from higher grades ) to improve the school s supervision and governance in clinical environment , to recognize problems , as soon as possible and react to them appropriately .
the paper scores according to the indices represented in table 2 are shown in the vertical axis .
the horizontal axis labels show the code of each paper ( the same as the reference number in the references section of this article ) and its publication year . | background : clinical education is a critical and complex component of nursing education that is influenced by many variables .
one of them is stress , which may disturb students learning , too .
stressors may differ according to the learning situation and environment , and recognizing them , seems to be essential for corrective interventions .
the present work was performed to identify stressors in clinical nursing education in iran , according to the published research reports.materials and methods : in this systematic review , all published research reports available in iranian and international web - based data bases and search engines were searched . also ,
the archives of peer reviewed iranian nursing and medical education journals ( published between 1989 and 2009 ) were hand searched . out of 1104 retrieved records ( by a more general terms of clinical education and nursing ) , after stepwise screening , 15 original research articles were selected for content analysis .
coded data were classified and their frequency was represented in tables.results:the following themes were obtained to classify main areas of importance for factors related to stress in clinical nursing education : a ) clinical competence and ability to play one s roles , b ) care load , or stress due to care , c ) main area of education , d ) interpersonal relationships and interactions , e ) clinical environment ( facilities and equipments , space , learning opportunities , etc , ) .
subthemes were also identified in each theme.conclusion:published studies in iran provide appropriate background evidences for planning and evaluating interventional programs to reduce stress among nursing students and instructors .
each identified theme in this study could be considered as a subject for planned interventions . among them , it seems that interpersonal relationships and interactions is of the highest priority . | I
M
Searching methods and search strategy
Data management and processing
R
Retrieved documents
Stressors in clinical nursing education, according to the instructors
Stressors in clinical nursing education, according to the students
D
This review faces some limitations |
PMC3071993 | patellofemoral pain syndrome ( pfps ) is one of the most common knee dysfunctions among physically active young women . despite its high incidence ,
rehabilitation is challenging , as the multifactorial etiology of this syndrome is not fully understood.13 pfps is believed to originate from a combination of extrinsic and intrinsic risk factors.1,4,5 the most commonly described extrinsic risk factors related to pfps include the surface used for running or physical activity practice , sport shoes and the volume and intensity of training.5 the most frequently cited intrinsic factors include femoral trochlear anatomic alterations , weakness and/or imbalance of the quadriceps , peripatelar soft - tissue tightness and lower extremity dynamic misalignments.1,4 the most commonly cited lower extremity dynamic misalignments are excessive hip adduction and internal rotation as well as excessive and/or prolonged rearfoot pronation during locomotion.6,7 pfps is believed to be related to a reduction in the contact area in the patellofemoral joint ; this reduction occurs due to alterations in the dynamic alignment of the tibiofemoral joint.8 one theory states that excessive and/or prolonged pronation of the rearfoot leads to excessive medial rotation of the tibia in a closed kinetic chain.7 this medial rotation of the tibia would induce a compensatory medial rotation of the femur to maintain the relative lateral rotation of the tibial plateau in relation to the femoral condyles , which are associated with knee extension during the midstance phase of gait . when the femur medially rotates , the compression between the lateral surface of the patella and the lateral femoral condyle rises . as a result , patellofemoral joint stress increases.6,9
kinematic studies that have investigated the relationship between excessive /prolonged foot pronation and rearfoot eversion with pfps have produced controversial findings .
some authors observed greater pronation in subjects with pfps during locomotor activities,1315 while others could not confirm the coexistence of excessive rearfoot pronation and pfps.5,16 one of the potential limitations of these studies was that rearfoot motion could not be differentiated from forefoot motion .
total foot pronation is composed of two events , rearfoot eversion during weight acceptance and midfoot / forefoot loading during early midstance .
models that show the foot as a rigid segment may miss information related to its flexibility during the foot rollover process in locomotor tasks.16 alternatively , an indirect way of evaluating the kinetic chain results of the foot rollover process during gait is to assess plantar pressure distribution during the gait subphases .
higher pressures on the medial areas of the plantar surface as well as excessive pronation during running were associated with the development of lower limb injuries.17 thijs et al.18 evaluated plantar pressure in soldiers during barefoot gait and observed a relationship between pfps and lateralized support of the feet , suggesting that individuals who developed pfps exhibited a heel strike in a less - pronated position and a foot rollover that was more directed toward the lateral side of the foot .
this study evaluated a specific military population that was going through an intense training period , and the authors commented that caution should be used when generalizing these findings .
the plantar pressure distribution findings and their relationship to lower limb injuries and pain suggest that there is not a consensus in the literature and more investigation is needed . to our knowledge
, no studies have investigated plantar pressure distribution during the subphases of the foot rollover process in a pfps population .
this investigation may identify the phases of the foot rollover process during which plantar loading and foot contact alterations occur and may contribute to elucidating foot mechanics in pfps individuals .
this study aimed to investigate the influence of patellofemoral pain syndrome on plantar pressure distribution during the initial heel contact , midstance and propulsion phases of gait .
fifty - seven adults of both sexes volunteered for the study and were divided into two groups : the control group ( cg ) ( n = 35 ; 297 yrs ; 164 8 cm ; 6011 kg ; 32 women ) and the pfps group ( pfps ) ( n = 22 ; 307 yrs ; 1659 cm ; 6312 kg ; 20 women ) .
the sample power calculation was based on the primary outcome ( the pressure variables ) with an expected proportion of pfps development of 30% , a power of 80% and an alpha error of 5%.19,20 the groups did not differ in mean age ( p = 0.698 ) , height ( p = 0.935 ) , or body mass ( p = 0.734 ) .
all participants gave their written informed consent , and the local ethics committee approved the study ( protocol n.1237/05 ) .
the pfps subjects experienced pain in the patellofemoral joint region for at least two months ( 43 years ) .
their pain occurred during at least one of the following situations : resisted contraction of the femoral quadriceps , squatting , prolonged sitting and descending or ascending stairs .
subjects were excluded from the study if they had undergone any previous knee surgery , had a history of patellar dislocation or had any other limitations that would influence gait .
the control subjects had no history or diagnosis of knee pathology or trauma , no knee pain with any of the activities mentioned and no limitations that would influence gait .
overall exclusion criteria for both groups were a discrepancy of 1 cm21 or greater in lower leg length and major foot deformities . the arch index was evaluated to exclude major arch alterations ( planus , equinus planus and extra cavus feet ) that could interfere with gait mechanics.22 knee pain intensity in individuals with pfps was measured with a visual analogue pain scale ( vas ) , in which subjects rated their current pain on a 10-cm horizontal scale that ranged from no pain on the left to worst pain imaginable on the right .
the vas was shown to be a reliable , valid and responsible means of assessing pain in studies of pfps.2325 the intensity of knee pain in the pfps group was 1.72.3 cm , and the control subjects presented a knee pain intensity equal to 0.0 cm . to better characterize knee function in pfps and cg subjects ,
each individual was evaluated with the lysholm functional knee scale ; the average ( median ) lysholm score was 70 for the pfps group and 98 for the cg.24,25 as pfps is common in physically active individuals , the subjects in both groups answered a questionnaire about how many time they have been physically active , frequency and duration of physical activity ; they were considered physically active when their physical activity frequency was at least three times a week.26 both groups had similar scores for physically activity ( gc = 51.3% , pfps = 40.0% ; p = 0.422 ) , practice time ( gc = 55 years , pfps = 52 years ; p = 0.330 ) , frequency ( gc = 31 days / week , pfps = 21 days / week ; p = 0.246 ) and duration ( gc = 8530 min , pfps = 6822 min ; p = 0.534 ) .
the contact area and peak pressure were evaluated during barefoot gait using the pedar - x system ( novel , munich , germany ) synchronized to the ankle sagittal angular variation .
this variation was evaluated by an electrogoniometer that was instrumented with a strain gage ( model sg110/a , biometrics , gwent , england ) and fixed to the ankle joint according to the manufacturer 's instructions ( goniometer and torsiometer operating manual .
the insoles of the pedar - x system were 2.5 mm thick and contained a matrix of 99 capacitive pressure sensors with a spatial resolution of 1.6 to 2.2 cm;2 the insoles were placed inside an anti - skid sock.27 prior to the tests , the insoles were calibrated according to the manufacturer 's instructions , and the zero setting procedure was performed as recommended by novel prior to data acquisition.28 the electrogoniometer was calibrated with the ankle in its mechanically neutral position while the subject stood in a relaxed posture with his or her body weight equally distributed in both feet and in stationary equilibrium ; the output value was defined as the zero angle of the goniometer . forward
motion of the lower segment was regarded as flexion ( positive values ) and backward motion as extension ( negative values ) . before data acquisition
, the subjects were instructed to walk freely in a self - selected cadence on a 10-meter walkway to reproduce their daily gait and adapt to the lab environment and to the equipment attached to their feet .
the self - selected cadence was converted into an audible digital signal using a metronome .
the participants kept a similar cadence between trials based on the audio feedback provided by the metronome ; this procedure helped avoid great differences in gait cadence between trials .
the cadences performed during the gait trials did not differ between the groups ( cg = 1083 steps / min ; pfps = 1073 steps / min ; p = 0.871 ) .
a total of 15 steps were recorded , and the mean was used for statistical purposes .
pedar and electrogoniometer data were acquired and synchronized using a 12-bit analog - to - digital converter ( amti , dt3002 ) at a sampling rate of 100 hz . both signals were synchronized by a ttl ( transistor - transistor logic ) signal that was emitted by the pedar sync box .
this box emitted 0 volts ( v ) when at least 20 kpa was detected by the pedar system while the foot was in contact with the surface and 5 v when the pressure was lower than 20kpa while the foot was in the swing phase .
although the pedar - x system is most appropriate for evaluating shod gait , we used the insoles to evaluate barefoot gait as reported by nurse and nigg.29 barefoot gait analysis was performed because our intention was to investigate the complex behavior of the foot - floor interaction30,31 without any other interference such as the subject 's shoes .
additionally , the pedar - x system could acquire multiple steps without requiring the subject to alter their gait to make contact with any platform.32 the contact area ( cm ) and peak pressure ( kpa ) were evaluated in six plantar areas that were adjusted proportionally to the length and width of each subject 's foot with the novel multiprojects software ( novel , munich , germany ) .
the plantar surface was first divided into three larger areas , the rearfoot ( 30% of the foot length ) , midfoot ( 30% of the foot length ) and forefoot ( 40% of the foot length ) , according to the scheme established by cavanagh and ulbrecht.33 the rearfoot was subdivided into the medial ( 30% of the rearfoot width ) , central ( 40% of the rearfoot width ) and lateral rearfoot ( 30% of the rearfoot width ) areas , and the forefoot was subdivided into the medial ( 55% of the forefoot width ) and lateral forefoot ( 45% of the forefoot width).34
fifty - seven adults of both sexes volunteered for the study and were divided into two groups : the control group ( cg ) ( n = 35 ; 297 yrs ; 164 8 cm ; 6011 kg ; 32 women ) and the pfps group ( pfps ) ( n = 22 ; 307 yrs ; 1659 cm ; 6312 kg ; 20 women ) .
the sample power calculation was based on the primary outcome ( the pressure variables ) with an expected proportion of pfps development of 30% , a power of 80% and an alpha error of 5%.19,20 the groups did not differ in mean age ( p = 0.698 ) , height ( p = 0.935 ) , or body mass ( p = 0.734 ) .
all participants gave their written informed consent , and the local ethics committee approved the study ( protocol n.1237/05 ) .
the pfps subjects experienced pain in the patellofemoral joint region for at least two months ( 43 years ) .
their pain occurred during at least one of the following situations : resisted contraction of the femoral quadriceps , squatting , prolonged sitting and descending or ascending stairs .
subjects were excluded from the study if they had undergone any previous knee surgery , had a history of patellar dislocation or had any other limitations that would influence gait .
the control subjects had no history or diagnosis of knee pathology or trauma , no knee pain with any of the activities mentioned and no limitations that would influence gait .
overall exclusion criteria for both groups were a discrepancy of 1 cm21 or greater in lower leg length and major foot deformities . the arch index was evaluated to exclude major arch alterations ( planus , equinus planus and extra cavus feet ) that could interfere with gait mechanics.22 knee pain intensity in individuals with pfps was measured with a visual analogue pain scale ( vas ) , in which subjects rated their current pain on a 10-cm horizontal scale that ranged from no pain on the left to worst pain imaginable on the right .
the vas was shown to be a reliable , valid and responsible means of assessing pain in studies of pfps.2325 the intensity of knee pain in the pfps group was 1.72.3 cm , and the control subjects presented a knee pain intensity equal to 0.0 cm . to better characterize knee function in pfps and cg subjects ,
each individual was evaluated with the lysholm functional knee scale ; the average ( median ) lysholm score was 70 for the pfps group and 98 for the cg.24,25 as pfps is common in physically active individuals , the subjects in both groups answered a questionnaire about how many time they have been physically active , frequency and duration of physical activity ; they were considered physically active when their physical activity frequency was at least three times a week.26 both groups had similar scores for physically activity ( gc = 51.3% , pfps = 40.0% ; p = 0.422 ) , practice time ( gc = 55 years , pfps = 52 years ; p = 0.330 ) , frequency ( gc = 31 days / week , pfps = 21 days / week ; p = 0.246 ) and duration ( gc = 8530 min , pfps = 6822 min ; p = 0.534 ) .
the contact area and peak pressure were evaluated during barefoot gait using the pedar - x system ( novel , munich , germany ) synchronized to the ankle sagittal angular variation .
this variation was evaluated by an electrogoniometer that was instrumented with a strain gage ( model sg110/a , biometrics , gwent , england ) and fixed to the ankle joint according to the manufacturer 's instructions ( goniometer and torsiometer operating manual .
the insoles of the pedar - x system were 2.5 mm thick and contained a matrix of 99 capacitive pressure sensors with a spatial resolution of 1.6 to 2.2 cm;2 the insoles were placed inside an anti - skid sock.27 prior to the tests , the insoles were calibrated according to the manufacturer 's instructions , and the zero setting procedure was performed as recommended by novel prior to data acquisition.28 the electrogoniometer was calibrated with the ankle in its mechanically neutral position while the subject stood in a relaxed posture with his or her body weight equally distributed in both feet and in stationary equilibrium ; the output value was defined as the zero angle of the goniometer . forward motion of the lower segment was regarded as flexion ( positive values ) and backward motion as extension ( negative values ) . before data acquisition , the subjects were instructed to walk freely in a self - selected cadence on a 10-meter walkway to reproduce their daily gait and adapt to the lab environment and to the equipment attached to their feet .
the self - selected cadence was converted into an audible digital signal using a metronome .
the participants kept a similar cadence between trials based on the audio feedback provided by the metronome ; this procedure helped avoid great differences in gait cadence between trials .
the cadences performed during the gait trials did not differ between the groups ( cg = 1083 steps / min ; pfps = 1073 steps / min ; p = 0.871 ) .
a total of 15 steps were recorded , and the mean was used for statistical purposes .
pedar and electrogoniometer data were acquired and synchronized using a 12-bit analog - to - digital converter ( amti , dt3002 ) at a sampling rate of 100 hz . both signals were synchronized by a ttl ( transistor - transistor logic ) signal that was emitted by the pedar sync box .
this box emitted 0 volts ( v ) when at least 20 kpa was detected by the pedar system while the foot was in contact with the surface and 5 v when the pressure was lower than 20kpa while the foot was in the swing phase .
although the pedar - x system is most appropriate for evaluating shod gait , we used the insoles to evaluate barefoot gait as reported by nurse and nigg.29 barefoot gait analysis was performed because our intention was to investigate the complex behavior of the foot - floor interaction30,31 without any other interference such as the subject 's shoes .
additionally , the pedar - x system could acquire multiple steps without requiring the subject to alter their gait to make contact with any platform.32 the contact area ( cm ) and peak pressure ( kpa ) were evaluated in six plantar areas that were adjusted proportionally to the length and width of each subject 's foot with the novel multiprojects software ( novel , munich , germany ) .
the plantar surface was first divided into three larger areas , the rearfoot ( 30% of the foot length ) , midfoot ( 30% of the foot length ) and forefoot ( 40% of the foot length ) , according to the scheme established by cavanagh and ulbrecht.33 the rearfoot was subdivided into the medial ( 30% of the rearfoot width ) , central ( 40% of the rearfoot width ) and lateral rearfoot ( 30% of the rearfoot width ) areas , and the forefoot was subdivided into the medial ( 55% of the forefoot width ) and lateral forefoot ( 45% of the forefoot width).34
the stance phase of the gait was divided into three subphases using a customized mathematical function developed using matlab software ( v.7.1 ) .
the initial heel contact phase was defined as the time interval between the first foot contact with the ground ( as seen in the pressure data ) and the deflection instant in the transition from extension to flexion of the ankle angular variation curve .
the midstance phase was defined as the time interval between the prior ankle deflection instant ( extension to flexion ) and the deflection instant in the transition from the flexion to extension of the ankle .
the propulsion phase was the interval between the last deflection instant from flexion to extension of the ankle and the toe - off instant ( as seen in the pressure data ) .
statistical inferential analysis was performed with statistica v.7 software ( statsoft inc . ) . for statistical purposes ,
pressure data from only one foot per subject were analyzed and compared . in the control group ,
a foot was randomly chosen for analysis , while in the pfps group , the chosen foot corresponded to the painful knee in subjects with unilateral pain and the most painful knee in subjects with bilateral pain .
plantar pressure variables followed a normal distribution ( shapiro - wilk 's test ) , and variances were homogeneous ( levene 's test ) .
groups and areas were compared using 2 three - way anovas ( 2x3x6 ) that considered the gait subphases ( 3 ) and the plantar areas ( 6 ) as repeated measurements ; these analyses were followed by a newman - keuls post - hoc test .
the level of significance was set at = 5% and marginal significance was set at 1% < < 5% .
the pfps group showed a greater contact area at the medial ( p = 0.004 ) and central ( p = 0.002 ) rearfoot during initial heel contact , a greater contact area at the medial ( p = 0.072 ) and lateral ( p = 0.005 ) forefoot in the midstance and a greater contact area at the lateral forefoot in the propulsion phase ( p = 0.079 ) .
the pfps group also presented a smaller peak pressure at the medial forefoot during propulsion than the cg ( p = 0.003 ) .
our main findings showed that subjects with pfps presented larger contact areas at the medial and central rearfoot during initial heel contact , at the medial and lateral forefoot in the midstance , and at the lateral forefoot during propulsion .
pfps individuals also presented a smaller peak pressure at the medial forefoot that was followed by a larger contact area at the lateral forefoot during propulsion .
these results suggest that pfps individuals exhibit a foot rollover process characterized by an initial heel contact that is performed more medially at the rearfoot and a propulsion phase that is performed more laterally at the forefoot . in the midstance ,
the larger contact area at both the medial and lateral forefoot suggests that pfps individuals have a greater excursion of the foot during this phase both medially and laterally .
according to willems et al.,17 this laterally directed support during propulsion , which causes the terminal push - off to occur laterally rather than predominantly across the hallux as expected , occurs because an increase in eversion during initial heel contact leads to a less stable foot .
consequently , a greater re - inversion is performed to provide a rigid lever for optimal push - off .
willems et al.17 performed a prospective study of physically active subjects who developed exercise - related injuries .
plantar pressure and rearfoot 3d kinematics were evaluated , and the researchers observed a foot rollover pattern during running that was similar to what we observed during gait .
subjects who developed injuries showed a more central initial contact that was associated with a more everted rearfoot as well as a more laterally directed propulsion . in our study , pfps individuals presented a more medially directed initial contact and more laterally directed support during propulsion .
this result may also be attributed to a greater evertion of the rearfoot at heel strike followed by an increase in supination during the propulsion phase .
the laterally directed propulsion observed in this study , inferred from the larger lateral forefoot contact area and the smaller peak pressure at the medial forefoot during propulsion , is compatible with the pattern reported by thijs et al.18 these authors prospectively evaluated plantar pressure during the gait of military subjects who developed pfps . they observed a more lateralized foot rollover pattern in subjects who developed pfps than in subjects who did not develop the disorder .
the present study contributes to discussions about the influence of pfps in the foot rollover mechanism during gait , revealing changes in this pattern that would be difficult to perceive through a clinical visual observation of gait .
the decision to divide the foot rollover mechanism into three phases allowed us to investigate in more detail what was happening at initial contact , midstance and propulsion in this population .
the rollover pattern observed in the pfps individuals was different from that observed in the healthy individuals and could induce alterations in the load attenuation within the kinetic chain of the lower limbs .
plantar pressure that is medially distributed at the initial contact and laterally distributed during propulsion would probably result in torque alterations in the lower kinetic chain . a plantar contact that is medially oriented in the rearfoot is probably related to an everted rearfoot and could lead to an excessive medial rotation of the tibia .
this rotation could induce a compensatory medial rotation of the femur and a lateralization of the patella in relation to the femur , increasing the patellofemoral joint stress.7 this medially directed contact in the rearfoot has already been detected in individuals with pfps during stair descent.34 plantar pressure that is laterally distributed during propulsion is probably related to a greater re - inversion that is performed to provide a rigid lever for push - off , as the more everted initial contact leads to a less stable foot.17 this event can possibly cause lower limb torque modification during gait , inducing alterations in the load attenuation within the kinetic chain .
this detailed characterization of the rocker mechanism is clinically relevant because these findings can be used to develop clinical interventions such as insoles , taping and therapeutic exercises useful for the rehabilitation of this dysfunction .
furthermore , this study evaluated a population that was predominantly female who participated in this study and exhibited similar levels of physical activity compared with the control group , which decreased the possibility that gender and variable levels of physical activity interfered with our results .
individuals with pfps exhibit a foot rollover pattern that is medially directed at the rearfoot during initial heel contact and laterally directed at the forefoot during propulsion .
these alterations during the foot rollover process can be used to develop clinical interventions that use insoles , taping and therapeutic exercise to rehabilitate this dysfunction . | background : patellofemoral pain syndrome is one of the most common knee disorders among physically active young women . despite its high incidence , the multifactorial etiology of this disorder is not fully understood.objectives:to investigate the influence of patellofemoral pain syndrome on plantar pressure distribution during the foot rollover process ( i.e. , the initial heel contact , midstance and propulsion phases ) of the gait.materials and methods : fifty - seven young adults , including 22 subjects with patellofemoral pain syndrome ( 30 7 years , 165 9 cm , 63 12 kg ) and 35 control subjects ( 29 7 years , 164 8 cm , 60 11 kg ) , volunteered for the study .
the contact area and peak pressure were evaluated using the pedar - x system ( novel , germany ) synchronized with ankle sagittal kinematics.results:subjects with patellofemoral pain syndrome showed a larger contact area over the medial ( p = 0.004 ) and central ( p = 0.002 ) rearfoot at the initial contact phase and a lower peak pressure over the medial forefoot ( p = 0.033 ) during propulsion when compared with control subjects.conclusions:patellofemoral pain syndrome is related to a foot rollover pattern that is medially directed at the rearfoot during initial heel contact and laterally directed at the forefoot during propulsion . these detected alterations in the foot rollover process during gait may be used to develop clinical interventions using insoles , taping and therapeutic exercise to rehabilitate this dysfunction . | INTRODUCTION
MATERIALS AND METHODS
Subjects
Gait Measurement
DATA ANALYSIS
RESULTS
DISCUSSION
CONCLUSION |
PMC3410331 | ever since the development of unfillied resin by bowen in 1962 , the concept of conservative restorative dentistry has grown rapidly . dental composite with dentine adhesive systems are currently the most popular materials for fabrication of restorations .
the main advantages of these include esthetic properties , conservative tooth preparation , and ability to be directly placed without laboratory procedures .
although composite resins have better esthetics and mechanical properties compared with glass ionomer cements , their inherent drawbacks are polymerization shrinkage and high coefficient of thermal expansion as compared with tooth structure .
microleakage may result in percolation , postoperative sensitivity , secondary marginal caries , and loss of esthetics .
also restoration of even a tiny lesion with composites is a long and complex procedure requiring acid etching , priming , bonding , light curing , placement of the restorative material and then light curing again . with the introduction of wetbond the time - consuming procedures
the embrace wetbond resin technology ( pulpdent , watertown , ma , usa ) is an advanced bonding chemistry and new generation of hydrophilic dental restorative material that chemically and micromechanically bonds to the tooth .
the materials are formulated from a unique dental resin that is self - priming , wet bonding , water miscible , hydrophilic , and hydrobalanced .
embrace class v is the first wet bonding restorative resin specially designed for class v restorations .
it incorporates di- , tri , and multifunctional acrylate monomers into a hydrophilic , resin acid integrating network ( r.a.i.n . ) .
traditional composite resins when used to restore class v lesions invariably result in leakage at gingival margin , whereas wetbond integrates with the tooth and provides excellent seal against microleakage .
this study compared the microleakage of traditional composite , that is , charisma / gluma comfort bond and self - priming resin , that is , embrace wetbond .
twenty noncarious extracted human premolars of patients in the age group of 2140 years were selected .
the teeth were cleaned of calculus , soft tissue , and debris with hand instrumentation and stored in a 1% chloramine solution for not more than 1 month after extraction .
each specimen provided 2 surfaces for preparation , facial and lingual , resulting in 40 samples for evaluation .
class v cavities ( 2 mm occlusogingivally , 2 mm mesiodistally , and 1.5 mm in depth ) were prepared with cylindrical diamond bur in an air-/water - cooled high - speed handpiece ( nsk pana air high - speed air turbine handpiece , nakanishi inc .
the occlusal margins of the cavities were located in the enamel and gingival margins in dentin .
dimensions of cavities were standardized using a template ( 2 2 mm ) prepared in a metal band strip and depth by marking the burs at 1.5 mm length prior to use .
the teeth were randomly assigned into 2 groups of 10 teeth each ( 20 class v cavities ) .
group 1 : teeth were restored with charisma and gluma comfort bond ( heraeus kulzer , llc , usa ) .
group 2 : teeth were restored with embrace wetbond ( pulpdent , watertown , ma , usa ) according to manufacturer 's instructions . in group 1 ( charisma / gluma comfort bond ) dentin and enamel were etched with 37% phosphoric acid gel for 15 s. the cavities were rinsed with water for 30 s. excess water was removed with blotting paper .
two coats of gluma comfort bond were applied with a brush to the prepared cavities , which were then left for 15 s , lightly air dried by a gentle stream of air for 10 s followed by light curing for 20 s. the restorative material charisma shade a2 was placed and cured . in group 2
( embrace wetbond ) only enamel was etched with 37% phosphoric acid gel for 15 s. the cavities were rinsed with water for 30 s and excess water was removed with blotting paper . no bonding agent was applied .
wetbond was placed and light cured for 20 s. after 24 h storage in distilled water at 37c , the restored teeth were subjected to 200 thermocycles between 2 baths at 5c and 55c .
the immersion time in each bath was 1 min and the time of transfer between baths was 10 s. after thermocycling the apices of the teeth were sealed with sticky wax .
all tooth surfaces , except for 1 mm around margins of each restoration , were sealed with 2 coats of nail polish .
the teeth were immersed in 0.2% methylene blue for 24 h. upon retrieval from the dye , the teeth were washed for 4 h under running water and left to dry for dye fixation .
each tooth was sectioned longitudinally in a buccolingual direction through the center of each restoration with a slow - speed diamond disk .
the cut surfaces were examined at the occlusal and gingival margins using a stereomicroscope at 10 magnification .
dye penetration at the composite - tooth interface was evaluated for both occlusal and gingival margins using the following scoring system .
no dye penetrationdye penetration less than half the length of the gingival or occlusal walldye penetration up to the full length of the gingival or occlusal walldye penetration along the gingival wall and extending to the axial wall
dye penetration less than half the length of the gingival or occlusal wall dye penetration up to the full length of the gingival or occlusal wall dye penetration along the gingival wall and extending to the axial wall the data was statistically analyzed using wilcoxon signed - rank test at the 0.05 level of significance .
table 1 shows the distribution of dye penetration scores at the occlusal and gingival margins in all groups . in group 1 ( charisma / gluma comfort bond )
mean microleakage score at the occlusal margin ( 1 0.9177 ) was less than the gingival margin ( 1.9 1.1653 ) . in group 2 ( embrace wetbond )
also mean microleakage score at the occlusal margin ( 0.25 0.4443 ) was less than the gingival margin ( 1.65 1.1821 ) . when microleakage at occlusal and gingival margins were compared , the results were statistically significant in both the groups ( p < 0.05 ) [ table 1 ] . when group 1 ( charisma / gluma comfort bond ) and group 2 ( embrace wetbond ) were compared , mean microleakage score on both occlusal and gingival margin was more in group 1 ( charisma / gluma comfort bond ) .
but till date it has a number of limitations , such as it requires a large number of steps and bonding of composite to enamel is reliable but dentin bonding is still not reliable .
research is going on to introduce a material to combat the drawbacks of composite resins .
therefore , wetbond has been introduced , which offers superior marginal sealing with fewer steps .
microleakage is defined as the clinically undetectable passage of bacteria , fluids , molecules , or ions between a cavity wall and the restorative materials applied to it .
microleakage around resin composite restorations results from marginal gaps due to shrinkage that resin composites undergo during polymerization .
oral environmental bacteria may invade a cavity via gaps at the margin of dental restoration resulting in inflammatory alteration of subjacent tissue , discoloration of the restoration , marginal breakdown , recurrent caries , pulpal inflammation , and postoperative sensitivity , which can affect the longevity of the restoration as well as the vitality of the dental pulp.[1012 ] in the present study , all the class v cavities had same dimensions .
( charisma / gluma comfort bond ) cavities were restored with conventional composite , which required acid etching of dentin as well as enamel , application of gluma comfort bond , and insertion of charisma composite resin . in group 2
( embrace wetbond ) teeth were restored with self - priming , wet bonding , water miscible , hydrophilic and hydrobalanced wetbond , which required only enamel etching and insertion of composite .
thermocycling is the in vitro process of subjecting the restoration on the tooth to temperature extremes compatible with the oral cavity .
this simulates introduction of hot and cold extremes in the oral cavity and shows the relationship between coefficient of thermal expansion between tooth and restorative material . in group 1 ( charisma / gluma comfort bond )
mean microleakage score at the occlusal margin ( 1 0.9177 ) was less than the gingival margin ( 1.9 1.1653 ) and the results were statistically significant ( p < 0.05 ) .
the results are in concurrence with the findings of other studies.[1416 ] when the teeth were restored with various composite resins , occlusal margin shows lower microleakage scores than the gingival margin.[1721 ] although the enamel is a reliable substrate for bonding , yet bonding to dentin is far more challenging because dentin is a living tissue , it is heterogenous and its tubular nature provides variable area through which dentinal fluid may flow to the surface on account of pulpal pressure and adversely affect adhesion . in group 2 ( embrace wetbond ) also mean microleakage score at the occlusal margin ( 0.25 0.4443 ) is less than the gingival margin ( 1.65 1.1821 ) and the results were statistically significant ( p < 0.05 ) .
when compared with group 1 ( charisma / gluma comfort bond ) , group 2 ( embrace wetbond ) exhibited less dye penetration at occlusal as well as gingival margin and the results were statistically significant ( p < 0.05 ) .
the difference could be attributed to bonding mechanism between tooth and wetbond as it chemically and micromechanically bond to the tooth structure .
but there is no substantial scientific proof that demonstrates the development of significant chemical bonding between dentin adhesives and tooth surface under intraoral conditions .
this can be augmented by secondary bonds , which have formed between hydrophilic resins ( as in embrace wetbond ) and tooth .
however , a larger sample size combined with in vivo studies seems to be necessary to further investigate the results .
significantly less microleakage was found in teeth restored with wetbond than traditional composite.more microleakage was observed in gingival margins than occlusal margins .
significantly less microleakage was found in teeth restored with wetbond than traditional composite . | background : the purpose of this study was to compare the microleakage of traditional composite ( charisma / gluma comfort bond ) and self - priming resin ( embrace wetbond).materials and methods : standardized class v cavities partly in enamel and cementum were prepared in 20 extracted human premolars .
teeth were divided into two groups .
group 1 was restored with charisma / gluma comfort bond and group 2 with embrace wetbond .
the specimens were stored in distilled water at room temperature for 24 h and then subjected to 200 thermocycles at 5c and 55c with a 1 min dwell time .
after thermocycling teeth were immersed in a 0.2% solution of methylene blue dye for 24 h. teeth were sectioned vertically approximately midway through the facial and lingual surfaces using a diamond saw blade .
microleakage was evaluated at enamel and cementum surfaces using 10 stereomicroscope .
the statistical analysis was performed using wilcoxon signed - rank test.results:wetbond showed less microleakage at occlusal and gingival margins as compared with charisma / gluma comfort bond and the results were statistically significant ( p < 0.05).conclusion : class v cavities restored with embrace wetbond with fewer steps and fewer materials offers greater protection against microleakage at the tooth restorative interface . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC4699501 | rheumatoid arthritis ( ra ) is a chronic autoimmune disorder characterized by inflammation
of synovial tissues that leads to joint swelling , stiffness , pain , and progressive joint
destruction with unpredictable course and wide variation in severity .
the one
percent of the world s population is affected by ra , suffer substantial morbidity as a
result of rheumatoid arthritis and early death as a result of comorbid diseases .
unfortunately , cure of ra is not possible , and management and control of this disease is
considered with current procedures , then it can be said that , for incurable diseases the
quality of care is considered very important .
quality of care has become increasingly critical in the evaluation of healthcare and
healthcare services .
however , little is
known about whether patients with ra receive appropriate care for their disease and few
tools exist to evaluate this question .
evaluation of the rheumatic healthcare quality is a major issue for detection of needed care
and long - term dependency on healthcare .
initially , the assessing of quality of care is often performed by healthcare professionals ;
however , patients perspectives on healthcare quality differ from the views of healthcare
professionals and policy makers .
quality of care from the perspective of patients is increasingly considered an important
component of comprehensive chronic disease management and as an instrument for evaluation of
health care quality . in many preceding studies , quality of care from the patient s
perspective has been considered as patient satisfaction .
the limitation of these
surveys was that the scores were extremely subjective , highly skewed ( > 90% are
satisfied ) , and influenced by personal preference and patient expectation .
therefore , a more refined and less subjective
instrument for evaluating healthcare quality from the patients perspective is needed .
donabedian , one of the pioneers in quality promotion , proposed a model to measure quality
of health care using technical quality ( tq ) , structure quality ( sq ) , and process quality
( pq ) .
lack of a comprehensive
measurement method in health care has created a miss understand regard to result of quality
assessment in health services . also , most
of studies on the quality of health care focus on the technical and service aspects of care ,
and the most important aspect in delivery of health services , customer quality , is
neglected . in this regard ,
, the quality of health services are assessed from three
dimensions : service , technical and customer quality .
service quality refers primarily to how the care are received and is
influenced by the physical , social , and cultural context .
technical quality refers to the
degree to which the delivered care meets scientific and professional standards that are
likely to optimize the benefits and minimize the risks .
this aspect of quality
reflects the knowledge , skill , and ability of the care giver for self - management .
customer
quality refers to the attributes of patients or health care consumers that enable them to
participate more effectively with health care delivery system in order to successfully
manage their own conditions .
quality
index ( qi ) is the combination of three aspects of quality of care and indicates an overall
value for quality of delivered care among patients ( figure
1 ) . the proposed model of comprehensive quality measurement in health care
( cqmh ) to the best of our knowledge , despite the importance of care among the arthritis
rheumatoid patients ,
so , our aim in
this study was to assess quality of delivered care among patients with rheumatoid arthritis
based on a model of comprehensive quality measurement in health care ( cqmh ) .
this was a cross - sectional study , conducted on 170 subjects with rheumatoid arthritis who
were received care from specialist clinics of isfahan university of medical sciences in
2013 . according to the convenience sampling subjects who were referred to clinics from
january to april 2013
quality of delivered care -tq , sq , and cq-
was measured by using the reformed cqmh questionnaire for ra patients . due to the lack of national instructions ,
international instructions were used for
measuring the technical quality of services . then standards was
categorized in five group of clinical care and services , paraclinical services , lifestyle
education , education about drugs , and assessment of drug side effects .
for each aspect of
service quality , respondents were asked to evaluate the importance of that aspect and to
determine their perception about the quality of care received in relation to that aspect
( performance ) , over the past year .
this instrument measures
customer empowerment in four important stages : 1 ) believing the patients role is important ,
2 ) having the confidence and knowledge necessary to take action , 3 ) actually taking action
to maintain and improve one s health , and 4 ) staying the course even under stress and
financial constraints . final section of study questionnaire was included demographic
information such as : age , education level , care provider , health insurance and rheumatoid
arthritis control status .
validity of the study questionnaires were reviewed and confirmed by 10 faculty members at
tabriz and isfahan university of medical sciences . also reliability was confirmed according
to cronbach 's alpha index ( sq ; = 0.721 , tq ; = 0.766 , cq ; = 0.803 ) , based on a pilot
study by participation of thirty subjects .
the scores of technical quality was determined as 0 ( worst adherence ) and 1(best
adherence ) according to the adherence of rheumatologist to care standards .
service quality
for each of twelve aspects was measured using the importance and performance scales based on
the netherlands institute for health services research methodology .
crude scores of cq normalized as 0 - 100 , and then active cq scores was calculated .
cq
active scores were categorized based on the study has been conducted by tabrizi et al .
final scores for each
dimension were transformed to 0 - 100 scales , and higher scores indicated more quality . based
on the study conducted by tabrizi et al .
, three dimensions of quality had same contribution ,
so the principal component analysis was used to calculate quality index ( qi ) , instead of
three dimensions . in the descriptive statistics ,
frequencies were used for qualitative variables and mean
( sd ) for continuous variables such as : cq , sq , tq and qi scores .
independent samples t - test
and anova test were conducted to compare cq , sq , tq and qi score between categorical
variables .
data were analyzed through the spss-13 statistical package ( spss , chicago , il ,
usa ) .
findings indicated that 82.7% of participants were female , and the majority ( 63% ) of
participants was housekeeper .
most of participants aged over 50 years old ( 41.4% ) , and the
mean disease duration was approximately 10 years .
also , 14.2% of patients were illiterate
and only 22.6% of them had educated in the university .
response to treat was assessed well by majority of participants ( 73.5% ) , and considerable
proportion of them ( 75.9% ) reported some kinds of complications . among these , the prevalence
of joint complications and arthritis among subjects were 32.7% and 43.8% , respectively .
findings showed that 41.4% of patients
also suffered from osteoporosis , 14.1% had a history of heart disease and 35.8% of
participants had ophthalmic problems .
whereas 73.5% of participants stated that
self - evaluation of disease control , were good and excellent .
regarding to self - reported customer quality score , all of the participants passed the
first stage of self management successfully .
the failure rate in second and third stage was
9.2% and 71% , respectively .
finally , forty - five subjects reached to last stage ( table 1 ) .
recommended care for patients within rheumatoid arthritis and received care based on the
reports of participants are presented in table 2 .
findings showed that , para - clinic examinations and lifestyle education with mean 80 ( 20.32 )
and 41.37 ( 27.51 ) had highest and lowest scores , respectively .
the average scores for importance , performance and service quality according to sq aspects
are shown in table 3 .
1 was the best and 0 is the worst , 10 was the best and 0 is
the worst,10 was the best and 0 is the worst findings indicated that , confidentiality had the highest score for performance and then
there is choice of care provider , accessibility and dignity , respectively .
basic amenities ,
communication , prevention , dignity and safety had the highest scores for importance aspect .
confidentiality and choice of care provider achieved the highest sq scores the overall
results have shown in table 4 . in all quality dimensions and the quality index , men had higher quality score than women ,
but this difference was not statistically significant ( p>0.05 ) .
study results indicated
that participations with higher educational level significantly had greater cq and quality
index scores ( p= 0.13 , and p<0.01 , respectively ) .
patients with complication also
significantly had greater tq and quality index scores ( p<0.01 ) .
similarly , there was a significantly positive correlation between age and tq score ( p
= 0.12 ) .
participants who had active disease reported higher cq score than who had n't active
disease ( p=0.03 ) .
there was also positive significant correlation between occupation and
quality index ( p = 0.02 ) .
in this study , quality index score was relatively low for subjects with rheumatoid
arthritis from patient 's perspective .
there is a significant gap between the quality of care
received by patients with ra with what was recommended in the guideline , in term of overall
service quality , and customer quality scores . according to patient 's report
similarly , delivered care to arthritis rheumatoid patients in canada were not met
standards .
the largest gap in this
study was related to lifestyle education and clinical care and services .
so , there are
considerable opportunities to improve ra care by paying more attention to nutritional
regimens , effective physical activity , and refer to orthopaedic surgery .
it is important to
state that these guidelines are minimum of standards which must be fully implemented , then
there is still room for considerable improvement .
there is much potential for self - management of arthritis rheumatoid with regard to long
term and chronic nature of disease and low ability of patients to manage their health under
stressful condition and financial constraints .
based on the instructions of american college of rheumatology , educating self - management
measures to patients was considered as main component of treatment .
exercise has become an important part of rehabilitation during the last decade which , main purpose for prescribing
therapeutic exercise to prevention of functional disability . study of van den ende indicated that dynamic exercise therapy
has a positive effect on physical capacity and has been shown to decrease the risk of heart disease ,
hypertension , patient - reported
disability and pain as well as
beneficial in improving symptoms and quality of life .
thus , our study suggests patient self - management education to improve
customer quality and ability in managing their daily problems and conditions that leads to
reduction in long - term and acute complications . in our study ,
service quality score was relatively low for people with rheumatoid
arthritis from the patient perspective .
of the 12 aspects of service quality , the scores of
" confidentiality " and " choice of care provider " were highest ( high 9 score ) , while the score
of " availability of support group " was lowest .
based on the service quality score ,
participants in the current study were less concerned about communication , autonomy ,
continuity of care , accessibility and dignity , while participants also reported inadequate
quality in term of basic amenities , timeliness , safety and prevention .
service quality and
non - health aspects of care has become most important factor in marketing and utilization of
services especially in industries with high customer like the health system . based on the experts ' viewpoint ,
service
quality is also defined as organization 's ability to meet growing expectations of
customer .
nevertheless , scores of
service quality was poor from the patient 's perspectives especially in " availability of
support group " domain . despite the important effect of the supportive groups to individual 's health ,
studies have been
done by doeglas et al . , and gottlieb et al . , showed that , participating in support groups
had valuable contribution to the empowerment of patients .
it 's important to be
noted that , these results could be helpful for decision makers to focus on important and
neglected aspects of service quality .
the customer quality based on the ra patients ' perspectives in the city of isfahan was
found to be moderate with an average score of 70.25 ( 13.20 ) .
a great part of the
participants ( 71% ) reported taking action while faced with ra - related health problems , and
only about 20% of patients were able to maintain needed actions even under stress and
financial constraints . in our study , highly educated subjects had high scores in customer
quality , self - management ability , and quality index ( table
4 ) .
these findings is consistent with other studies that showed , education level is positively
associated with level of patient involvement in medical decisions and patients with inactive
disease had better c.q score than active ones .
shokri also has stated
increased anxiety and depression in patients with active disease .
self - management and stress management education is a essential base for the empowering
patients and is a basic ability for patients to effectively manage their diseases and to
make appropriate decisions in all conditions .
koehn and newman defined
self - management as the individual s ability to manage the symptoms , physical , psychological
consequences and life - style changes inherent in living with a chronic condition. this study has some limitation such as the high number of questions , time consuming
interviews , and lack of appropriate places to interview with patients .
this model shows that from the patients perspective the quality of service delivery for
ar patients in terms of three dimensions were almost low in isfahan .
this means that , despite the improvement in the status of ra patients in the last decade ,
the quality of services is still worrying .
so , regarding to technical quality , life style
trainings should be considered for ra patients . also , there is need for focusing on clinical
services through providing related equipment , physiotherapy programs , regimes , and need
assessment of patients for referring to orthopedist .
supportive assembly for ra patients ,
high level facilities , preventive measures , and safety in service delivery should be
considered to improve the service quality .
also , customer quality could be promoted through involving the ra patients in treatment
process and educational programs such as knowledge , skill , and self - esteem increment for
self - management of disease .
finally , it is no table that total quality of service could be
improved through providing intelligible information for low educated subjects , more
attention on the complication of disease and treating of it .
we would like to thank all experts in department of health services management for their
useful comments .
also special thanks to all ra patients for their patient and participation
in this study . |
introduction : quality of care has become increasingly critical in the evaluation of healthcare
and healthcare services .
the aim of this study was to assess quality of delivered care
among patients with rheumatoid arthritis using a model of comprehensive quality
measurement in health care ( cqmh ) .
methods : this cross - sectional study was conducted on 172 patients with rheumatoid
arthritis ( ra ) who were received care from private clinics of isfahan university of
medical sciences in 2013 .
cqmh questionnaires were used for assessing the quality of care .
data were analyzed using spss for windows .
results : the mean scores of quality index , service quality ( sq ) , technical quality ( tq ) ,
and costumer quality ( cq ) were 72.70 , 79.09 , 68.54 and 70.25 out of 100 , respectively . for
cq only 19.8% of participations staying the course of action even under stress and
financial constraints , there is a significant gap between what ra care they received with
what was recommended in the guideline for tq .
scores of service quality was low in
majority of aspects especially in " availability of support group " section .
conclusion : study shows paradoxical findings and expresses that quality scores of service
delivery for patients with arthritis rheumatoid from patient 's perspective is relatively
low .
therefore , for fixing this paradoxical problem , improving the participation of
patients and their family and empowering them for self - management and decision should be
regarded by health systems . | Introduction
Materials and methods
Results
Discussion
Conclusion
Acknowledgments
Ethical issues
Conflict of interest |
PMC3659856 | demands for esthetic restorations are increased . for achieving a long - term high performance composite restoration , a good isolation especially near or at the gingival margin is needed .
in a situation such as sub - gingival caries , class v cavity preparation , taking impression , cementation of all - ceramic restorations with margins near the gingiva , and chronic gingival inflammation preventing or eliminating gingival bleeding or contamination by crevicular fluid is very critical for the longevity of the restoration .
blood has high - protein content ( approximately 6.7% ) and fibrinogen macromolecules , on the other hand , because of protein attraction property of dentin , blood proteins can form a layer , which prevents resin infiltration into the dentin structure and reduces 30 - 70% of bond strength . in the other words , in blood contamination situation
, some reactions have occurred between dentin organic layer and the blood proteins that influence resin infiltration . in recent years , use of hemostatic agents for controlling of gingival bleeding and reducing sulcular fluid are indicated .
they have presented in various formulations with different mechanism of action , such as aluminum chloride , ferric sulfate compounds , iron solution , aluminum and potassium sulfate and epinephrine 0.1% .
recent studies showed that most of them have acidic property ( ph = 0.7 - 3 ) and hydrophilic characteristics that can contaminate each stage of bonding procedure . by hemostatic agents application ,
smear layer was removed or some change in dentin surface happened that could affect the hybrid layer quality after the self - etch or etch - and - rinse adhesives usage .
therefore , the efficacy of self - etch and etch - and - rinse adhesive systems should be considered first and then recommended for clinical uses .
this study evaluated the shear bond strength of one etch - and - rinse and two self - etch adhesives to human dentin contaminated with a ferric sulfate hemostatic agent .
the null hypothesis of this study was that contamination with ferric sulfate 20% may not have any effect on shear bond strength of etch - and - rinse and self - etch adhesive systems to dentin .
in this in vitro experimental study , 60 intact caries - free human third molars were selected and cleaned up of soft tissues . then , they were stored in a 0.5% chloramine - t ( fisher chemical , fair lawn , nj , usa ) solution for 24 h. after they were rinsed , about 1.5 - 2 mm of buccal or lingual tooth structure ( enamel and dentin ) were removed by using a diamond bur ( ss white , great white series , lakewood , nj , usa ) to create flat surfaces of superficial dentin for testing ( each bur was discarded after each five preparation ) .
dentin surfaces were ground with 400 and 600 grit sand paper under running water to produce a standardized smear layer .
utah , usa ) was applied for 60 s and then it was rinsed for 60 s thoroughly .
then dentin surfaces were etched with 37% phosphoric acid ( ivoclar / vivadent , schaan , liechtenstein ) for 10 s , were rinsed for 30 s , were blotted dried and were followed by application of one coat of tetric n - bond ( ivoclar / vivadent , schaan , liechtenstein ) for 10 s , gently air dried and were light cured for 20 s with a quartz - tungsten - halogen unit with 600 mw / cm intensity ( demetron lc , kerr , usa)group 2 : no viscostat application was done prior to etching .
etching and bonding were done as in group 1group 3 : after viscostat application as in group 1 and 60 s rinsing and blot drying , primer bottle of adhese ( ivoclar / vivadent , schaan , liechtenstein ) was applied for 30 s , air drying was done and adhesive bottle of adhese was applied for 20 s , gently air dried and light cured for 20 sgroup 4 : no viscostat application was done . adhese bonding system was used same as in group 3group 5 : after viscostat application as in group 1 and 60 s rinsing and blot drying , adhese one f ( ivoclar / vivadent , schaan , liechtenstein ) was applied for 20 s , gently air dried and light cured for 20 sgroup 6 : no viscostat application was done .
adhese one fadhese one fa plastic mold ( 2 mm in internal diameter and 4 mm in height ) were used to prepare the composite ( tetric n - ceram , ivoclar / vivadent , schaan , liechtenstein ) cylinders on dentin surfaces .
composite were placed in two increments and each increment was light cured for 40 s. all samples were stored in distilled water for 24 h at room temperature , then were thermocycled for 500 cycles between 5c and 55c ( with a 60 s dwell time and a 15 s transfer time ) .
group 1 : viscostat hemostatic agent ( ultradent product inc . , utah , usa ) was applied for 60 s and then it was rinsed for 60 s thoroughly .
then dentin surfaces were etched with 37% phosphoric acid ( ivoclar / vivadent , schaan , liechtenstein ) for 10 s , were rinsed for 30 s , were blotted dried and were followed by application of one coat of tetric n - bond ( ivoclar / vivadent , schaan , liechtenstein ) for 10 s , gently air dried and were light cured for 20 s with a quartz - tungsten - halogen unit with 600 mw / cm intensity ( demetron lc , kerr , usa ) group 2 : no viscostat application was done prior to etching .
etching and bonding were done as in group 1 group 3 : after viscostat application as in group 1 and 60 s rinsing and blot drying , primer bottle of adhese ( ivoclar / vivadent , schaan , liechtenstein ) was applied for 30 s , air drying was done and adhesive bottle of adhese was applied for 20 s , gently air dried and light cured for 20 s group 4 : no viscostat application was done .
adhese bonding system was used same as in group 3 group 5 : after viscostat application as in group 1 and 60 s rinsing and blot drying , adhese one f ( ivoclar / vivadent , schaan , liechtenstein ) was applied for 20 s , gently air dried and light cured for 20 s group 6 : no viscostat application was done .
the shear bond test was done using a universal testing machine ( m350 - 10ct testometric , lancashire , united kingdom ) at a cross head speed of 0.5 mm / min .
two examiners evaluated the debonded surfaces at 40 magnifications by using a stereomicroscope ( olympus , dp 12 , germany ) to identify the mode of bond failure whether adhesive , cohesive or mixed .
statistical analysis was done using one - way anova and unpaired t - test with p < 0.05 as the level of significance .
mean and standard deviation values of shear bond strength test are shown in table 1 . shear bond strength data in mega pascal ( meansd ) for each tested group in comparison with case and control groups , only there was statistically significant reduction in the shear bond strength value in adhese groups ( p < 0.0001 ) .
in tetric n - bond and adhese one f there was no significant reduction in shear bond strength . in comparison with viscostat applied groups ,
tetric n - bond had the highest ( p < 0.0001 ) and adhese one f had the lowest ( p < 0.0001 ) shear bond strength value .
ferric sulfate as a hemostatic agent can coagulate collagen or plasma proteins in dentinal fluids ; changes dentinal surface structure due to its acidity and may reduce the shear bond strength .
the null hypothesis was confirmed for tetric n - bond and adhese one f adhesives , which ferric sulfate 20% contamination had no effect on shear bond strength of these adhesives to dentin , significantly .
phosphoric acid 37% because of its strong acidity ( ph = 0.5 ) accompany with demineralization , can eliminate surface viscostat contamination .
furthermore , according to fischer 's study , phosphoric acid can decompose and destroy ferric sulfate .
study showed that rinsing contaminated dentin surfaces with viscostat and viscostat plus after blood contamination did not alter shear bond strength of optibond solo plus ( kerr , orange , ca , usa ) to dentin .
has shown that there were no differences in dentinal content between aluminum chloride ( a hemostatic agent ) contaminated dentin and non - contaminated one after acid etching . in adhese groups , results showed that rinsing viscostat , alone , can not eliminate surfaces contamination and thus the remnant ferric sulfate interfered with diffusion of adhese in dentinal tubules and reduce shear bond strength significantly . in a scanning electron microscope - energy dispersive x - ray spectroscopy ( sem - edx ) study by ayo - yusuf , contaminated dentinal surfaces with different hemostatic agents were studied .
it was mentioned that hemostatic agents because of their acidic property ( ph = 0.7 - 3 ) , can dissolve smear layer and also obturated dentinal tubules orifices .
they form an amorphous layer or a granular precipitation on the surfaces . because of the weak acidity of the primer of self - etch adhesives in comparison with phosphoric acid , it was predictable that they could not dissolve viscostat contamination so that their penetration to deeper areas of dentin was impossible .
several studies emphasize that hemostatic agent can cause derangement in bonding procedure because of dentinal tubules obturation and dentinal surface was demineralized in different values .
study showed that rinsing ferric sulfate and aluminum chloride hemostatic agents with water before using self - etch adhesives causes higher bond strength than non - rinsing ones .
adhese one f had moderate acidity strength ( ph = 1.5 ) with 0.05 penetration depth in dentin .
on the other hand , it seems that demineralized property of viscostat could be increased adhese one f dentinal diffusion depth and may be viscostat dissolved smear layer and smear plugs , which helps to better diffusion of adhese one f ( or not interfered with adhese one f diffusion ) .
furthermore , in one study , it seems that despite control group that had a thick smear layer , in group with aluminum chloride hemostatic agent contamination , the smear layer was partially removed . in our study , there were significant differences between contaminated groups . according to manufacturer 's claim , viscostat is a water soluble gel and rinsing with water before bonding procedure is necessary . in etch - and - rinse adhesives because of lower ph of phosphoric acid and its capability for removing contaminants that lead to better diffusion of adhesive , the shear bond strength was higher than the others , where the demineralization effect on contaminated dentin was inhibited
also , there were significant differences among control groups where tetric n - bond had the highest and adhese one f had the lowest bond strength value .
phosphoric acid could cause much more microporosities in dentin than achieving better contact between adhesives and dentin substrate . removing more smear layer resulted in more mechanical retention and bond strength , which can explain the higher bond strength in tetric n - bond in comparison with adhese and adhese one f. hybrid layer in self - etch adhesives were uniform cylindrical resin tags but in etch - and - rinse adhesives were thicker with funnel - shaped resin tags .
methacrylates in most of the self - etch adhesives ( such as adhese ) in acidic solutions are not stable , because of hydrolysis of ester groups that causes weaker cross - linking , an inadequate polymerization , lower bond strength and lower bonding durability .
the presence of hema in adhese composition , prevents phase separation and collagen collapse in adhesive bottle and helps better adhesive diffusion unlike it can maintain water in adhesive layer and weakened mechanical strength in long - term hence to prevent this problem , hema free adhesives were introduced ( such as adhese one f ) . in hema free adhesives , higher concentration of solvents was necessary but after solvent evaporation , homogeneity of one - step self - etch adhesives was disturbed and phase separation and water blister formation happened .
these water blisters promoted to reduce hydrolytic stability of adhesive even after polymerization and bond strength reduction happened .
hybrid layer in one - step and two - step self - etch adhesives were semi permeable that water can transfer through the tooth - composite interface even after polymerization .
the differences in depth of penetration in dentin ( adhese : 1.2 - 2.2 , adhese one f : 0.05 ) could explain the differences of their bond strength , which resulted from this study .
the presence of hydrophobic monomers and hydrophilic acids in one bottle in one - step self - etch adhesives could compromise polymerization steps and also waters in dentinal tubules , caused incomplete polymerization at the interface .
all these problems can explain lower bond strength in adhese one f group in comparison with adhese and these findings were in agreement with grgoire et al . and knobloch et al . studies .
in filled adhesives such as adhese and adhese one f , polymerization shrinkage reduced but because of lesser diffusion in dentin , bond strength reduced as well . according to mousavinasab and kavian study , one - bottle adhesive ( such as adhese one f ) had a very thin hybrid layer where polymerization may be damaged because of the oxygen inhibiting layer formation .
some self - etch adhesives could not diffuse through smear layer because of their weak acidity in which they buffered with smear layer , enamel and dentin . buffered primer or adhesive had lower penetration .
tooth demineralization with self - etch adhesives has contrary with tooth buffering capacity and these adhesives could show different behavior .
adhese one f was one of the new presented products , which had an unknown behavior in adhesion . according to proena
et al . study in 2009 , lower degree of conversion of self - etch adhesive and water trees builds up ( pertain to water diffusion from underlying dentin to hybrid layer ) was the main reason of lower bond strength of self - etch adhesive in comparison with etch - and - rinse adhesives . if bond strength to dentin or enamel was more than 20 mpa , mode of failure dominantly happened cohesively in tooth structure or composite . in this study ,
cohesive failure was seen in etch - and - rinse groups more than self - etch groups .
within the limitations of this study , it can be concluded that dentin contamination with viscostat hemostatic agent had an adverse effect on the shear bond strength of two - step self - etch adhesive , adhese , and reduced it . | aim : this study investigated the effect of one hemostatic agent on the shear bond strength of self - etch and etch - and - rinse adhesive systems.materials and methods : sixty extracted third molars were selected . after preparing a flat surface of superficial dentin
, they were randomly divided into six groups .
adhesives were tetric n - bond , adhese , and adhese one f. before applying adhesives , surfaces were contaminated with viscostat for 60 s in three groups and rinsed . then composite were attached to surfaces and light cured .
after thermocycling , the bond strength was calculated and failure modes were determined by stereomicroscope .
the data were analyzed by t - test and one - way anova with p < 0.05 as the level of significance.results:viscostat had significantly decreased the shear bond strength of adhese ( p < 0.0001 ) to dentin .
modes of failures in all groups were mainly adhesive.conclusion:contamination had an adverse effect on the shear bond strength of adhese and reduced it . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC3621223 | there is a great physiological variability in origin , course , and distribution of these vessels , and we can assure that do not exist two equal coronary trees .
coronary artery anomalies are often not associated with clinical signs , symptoms , or complications , and they are usually compatible with normal heart development and function , even allowing normal physical activity .
nevertheless , this condition can be fatal , typically in young and previously healthy athletes .
in fact , as an exception to angina and syncope , sudden death is frequently the only manifestation of anatomic subset . according to the sudden death committee of the american heart association ,
coronary artery anomalies may also be associated with congenital heart diseases such as great arteries transposition , single ventricle , or tetralogy of fallot . however , coronary artery anomalies are more often discovered as incidental findings at the time of coronary angiography ( ca ) or autopsy . according to the literature ,
the clinical relevance of coronary artery anomalies is closely related to the functional ability of anomalous vessels to provide adequate blood supply to the myocardial tissue , with or without significant obstructive coronary disease .
we present the case of a 54 years old woman , who was a smoker and was affected by obesity , type 1 diabetes , and hypertension .
eight years before , she had undergone surgical aortic valve replacement with mechanical aortic valve .
the patient was admitted to hospital to perform elective surgery for ascending aortic aneurysm . in the last 6 months , she experienced several episodes of typical angina and chest tightness not related to exertion or emotional stress .
echocardiography showed global and regional left ventricular contractile function ( left ventricular ejection fraction 56% ) , mild mitral and tricuspid regurgitation , and ascending aortic aneurysm with maximum diameter of 71 mm .
the patient was in coronary angiography ( ca ) . at ca , there was no significant coronary artery stenosis or dynamic obstructions , but we incidentally found a congenital coronary anomaly in the form of peculiar dual left anterior descending artery ( lad ) anatomy .
lad arising from the left sinus of valsalva was short and gave rise to one septal branch , a little intermedius branch , and one diagonal branch [ figure 1 ] .
the left circumflex artery ( lcx ) was aplastic [ figure 2 ] and the right coronary artery ( rca ) was dominant and normal in origin and course [ figures 3 and 4 ] .
a second lad was seen arising from the right sinus of valsalva , with a third ostium different from rca [ figure 5 ] .
this second lad was long , run over the right ventricle , and gave rise to septal and diagonal branches , supplying the mid and distal territories of septal , antero - lateral wall , and apex [ figure 6 ] .
short lad arises from the left sinus of valsalva and gives rise to one septal branch , little intermedius branch , and one diagonal branch circumflex artery ( lcx ) is aplastic , as shown in the left caudal angiographic projection right coronary artery ( rca ) is normal in its origin and course right coronary artery ( rca ) is normal in its origin and course long lad arises from the right sinus of valsalva with a separate ostium , different from rca .
this lad is longer than the first , runs on the right ventricle , and gives rise to septal and diagonal branches , supplying the mid and distal territories of septal , anterior , lateral wall , and apex overlapping figures 1 and 5 , we can see the entire course of lad along interventricular sulcus to the apex
this specific anatomy , for some features , could be seen in type iv dual lad , referring to spindola - franco et al .
moreover , in our case , we can identify a number of anatomical peculiarities which might be useful to define a distinct coronary artery anomaly . among the coronary arteries ,
anomalies like dual lad anatomy are rare , in particular , the anomalous origin of lad from the right sinus of valsalva or proximal portion of rca is extremely rare , ranging between 1.2% and 6.1% of all coronary anomalies .
have proposed an angiographic classification for dual lad , consisting of four types of lad anomaly . in the first three types
, lad is divided into two branches : a short branch that supplies the proximal third of the septum and a long branch that can take several courses as shown below .
type i : running in the anterior interventricular sulcus ( aivs ) , the short lad is generally the source of all the major proximal septal perforators .
the long lad also runs in the aivs , descending on the left ventricular side of the aivs , and then reentering the distal aivs in order to reach the apex.type ii : the short lad is the same as in type i. the long lad descends over the right ventricular side before reentering the aivs.type iii : the short lad is consistent with that described in types i and ii .
the long lad travels intramyocardially in the ventricular septum.type iv : high in the aivs , a very short vessel is formed by the lad proper and the short lad . from this vessel ,
type i : running in the anterior interventricular sulcus ( aivs ) , the short lad is generally the source of all the major proximal septal perforators .
the long lad also runs in the aivs , descending on the left ventricular side of the aivs , and then reentering the distal aivs in order to reach the apex .
type ii : the short lad is the same as in type i. the long lad descends over the right ventricular side before reentering the aivs .
type iii : the short lad is consistent with that described in types i and ii .
type iv : high in the aivs , a very short vessel is formed by the lad proper and the short lad . from this vessel ,
, we report a type of dual lad anatomy that is not included in the angiographic classification previously proposed .
it could be considered as a variant of type iv , with anomalous lad origin from an ostium located in the right sinus of valsalva , separated by the right coronary ostium .
furthermore , the short lad is a unique vessel with the left main ( lm ) , without lcx wich appears aplastic .
isolated dual lad , in absence of significant stenosis , has usually little clinical relevance .
in fact despite its anomalous anatomy , functional status is often not compromised and does not occur flow reduction , except for rare conditions associated with its route , like ab extrinseco compression , inter - arteriosus course between aorta and pulmonary artery , intramyocardial tract , kinking , or pivot point .
we describe for the first time in the literature , a dual lad anomaly associated with lcx aplasia
it would be important to know this anatomical subset , not only for its peculiar anatomical features , but also , above all , for the implication in terms of any surgical or interventional coronary approach when it should prove necessary . | coronary artery anomalies are uncommon disorders . according to the literature , 1% of the general population
is affected by a coronary artery abnormality .
coronary artery anomalies are often not associated with clinical signs , symptoms , or complications ; nevertheless , they can be associated with congenital heart diseases and lead to sudden death . however , these anomalies are more often discovered as incidental findings at the time of coronary angiography or autopsy .
the clinical relevance of coronary artery anomalies is closely related to the functional ability to provide adequate blood supply to the myocardial tissue .
we describe a complex left coronary artery anomaly , not previously reported in medical literature , involving origin , course , and distribution of this vessel . | INTRODUCTION
Case Report
DISCUSSION
CONCLUSION |
PMC4987909 | functional genomics experiments are widely used to gain insights into biological processes . a typical experiment measures gene expression in a specific ( perturbed ) condition and a control from which a list of differentially expressed genes is calculated .
this list does not normally give much direct insight as the differentially expressed genes may represent a mix of different biochemical functions and categories .
however , if they are known to interact with each other in a pathway then this pathway is clearly affected .
a range of pathway databases exist ( 1 ) , but the most general ones are kyoto encyclopedia of genes and genomes ( kegg ) ( 2 ) and gene ontology ( go ) ( 3 ) .
a large number of tools are available to assess whether a gene list is associated with a pathway or not ( see ( 4 ) for a review ) .
they typically apply a statistical measure such as the fisher 's exact test to assess the significance of the gene overlap between the gene list and pathway . however , because the pathway databases are far from complete , many true associations will be missed .
furthermore , the fisher exact test generally overestimates the significance because it assumes that all genes are independent of each other , but this is not true as they interact with each other ( 5 ) .
the problem can be reduced with for instance the ease score ( 6 ) but still remains paramount . in summary ,
gene overlap methods produce high levels of false negatives and false positives , and there is a great need to improve this situation .
a solution has recently been proposed by network - based approaches such as nea ( 7 ) , enrichnet ( 8) , crosstalkz ( 9 ) and binox ( ogris et al . ,
these methods analyse enrichment of network links between gene sets rather than the gene overlap .
if employing a dense comprehensive network of functional gene associations such as funcoup ( 10,11 ) or string ( 12 ) , the relation between gene list and pathway can be analysed using a lot more data than are provided by the gene overlap .
using crosstalk analysis one can also detect significant depletion of crosstalk relative to what is expected , which is never possible with gene overlap analysis . however , network - based approaches face two main challenges : ( 1 ) which statistical model to assess significance , and ( 2 ) if employing iterative network rewiring to estimate a null model , how to avoid an excessive compute time ?
enrichnet solves the latter by random walk with restart instead of randomizing the whole network .
nea and crosstalkz both use the normal distribution as model , but this is often unsuitable and leads to a high false positive rate for nea and a high false negative rate for crosstalkz .
the binomial distribution , which is used by binox , is more suitable and gives much lower false positive and false negative rates ( ogris et al . , 2016 ,
the binox algorithm employs monte - carlo sampling of randomized networks ( while preserving topological properties ) to estimate parameters of a binomial distribution which is used to calculate the statistical significance of an observed crosstalk .
two gene sets are considered to have significant enrichment if the number of network connections is significantly higher in the real network than expected from the random model .
if the groups have fewer connections in the real network than expected from the random model , they have crosstalk depletion .
the earlier methods , crosstalkz and nea , need to generate and analyse hundreds of randomized networks to calculate the statistical significance of a crosstalk for every query , and this is too time - consuming to be done interactively in a web site . to make it possible to obtain fast network crosstalk results
, binox employs a very efficient statistical method based on pre - sampled randomized networks that are stored in a database , including information about up - to - date curated pathways . to use binox
, it is necessary to first obtain a large network and a set of pathways , and this can be a hurdle for some users . to make binox readily available to the public , we developed the pathwax ( pathway analysis with crosstalk ) web server .
it contains kegg pathway information and genome - wide association networks of 11 well - studied model organisms . to minimize compute time , we have pre - randomized the networks , which gives run times for single gene sets of half a minute up to a few minutes .
the pathwax web site thus provides interactive online network crosstalk based pathway annotation that has a high chance of discovering affected pathways .
the system relies on loading data dynamically allowing a multithreaded interplay between client and server modules .
the server is running python 2.7 cgi scripts optimized for fetching and serving data , while the client side ( the browser ) is used for integrating data and estimating the statistical significance of crosstalk .
the web service is based on javascript using the libraries jquery v2.1.4 and jstat for efficient data handling and calculation . for visualization ,
the platform is optimized for the chrome browser ; slightly more compute time should be expected using the browsers firefox , safari , edge or opera .
due to lack of support for some of the used javascript libraries , pathwax is not compatible with internet explorer .
the binox workflow was adapted for pathwax , where an annotation request is divided into four main stages , see figure 1 . during the first stage
, pathwax translates the query genes to internal ids using the funcoup web service , and a subnetwork including valid query genes and their adjacent network genes is returned to the client .
the second stage includes requesting relevant pathways , i.e. pathways having at least one connection to the query within the present subnetwork , as well as the total number of outgoing connections for the query gene set and each pathway .
after combining the pathway information with the subnetwork , it is possible to count the number of network connections k between the query gene set and each pathway . in the third step , the client requests for each query - pathway pair the average number of connections k in the randomized networks , which is used as an estimate of the expected connections within a randomized environment , e(k ) .
the pathwax client assumes the binomial distribution to employ alternative hypothesis testing to calculate the statistical significance of observing k. the binomial distribution for the alternative hypothesis depends on n , the maximum possible connections between gene set and pathway , and p , the probability of observing k. here p can be approximated by e(k)/n. finally , pathwax uses the benjamini - hochberg procedure to account for multiple testing and calculates corrected false discovery rate ( fdr ) values .
after the user submits an input query , a subnetwork containing all query genes and their neighbours are requested from the server .
a second request looks up all pathways sharing at least one gene with the subnetwork . in the final call
the browser gets the parameters of the randomized connections between the pathways and the query gene .
once the browser has obtained the subnetwork , pathways of interest and the randomized connection parameters , it calculates the crosstalk statistics and displays these in the browser . during each stage ,
pathwax requests additional information of gene ids , pathway names , pathway components , etc .
the additional information is rendered together with the estimated fdr in the final summary to give the user an overview of the pathway annotation .
pathwax incorporates 1930 pathways from the kegg database ( release 70.1 ) distributed among 11 model organisms ( see table 1 for details ) .
the funcoup database provides a genome - wide functional association network for each species ( link confidence score > 0.8 ) .
the networks are undirected , scale free and dense ; they were constructed by integrating nine different evidence types : mrna co - expression , phylogenetic profile similarity , protein
protein interaction , subcellular co - localization , co - microrna regulation , domain interaction , protein co - expression , shared tf binding and genetic interaction profile similarity . across all species networks ,
the densest network is arabidopsis with 154 neighbours on average for each gene whereas yeast is the sparsest with 45 links on average . using the binox algorithm ,
pathwax supports a variety of different identifier types including ensembl gene , protein as well as transcript ids , ncbi gene ids , refseq ids and uniprot ids .
network genes are defined as protein coding genes having at least one connection within the network .
the input of pathwax is a list of genes with identifier types as above , and the output is a table of significantly enriched or depleted kegg pathways , together with graphical displays of pathway categories and the network connections between query genes and each pathway as a matrix .
the gene list input size is limited to 400 as larger sizes compromises the rendering of the network connectivity matrix which shows graphically each query gene 's connectivity to each pathway and provides hyperlinks to view these links in the funcoup network .
we want to ensure that this key functionality of the web site works . to illustrate pathwax , we chose an example from an experiment with a well - known phenotype : the gene list lopez_mesothelioma_survival_worst_vs_best_up ( 13 ) from msigdb v3.0 collection ( 14 ) .
these 14 genes were expressed higher in the worst 25 survivors compared to the 25 best survivors , in a study of 99 pleural mesothelioma patients , and thus reflect processes that make this cancer more lethal .
pathwax identified 31 significant ( fdr 0.05 ) pathways of which only 15 had any gene overlap ( 1 or 2 genes ) with the query set , see figure 2 .
the largest category , with 12 pathways , is human diseases , and four of these are cancer pathways .
this particular form of cancer affects cells of the pleural mesothelium , the protective lining of the lungs and chest wall .
it is therefore not surprising that many of the significant pathways are related to cell adhesion , including the most significant ( focal adhesion ) and the third most significant ( leukocyte transendothelial migration ) .
the latter pathway received an fdr of 8.9e-9 , yet it would not be discoverable using traditional gene overlap methods as there is no overlap between it and the query gene list .
this pathway contains 117 genes ; 26 network links were observed between it and the query gene list , yet only 4.8 links are expected by chance .
it is of clinical interest that four pathways related to heart disease have significant crosstalk with this gene list , which manifests poor survival .
pathwax results for the 14 genes in the human gene set lopez_mesothelioma_survival_worst_vs_best_up in msigdb ( plxna3 , psrc1 , dlgap4 , hn1 , cdc25c , flnb , c20orf20 , ccnd1 , acot7 , flj20674 , tgfb1i1 , lox , ddah1 , cdc42ep3 ) .
the upper table and pie diagram summarize the results and visualize the distribution of pathway classes .
the lower table lists all enriched ( blue ) and depleted ( red ) pathways for the query that are significant for the chosen cutoff ( only top part shown ) .
the pathways may be restricted to a class by clicking on one in the upper table .
each gene is shown as a coloured box and mouseover shows its number of links to the pathway .
green boxes represent query genes linked to the pathway and purple boxes indicate genes which are part of the pathway .
in this paper we present pathwax , a new web server for pathway annotation based on network crosstalk . the statistical model used for calculating significance
this increases sensitivity , accuracy and speed , and allows to combine network crosstalk pathway analysis with interactive web usage .
compared to traditional gene overlap enrichment analysis , pathwax offers a substantial improvement in sensitivity and specificity .
for instance , applying gene overlap enrichment analysis using the ease score ( 6 ) , which is employed by the popular david web site ( 15 ) , to the example in figure 2 is only possible for three of the 31 pathways found by pathwax .
the other 28 pathways can not be tested because they have a gene overlap of less than two .
two is the minimum overlap to calculate the ease score , which performs the fisher exact test on the overlap minus 1 .
this is done in order to reduce the high false positive rate of the pure fisher exact test .
only about a third of all human genes are annotated in kegg , hence the chance of an overlap is small .
usually more than 90% of all pathway gene set pairs overlap by less than two genes , and can therefore not be tested with the ease score . in the example in figure 2 , the three pathways that can be tested with ease had an overlap of exactly two genes .
however , such a small overlap can often happen by chance , which would result in false positives .
pathwax can analyse both enriched and depleted network crosstalk . what does a significantly depleted crosstalk imply ?
technically it means that there are significantly fewer links than expected , and the implication is that there is statistical evidence that the gene set is not affected by a depleted pathway .
one should thus not misinterpret it as an indication that the pathway is turned off. in the example in figure 2 , the pathway oxidative phosphorylation was significantly depleted .
this makes sense because cancer cells are mostly performing anaerobic energy production by glycolysis instead of oxidative phosphorylation , which is the aerobic energy production mostly used in healthy cells .
we chose the funcoup network because of its comprehensiveness , which is paramount for crosstalk analysis .
although a variety of pathway databases exist , kegg has the advantage of well - defined and relatively distinct pathways , which gives results that are easy to interpret , and it has good coverage for the species that we support .
methodologically one could consider using gene expression values more beyond just to extracting a list of differentially expressed genes .
methods exist that use such values to weight the relations to pathways ( 17,18 ) .
however , most of the information is captured by the list of significant differentially expressed genes and not much can be gained from including less informative data . also , absolute gene expression levels are highly variable and it may be unwise to trust these too much . in practice ,
methods that use expression profiles are not as widely used as traditional gene overlap methods , possibly due to instability of the results and less interpretability . in a recent benchmark ,
another possibility would be to compare the pattern of crosstalk with the known wiring of genes within pathways to give higher weight to crosstalks with interacting genes
. this may be a way to better rank the pathways , but we believe that the current level of biological knowledge is too fragmentary to dismiss a crosstalk based on poor consistency with the annotated wiring of the pathway .
the web server is a virtual machine running scientific linux 6.7 with 2 gb ram and 2 intel xeon e5 - 2630v2 2.60 ghz cores . | pathway annotation of gene lists is often used to functionally analyse biomolecular data such as gene expression in order to establish which processes are activated in a given experiment .
databases such as kegg or go represent collections of how genes are known to be organized in pathways , and the challenge is to compare a given gene list with the known pathways such that all true relations are identified .
most tools apply statistical measures to the gene overlap between the gene list and pathway .
it is however problematic to avoid false negatives and false positives when only using the gene overlap .
the pathwax web server ( http://pathwax.sbc.su.se/ ) applies a different approach which is based on network crosstalk .
it uses the comprehensive network funcoup to analyse network crosstalk between a query gene list and kegg pathways .
pathwax runs the binox algorithm , which employs monte - carlo sampling of randomized networks and estimates a binomial distribution , for estimating the statistical significance of the crosstalk .
this results in substantially higher accuracy than gene overlap methods .
the system was optimized for speed and allows interactive web usage .
we illustrate the usage and output of pathwax . | INTRODUCTION
IMPLEMENTATION
DATA
USING PATHWAX
DISCUSSION
SERVER INFORMATION
FUNDING |
PMC4262851 | this informed the need to undertake a critical appraisal of the uptake of the scheme by these important stakeholders .
however of importance is the uptake of the scheme by the health care providers who are the main operators of the scheme . in the south - east ,
okaro et al study in 2010 reported that almost all the hospitals studied had registered with the scheme.4 result from the study of sanusi and awe in 2009 showed that about 83% of the respondents have accepted the programme.5 however , the olugbenga - bello study of 2008 reported only 0.3%.6 and this is in contradistinction to the rate of uptake of the scheme in lagos which increased from 4.5% in 2000 and 2003 to 13.6% in 2004 , 27.6% in 2005 and 31.6% in 2006.7 the federal mandate to the present nhis administration is to achieve population coverage of 30% by 2015.8 this is in contrast to france which operates a health insurance policy that is compulsory.9 ghana national health insurance act which was promulgated in 2003 with the aim of making health care available to all residents of ghana recorded an uptake of 62% according to the 2009 annual report of the nhis of ghana .
however , a decline of 34% in 2010 and 33% in 2011 were recorded in the respective annual nhis ghana reports.101112 this study assessed the level of uptake of nhis by private health care practitioners in a local government area of lagos state .
the objective was to assess the awareness , knowledge and uptake of nhis among private health care providers .
multistage random sampling was used to recruit 180 participants with a response rate of 88.9% which was made up of 111 doctors and 49 nurses ( 160 ) .
stratification of health facilities into large ( 12 ) , medium ( 127 ) and small ( 8) out of the 104 hospitals , 30 clinics and 26 maternity homes .
there were 12 big hospitals with an average of six healthcare providers ( doctors and nurses ) , 127 medium hospitals with average of two healthcare providers ( doctors and nurses ) and eight small hospitals with average of two health care providers ( only nurses ) .
+ ( 127 2 ) + ( 8 2 ) = 342 . proportionate allocation of sample to determine number of questionnaires for each stratum .
this was calculated thus : the estimated health care providers in all the health facilities were 342 . for the large health facility ,
the same procedure was carried out to obtain 119 for medium , and small gave 7 health facilities .
selection of health care facilities : all the large health facilities ( 12 ) were included in the study while 64 and 4 were randomly selected from medium and small health facilities respectively using simple random sampling by balloting .
selection of participants : simple random sampling was used to select eligible respondents using balloting until the desired numbers were obtained .
overall knowledge of the study participants about nhis was assessed using the 31 responses in the knowledge section of the questionnaire . after assessing normality to the score using histogram ,
the composite score was dichotomised using mean as a cut - off value so that higher value coded as 1 showing higher overall knowledge of nhis .
a score of one was given to correct responses , zero being used for incorrect / do not know responses .
based on the mean score of the composite variable ( mean = 19.5 ) , the responses were categorised into good ( score above mean value ) and poor ( score below mean value ) knowledge of nhis .
data was analysed using microsoft excel and spss , and presented in tabular form , level of significance of results was tested using chi - square , with p - value set at 0.05 .
ethical clearance was obtained from the ethics and research committee of the lagos university teaching hospital .
stratification of health facilities into large ( 12 ) , medium ( 127 ) and small ( 8) out of the 104 hospitals , 30 clinics and 26 maternity homes .
there were 12 big hospitals with an average of six healthcare providers ( doctors and nurses ) , 127 medium hospitals with average of two healthcare providers ( doctors and nurses ) and eight small hospitals with average of two health care providers ( only nurses ) .
total healthcare providers was ( 6 12 ) + ( 127 2 ) + ( 8 2 ) = 342 .
this was calculated thus : the estimated health care providers in all the health facilities were 342 . for the large health facility ,
the same procedure was carried out to obtain 119 for medium , and small gave 7 health facilities .
selection of health care facilities : all the large health facilities ( 12 ) were included in the study while 64 and 4 were randomly selected from medium and small health facilities respectively using simple random sampling by balloting .
selection of participants : simple random sampling was used to select eligible respondents using balloting until the desired numbers were obtained .
overall knowledge of the study participants about nhis was assessed using the 31 responses in the knowledge section of the questionnaire . after assessing normality to the score using histogram ,
the composite score was dichotomised using mean as a cut - off value so that higher value coded as 1 showing higher overall knowledge of nhis .
a score of one was given to correct responses , zero being used for incorrect / do not know responses .
based on the mean score of the composite variable ( mean = 19.5 ) , the responses were categorised into good ( score above mean value ) and poor ( score below mean value ) knowledge of nhis .
data was analysed using microsoft excel and spss , and presented in tabular form , level of significance of results was tested using chi - square , with p - value set at 0.05 .
ethical clearance was obtained from the ethics and research committee of the lagos university teaching hospital .
up to 180 private health care practitioners were recruited into the study with a response rate of 88.9% .
this predominantly christian ( 135 , 84.4% ) , married ( 132 , 82.5% ) respondents had a mean age of 42.55 10.6 years .
there were more males ( 91 , 56.9% ) than females and more doctors ( 111 , 69.4% ) than nurses [ table 1 ] .
socio - demographic characteristics of respondents almost all the respondents ( 97.5% ) 156 had heard of nhis , mainly through seminars and conferences ( 58.1% ) 93 ; news papers ( 45.6% ) 73 ; friends and colleagues ( 40.6% ) 65 and television and radio ( 35% ) 56 . up to 83.8% ( 134 ) cited funding , 60.0 % ( 96 ) insufficient health facilities , 48.8% ( 78 ) inadequate health manpower as the key problems meant to be addressed by the nhis . up to 95.6% ( 153 )
correctly indicated access to good healthcare , efficiency in healthcare services ( 68.8% ) 110 and high quality of healthcare ( 62.5% ) 100 , as objectives of nhis .
more than half of the respondents 65.0% ( 104 ) and 61.9% ( 99 ) associated pooling of resources and sharing of risk with concepts of nhis , respectively , 14.4% ( 23 ) with paying of tax and 30.6% ( 49 ) had no clue .
as pertained to payment strategy , 68.1% ( 109 ) identified capitation , 23.8% ( 38 ) fee for service and 22.5% ( 36 ) case payment as provider payment mechanism .
the overall knowledge was good as 68.8% of the respondents had good knowledge [ table 2 ] . a large proportion ( 74.4% )
119 believed that nhis is the best healthcare financing option , 84.4% ( 135 ) agreed that healthcare services are inadequate in nigeria . up to 57 ( 35.6% ) expressed dissatisfaction with level of nhis publicity [ table 3 ]
awareness and knowledge of nhis among respondents attitudes toward nhis among the respondents almost two - third of the health facilities 97 ( 60.6% ) had been registered with nhis . of the 63 ( 39.4% ) that did not register , 9 ( 14.3% ) ; 8 ( 12.7% ) 14 ( 22.2% ) cited capitation not understood ; capitation not adequate and insufficient clients as reasons for not registering respectively .
almost one third 45 ( 28.1% ) of the respondents were satisfied with the scheme .
fifty - seven ( 35.6% ) accepted the operation of the scheme of which 16(28.1% ) had regrets of accepting the nhis .
reasons for regrets were making loses in the scheme 8(50.0% ) and late payment by hmo13 ( 81.3% ) [ table 4 ] .
uptake of nhis among respondents there was a statistically significant association between awareness , level of education , knowledge of nhis , attitude and registration , p - value < 0.05 [ table 5 ] .
the sustainability and viability of a country 's economic and social growth depend largely on vibrant healthcare sector of that nation , hence nigerian national health insurance scheme ( nhis ) was launched with the major aim of improving accessibility and equity in healthcare delivery .
this study revealed that 33.1% of respondents fell within the age bracket of 30 - 39 years .
this implies that majority of respondents were within the ( active age ) working class of the population , relating closely with the 30 - 39 years ( 54% ) obtained in enugu study.4 the mean age of the respondents was 42.6 10.6 years in line with the study subjects with active age group in the middle range.13 the high percentage of males ( 56.9% ) in the study reflects the higher proportion of males in the parent population of study subjects ( 50.3% ) , as obtained by the 2005 national population census and the fact that men have traditionally constituted the bulk of the workforce , further buttressed by the 51.6% and 67.6% found in both oyo and enugu studies respectively.5414 the above assertion is , however , not tantamount to 60.4% of respondents being females reported in the minna study .
an observation that can be attributed to 74.1% of respondents being nurses and/or midwives , a profession generally skewed towards the female gender.3 the married respondents of 82.5% obtained in this study is high , probably because the age bracket of the study is in reproductive age compared to 75.7% of enugu study .
the other demographic features of respondents also echo those of the parent population , with most ( 68.1% ) being yoruba by tribe and reverse for christians ( 84.4%).14 the study revealed that 97.5% of the respondents had heard of the national health insurance scheme .
this is slightly close to a similar study in enugu state carried out in 2009 by okaro which showed an awareness level of 100%.4 this is as a result of the fact that the establishment of the scheme is almost running to 7 years now .
hence , awareness did translate to good uptake as many respondent 97 ( 60.6% ) had registered with the scheme [ table 4 ] .
the main sources of information were friends and colleagues 40.6% , radio / tv 35.0% , newspaper 45.6% , seminars and conferences 58.1% .
short intensive course of study ( seminars and conferences ) as the principal source of information about health insurance in this study is in contrast to the niger and lagos studies with 11.6% and 13.5% , respectively.1516 respondents in this study exhibited a high level of knowledge of nhis .
the knowledge of provider payment mechanism and capitation was higher ( 68.1% ) than the studies in niger and osun states with 13.7% and 26.5% , respectively .
the overall knowledge of the respondents in this study was high as demonstrated by the grading using the mean knowledge of 19.5 4.6 : good 110 ( 68.8% ) and poor 50(31.3% ) [ table 2 ] .
the increasing trend of awareness to health insurance from the studies may be attributed to increased publicity of the nhis following its formal launching in 2000 and commencement of operation in 2001 .
six out of 10 ( 60.6% ) of the respondents in this study had registered with the scheme .
this is slightly lower than a similar study by sanusi and awe in 2009 in oyo state , where about 83% of the respondents were registered .
just about half ( 53.4% ) accepted the operation of the scheme , and reasons for acceptance of the operation ranged from ; reasonable offer ( 13.85 ) , making of profit ( 13.87% ) and not to be left out of the scheme ( 16.95% ) .
however , as high as 57.2% expressed their regrets of accepting the scheme , major reasons for regret bordered on finance ; in terms of failure of payment by hmos ( 59.4% ) and making losses in the scheme ( 30.6% ) .
the study revealed that private healthcare providers in the local government area were aware of the existence of nhis in nigeria .
there was good demonstration of adequate knowledge with regards to principles of operation of health insurance scheme . however , more than half of the participants regretted keying into the scheme mainly due to late payment by hmos and running the scheme at a loss . | background : national health insurance scheme ( nhis ) is one of the health financing options adopted by nigeria for improved healthcare access especially to the low income earners .
one of the key operators of the scheme is the health care providers , thus their uptake of the scheme is fundamental to the survival of the scheme .
the study reviewed the uptake of the nhis by private health care providers in a local government area in lagos state.objective:to assess the uptake of the nhis by private healthcare practitioners.materials and methods : this descriptive cross - sectional study recruited 180 private healthcare providers selected by multistage sampling technique with a response rate of 88.9%.result : awareness , knowledge and uptake of nhis were 156 ( 97.5% ) , 110 ( 66.8% ) and 97 ( 60.6% ) , respectively .
half of the respondents 82 ( 51.3% ) were dissatisfied with the operations of the scheme .
major reasons were failure of entitlement payment by health maintenance organisations 13 ( 81.3% ) and their incurring losses in participating in the scheme 8(50% ) .
there was a significant association between awareness , level of education , knowledge of nhis and registration into scheme by the respondents p - value < 0.05.conclusions:awareness and knowledge of nhis were commendable among the private health care providers .
six out of 10 had registered with the nhis but half of the respondents 82 ( 51.3% ) were dissatisfied with the scheme and 83 ( 57.2% ) regretted participating in the scheme .
there is need to improve payment modalities and ensure strict adherence to laid down policies . | INTRODUCTION
MATERIALS AND METHODS
Stage 1
Stage II
Stage III
Stage IV
Scoring system
RESULTS
DISCUSSION
CONCLUSION AND RECOMMENDATION |
PMC2868190 | a metabolic network can be represented with a stoichiometric matrix n , where rows correspond to the m metabolites and columns to the n reactions . assuming that intracellular metabolites are at steady state , material balances can be formulated as follows :
( 1)nv=0 ,
where v = ( v1,v2, ,vn ) is the n - dimensional vector of flux through each reaction .
each feasible steady state is represented by a flux vector v. taking into account these mass balances and the irreversibility of certain reactions , the space of feasible steady state flux distributions , or flux space , can be defined as follows ( see glossary for words in italics ) :
( 2)p={vrn : nv=0 , dv0 } ,
where d is a diagonal n n - matrix with dii = 1 if the flux i is irreversible ( otherwise 0 ) . the concept of the flux space is the cornerstone of constraint - based modeling , an approach supported by the fact that cells are subject to governing constraints that limit their behavior [ 2 , 3 ] . in this context
, network - based pathways are used to investigate the modeled metabolism by the analysis of a finite set of relevant pathways , which ideally represents all of the metabolic states that a cell can show .
however , there is not a unique set of network - based pathways to be tagged as
relevant and different proposals have been applied with success : extreme currents , elementary modes , extreme pathways , and minimal generators .
there are three major properties that a set of network - based pathways can hold : ( p1 ) they can generate the flux space p ,
( p2 ) they can be the minimal set of vectors fulfilling the first property , and ( p3 ) they can be all the non - decomposable pathways in the network .
the fact that all of the network - based pathways elementary modes , extreme pathways , and so forth fulfill the first property but not the others has been a source of errors , imprecisin , and misunderstandings .
this paper discusses the relationship between the different network - based pathways from a theoretical point of view .
we will start defining four pathway concepts and then we will perform a comparison among them . finally , we will present some examples and outline the major conclusions .
the first attempts to systemically extract a set of pathways from a given metabolic network were based on the assumption that all of the fluxes were irreversible , or more precisely , that its dominant direction could be presumed .
convex algebra shows that in this case the flux space p is a pointed convex polyhedral cone in the positive orthant r+ , which can be generated by non - negative combination of certain vectors , its edges , or extreme rays .
these extreme rays were flux vectors , or pathways , with a remarkable property ( p1 ) : the extreme rays generate the flux space p ; that is , every flux distribution v in p can be represented as a non - negative combination of fluxes through these pathways ( ek denotes the extreme rays ) :
( 3)p=(v : v=kewkek , wk0 ) .
notice that , in general , a given v can not be uniquely decomposed into an activity pattern w , but a space of valid solutions exists [ 22 , 23 ] .
this is also true for the rest of generating sets that will be introduced in subsequent sections .
moreover , the set of extreme rays had two additional properties : ( p2 ) it was the smallest ( minimal ) generating set of p , and ( p3 ) the extreme rays were all the non - decomposable vectors in p , those that can not be decomposed in simpler vectors .
a non - decomposable vector is a minimal set of reactions that form a functional unit ; if any of its participant reactions is not carrying flux , the others can not operate alone .
these functional units are the simplest steady - state flux distributions that cells can show , and the rest of feasible states can be seen as the aggregated action of these units .
this property makes it possible to investigate the infinite behaviors that cells can show by inspection of the finite set of non - decomposable vectors .
moreover , a set of vectors holding the three properties simultaneously ( p1 , p2 , and p3 ) will not exist ; there will be sets fulfilling p1 and p2 , or p1 and p3 , but not p2 and p3 in a general case .
extreme currents are probably the first attempt to define a set of network - based pathways . their computation is based on splitting up each reversible reaction into two irreversible ones .
thus , if fluxes are reordered to separate the irreversible fluxes vi and the reversible ones vr , the flux space ( 2 ) is augmented ( n = [ ninr ] ) :
( 4)prc={vrn+r:(ninrnr)(vivrvr)=0,(vivrvr)0}.
the extreme rays of the cone prc are defined as the extreme currents of p. notice that prc is a pointed cone in the positive orthant r , so its extreme rays have all of the properties mentioned above ( p1p3 ) .
however , prc lives in a higher - dimensional vector space ( augmented in one dimension for each split reversible reaction ) and the extreme currents lose their properties when they are translated to the original vector - space .
in fact , it has been recently shown that the set of extreme currents ( ecs ) coincides with the set of elementary modes , which will be introduced below , when it is translated to the original vector - space when computing the ecs a set of r spurious cycles appear ( pathways formed by the forward and backward reaction of each reversible flux ) ; however , these pathways are not considered meaningful in most applications and they disappear when the ecs are expressed in the original vector - space r. the concept of elementary modes was introduced to extend the property of non - decomposability of the extreme rays ( p3 ) to networks with reversible fluxes [ 7 , 8 ] .
hence , a flux vector e is an elementary mode ( em ) if and only if there is no nonzero vector v p such that the support of v supp(v ) is a proper subset of the support of e supp(e ) . in other words
, e can not be decomposed as a positive combination of two simpler vectors v and v in p that contain zero elements wherever e does and include at least one additional zero component each .
this condition is the so - called nondecomposability , simplicity , or genetic independence .
thereby , the set of elementary modes ( ems ) is defined as the set of all the nondecomposable vectors in the flux space ( p3 ) .
this definition implies that the ems fulfills property p1 , as in ( 3 ) , but also a more restrictive condition due to c2 : each flux distribution can always be represented as a non - negative combination of elementary modes without cancelations :
( 5)p=(v : v=kewkek , wk0 ) without cancelations ( ) .
( * ) if the sum runs over two or more indices k , all of the ek have zero components wherever v has zero components and include at least one additional zero each .
that means that the elementary modes are all the simple states , or functional units , that a cell can show ( the non - decomposable vectors ) and the rest of feasible states can be seen as its strictly aggregated action , that is , its aggregated action without cancelations .
the no cancelation rule is relevant for several applications of network - based pathways .
the no cancelation rule is what makes it possible to investigate the infinite behaviors that cells can show by simply inspection of the finite set of elementary modes , because there is no possibility of cancelations of reversible fluxes .
this allows to answer many interesting questions in an easy manner ; consider , for example the following : which reactions are essential to produce the compound y ? those that participate in all of the elementary modes producing y. is there a route connecting the educt a with the product y ? only if there is an elementary mode connecting them .
which are the capabilities of the network if a reaction r is not carrying flux or has been knocked - out ?
the feasible states in these circumstances are only those that result from aggregating , with no cancelations , the elementary modes not involving r ( i.e. , the consequences of r not carrying flux can be directly predicted ignoring the elementary modes participated by r ) . which is the optimal yield to produce y from a ?
the ( stoichiometrically ) optimal pathway is the elementary mode consuming a and producing y with the best yield .
as we will see in subsequent sections ,
the main difference among network - based pathways is that all of them satisfy ( 3 ) , but only the elementary modes satisfy ( 5 ) , and this fact determines their practical applications .
we have seen that the elementary modes generate the flux space , as in ( 3 ) , but usually they are not the smallest set satisfying this condition because they have to fulfill the most exigent condition of ( 5 ) . which is then the minimal set of vectors that generates p by non - negative combination ?
the term minimal generating set ( mgs ) has been recently coined to refer to this set .
it was also shown how to obtain an mgs that is subset of ems . however , in general there is not a unique minimal generating set : different mgss may exist within the ems , and even vectors that are not ems can be part of an mgs
yet , the concept of the minimal generating set also arises from a different point of view .
it is well known that the elementary modes are not systemically independent because it is possible to represent some modes as non - negative combination of others .
clearly , dependent modes that are not necessary to fulfill ( 3 ) can be removed .
thus , any resultant irreducible subset of the elementary modes is a minimal generating set . in summary ,
a set of minimal generators fulfill properties p1 and p2 , whereas the elementary modes fulfill p1 and p3 .
the elementary modes include additional non - decomposable vectors to fulfill p3 , which are redundant in ( 3 ) but necessary in ( 5 ) .
the fact that an mgs does not fulfill ( 5 ) reduces its usability for analysis of the underlying metabolism .
remarkably , the questions mentioned in the previous section can not be easily addressed using the mgs because the cancelation of reversible fluxes hides simple pathways .
for example , the mgs has to be recalculated after a gene deletion , and similar difficulties arise in other applications .
the advantage of the mgss against the ems is its reduced size : considering the central carbon metabolism of e. coli , the computation of the ems returns more than 500 000 ems , whereas an mgs contains around 3000 mgs .
thereby , the mgs will be preferred in those applications that just require a set of vectors generating the fluxspace . for instance
, the mgs has been used to perform phenotype phase - plane analysis and it can be used to extract the minimal connections between extracellular compounds , information that can then be used to develop unstructured , kinetic models [ 2931 , 38 ] . as it happens with the extreme currents ,
extreme pathways are obtained in an augmented vector - space ; however , only the internal fluxes are decomposed in both forward and backward directions ( the exchange fluxes , those that connect internal and external metabolites with one - to - one correspondence , are kept as reversible ) .
hence , if fluxes are reordered to separate the irreversible internal fluxes vi , the reversible ones vr , and the exchange fluxes vb , as v = [ vi vb vr ] , the flux space ( 2 ) can be reformulated as follows ( where n = [ ni nb nr ] ) :
( 6)prc={vrn+r:(ninbnrnr)(vivbvrvr)=0,(vivbvrvr)0}.
in this augmented vector - space , and only there , the set of extreme pathways ( eps ) is a subset of the elementary modes that is systemically independent . the eps are still capable of generating p , as in ( 3 ) , because only dependent elementary modes are discarded .
however , the extreme pathways are not systemically independent in the original one ( and even the ecs , which are equivalent to the ems , are systemically independent in the augmented space where they are obtained ) .
therefore , they are not the irreducible subset of the elementary modes in the original vector - space ; that is , they are not the minimal generating set .
the extreme pathways fulfill property p1 , but not p2 nor p3 in the original vector - space . as it happens with the mgs , the fact that the eps does not fulfill ( 5 ) reduces its usability in certain applications .
their advantage with respect to the ems is its smaller size , but it must be kept in mind that , in general , the mgs will be smaller than the eps ( and never larger ) .
example : two different vector - spacesconsider the small network depicted in figure 2 , case 2a
. the three eps of this network represented in the augmented vector - space { v1 , v2 , v3 , v3 } are e1 = ( 1 0 1 0 ) , e2 = ( 0 1 0 1 ) , and e3 = ( 1 1 0 0 ) . these three vectors are systemically independent .
however , when translated to the original vector - space { v1 , v2 , v3 } , these vectors are e1 = ( 1 0 1 ) , e2 = ( 0 1 1 ) and e3 = ( 1 1 0 ) , which are not longer systemically independent , since e1 = e2 + e3 .
the three eps of this network represented in the augmented vector - space { v1 , v2 , v3 , v3 } are e1 = ( 1 0 1 0 ) , e2 = ( 0 1 0 1 ) , and e3 = ( 1 1 0 0 ) .
these three vectors are systemically independent . however , when translated to the original vector - space { v1 , v2 , v3 } , these vectors are e1 = ( 1 0 1 ) , e2 = ( 0 1 1 ) and e3 = ( 1 1 0 ) , which are not longer systemically independent , since e1 = e2 + e3 . figure 2 also illustrates the systemic dependancy of the eps .
this section is devoted to the comparison of the network - based pathways described above : extreme currents , minimal generators , elementary modes and extreme pathways .
the case where all of the fluxes are irreversible will be introduced first to contextualize the problem ; then , the presence of reversible fluxes will be considered and the differences will become apparent ( see figure 2 ) .
reference vector - spacehereinafter we consider the original vector - space as the reference one : all of the generating sets will be expressed as elements of the vector - space r where each flux corresponds to an axis .
we choose r because it is the original space of the fluxes that connect the metabolites of the network , and thus it is the meaningful one .
for instance , in the previous example the eps expressed in the augmented vector - space were unable to capture the fact that pathway e1 can be seen as a combination of e2 and e3 ( e1 = e2 + e3 ) .
notice also that the relevant difference between equations ( 3 ) and ( 5 ) , which depends on the cancelation of reversible fluxes , can not be easily observed in the augmented vector - spaces . since ecs and
eps are computed in augmented vector - spaces , once obtained , they have to be translated to r , simply merging again the decomposed reversible fluxes .
this process also removes the spurious cycles , those pathways formed only by the forward and backward reaction of each reversible flux and appearing as eps and ecs in the augmented vector - spaces .
hereinafter we consider the original vector - space as the reference one : all of the generating sets will be expressed as elements of the vector - space r where each flux corresponds to an axis .
we choose r because it is the original space of the fluxes that connect the metabolites of the network , and thus it is the meaningful one .
for instance , in the previous example the eps expressed in the augmented vector - space were unable to capture the fact that pathway e1 can be seen as a combination of e2 and e3 ( e1 = e2 + e3 ) .
notice also that the relevant difference between equations ( 3 ) and ( 5 ) , which depends on the cancelation of reversible fluxes , can not be easily observed in the augmented vector - spaces . since ecs and
eps are computed in augmented vector - spaces , once obtained , they have to be translated to r , simply merging again the decomposed reversible fluxes .
this process also removes the spurious cycles , those pathways formed only by the forward and backward reaction of each reversible flux and appearing as eps and ecs in the augmented vector - spaces . as explained in a previous section , when all of the reactions are irreversible
, the flux space p is a convex cone that satisfies two conditions : ( a ) it is in the positive orthant r and ( b ) it is a pointed cone .
condition ( b ) implies that p can be generated by non - negative combination of its extreme rays ( 3 ) ( more details below ) .
in fact , the extreme rays always belong to every generating set because by definition they can not be generated by non - negative combination of other vectors within the cone ( see glossary ) .
thus , if the extreme rays are able to generate the cone , as it happens in this case , they are necessarily the minimal generating set . on the other hand ,
the extreme rays are always non - decomposable vectors of p ( since they can not be generated by non - negative combination ) .
moreover , condition ( a ) implies that the intersections of the cone with the ( positive or negative ) axis of the vector - space , which are potential non - decomposable vectors , can not be interior points of p. thus , the extreme rays will be all the non - decomposable vectors in p. these two conditions imply that in this case the extreme rays are not only the minimal generating set of the flux space ( p1 and p2 ) , but also the set of all non - decomposable vectors ( p3 ) . since the ecs and the eps are the extreme rays of two cones defined in augmented vector - spaces where the reversible fluxes are decomposed , it is obvious that , if there are no reactions to be decomposed , the ecs and eps are the extreme rays of the original cone p. therefore the following is maintained .
rule 1if all fluxes are irreversible , all the generating sets are equivalent , ems = ecs = eps = mgs , and coincide with the extreme rays of the flux space p.
if all fluxes are irreversible , all the generating sets are equivalent , ems = ecs = eps = mgs , and coincide with the extreme rays of the flux space p. now we consider the situation where certain fluxes are reversible .
the flux space p is still a convex cone , but it is not necessarily in the positive orthant r and it can be non - pointed .
if one or more reversible reactions are effectively reversible that is , both forward and backward directions can be realized by flux distributions the cone will not be in the positive orthant ( otherwise p would remain a pointed one in the positive orthant as in case 1 ) . then , two situations are possible : case 2a , the cone is pointed , and case 2b , it is not .
consider the lineality space of p , defined as lin.space(p ) : = { x r | a x = 0}. it represents the linear subspace contained in the cone and allows to characterize the cone as follows : p is pointed if lin.space ( p ) = { 0 } , otherwise non - pointed .
hence , p will be a non - pointed cone if a vector x and its opposite x exist in p. these vectors would involve only reversible fluxes and represent reversible vectors that can operate in both directions .
thus p is non - pointed cone if and only if it contains a reversible vector .
it is also possible to check whether a cone is pointed inspecting k , the kernel of n , arranged in a suitable way ( see for details ) .
the more important consequence of this classification is the following : a pointed cone p can be generated by non - negative combination of its extreme rays , but this no longer true for a non - pointed one .
a non - pointed cone still can be generated by non - negative combination , but a unique mgs will not exist . if there are reversible fluxes but not a reversible vector , the flux - space p is still a pointed cone and it can be generated by its extreme rays . as explained above , if the extreme rays generate the cone , they are necessarily the minimal generating set because they belong to every generating set by definition .
rule 2if the flux space p does not contain a reversible vector , a unique mgs exists and it coincides with the extreme rays of p.
if the flux space p does not contain a reversible vector , a unique mgs exists and it coincides with the extreme rays of p. however , if there are reversible fluxes , and they are effectively used in both directions , the cone is not restricted to the positive orthant r. this implies that the intersections of vector - space axis with the cone will be non - decomposable vectors of p. that is , there are non - decomposable vectors in p that are not extreme rays .
the ems sill contains the extreme rays , which are always non - decomposable , but could also contain other non - decomposable vectors .
notice that these extra ems are necessary to generate the flux space p without cancelations ( 5 ) , but can be redundant to fulfill ( 3 ) .
rule 3the ems ( ecs ) is always a superset of the extreme rays of the flux space p. if there are reversible fluxes , more ems than extreme rays may exist .
the ems ( ecs ) is always a superset of the extreme rays of the flux space p. if there are reversible fluxes , more ems than extreme rays may exist . by rules 2 and 3 it follows that , if the flux space p does not contain a reversible vector , the mgs is a subset of the ems .
moreover , those ems not belonging to the mgs will be systemically dependent and the mgs will be the unique irreducible subset of the ems .
rule 4if the flux space p does not contain a reversible vector , the unique mgs is the irreducible subset of the ems .
it can be extracted from the ems selecting the systemically independent vectors ( see the appendix ) .
if the flux space p does not contain a reversible vector , the unique mgs is the irreducible subset of the ems .
it can be extracted from the ems selecting the systemically independent vectors ( see the appendix ) .
this property was incorrectly assigned to the extreme pathways in the past , but these are systemically independent only in an augmented vector - space and not in the original one ( see example below ) .
the eps are the extreme rays of the cone obtained when the internal , reversible reactions are split , whereas the ems ( ecs ) are the extreme rays of the cone obtained when all of the reversible reactions are split .
rule 5if the flux space p does not contain a reversible vector , the eps can be a subset of the ems , but in general it is not the mgs .
that is , ems ( ecs ) eps mgs , and the following two particular cases exist.if all exchange fluxes are irreversible , ems ( ecs ) = eps.if all internal fluxes are irreversible , eps = mgs.the two rules can be rephrased as followseps can be a proper subset of the ems there are reversible exchange fluxes.mgs can be a proper subset of the eps there are reversible internal fluxes .
if the flux space p does not contain a reversible vector , the eps can be a subset of the ems , but in general it is not the mgs .
that is , ems ( ecs ) eps mgs , and the following two particular cases exist.if all exchange fluxes are irreversible , ems ( ecs ) = eps.if all internal fluxes are irreversible , eps = mgs.the two rules can be rephrased as followseps can be a proper subset of the ems there are reversible exchange fluxes.mgs can be a proper subset of the eps there are reversible internal fluxes .
eps can be a proper subset of the ems there are reversible exchange fluxes .
mgs can be a proper subset of the eps there are reversible internal fluxes .
proof outline(a ) if all of the reversible fluxes are internal , the eps and the ecs ( ems ) are the extreme rays of the same cone .
( b ) if all of the internal fluxes are irreversible , the eps are the extreme rays of the original cone , which coincide with the extreme rays due to rule 2 .
( a ) if all of the reversible fluxes are internal , the eps and the ecs ( ems ) are the extreme rays of the same cone .
( b ) if all of the internal fluxes are irreversible , the eps are the extreme rays of the original cone , which coincide with the extreme rays due to rule 2 . if the reversible fluxes form a reversible vector , the convex cone pr is non - pointed .
a non - pointed cone can be represented as pr = h + q , where h is the linear space lin.space(pr ) , and q is a pointed subcone , with q h ( h denotes the orthogonal complement of h ) . in fact
, this is the general representation of a convex polyhedral cone and cases 1 and 2a are particular cases where h = { 0}. thus , a non - pointed cone can be generated as follows :
( 7)pr={v : v=knfkfk+jnbjxj , k0 } ,
where fk are the
irreversible generating vectors , for which its opposites are not contained in pr , and xj are the reversible ones , for which its opposite xj is also contained in pr .
vectors xj must form a base of h , whereas vectors fk generate the sub - cone q. notice that pr can still be generated by non - negative combination , as in ( 3 ) , using fk , xj , and xj as generating vectors .
unfortunately , there is a price to pay for the cone being non - pointed : the set of minimal generating vectors is not unique anymore .
in fact , a minimal generating set of pr is obtained choosing an arbitrary base { xj } of h , and taking one arbitrary ray fk from each minimal proper face of the cone . when the cone is pointed , there are no vectors { xj } and the minimal proper faces are the extreme rays , so they are uniquely defined
. the extreme rays of pr will be present in any generating set ems , eps , or an mgs because they can not be represented as non - negative combination of other vectors in pr .
however , they are insufficient to generate a non - pointed cone , they could even not exist ( e.g. , if all fluxes are reversible , the cone is an n - dimensional vector - space generated only by vectors xj and xj ) .
additional vectors { xj } and { fk } must be combined with the extreme rays to form an mgs , but the choice is not unique .
rule 6if the flux space pr contains a reversible vector , its extreme rays are not a complete generating set and there is not a unique mgs .
if the flux space pr contains a reversible vector , its extreme rays are not a complete generating set and there is not a unique mgs .
however , it is still possible to define a mgs containing only non - decomposable vectors , and thus being a subset of the ems .
this kind of mgs can be obtained with a lexico - smallest representation or extracted from the set of ems , as explained in the appendix .
rule 7
if the flux space
pr contains a reversible vector , an irreducible subset of the ems constitutes an mgs formed only with non - decomposable vectors .
if the flux space
pr contains a reversible vector , an irreducible subset of the ems constitutes an mgs formed only with non - decomposable vectors .
notice that other mgss exist . indeed , even more than one mgs formed with different non
- decomposable vectors may exist , since there is not necessarily a unique irreducible subset of ems .
both situations will be illustrated in subsequent examples . with respect to the eps , rule 5
moreover , since a reversible vector will be ( a ) always participated by at least one internal flux a reversible vector only with external fluxes has little sense and ( b ) in most cases also participated by external ones ( except that if all of the reversible vectors are futile cycles ) , a common situation arises where ems ( ecs ) eps an mgs .
rule 8
if the flux - space
pr
contains a reversible vector , the eps can be a subset of the ems , but in general the eps is not an mgs .
the most common case will be ems ( ecs ) eps an mgs .
if the flux - space
pr
contains a reversible vector , the eps can be a subset of the ems , but in general the eps is not an mgs
. the most common case will be ems ( ecs ) eps an mgs .
the first examples ( 1 to 5 ) use a simple network taken from papin et al . .
the network has 6 reactions3 internal and 3 exchange and three metabolites , so it has 3 degrees of freedom .
if all of the reactions were reversible , the kernel of n would provide a basis of the flux space formed by three reversible vectors .
herein we consider five cases where different reactions are irreversible ( results are depicted in figure 4 ) .
example 1 in the first example all fluxes are assumed to be irreversible ( case 1 ) . in this case , the flux space is a pointed cone in r and all of the network - based pathways
ecs , ems , eps , and mgs are equivalent .
in the first example
all fluxes are assumed to be irreversible ( case 1 ) . in this case , the flux space is a pointed cone in r and all of the network - based pathways
this example corresponds to case 2a ( the flux space is a pointed cone not in r ) . in this case
the ems can be a superset of the mgs , as indeed happens in this example : em4 is systemically dependent ( em4 = mg1 + mg2 ) , so it is an em but not a mg .
on the other hand , the eps is equal to the mgs because the internal fluxes are all irreversible .
em4 is not an ep because the reversible flux being cancelled in mg1 + mg2 is an exchange , so em4 is systemically dependent in the vector - space where eps are computed .
this example corresponds to case 2a ( the flux space is a pointed cone not in r ) . in this case
the ems can be a superset of the mgs , as indeed happens in this example : em4 is systemically dependent ( em4 = mg1 + mg2 ) , so it is an em but not a mg .
on the other hand , the eps is equal to the mgs because the internal fluxes are all irreversible .
em4 is not an ep because the reversible flux being cancelled in mg1 + mg2 is an exchange , so em4 is systemically dependent in the vector - space where eps are computed .
example 3 in this third example the exchange flux v4 and the internal flux v2 are reversible .
em5 is neither an ep nor an mg ( em5 = mg1 + mg2 ) .
em4 is not a mg ( em4 = mg3 + mg2 ) , but it is an ep ; one of the fluxes cancelled in mg3 + mg2 is an internal flux , so this cancelation can not be done in the augmented vector - space where the eps are computed .
in this third example
em5 is neither an ep nor an mg ( em5 = mg1 + mg2 ) .
em4 is not a mg ( em4 = mg3 + mg2 ) , but it is an ep ; one of the fluxes cancelled in mg3 + mg2 is an internal flux , so this cancelation can not be done in the augmented vector - space where the eps are computed .
example 4 in this example only two internal fluxes , v1 and v3 , are reversible .
again , the ems is a superset of the mgs : em4 is not an mg because it is systemically dependent ( em4 = mg3 + mg2 ) . on the other hand , as all of the reversible fluxes are internal , the eps and the ems are necessarily equivalent
.
in this example only two internal fluxes , v1 and v3 , are reversible .
again , the ems is a superset of the mgs : em4 is not an mg because it is systemically dependent ( em4 = mg3 + mg2 ) . on the other hand , as all of the reversible fluxes are internal , the eps and the ems are necessarily equivalent .
this corresponds to case 2b , where the flux space is a non - pointed cone .
the two vectors that form the reversible vector are extreme rays in this example . to form an mgs they need to be combined with 2 other vectors , but
for instance , 2 subsets of ems are minimal generating sets , mgs1 and mgs2 .
this corresponds to case 2b , where the flux space is a non - pointed cone .
to form an mgs they need to be combined with 2 other vectors , but the choice is not unique .
for instance , 2 subsets of ems are minimal generating sets , mgs1 and mgs2 .
example 6klamt and stelling use a simple example , referred to as n2 in their article , to investigate the relationship between the ems and the eps .
after computing the ems , the eps , and the mgs , it turns out that there are 8 ems and 5 eps ( the extra em9/ep6 in disappears in the original vector - space because it is a spurious cycle caused by decomposing the reversible fluxes ) .
yet , the mgs contains only 4 vectors , indicating that there is an ep that is not systemically independent : it can be checked by simple inspection that ep1 = ep2 + ep4 ( when they are represented in the original vector - space ) .
klamt and stelling use a simple example , referred to as n2 in their article , to investigate the relationship between the ems and the eps .
after computing the ems , the eps , and the mgs , it turns out that there are 8 ems and 5 eps ( the extra em9/ep6 in disappears in the original vector - space because it is a spurious cycle caused by decomposing the reversible fluxes ) . yet , the mgs contains only 4 vectors , indicating that there is an ep that is not systemically independent : it can be checked by simple inspection that ep1 = ep2 + ep4 ( when they are represented in the original vector - space ) .
example 7another example to be analyzed is the small network used by schilling et al . .
again , the eps are not systemically independent when translated to the original vector - space ( ep2 = ep3 + ep5 ) and 4 vectors are sufficient to form an mgs .
it turns out that the mgs is not unique because there is a reversible vector in the flux - space ( in fact , the reversible vector defines two eps : ep3 and ep4 use the same reactions but in opposite directions ) .
another example to be analyzed is the small network used by schilling et al . .
again , the eps are not systemically independent when translated to the original vector - space ( ep2 = ep3 + ep5 ) and 4 vectors are sufficient to form an mgs .
it turns out that the mgs is not unique because there is a reversible vector in the flux - space ( in fact , the reversible vector defines two eps : ep3 and ep4 use the same reactions but in opposite directions ) .
the network has 24 reactions ( 9 reversible ) and 18 internal metabolites , so it has 6 degrees of freedom .
there are 18 ems and 8 eps , but only 6 vectors form the unique mgs ( see supplementary file , figure 2 in supplementry material available online at doi:10.1155/2010/753904 ) .
the metabolic pathways that correspond to the mgs are given in the supplementary file , figure 1 .
the network has 24 reactions ( 9 reversible ) and 18 internal metabolites , so it has 6 degrees of freedom .
there are 18 ems and 8 eps , but only 6 vectors form the unique mgs ( see supplementary file , figure 2 in supplementry material available online at doi:10.1155/2010/753904 ) .
the metabolic pathways that correspond to the mgs are given in the supplementary file , figure 1 .
the purpose of network - based pathways analysis is to identify a finite set of systemic pathways in a metabolic network , and then use these pathways to study the cell metabolism . in this paper four similar definitions of network - based pathways
we have seen that all of the flux states of a given metabolic network can be represented as an aggregation of flux through its elementary modes , which are all the simple , or non - decomposable , pathways in the network .
nevertheless , the set of elementary modes is not the smallest set of pathways fulfilling this property ; this role corresponds to the so - called minimal generating sets . in certain cases
there is a unique minimal generating set , but in general there are several alternatives .
interestingly , the set of elementary modes can be reduced by eliminating modes that are systemically dependent , resulting in a minimal generating set formed only with elementary modes .
it has been also highlighted that , contrarily to what has sometimes been stated , the extreme pathways are not the minimal generating set , because they are usually systemically dependent in the original vector - space .
the minimal generating sets can be of use in applications where a set of generating vectors are required . in these cases
they will be preferred due to its reduced size and because their computation is more efficient .
for instance , minimal generators are suitable for extracting the fundamental connections between extracellular compounds , information that can be used to develop unstructured , kinetic models [ 2931 , 38 ] .
the fact that the set of elementary modes comprises all of the simple pathways in the network its functional states makes it possible to investigate the infinite behaviors that cells can show by simply inspecting them .
this makes it easy to answer several questions : which reactions are essential to produce a certain compound , which will be the capabilities of the network if a reaction is knock - out , and so forth .
answering these questions using the minimal generators or the extreme pathways may be difficult because one has to take into account the possible cancelations of reversible fluxes .
significant efforts are being done to improve network - based pathways analysis , particularly in the context of genome - scale metabolic network , where their more critical limitation appears : when the number of reactions in the network grows , the number of pathways dramatically increases , reducing understandability and even becoming not computable [ 5 , 6 ] .
recent works have improved the computation algorithms [ 41 , 42 ] , and proposed methods to get particular subsets of pathways and to decompose large networks in modules [ 44 , 45 ] .
elementary flux patterns , which explicitly takes into account possible steady - states fluxes through a genome - scale network when analyzing pathways through a subsystem , thus allowing the application of many ( not all ) elementary - mode - based tools to genome - scale networks .
have used monte carlo sampling in conjunction with principal component analysis to obtain a low - dimensional set of pathways generating the flux space of genome - scale networks .
most applications of network - based pathway analysis are found not only in the context of microbial production [ 9 , 11 , 12 , 17 , 20 ] , but also in botany [ 48 , 49 ] or in biomedicine [ 50 , 51 ] .
the number of applications increases steadily , and we believe that this will continue in the foreseeable future .
flux distributionthe values of every metabolic flux of a given network at a particular ( steady ) state form a flux distribution .
the values of every metabolic flux of a given network at a particular ( steady ) state form a flux distribution .
flux spacethe space p that contains all of the feasible flux distributions of a given metabolic network is the flux space .
the space p that contains all of the feasible flux distributions of a given metabolic network is the flux space .
r is a convex cone if and only if any non - negative combination of elements from p remains in p. a convex cone p is polyhedral if , for some matrix a , p = { x r | a x 0}. a convex polyhedral cone p is the set of solutions of a homogeneous system of inequalities ( or the intersection of finitely many affine half - spaces ) .
a nonempty set of points p
r is a convex cone if and only if any non - negative combination of elements from p remains in p. a convex cone p is polyhedral if , for some matrix a , p = { x r | a x 0}. a convex polyhedral cone p is the set of solutions of a homogeneous system of inequalities ( or the intersection of finitely many affine half - spaces ) .
conefor brevity , we use the term cone to refer to a convex polyhedral cone .
for brevity
, we use the term cone to refer to a convex polyhedral cone .
nonnegative generationthe farkas theorem asserts that a convex cone is polyhedral if and only if it is finitely generated .
a cone is finitely generated if there exist a set of vectors g = { gi } that generate it by non - negative combination .
the farkas theorem asserts that a convex cone is polyhedral if and only if it is finitely generated .
a cone is finitely generated if there exist a set of vectors g = { gi } that generate it by non - negative combination .
generating setany set of vectors g in p that generates p by non - negative combination is a generating set of p. the ems , the ecs , the eps , and the mgss are generating sets of p.
any set of vectors g in p that generates p by non - negative combination is a generating set of p. the ems , the ecs , the eps , and the mgss are generating sets of p.
lineality spacelet p be a convex polyhedral cone , p = { x r | a x 0}. then , lin.space(p ) = { x r | a x = 0 } is called the lineality space of p. it is the t - dimensional linear subspace contained in the cone .
let p be a convex polyhedral cone , p = { x r | a x 0}. then , lin.space(p ) = { x r | a x = 0 } is called the lineality space of p. it is the t - dimensional linear subspace contained in the cone .
reversible vectorsthe lineality space , lin.space(p ) , contains the nonzero vectors r in p whose opposite r is also in p. these involve only reversible fluxes and represent flux distributions that can operate in both directions .
the lineality space , lin.space(p ) , contains the nonzero vectors r in p whose opposite r is also in p. these involve only reversible fluxes and represent flux distributions that can operate in both directions .
.
pointed conea convex polyhedral cone p is said to be pointed if lin.space(p ) = { 0}. in other words , a cone is non - pointed if it contains a reversible vector , and pointed otherwise .
a convex polyhedral cone p is said to be pointed if lin.space(p ) = { 0}. in other words , a cone is non - pointed if it contains a reversible vector , and pointed otherwise .
generation of a pointed conea pointed cone p can be generated by non - negative combination of its extreme rays , which is the unique , minimal generating set of p ( mgs ) .
a pointed cone p can be generated by non - negative combination of its extreme rays , which is the unique , minimal generating set of p ( mgs ) .
generation of a nonpointed conea non - pointed cone p can still be generated by convex combination of a set of vectors , but there is no longer a unique mgs .
a non - pointed cone p can still be generated by convex combination of a set of vectors , but there is no longer a unique mgs .
extreme rays or edgesa vector d is a ray of the convex polyhedral cone p if for all x p , x + d p for each 0 .
if a ray d can not be expressed as non - negative combination of other rays in p , it is an extreme ray . in metabolic pathway analysis ,
extreme rays are important because ( i ) if the cone is pointed , the extreme rays are the unique mgs . in the general case ,
the extreme rays ( ii ) belong to every generating set of p , and ( iii ) they are always non - decomposable .
a vector d is a ray of the convex polyhedral cone p if for all x p , x + d p for each 0 .
if a ray d can not be expressed as non - negative combination of other rays in p , it is an extreme ray . in metabolic pathway analysis ,
extreme rays are important because ( i ) if the cone is pointed , the extreme rays are the unique mgs . in the general case ,
the extreme rays ( ii ) belong to every generating set of p , and ( iii ) they are always non - decomposable .
nondecomposabilitygiven a cone p , a vector n p is non - decomposable if it can not be represented as a positive combination of two vectors v and v in p that contain zero elements wherever n does and include at least one additional zero each .
these vectors represent the simple states or functional units that a cell can show ; the rest of feasible states can be seen as its aggregated action without cancelations .
all the generating set may contain non - decomposable vectors , but only the ems is the set of all of the non - decomposable vectors in p.
given a cone p , a vector n p is non - decomposable if it can not be represented as a positive combination of two vectors v and v in p that contain zero elements wherever n does and include at least one additional zero each .
these vectors represent the simple states or functional units that a cell can show ; the rest of feasible states can be seen as its aggregated action without cancelations .
all the generating set may contain non - decomposable vectors , but only the ems is the set of all of the non - decomposable vectors in p.
systemic independencea set of vectors i are systemically independent if no vector in i can be represented as a non - negative combination of others .
the extreme rays are always a systemically independent set of vectors .
a set of vectors
i are systemically independent if no vector in i can be represented as a non - negative combination of others .
irreducible subsetgiven a generating set g of p that is not systemically independent , every smallest subset of g that generates g , and therefore p , is an irreducible subset of g. if the cone is pointed , there is a unique irreducible subset for every generating set and it coincides with the extreme rays of the cone , but if the cone is non - pointed , several irreducible subsets may exist .
given a generating set g of p that is not systemically independent
, every smallest subset of g that generates g , and therefore p , is an irreducible subset of g. if the cone is pointed , there is a unique irreducible subset for every generating set and it coincides with the extreme rays of the cone , but if the cone is non - pointed , several irreducible subsets may exist .
( flux ) vector - spacethe term ( flux ) vector - space refers to the space with the metabolic fluxes as axis .
the original flux vector - space has dimensions n ( n is number of reaction in the network ) , but some network - based pathways are computed in auxiliary vector - spaces of higher dimension .
the term ( flux ) vector - space refers to the space with the metabolic fluxes as axis .
the original flux vector - space has dimensions n ( n is number of reaction in the network ) , but some network - based pathways are computed in auxiliary vector - spaces of higher dimension .
the elementary modes can be computed with metatool and cellnetanalyzer , both running under matlab .
minimal generating sets can be obtained using sna , a software package running under mathematica , or using ccd as reported in .
in addition , we describe a simple method to get an mgs from the ems extracting an irreducible subset .
extracting an mgs from the emsthe procedure
can be outlined with the following pseudocode : for each elementary mode ei in e define a = [ m er ] if ( there is no w 0 | a w = e ) then : add ei to m endwhere e is the matrix formed with ems as columns , er is the submatrix of e with only columns after i , and m is the matrix collecting the mgs ( thus empty on first iteration ) .
the procedure can be outlined with the following pseudocode : for each elementary mode ei in e define a = [ m er ] if ( there is no w 0 | a w = e ) then : add ei to m endwhere e is the matrix formed with ems as columns , er is the submatrix of e with only columns after i , and m is the matrix collecting the mgs ( thus empty on first iteration ) .
for each elementary mode ei in e if ( there is no w 0 | a w = e ) then : add ei to m if the cone is pointed , the resultant set is the unique mgs ( and coincides with the extreme rays of the cone ) . | important efforts are being done to systematically identify the relevant pathways in a metabolic network .
unsurprisingly , there is not a unique set of network - based pathways to be tagged as relevant , and at least four related concepts have been proposed : extreme currents , elementary modes , extreme pathways , and minimal generators .
basically , there are two properties that these sets of pathways can hold : they can generate the flux space if every feasible flux distribution can be represented as a nonnegative combination of flux through them or they can comprise all the nondecomposable pathways in the network .
the four concepts fulfill the first property , but only the elementary modes fulfill the second one .
this subtle difference has been a source of errors and misunderstandings .
this paper attempts to clarify the intricate relationship between the network - based pathways performing a comparison among them . | 1. Introduction
2. Results and Discussion
3. Different Network-Based Pathways
4. Comparison among Network-Based Pathways
5. Conclusions
Glossary Box
Computation of Network-Based Pathways |
PMC3948956 | needle stick injuries ( nsi ) has always been one of the most important risk factor for healthcare workers ( hcws ) for transmission of various infections such as hepatitis b , hepatitis c and human immunodeficiency virus ( hiv).1 as per the 2008 - 2009 hiv estimates , there are an estimated 23.9 lakh people currently living with hiv / aids in india with an adult prevalence of 0.31% in 2009.2 variety of procedures like needle recapping , injuries sustained in the operating room , blood collection or intravenous line administration , suturing and checking blood sugar can lead to accidental nsi [ table 1].34 the probability of transmission varies depending on whether the exposure is with a hollow - bore needle or a solid needle due to higher fluid content and pathogen load .
although modern dentistry has been cited as the least hazardous of the all the occupations , risks like nsi still challenge the status of this occupation.5 compared to many other healthcare settings , dental professionals are at higher risk of acquiring infections due to the fact that dentists work in a limited - access and restricted - visibility field and frequently use sharp devices.6 various determinants of nsis every year , about 16 billion injections are administered in developing and transitional nations and approximately 3 million individuals are injured due to needle stick and sharp injuries.7 these types of blood - borne exposures can be career and life - ending .
there is gross under reporting of nsi and , therefore , the incidence of nsi is higher than the current estimates.8 according to recent estimates , approximately 10,00,000 hcws in us and 1,00,000 hcws in uk receive nsi from conventional needles and sharps every year.3 it was also found that the use of glass syringe was constantly associated with higher degree of unsafety.9 in developing countries like ghana , indonesia and uganda , 80 - 90% of patients who visited a health centre received one or more injections per visit.10 a positive correlation has been reported between the frequency of injections and the prevalence of blood - borne infections in the population .
another study reported a high incidence of needle stick and sharp injuries among hcws in jordan.11 in south africa , 91% of junior doctors reported sustaining a nsi in the previous year.12 results of another cross - sectional study conducted in iran among medical and dental students showed that 74.3% had experienced nsis , and the highest incidence among the dental students was seen in endodontics , surgery and periodontics departments.13 according to another study conducted in a dental institute in london , it was found that oral surgery clinics were the major source of reporting of nsis compared with other specialised dental clinics within the institute.14 in a country like india , in spite of large number of awareness programs , it is not possible to estimate the annual incidence of nsi in different occupations because of the scarcity of data.1 moreover , data regarding the prevalence of nsi in dental profession is almost non - existent in india as compared to that in other countries .
as many dental professionals and students are not aware of the preventive and immediate prophylactic measures to be taken in case of such happenings,1516 the present systematic review was conducted : reporting the knowledge and awareness of the dental health professionals ( students ) regarding transmission of infection through nsi.reporting about the various practices employed by dental students such as methods to dispose the needles after use.reporting on the post - exposure prophylaxis ( pep ) taken by them in case of any accidental nsi.comparing the level of awareness and injuries sustained among the undergraduate and postgraduate dental students .
reporting the knowledge and awareness of the dental health professionals ( students ) regarding transmission of infection through nsi . reporting about the various practices employed by dental students such as methods to dispose the needles after use . reporting on the post - exposure prophylaxis ( pep ) taken by them in case of any accidental nsi . comparing the level of awareness and injuries sustained among the undergraduate and postgraduate dental students .
the present paper deals with the systematic review on nsi , but the authors have not attempted to conduct a thorough meta - analysis . study selection was based on the following inclusion criteria : 1 ) studies conducted in india ; 2 ) subjects limited to dental hcws in hospitals and dental colleges ; 3 ) studies conducted on dental students ; 4 ) studies published in english language ; 5 ) studies evaluating the knowledge , awareness and practice regarding nsi as outcome measures ; and 6 ) observational studies .
the studies that were excluded from the present review were 1 ) studies not conducted in india ; 2 ) reviews ; 3 ) studies engaging medical hcws ; and 4 ) studies on all hcws that did not differentiate between different healthcare professionals ( dental , medical or any other ) . initial electronic search for nsi in hcws yielded 92 references and only four were retained .
full texts of all four articles were extracted by electronic and manual search from pgimer library and national library .
the present review was carried out based on the protocol and guidelines for preparation from a previous study.17 a comprehensive literature search of english articles using pubmed , medline , embase and cochrane library was carried out irrespective of the date of publication using the appropriate terms and key words .
various key words and their combination were used to build - up the search strategy [ table 2 ] .
various key words and their combinations used experts and authors were also contacted for obtaining missing or unclear data whenever deemed essential .
articles were manually retrieved from pgimer library , chandigarh and national medical library , new delhi .
two authors ( vk and rsg ) independently identified studies that were included in the present review .
initially , titles and abstracts of the records retrieved by the search were assessed in order to exclude those studies that were inappropriate . for the remaining studies ,
selected studies were screened using strobe checklist for observational studies.18 the strobe statement is a checklist of items that should be addressed in articles reporting on the three main study designs of analytical epidemiology : cohort , case - control and cross - sectional studies .
these items relate to the article 's title and abstract , the introduction , methods , results and discussion sections and other information .
following issues were included in the risk of bias or quality assessment in the present systematic review : ( 1 ) completeness of reporting information regarding nsis , ( 2 ) selective outcome reporting , ( 3 ) choice of outcome measures ( knowledge or awareness levels , practices adopted and pep measures ) , ( 4 ) study design and ( 5 ) conflict of interest in the conduct of the study .
an overall estimation of plausible risk of bias ( low , moderate or high ) was performed for each of the selected studies .
this review was done according to the guidelines set forth by preferred reporting items for systematic reviews and meta - analyses ( prisma).19 two of the authors ( ss and sg ) were given the responsibility of extracting data from the studies .
pre - specified data was extracted from each of the studies , including the study design , sample size , prevalence of nsis among the study subjects , awareness and knowledge regarding the nsis occurring at the work place and other study characteristics .
any kind of disagreement regarding article screening and extraction was sorted out by the other author .
the original search identified 92 potentially eligible citations , of which only four were finally included in the present review.20212223 on screening by strobe checklist , all the four studies fulfilled the requirements of reporting of observational studies .
the study population in all the four studies comprised of dental students enrolled in various dental colleges of india [ table 3 ] .
two of the studies were conducted in the state of maharashtra and one each was conducted in karnataka and andhra pradesh .
all the studies were cross - sectional in nature and used a closed- or open - ended questionnaire for gathering the relevant data on nsis from the subjects . various study characteristics on needle stick injuries included in the review knowledge and awareness level of dental students regarding nsi was the main outcome in the study conducted by rajiv saini,20 whereas guruprasad et al.,21 assessed the knowledge , attitude and practice regarding risk of human immunodeficiency virus ( hiv ) transmission through nsi among dental students .
hussain et al.,22 determined the prevalence and factors associated with sharp instrument injuries among different healthcare students ( dental , medical and nursing ) .
awareness and prevalence of accidental blood - borne exposures among undergraduate and postgraduate dental students was the main outcome in the study done by anjum et al.23 on an average , 89.23% of the students had correct knowledge about nsi and 91.55% of the students had adequate level of awareness regarding its management in one of the studies ( p < 0.05).20
figure 2 depicts the knowledge regarding transmission of infection through infected needle among students in different studies .
guruprasad et al.,21 reported in their study that 89% of the students were aware of the fact that the virus could be transmitted through infected needles and among the students who were unaware , 22.5% were third year students .
only 39.8% of the healthcare students could correctly define sharp instrument injury in the study conducted by hussain et al.,22 ( did not specify the field of healthcare for this particular question ) . according to another study
reports , 88% of the dental students were aware of the occupational blood - borne diseases ( p < 0.05);23 33% of the respondents were aware about universal precaution guidelines that was found to be statistically significant among postgraduate dental students ( p = 0.00 ) .
postgraduate students had more awareness as compared to undergraduate students.23 knowledge regarding transmission of infection through infected needle among dental students guruprasad et al.,21 reported on the practice of needle destruction after use and found that 44% of the students destroyed the needle using needle destroyer and 15% destroyed them in puncture - resistant container with disinfectant .
when enquiring about pep , 26% reported washing the site of injury with surgical spirit and the same number of students said that they promote active bleeding at the site of injury .
maximum numbers of students ( 89% ) in the study conducted by saini20 were aware of taking pep after an accidental nsi as compared to other studies [ figure 3 ] .
hussain et al.,22 reported 2.3% of the sharp instrument injuries occurred during administration of local anaesthetic injection ( p < 0.05 ) . in the study conducted by anjum et al.,23 majority of the accidents ( 56% )
were reported while performing injections and were found to be more among third year students ( p = 0.02 ) .
the results in the present systematic review have been prepared and discussed regarding nsi among dental students in india .
the present review shows that the knowledge and awareness level of dental students regarding nsi is adequate , which is evident from different studies , although there is considerable inadequacy and variation in the practice and management of nsi among dental students .
the knowledge and awareness level varies in different studies , which can be attributed to the difference in sample size and different study settings .
there was only one study that involved postgraduate dental students as compared to rest of the studies , which targeted only undergraduate students and interns.23 all the studies used a closed- or open - ended questionnaire for gathering information about nsi from the dental students . in comparison with different groups ,
it was reported in one of the studies that postgraduate students had good awareness , but alarmingly they also reported higher number of injuries.23 this could be due to the fact that postgraduate students have more clinical load as compared to the undergraduate students and thus are generally at higher risk of such hazards.14 only 31% of the third year dental students were in favour of washing the wound with soap and running water after accidental injury in one study21 as compared to 80% in another study reports.20 different statistical tools were used in the studies to assess knowledge and awareness regarding nsi among dental students .
one of the studies used z test and standard deviation,20 while three of the studies employed chi - square test for associations between different attributes.212223 statistical significance was set to p < 0.05 to compare knowledge , awareness and practice in all the studies .
two of the studies did not mention the year of the study course in which the students were enrolled , as this can have a significant impact on the knowledge and awareness level of the students and may be a source of potential bias when the results are interpreted.2022 a self - reported questionnaire was used for gathering information from the students regarding nsi . this can increase the risk of bias while evaluating studies on knowledge and awareness .
it was based on a review of earlier studies that were conducted in different time periods by different authors .
therefore , the generalizability may be inaccurate , although the studies were screened using strobe checklist for observational studies .
all the studies included in the present review were conducted on dental students in india .
studies conducted on knowledge and awareness levels of dentists or other dental professionals like dental assistants and dental nurses were almost non - existent .
these personnel are in immediate contact with diverse patients on a daily basis and consequently are prone to more risk .
the questions that were formulated to extract information regarding nsis from the study participants in all the studies were diverse ; therefore , it was difficult to compare the results of the selected studies and common findings were reported .
furthermore , attempts were made to include all the published literature on the knowledge , awareness and practice of nsi in dental profession , but it is possible that some relevant data may have overlooked in terms of fugitive literature ( e.g. , conference proceedings , dissertations ) and important information will undoubtedly be overlooked with the type of literature search strategy used to conduct the present review .
the knowledge and awareness of the dental students in the present review is adequate , although there is considerable variation in the practice and management of nsi among different studies . not
every nsi can be preventable , but , according to a research , 83% of injuries from hollow bore needles can be prevented.24 the present review also concludes that , for prevention of nsi , knowledge and awareness among dental hcws should be increased .
more studies should be conducted involving dentists and other dental professionals , as there is scarcity of literature on knowledge and awareness levels of dentists in india .
various health and safety measures can be adopted to decrease the incidence of nsi , as follows : workers should be properly trainedpersonal protective equipment and clothing should be providedan effective occupational health and safety program should be established that includes immunization , pep , medical and dental surveillance workers should be properly trained personal protective equipment and clothing should be provided an effective occupational health and safety program should be established that includes immunization , pep , medical and dental surveillance | background : hundreds of thousands of healthcare workers remain susceptible to deadly viruses throughout the year , including blood - borne pathogens .
needle stick injuries ( nsi ) constitute one of the most common occupational health hazards in healthcare profession .
dental professionals are at more risk of acquiring nsi due to their limited and restricted working area .
the present systematic review was carried out to determine knowledge , awareness and practice regarding nsi in dental professionals and students in india.materials and methods : a systematic review of cross - sectional studies available in the literature on the knowledge , attitude and practice among dental professionals and students in india was carried out .
we identified relevant articles through electronic and manual search such as pubmed and medline .
two investigators independently identified studies that were included in the review.results:four articles were finally selected for inclusion in the review .
all the studies were done involving dental students ; 89.23% of the students had correct knowledge about nsi and 91.55% of the students had adequate level of awareness regarding its management in one of the studies .
also , 89% of students in one of the studies were aware of taking post - exposure prophylaxis after accidental nsi . in another study
, 44% of the students destroyed the needles using a needle destroyer and 15% destroyed them in puncture - resistant containers.conclusion:the knowledge and awareness of the dental students is adequate , although there is considerable variation in practice and management of nsi among different studies .
there is a need for more studies involving dental professionals . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION AND RECOMMENDATIONS |
PMC4688033 | local excitatory synaptic connections between cortical pyramidal neurons are critical for sensory perception , cognition , and memory and form the backbone of massive - scale modeling and mapping efforts of the mammalian brain
. fundamental properties of monosynaptic excitatory glutamatergic transmission have been well characterized in cortical slice studies ( feldmeyer et al . , 2006 ,
, 2005 , thomson and lamy , 2007 ) . in vitro work has demonstrated that monosynaptic connections between neighboring pyramidal neurons are typically small in amplitude , are highly reliable with little synaptic failure , and show short - term depression .
the probability of finding a connection between two neighboring pyramidal neurons is low , about 10% in somatosensory cortex ( holmgren et al . , 2003 ,
, 2009 , markram et al . , 1997 ) and 10%20% in visual cortex ( cossell et al . , 2015 , ko et al .
yoshimura et al . , 2005 ) , and previous studies have identified non - random patterns of connectivity between pyramidal neurons in some cortical regions ( cossell et al .
, 2015 , ko et al . , 2011 , markram et al . , 1997 ,
however , because of axonal slicing and dendritic spine growth ( kirov et al . , 1999 ) , as well as differences in firing rates , extracellular calcium ( borst , 2010 ) , and neuromodulator concentrations in brain slices , it is unclear whether these features of cortical excitatory monosynaptic connections are also found in vivo .
little is known about properties of cortical synaptic transmission in vivo , in part because of the technical difficulty of performing membrane potential ( vm ) recordings from multiple , neighboring neurons .
cortical slice work has shown that the probability of identifying a connected pair of pyramidal neurons is higher if the cells somata are within 200 m of each other ( holmgren et al . , 2003 , perin et al . ,
thus , to identify a connection without prior knowledge of which cells are connected , recordings should ideally be targeted to nearby cell somata .
previous in vivo studies of synaptic connectivity , however , have used blind recording methods including : dual extracellular recordings ( fujisawa et al . , 2008 ,
reid and alonso , 1995 , swadlow and gusev , 2002 ) , a combination of extracellular and intracellular recordings ( bruno and sakmann , 2006 , london et al .
, 2010 , matsumura et al . , 1996 , yu and ferster , 2013 ) , or sharp microelectrode recordings ( crochet et al . , 2005 ) .
more recently , optogenetics with targeted whole - cell recordings was used to identify excitatory connections to cortical gabaergic interneurons ( pala and petersen , 2015 ) . here , we used in vivo two - photon targeted whole - cell recordings from two to four neighboring , layer 2 ( l2 ) pyramidal neurons in primary somatosensory cortex of anesthetized mice .
our approach allowed us to test for unidirectional and bidirectional connections and examine fundamental properties of unitary excitatory connections between pyramidal neurons .
we made 185 dual , 84 triple , and 2 quadruple vm recordings from 630 neighboring ( horizontal distance between soma centers , 41.01 0.74 m ; n = 878 tested connections ) pyramidal neurons in the primary somatosensory whisker cortex ( s1 ) of urethane anesthetized mice ( age , postnatal day 22 0.2 days ; n = 188 mice ) ( figure 1a ) .
all recordings were targeted to l2 pyramidal neurons within the first dense layer of pyramidal neurons below the pial surface ( mean depth , 177.13 0.89 m ; range , 111.41 to 256.77 m ) .
the lower border of l2 in mouse barrel cortex is 269 m ( lefort et al . , 2009 ) .
l2 pyramidal neurons were visually targeted either by fluorescent genetic labeling or by shadowpatching ( kitamura et al . , 2008 ) , where cell somata appeared as dark shadows against the extracellular space filled with a fluorescent dye ( figure s1 ) .
all neurons recorded in wild - type mice showed a regular spiking firing pattern . furthermore , 71% ( 150/212 ) of wild - type neurons were identified as pyramidal neurons by in vivo visualization of dendritic spines and/or post hoc biocytin staining ( figures 1b and 1c ) . to identify a synaptic connection
, we injected 10 to 20 ms depolarizing current steps into each cell to trigger single action potentials ( aps ) at 0.5 or 1 hz ( figures 1d and 1e ) . under urethane anesthesia ,
cortical neurons oscillate between epochs of depolarized , synaptically active upstates and hyperpolarized downstates with low levels of excitatory input ( steriade et al . , 1993 ) that are thought to resemble the quiescent synaptic activity in cortical slices .
we restricted our analysis to downstates and averaged the postsynaptic responses triggered by single aps . across our entire dataset , 59 unitary excitatory postsynaptic potentials ( uepsps )
were identified from 878 tested connections , giving an overall connectivity probability of 6.7% , a significantly lower rate than reported by a previous in vitro study of l2 pyramidal neurons in mouse barrel cortex ( 9.3% , 88 connections/950 tested , p < 0.01 ; lefort et al . , 2009 ) .
notably , 14 of the 59 identified connections were part of a bidirectionally connected pair of cells , a proportion higher than expected from a randomly connected network ( seven identified bidirectional pairs , 6.7% 6.7% 858 = 3.85 expected bidirectional pairs , p < 0.005 , see also experimental procedures ) ( figures 1f and 1 g ) .
there was no correlation between the probability of identifying a connection and the distance between the cell somata in pairs 1080 m apart ( n = 878 connections , p = 0.542 ; figure 1h ) .
likewise , there was no significant correlation between connectivity rate and depth of recording between 100 and 230 m from the pial surface ( n = 878 connections , p = 0.084 ; figure 1i ) .
therefore , we next determined the kinetics and amplitude of the averaged downstate uepsps ( figures 2a and 2b ; table s1 ; n = 59 ) .
two connections , however , exhibited a latency > 4 ms ( figure s2 ) , which may result from a disynaptic input , as observed between l4 pairs in rats ( feldmeyer et al . , 1999 ) and between l2/3 human cortical neurons ( molnr et al .
the population uepsp had a rise time of 1.97 0.13 ms ( figure 2d ) , peaked in amplitude at 6.66 0.31 ms after the presynaptic ap ( figure 2e ) , and had a half - width of 15.52 0.97 ms ( figure 2f ) and decay time of 16.22 1.24 ms ( figure 2 g ) . across our entire dataset ,
uepsp amplitude ranged from 0.05 to 2.59 mv , with a mean value ( 0.43 0.07 mv ) lower than that in in vitro studies ( feldmeyer et al .
amplitudes revealed a skewed distribution , with the majority ( 76.3% ) of uepsps being < 0.5 mv and a small minority ( 10.2% ) being > 1 mv ( figure 2h ) .
this resembled the distribution of the amplitude of excitatory connections in vitro in different cortical areas both within l2/3 ( cossell et al .
lefort et al . , 2009 ) and between pyramidal neurons within and across other layers ( feldmeyer et al .
bidirectional connections were not significantly larger in amplitude than unidirectional connections ( bidirectional , 0.53 0.18 mv , n = 14 ; unidirectional , 0.40 0.07 mv , n = 45 ; p = 0.120 ) .
the reliability of a synaptic connection will profoundly influence subthreshold response and information flow across the network .
cortical slice work has shown that synaptic failures are extremely rare and that reliability is high ( feldmeyer et al . , 2006 , frick et al . , 2008 ,
2009 ) . however , one recent study , using a modified extracellular solution , showed higher failure rates between l2/3 pyramidal neurons ( urban - ciecko et al . , 2015 ) .
to measure the reliability of synaptic transmission , we quantified the coefficient of variation ( cv ) and the synaptic failure rate ( figures 3a3d ) .
the mean cv was 0.74 0.06 , and the mean failure rate was 21.91 0.30% .
the mean amplitude and vm was stable over a longer time course ( figure s3 ) , suggesting that our stimulus protocol was not inducing long - lasting synaptic change during the testing period .
together , these measurements show that , in vivo , during cortical downstates , excitatory connections were reliable .
however , our measurements of cv and failure rate were higher than in in vitro studies . a clear relationship between the amplitude and both the cv and failure rate has been observed in many cortical slice studies ( feldmeyer et al . , 2006 , frick et al . , 2008 ,
therefore , we next plotted the cv and failure rate as functions of the amplitude of the connection ( figures 3e and 3f ) .
we observed a significant inverse relationship between amplitude and cv and between amplitude and failure rate ( cv versus amplitude , p < 0.0001 ; failure versus amplitude , p < 0.0001 ) .
interestingly , removing the failure trials from the dataset significantly reduced the mean cv ( cv with failure , 0.74 0.06 ; cv without failure , 0.42 0.03 ; n = 59 connections , p < 0.0001 ) and the correlation between cv and amplitude ( figures 3 g and 3h ) .
thus , our data show that while smaller amplitude connections are less reliable than larger amplitude connections , the cv is largely accounted for by failure trials . in vivo , l2/3 pyramidal neurons often fire in bursts of two aps , with an inter - spike interval ( isi ) of < 20 ms ( poulet and petersen , 2008 )
. short - term synaptic depression of the second of two uepsps to consecutive aps with a paired - pulse ratio ( ppr ) of < 1 is a hallmark of cortical synaptic transmission in quiescent cortical slices and consistent with a high probability of presynaptic release ( feldmeyer et al . , 2006 , markram et al . , 1997 , thomson and lamy , 2007 ) .
in contrast , paired - pulse extracellular cortical stimulation in vivo during downstates triggers synaptic responses in neighboring cortical neurons with a ppr of 1 ( crochet et al .
extracellular electrical stimulation , however , could evoke aps in axons from multiple neurons , so it is unclear whether short - term synaptic depression is present in vivo at the level of a monosynaptic connection . to examine the ppr in vivo , we evoked doublets of aps with a mean isi of 14.00 0.84 ms and low jitter ( sd of the isi , n = 16 connections , 1.88 0.21 ms )
next , we averaged the synaptic response from ap1 and ap2 separately and measured the ppr ( figures 4a4 g ) .
we observed a range of pprs from facilitation ( ppr > 1 ; figure 4c ) through no change ( ppr 1 ; figure 4d ) to depression ( ppr < 1 ; figure 4e ) .
overall , there was no significant difference in the amplitude of responses to ap1 and ap2 ( uepsp1 , 0.78 0.20 mv ; uepsp2 , 0.77 0.16 mv ; n = 16 connections , p = 0.744 ) , and the mean ppr was 1.15 0.09 mv .
thus , in vivo excitatory connections between l2 pyramidal neurons in barrel cortex show less depression than prior in vitro measurements ( feldmeyer et al . , 2006 , urban - ciecko et al . , 2015 ) .
the correlation of epsp1 amplitude and ppr had a negative slope ( r = 0.48 ) but was not significant ( p = 0.055 ; figure 4h ) .
we did , however , observe a significant positive correlation between the cv and the ppr ( r = 0.54 , p = 0.030 ; figure 4i ) ; i.e. , more reliable connections had a lower ppr than less reliable connections .
overall , while monosynaptic excitatory connections in vivo have a population average ppr near unity , individual connections exhibit short - term plasticity .
we used multiple in vivo two - photon targeted whole - cell recordings to investigate excitatory monosynaptic transmission between l2 pyramidal neurons in anesthetized mouse somatosensory cortex . in agreement with previous measurements of pyramid - to - pyramid connections in cortical slices ,
our data show a sparsely connected network linked by mostly small but occasionally large amplitude connections . however , we observed a higher synaptic failure rate and less synaptic depression than typically observed in vitro . a critical step to routinely identify monosynaptic connections in vivo was to visually target multiple recordings to neighboring neurons .
two - photon microscopy allowed visualization of genetically identified pyramidal neurons , and whole - cell recordings provided a tool to control presynaptic ap timing and record small - amplitude , subthreshold synaptic inputs .
moreover , to increase the number of testable connections , a value that increases exponentially with the number of recorded cells , we developed a configuration to insert up to four pipettes per recording attempt . in the future , multiple targeted patch - clamp recordings in vivo will allow investigations into the link between monosynaptic connectivity , subthreshold correlations , and synaptic computation during sensory processing , which are not yet possible with extracellular recording methods .
we hypothesized that connection rates in vivo would be greater than rates in vitro , since the preparation of acute brain slices truncates projections between neurons .
thus , it was surprising that the probability of two pyramidal neurons forming a connection in vivo ( 6.7% ) was slightly lower than the rate determined from in vitro measurements of the same neuronal population in mouse barrel cortex ( 9.3% ) ( lefort et al . , 2009 ) .
it was also lower than the connectivity rates identified between l2/3 neurons in cortical slice studies of mouse visual cortex ( cossell et al .
, 2015 , ko et al . , 2011 , yoshimura et al . , 2005 ) , but similar to that in a study including data from visual and somatosensory cortex ( 6.2% ) ( holmgren et al . , 2003 ) .
this difference may be due to a lower number of synaptic contacts in vivo or reflect the higher failure rates , leading to an underestimation of connectivity
. a higher number of connections than expected were bidirectional , in agreement with in vitro recordings in visual cortex ( cossell et al . , 2015 , ko et al . , 2011 , song et al . , 2005 ) and somatosensory cortex ( markram et al . , 1997 , perin et al . ,
2011 ) , but in contrast to findings from a study of barrel cortex ( lefort et al . , 2009 ) .
interestingly , bidirectional connections are more prominent between pyramidal neurons with similar functional response properties ( cossell et al . , 2015 ) , suggesting that connected l2 neurons may share receptive field properties ( jouhanneau et al . , 2014 )
. it will be intriguing to compare bidirectional connectivity in vivo across cortical regions to see whether their formation is correlated to distinct temporal coding strategies ( clopath et al . ,
the mean uepsp latency , rise , and peak time had values similar to those measured in cortical slice studies of monosynaptic connections between somatosensory cortex l2/3 pyramidal neurons , but with broader distributions ( feldmeyer et al .
the half - width and decay time in vivo were slightly shorter than some in vitro measurements ( holmgren et al .
1997 ) , which could reflect differences in the level of background synaptic activity or neuromodulator tone .
identification of the location of dendritic synaptic contacts between connected pairs or dendritic stimulation will help us understand whether the synaptic location site determines the in vivo kinetic properties .
similar to many in vitro and in vivo studies , synaptic connections were mostly small in amplitude , with infrequent , larger amplitude connections forming a skewed distribution with a long tail ( cossell et al .
, 2006 , frick et al . , 2008 , holmgren et al . , 2003 , lefort et al .
, 1997 , pala and petersen , 2015 , perin et al . , 2011 ,
however , the mean connection amplitude in vivo ( 0.43 mv ) was lower than prior slice work on l2/3 pyramidal neurons ( feldmeyer et al . , 2006 , lefort et al . , 2009 ) .
this could result from higher postsynaptic levels of synaptic activity and shunting conductances , reduced presynaptic release probability , or differences in the number of synaptic contacts .
cortical slice measurements in somatosensory cortex have shown that unitary excitatory connections between pyramidal neurons typically have very low failure rates and low trial - by - trial variability with a cv < 0.6 ( feldmeyer et al . , 2006 , frick et al . , 2008 , lefort et al . , 2009 ,
in contrast , we found a higher cv of 0.7 and a failure rate of 22% .
1997 ) , the cv and failure rate were negatively correlated with the amplitude of the connection .
interestingly , we never observed a failure in connections > 1 mv . when synaptic failure trials were removed , the cv was smaller ( 0.4 ) and the correlation of cv with amplitude was weaker , showing that most of the cv could be accounted for by synaptic failures . while transmission between l2 neurons can fail , it is reliable once it occurs .
the higher failure rates and cv in vivo may result from postsynaptic changes to the propagation of subthreshold activity along dendrites due to shunting conductances , or may result from a reduction in the probability of presynaptic glutamate release ( borst , 2010 , crochet et al . , 2005 , urban - ciecko et al . ,
2015 ) . an indirect way of assessing the probability of presynaptic release is through measurement of the ppr .
a ppr < 1 , indicating short - term synaptic depression , is typical of excitatory synaptic connections between pyramidal neurons in cortical slices ( feldmeyer et al . , 2006 ,
this is thought to result from a high probability of presynaptic release of glutamate to the first of two aps that depletes the glutamatergic vesicle pool , leading to reduced release and a smaller epsp to the second ap . here
, we made the first measurements of the ppr between monosynaptically connected excitatory pyramidal neurons in vivo and show an overall ppr of 1.15 .
moreover , we observed a correlation between the cv and the ppr , showing that more reliable connections are more likely to show synaptic depression .
other in vivo studies of synaptic connections that are known to depress in vitro , including the calyx of held ( lorteije et al . ,
2005 , reig and sanchez - vives , 2007 ) , and excitatory connections to parvalbumin - expressing gabaergic interneurons ( pala and petersen , 2015 ) , have also observed less depression and a ppr near or above 1 .
this has been attributed to a reduction in presynaptic release of glutamate in vivo , which may result from lower extracellular ca concentrations ( borst , 2010 , crochet et al . , 2005 ) , higher in vivo firing rates , differences in preparation ( e.g. , age , temperature ) , or active suppression of release through gabab and other metabotropic receptors ( urban - ciecko et al . , 2015 ) .
whole - cell recordings , axonal and dendritic functional imaging , and direct optogenetic or glutamatergic stimulation of postsynaptic structures could now be used to address the relative contribution of presynaptic release and postsynaptic integration on the higher ppr and failure rate values observed in vivo .
somatic whole - cell recordings have revealed that aps in l2/3 pyramidal neurons in awake mice are triggered by large - amplitude , cell - specific , depolarizing synaptic input ( poulet and petersen , 2008 ) .
is this input the result of the summation of synchronous small - amplitude uepsps or sparse but large - amplitude inputs ?
interestingly , in visual cortex , the amplitude of a connection is correlated with the similarity of sensory tuning between two pyramidal neurons ( cossell et al . , 2015 ) .
furthermore , modeling suggests that reliable , large - amplitude connections play an important role in cortical computation ( cossell et al . , 2015 , lefort et al . , 2009 ) .
our approach provides a way to investigate monosynaptic transmission in genetically identified cell types in vivo and to assess the impact of uepsps of different amplitudes on the sensory response and ap generation . in vivo
synaptic transmission occurs on a background of depolarized synaptic activity during active brain states , which has been suggested to alter synaptic transmission to different degrees ( destexhe and par , 1999 , waters and helmchen , 2006 ) .
thus , a next critical step will be to investigate how synaptic transmission , integration , and plasticity are modified by brain state , sensory input , and neuromodulators in vivo .
all experimental procedures were carried out in accordance with german regulations on animal welfare and the berlin animal welfare office ( landesamt fr gesundheit und soziales ; lageso ) . p18p30 male c57bl6j ( 212 neurons recorded ; 20 connections/288 tested ) , nex - cre ( goebbels et al . , 2006 ) ai9 ( madisen et al . , 2010 )
( 238 neurons recorded ; 22 connections identified/364 connections tested ) , fosgfp ( barth et al . , 2004 ) ( 92 neurons recorded ; 11 connections identified/106 connections tested ) , or gad67-gfp ( tamamaki et al . , 2003 ) ( 88 neurons recorded ; 6 connections identified/120 connections tested ) mice were anesthetized with 1.5 g / kg urethane or 1.5%2% isoflurane .
mouse core body temperature was constantly monitored with a rectal probe and kept at 37c with a heating pad .
a lightweight metal head support was implanted onto the skull with glue and dental cement .
the skull was covered with ringer s solution ( in mm ) : 135 nacl , 5 kcl , 5 hepes , 1.8 cacl2 , 1 mgcl2 ; and a small craniotomy ( about 1 mm in diameter ) was drilled over primary somatosensory whisker barrel cortex identified either with intrinsic optical imaging during stimulation of the c2 whisker or stereotactic coordinates ( 1.2 mm posterior , 3.5 mm lateral to bregma ) to expose the brain .
mice were placed under a femto2d in vivo two - photon laser - scanning microscope ( femtonics ) and illuminated with a chameleon ultra ii ( coherent ) pulsed laser - light source at 820 nm ( for shadowpatching and gad67-gfp ) and 950 nm ( for nex - cre ai9 imaging ) via a 40 olympus water immersion objective ( lumplfln 40xw , na 0.8 , working distance 3.3 mm ) .
imaging was controlled with mes software ( femtonics ) running in matlab ( mathworks ) .
whole - cell patch - clamp recordings were made with 2 mm diameter borosilicate glass pipettes ( hilgenberg ) with a resistance of 57 m. three to four pipettes were filled with intracellular solution containing the following , in millimolar : 135 potassium gluconate , 4 kcl , 10 hepes , 10 phosphocreatine , 4 mgatp , 0.3 na3gtp ( adjusted to a ph of 7.3 with koh ) , 30 m alexa fluor 594 ( invitrogen ) , and 2 mg / ml biocytin for anatomical reconstruction .
whole - cell recordings were made from an axon multiclamp 700b amplifier ( molecular devices ) in current clamp mode .
three or four pipettes were inserted through the pia at 150190 mbar , avoiding blood vessels .
next , at 510 m depth , the pressure was reduced to 100 mbar , and the pipettes were moved to 100 m depth . at this point ,
the pressure was reduced again to 50 mbar until reaching the l1/2 border , at which point , the pressure was reduced to 30 mbar .
the red fluorescent somatic signal in nex - cre ai9 mice was used to visually target excitatory neurons . in wild - type mice ,
pyramidal somata were targeted using the shadowpatch method , whereby alexa fluor 594 was puffed out of the recording electrodes to fill the extracellular space , leaving cell somata as dark shadows . in gad67-gfp mice , the gfp label of the gaba - ergic interneurons was used alongside the shadow signal to target pyramidal neurons .
then , we contacted each neuron and formed a gigaseal between the patch electrode and the cell membrane one cell at a time .
contact was monitored by resistance changes visualized on an oscilloscope ( tektronix tds2024c ) and live two - photon images . finally , we ruptured each cell membrane with negative pressure to achieve whole - cell configuration .
this sequence reduced tissue movement during the final stages of patching and helped achieve a successful multiple recording ( figure s1 ) .
recordings were digitized at 20 khz , filtered at 10 khz , and recorded via an itc-18 ( heka elektronik ) analog - to - digital converter connected to a pc under the control of igorpro ( wavemetrics ) . to test for a synaptic connection , first , the current threshold needed to trigger a single spike in each recorded cell was found manually using the multiclamp stimulus command .
next , under the control of igorpro , square current pulses with a duration of 1020 ms and an amplitude of 100400 pa were injected into each cell at 0.5 or 1 hz in sweeps of 60 s to trigger single aps . in some experiments , single - current pulses of longer duration
, 2050 ms , were used to evoke a doublet of spikes . in vivo z stack images ( 2 m per slice ) were made after the termination of the recording to check for the presence of dendritic spines .
depth and cell soma distance were calculated using images of targeted cells taken before the recording . after recording ,
the brain was removed , fixed in 4% pfa overnight , and stored in phosphate buffer at 4c before histological processing for all brains .
subsequently , 100-m - thick tangential slices were made using a leica vt1000 s vibrating microtome .
slices were stained for cytochrome oxidase to reveal the barrel cortex map and then for biocytin with a standard abc kit ( vectastain ) , with dab enhancement to reveal the recorded neurons .
pyramidal cells were identified using the in vivo z stack images showing dendritic spines and the post hoc biocytin fill .
a tested connection was included in the dataset if the postsynaptic downstate vm was less than 50 mv . of a possible 898 connections , we could theoretically test ( from 185 dual , 84 triple , and 2 quadruple recordings ) , in 20 cases , one cell in the pair had a resting vm greater than 50 mv , so it was excluded as postsynaptic recipient but was kept as a presynaptic partner , as we were able to trigger aps . in more detail
, connections from six pairs in 185 dual recordings were only tested one way ( instead of two ) , and connections from seven pairs in 84 triple recordings were only tested four ways ( instead of six ) . as a result , we tested 878 connections in total and identified 59 synaptic connections .
only data from pairs of cells that we could test for connections both ways were included in the test for overrepresentation of bilateral connectivity and shown in figure 1 g ( i.e. , 858 connections ) .
current evoked aps and the corresponding postsynaptic response were selected and separated based on the vm of the postsynaptic cell in two time windows , 1020 ms before and 100150 ms after the peak time of the presynaptic spike .
downstate responses were identified as those where the mean vm was not more than 34 mv more depolarized than the most negative vm value .
then , response amplitudes were measured from each trial as the difference between the average vm 0.5 ms around the peak response and the average vm 0.5 ms around latency .
next , shuffled amplitude measurements were made with the same time interval apart as in the response trials ( i.e. , peak time latency ) during the downstate .
statistical significance of a postsynaptic response was finally assessed by comparing amplitudes of the response and shuffled measurements using a one - tailed wilcoxon - mann - whitney two - sample rank test and a bootstrap test ( see statistical analysis ) .
we measured latency as the crossing point of the extrapolation of two linear fits : the first , from 5 ms to 2 ms prior to the presynaptic spike ; and the second , between time points on the rising phase of the uepsp corresponding to 20% and 80% of the uepsp amplitude . in some cases , we removed a presynaptic spike artifact in the postsynaptic response by subtracting the averaged response to downstate synaptic failures with low noise .
rise time was calculated as the difference in time between 20% and 80% of the peak of the uepsp on the rising phase .
the half - width was calculated as the difference in time between the rising phase and falling phase of the uepsp at 50% of peak response amplitude .
decay time was defined as the difference in time on the decay phase between 20% and 80% of the peak amplitude , as measured on the rising phase .
time to peak was measured as the time difference between peak amplitude and latency . to calculate the cv , we first measured the amplitude of the trial - by - trial downstate uepsps .
next , we measured the amplitude between two random time points during the downstate ( background uepsp ) with the time difference between these two points being the same as for the uepsps .
we then subtracted the mean variance of the background uepsps from the uepsp variance and divided the result by the mean uepsp amplitude .
this calculation was repeated 100 times , and an average value taken for the final cv value .
a synaptic failure was defined as a subthreshold response to a presynaptic ap that was smaller than 1.5 the sd of the baseline vm .
the averaged response of failure events in a connection was visually inspected and resulted in a flat vm trace . to measure the ppr
, we had to measure the amplitude of two epsps ( uepsp1 and uepsp2 ) evoked in response to the first and second ap ( ap1 and ap2 ) in the doublet .
then , the amplitude of epsp1 was measured as the difference between the average vm in a 0.5 ms window around the ap1 peak response and the vm at latency .
however , uepsp2 is riding on top of the decaying phase of uepsp1 , so , to calculate the amplitude of uepsp2 , we first measured the postsynaptic response to single ap responses from the same connection
. then , we used the isis between ap1 and ap2 to shift the averaging process of the single - spike response and construct a model synaptic response ( uepsp1 m ) with the same decay dynamics as for uepsp1 when centered on ap2 .
uepsp1 m was then overlaid with the postsynaptic response to ap2 and normalized to the amplitude of uepsp1 .
decay phases of uepsp1 m are shown as dashed lines in figures 4c4e ( middle columns ) .
the amplitude of uepsp2 was finally taken as the difference between the average vm in a 0.5 ms window around the uepsp2 peak response and the average uepsp1 m vm in a 0.5 ms window at the same time point .
we used the non - parametric wilcoxon mann - whitney two - sample rank test for unpaired data or a non - parametric wilcoxon signed - rank test for paired data .
all tests were two - tailed except where stated . bootstrapping to test for significance of a connection
was performed by randomly selecting , with replacement , amplitudes from the trial - by - trial response amplitudes and from the shuffled amplitude measurements .
then , we calculated the average response amplitude and the average noise amplitude from this bootstrapped dataset and repeated this process 10,000 times to obtain the 95% confidence intervals . to compare connectivity rates between in vivo and in vitro and to test whether bidirectional connections are overrepresented ( i.e. , non - random connectivity ) , we performed two - tailed chi - square tests . to test for correlations between features of uepsps , we used pearson s correlation coefficient r with t statistics .
correlations in figures 3e , 3f , and 3h , figures 4h and 4i , and figure s4d were analyzed on a log scale . | summarylittle is known about the properties of monosynaptic connections between identified neurons in vivo .
we made multiple ( two to four ) two - photon targeted whole - cell recordings from neighboring layer 2 mouse somatosensory barrel cortex pyramidal neurons in vivo to investigate excitatory monosynaptic transmission in the hyperpolarized downstate .
we report that pyramidal neurons form a sparsely connected ( 6.7% connectivity ) network with an overrepresentation of bidirectional connections .
the majority of unitary excitatory postsynaptic potentials were small in amplitude ( < 0.5 mv ) , with a small minority > 1 mv .
the coefficient of variation ( cv = 0.74 ) could largely be explained by the presence of synaptic failures ( 22% ) .
both the cv and failure rates were reduced with increasing amplitude .
the mean paired - pulse ratio was 1.15 and positively correlated with the cv .
our approach will help bridge the gap between connectivity and function and allow investigations into the impact of brain state on monosynaptic transmission and integration . | Introduction
Results
Discussion
Experimental Procedures
Author Contributions |
PMC5407233 | sexual dysfunction is defined as continuous or repetitive decrease of sexual desire , arousal and pain during intercourse and failure in achieving sexual orgasm ( 1 , 2 ) .
this common problem is thought to be age related and progressive ( 3 , 4 ) .
regarding the prevalence of female sexual dysfunction is variable in different cultures , age groups or health conditions such as cardiovascular or psychological disease , every study about this issue from different parts of the world seem unique in nature , and their findings can not be particularly compared with each other ( 5 ) .
studies have shown that sexual dysfunction has an important effect on women s life quality as well as life after marriage ( 6 ) . in a most recent study in our region ,
the prevalence of female sexual dysfunction was reported to be approximately 62% in healthy women ( 7 ) .
pregnancy and child birth are two important and common factors which are reported to have controversial effects on sexual function ( 811 ) .
sexual function fluctuates within 6 months after delivery and will gradually improve ( 12 ) .
pelvic floor dysfunction can be manifested as urinary or fecal incontinence , female reproductive organ prolapse , and pleasure and sexuality disturbances .
hormonal changes , as well as mechanical forces during pregnancy and also the pressure of labor process on pelvic floor muscles , can provide neural and muscular damage to pelvic floor organs ; leading to further sexual dysfunction ( 13 ) . in our country , the rate of cesarean section ( c / s ) with no medical indication is growing , and women show tendency toward c / s because of personal beliefs including fear of pain and injury during child birth , and complications of normal vaginal delivery ( nvd ) ( 14 ) . according to the rise in tendency toward c / s and because of antithesis about the long - term effects of type of delivery on female sexual function , we decided to evaluate the long - term effect of delivery type on female sexual function in an iranian population .
this cross - sectional study took place in healthcare facilities of sabzevar university of medical sciences in 2014 .
the study protocol was approved by sabzevar university of medical science ethic committee ( research code : 90008 ) .
study population was chosen by random sampling from every primipara woman who had delivered 2 years ago , in sabzevar hospitals .
none of the study participants had any previous mental or physical illness within 2 years after delivery and also did not take any medication for longer than a month within this 2-year period .
women who had preterm or post term labor , non - elective cesarean section ( c / s ) and instrumental vaginal delivery , or those who were multi partner were excluded .
eligible women filled the informed consent , and were given two separate questionnaires for assessing sexual function and demographic data .
this questionnaire evaluates six main domains of sexual functions including sexual desire , lubrication , sexual satisfaction , sexual arousal , orgasm and sexual pain over the previous month ( 7 , 15 ) .
persian translate of fsfi questionnaire has been previously validated in an iranian study ( 16 ) . in this study ,
cut - off points used for sexual dysfunction were similar to previous study in sabzevar university of medical sciences with the same questionnaire ( 7 ) .
score cut off points were as follow : sexual pain : 3.8 , sexual desire : 3.3 , sexual arousal : 3.4 , lubrication : 3.7 , orgasm : 3.4 and sexual satisfaction : 3.8 ( 7 , 17 ) .
sensitivity analysis for persian version of fsfi reported the score of 28 as the best cut - off point ( sensitivity : 83% , specificity : 82% ) ( 17 ) .
demographic questionnaire included questions about participant s age , marriage duration , type of delivery and educational status .
urinary and fecal incontinency were documented according to patients medical record , which was completed by their gynecologist .
participants who were diagnosed with sexual dysfunction in the study were advised to consult a gynecologist .
study data was analyzed by the statistical package for social sciences ( spss ) software version 19 .
chi square , student t - test and fisher exact test were used for comparing groups .
confidence limit was set at 0.95 , and p - value lesser than 0.05 was considered as statistically significant .
one hundred and seventy - seven women were eligible to enroll in this study . mean age of participants was 31.816.31 years and mean age of marriage was 11.76.15 years .
participants in both nvd and c / s groups were similar in terms of demographic data .
mean score of sexual function was not statistically significant between the study groups ( p=0.23 ) .
however , participants in c / s group showed significantly higher sexual arousal score than nvd group ( p=0.04 ) ( table 1 ) .
no significant relation was observed between urinary and fecal incontinence and labor type ( p=0.07 and p=0.6 respectively ) .
participants with both types of incontinency had weaker sexual function ( table 2 ) . out of a total of 123 nvd participants ,
56 had sutured lacerations ; however , statistical analysis did not reveal significant difference in study variables in comparison with labor with intact perineum ( p=0.1 ) .
our study shows that the overall fsfi score was not significantly different in women undergoing nvd or c / s .
however , participants in c / s group showed a significantly higher sexual arousal score than nvd group .
approximately 40% of the female population experience sexual problems in their lifetime , and decrease in sexual desire is the most common disturbance in women of all ages ( 6 ) . the rate of sexual dysfunction changes during different periods of lifetime .
total fsfi score in our study is approximately the same as another study which took place in our city a year ago ; however , the previous study evaluated all women referring to healthcare centers ( 7 ) .
a recent study in our country , showed that women who experienced child birth had lower marital satisfaction than women without children ( 18 ) . during the first three months after delivery
, women may suffer from sexual dysfunction as decrease in libido , dyspareunia or difficulty achieving orgasm .
the extent of damage to pelvic and genital organs are varied depending on type of delivery .
nvd may result in pudendal nerve damage and also vaginal prolapse due to hypotonic muscle .
it is believed that c / s has lesser effect on anatomical and structural arrangement of pelvic floor and organs ( 19 ) .
concluded that vaginal delivery had significant effect on reducing desire , arousal and lubrication within the first 3 months after delivery .
also , elective c / s was found to be associated with decrease in desire ( 10 ) .
although episiotomy is associated with dyspareunia , long term studies reported that the role of this issue on sexual function is inconclusive ( 12 ) .
study showed that there is no difference between nvd with through midiolateral episiotomy and c / s in primipara women in terms of sexual dysfunction .
their study assessed sexual function at two different stages ; once before pregnancy and the other time within 3 to 6 months postpartum . while the overall fsfi score in groups was not significantly different , desire , orgasm , arousal and satisfaction were lower than that of before pregnancy period .
they also mentioned that regarding to preserving normal sexual function , c / s is not preferred to nvd ( 20 ) .
de souza et al . also evaluated sexual function in two stages ( 6 and 12 months after delivery ) , and reported a significant effect of episiotomy on orgasm score .
furthermore , episiotomy had greater effect on lowering orgasm score than first and third degree tears .
they found arousal improved in women with perineal tear or episiotomy within first year postpartum ( 8) . we could not find any study assessing perineal injury 2 years after delivery , however ; our results were in line with short term studies , indicating that episiotomy does not have negative effect on female sexual function 1 year after delivery .
another finding of our study was that no significant relation was found between delivery method and urinary and fecal incontinency .
. the risk of urinary incontinence will increase by 67% 20 years after delivery ( 21 ) . also , pelvic floor dysfunction is two times more common in women with urinary incontinence during pregnancy and shortly after delivery ( 22 ) .
chan et al . stated that prevalence of stress urinary incontinence , urge urinary incontinence and fecal incontinence were 25.9% , 8.2% and 4.0% respectively after one year of delivery ( 23 ) .
there is a scarcity of data available about the effect of urinary and fecal incontinence following pregnancy on female sexual function .
one reason could be lack of validated questionnaires till recent years . despite this weakness in literature ,
women with fecal incontinence are as likely to engage sexual relation as healthy women ; however , they show poorer sexual function than healthy women ( 24 ) .
similar to the findings of our study , a cross sectional study by imhoff et al
. also showed a greater risk for having poor sexual life in women with fecal incontinence compared to normal women ( 25 ) . despite structural disturbances and traumas
, pregnancy may have a hormonal effect on sexual function within a few months after delivery , which could be due to lactation , relational and emotional changes ( 20 ) .
although not sufficient , we tried to minimize possible bias about hormonal effect on sexual function by evaluating women who have 2 year - old children and do not breast feed them . furthermore , we relied on gynecological diagnosis regarding urinary and fecal incontinence , instead of using questionnaires .
it is possible that some of the participants had received non - medical treatment for their incontinence and thus , their sexual functions were affected by exercises and not by medication or surgery .
the results of this study can be used by healthcare professionals , especially midwives , to design and develop effective counseling programs and appropriate education about sexual function after delivery , and the effect of delivery on sexual function . because midwives are responsible for the health of mothers in health centers , and provide correct information and real help for mothers .
the limitations of this study include lack of determining sexual satisfaction of spouses , lack of examining the sexual function of multipara women compared with nullipara women and self - completion of the questionnaire are among the limitations in this study .
present study did not find any relation between type of delivery and female sexual dysfunction .
this could be a document to prove the fallacy of the common belief in our region that c / s could prevent sexual problems . despite this
, we concluded that there was an association between urinary and fecal incontinency in primipara women . according to these results
, we suggest that health providers educate women about the best delivery type and possible sexual dysfunction in early months after delivery which is not going to be persistent or affect their sexual life . | introductionpregnancy and birth giving are two of the most important and common effective factors influencing female sexual function . sexual dysfunction can affect women s quality of life and marriage .
this research tried to determine the relation between labor type and long term sexual function in primipara women of sabzevar , iran.methodsthis cross - sectional study took place in sabzevar healthcare system in 2014 .
study population was chosen by random sampling from every primipara woman who had delivered 2 years ago , in sabzevar hospitals .
a total number of 177 primipara women were enrolled in this study after signing an informed contest .
sexual function of mothers was determined by validated persian version of female sexual function index ( fsfi ) .
spss statistical software version 19 and descriptive and inferential statistics such as percentage , mean , standard deviation , chi square , student t - test and fisher exact test were used to analyze data.resultsamong the study population , 69.5% had natural vaginal delivery ( nvd ) and 30.5% had caesarean section ( c / s ) .
the overall fsfi score was not significantly different in women undergoing nvd or c / s ( p=0.23 ) .
also , no significant relation was found between delivery method and urinary ( p=0.07 ) and fecal incontinency ( p=0.6 ) .
female sexual function was prominent in women with urinary ( p=0.015 ) and fecal incontinency ( p=0.018).conclusionthe results of present study showed that delivery method has no long - term effect on female sexual function and appropriate education about the sexual issues after delivery and effect of birth giving on sexual function are necessary for this group of society . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions |
PMC4411731 | acute loss of sensory function of the infraorbital nerve following orbitozygomatic complex fractures is often seen because of their close proximity as the nerve passes through the infraorbital sulcus in the floor of the orbit to exit through the infraorbital foramen1 .
traumatic injury to the infraorbital nerve may be due to compression , edema , ischemia , or laceration2 .
persistent hypoesthesia following correction of mid - face and zygomatic complex fractures indicates injury to the infraorbital nerve .
although hyperesthesia along the distribution of the infraorbital nerve has been documented , it is comparatively rare in occurrence3,4 .
the incidence of long - term neurosensory deficits in different studies varies from 10% to 50%1,5 .
the incidence of mid - face and zygomatic complex fractures presenting with fracture lines running through the inferior orbital foramen resulting in post - traumatic sensory disturbance has been well studied and documented through the years1,6,7,8,9 .
the most commonly documented cause for such sensory disturbance is nerve impingement by fractured segments which have been reduced or fixed inadequately2 .
the other cause is formation of fibrous or calcified tissue around the infraorbital nerve postoperatively , leading to nerve compression .
. however the documented successful outcomes are not reported with individual techniques , which could result in temporary or permanent sensory loss . to overcome this
, we describe a new technique of repositioning the infraorbital nerve into the orbital floor before reduction and stabilization of the fractured zygomatic complex .
two male and one female patients with history of road traffic accidents reported to the hospital emergency room . clinical and radiological examination ( fig .
1 ) revealed a displaced zygomaticomaxillary complex fracture on the left side for one of the patients and on the right for another.(fig . 2 , 3 )
the third patient had a lefort iii fracture on the left and lefort ii an intraoral approach was used to expose the zygomatic complex .
two percent lignocaine hydrochloride with adrenaline ( 1 : 80,000 ) was infiltrated at the site of incision .
the incision was made in the buccal vestibule in the high mucobuccal fold area extending from canine to second molar region .
sharp dissection was carried out subperiosteally to expose fractured segments in the infraorbital and zygomatic buttress region .
the fracture line was seen running through the infraorbital foramen , and the neurovascular bundle was found entrapped between the fracture lines in the described cases.(fig . 2 , 3 , 4 ) with great care taken to minimize direct trauma to the infraorbital nerve ,
5 ) sharp bony spicules of the fractured segment impinging on the nerve were removed , and the nerve was dissected off the surrounding soft tissue along its long axis with the help of a nerve hook.(fig .
6 ) then the nerve was mobilized sufficiently to check for any adhesions and was transpositioned to a higher level in the orbit at the floor level .
this was done to gain access to the fracture lines for the placement of a fixation device near the infraorbital margin .
7 ) the surgical site was closed with continuous suturing using 3 - 0 vicryl suture material ( johnson & johnson , aurangabad , india ) , and patients were reversed and extubated uneventfully .
postoperatively , patients ' eyes were examined using an orbital chart for signs of visual and sensory impairment .
the patients recovered uneventfully and had no evidence of sensory disturbance during their three - month follow - up .
two - point discrimination testing was performed in the extraoral area of the infraorbital nerve distribution both preoperatively and postoperatively , and on both the affected and contralateral sides as a control .
two percent lignocaine hydrochloride with adrenaline ( 1 : 80,000 ) was infiltrated at the site of incision .
the incision was made in the buccal vestibule in the high mucobuccal fold area extending from canine to second molar region .
sharp dissection was carried out subperiosteally to expose fractured segments in the infraorbital and zygomatic buttress region .
the fracture line was seen running through the infraorbital foramen , and the neurovascular bundle was found entrapped between the fracture lines in the described cases.(fig . 2 , 3 , 4 ) with great care taken to minimize direct trauma to the infraorbital nerve
5 ) sharp bony spicules of the fractured segment impinging on the nerve were removed , and the nerve was dissected off the surrounding soft tissue along its long axis with the help of a nerve hook.(fig .
6 ) then the nerve was mobilized sufficiently to check for any adhesions and was transpositioned to a higher level in the orbit at the floor level .
this was done to gain access to the fracture lines for the placement of a fixation device near the infraorbital margin .
7 ) the surgical site was closed with continuous suturing using 3 - 0 vicryl suture material ( johnson & johnson , aurangabad , india ) , and patients were reversed and extubated uneventfully .
postoperatively , patients ' eyes were examined using an orbital chart for signs of visual and sensory impairment .
the patients recovered uneventfully and had no evidence of sensory disturbance during their three - month follow - up .
two - point discrimination testing was performed in the extraoral area of the infraorbital nerve distribution both preoperatively and postoperatively , and on both the affected and contralateral sides as a control .
there was no evidence of sensory disturbance during their three month follow - up in any of the patient .
chronic sensory disturbance in the form of hypo- or hyperesthesia is a common postoperative symptom in patients with surgically repaired mid - face fractures1,4 . in isolated posterior fractures , the infraorbital nerve may block the surgical field , preventing visualization of the fracture site . rough dissection of this area may cause unexpected bleeding due to rupture of the orbital branch of the infraorbital artery12 .
these complications may cause surgeons to avoid manipulation around the nerve , which can lead to incomplete reduction of orbital floor fractures11 .
this , in turn , leads to restriction of ocular movement due to remnant entrapped periorbital tissue , and also postoperative enophthalmos due to undercorrection of the intraorbital volume11 .
although various operative alternatives have been tested to avoid these residual symptoms , satisfactory or definitive results have not been achieved8 .
we report three cases of mid - face trauma in which reduction of the fracture segment was performed after nerve transposition .
this approach defers the need for a second surgery to correct post - traumatic sensory loss . avoiding re - entry not only saves the patient 's time and money , but also limits the edema and inflammatory response that may worsen nerve compression after a second surgery .
hence , we recommend intraoperative transpositioning of the infraorbital nerve to the orbital floor in every mid - face fracture with fracture line running through the facial course of the nerve .
infraorbital nerve transposition is very effective in preventing paresthesia in patients which fracture line involving the infraorbital nerve . | objectivestranspositioning of the inferior alveolar nerve to prevent injury in lower jaw has been advocated for orthognathic , pre - prosthetic and for implant placement procedures .
however , the concept of infra - orbital nerve repositioning in cases of mid - face fractures remains unexplored .
the infraorbital nerve may be involved in trauma to the zygomatic complex which often results in sensory disturbance of the area innervated by it .
ten patients with infraorbital nerve entrapment were treated in similar way at our maxillofacial surgery centre.materials and methodsin this article we are reporting three cases of zygomatico - maxillary complex fracture in which intra - operative repositioning of infra - orbital nerve into the orbital floor was done .
this was done to release the nerve from fractured segments and to reduce the postoperative neural complications , to gain better access to fracture site and ease in plate fixation .
this procedure also decompresses the nerve which releases it off the soft tissue entrapment caused due to trauma and the organized clot at the fractured site.resultsthere was no evidence of sensory disturbance during their three month follow - up in any of the patient.conclusioninfraorbital nerve transposition is very effective in preventing paresthesia in patients which fracture line involving the infraorbital nerve . | I. Introduction
II. Materials and Methods
Surgical procedure
III. Results
IV. Discussion
V. Conclusion |
PMC4932094 | the suit was
developed for the maintenance of muscle tone in a weightless environment , such as that of a
spaceship worker1 .
it was designed to
create a normal framework of forces on the body for stabilizing the torso to allow for more
fluent and coordinated movement of all limbs .
it uses a system of elastic bands and pulleys
that create artificial forces against which the body can work in order to prevent muscular
atrophy .
the adeli suit treatment ( ast ) has been proposed as an intensive exercise protocol
for use in the management of cerebral palsy ( cp ) and other neuromuscular disorders1 .
the suit is composed of a cap , a vest ,
shorts , knee pads , shoes with attached auxiliary equipment , and a bungee cord to connect
auxiliary equipment .
the cords are
adjustable for the application of varying degrees of tension to the child s different muscle
groups .
the bungee cords are positioned to keep the body properly aligned and to forcibly
encourage movement within a normal range of motion2 . to reposition the limbs in order to correct for abnormal muscle
alignment ,
the bungee cords are adjusted by therapists to mimic normal flexor and extensor
patterns of the major muscle groups1 .
the mechanism behind the adeli suit is that through active movement therapy , the brain is
stimulated and thus is retrained to recognize , and eventually initiate , correct movement of
the muscles .
once the body is in proper alignment , aggressive movement therapy that will
reeducate the brain to recognize correct movement of the muscles can be performed .
the suit
enhances communication between the brain and peripheral muscles , particularly , the head and
trunk control muscles , and the locomotor functions of the legs1 , 3 .
rehabilitative gains in
motor control through adeli suit treatment are typically retained after the intensive course
is completed .
studies on groups of children with ast have shown long - term retention of
skills after the completion of the therapy program .
ast is often used as part of a
comprehensive physical therapy program and might improve mechanical efficiency without a
corresponding gain in gross motor skills , especially in children with higher levels of motor
function4 . in this study , we compared changes in gross motor function , spatiotemporal parameters , and
functional ambulation performance score ( faps ) in children with cp who were assigned to
receive ast and underwent training for a period of 4 weeks .
the results suggest the
importance of the development of ast programs to improve gait ability in children with
cp .
the subjects consisted of a 6-year - old boy ( height , 112 cm ; weight , 18 kg ; gross motor
function classification system [ gmfcs ] , level ii ) and a 3-year - old boy ( height , 89 cm ;
weight , 10 kg ; gmfcs , level ii ) with spastic diplegic cp recruited from an s rehabilitation
center in korea .
the subjects with cp had no history of newly developed neurological
problems , musculoskeletal disorders , or botulinum toxin injections in the previous 6 months .
the study was approved by the sahmyook university institutional review board
( syuirb2015 - 109 ) .
the objective of the study and its requirements were explained to the
subjects , and all of the participants parents provided written consent , in accordance with
the ethical principles of the declaration of helsinki .
ast was applied in two subjects for a period of 4 weeks , 5 times per week , in 60-minute
sessions per day .
ast included intensive multiple movements combined with the wearing of a
fitted garment ( adeli suit ) , which provided resistance during the multiple movements .
this
suit comprised a vest , shorts , knee pads , and specially designed shoes .
it was connected by
hooks , rings , and elastic bands that could be adjusted to provide pressure and support to
joints and muscle groups .
the bungee - like cords were adjusted by a physical therapist to
mimic normal flexor and extensor patterns of the major muscle groups in an attempt to
reposition limbs in order to correct abnormal muscle alignment .
each session included
multiple movements based on the children s abilities and included standing up , sitting ,
playing with balls while standing , jumping on the trampoline , walking on different
geographical features , and climbing stairs or ladders5 . to assess gross motor function
gmfm is
a tool for measuring and recording changes in exercise level during the process of treatment
or elapsed time .
the gmfm-88 consists of 88 items grouped
into five domains as follows : domain a ( lying and rolling , 17 items ) , domain b ( sitting , 20
items ) , domain c ( crawling and kneeling , 14 items ) , domain d ( standing , 13 items ) , and
domain e ( walking , running , and jumping , 24 items)7 ,
8 .
each item is worth 3 points , and the
points earned from each domain are multiplied by 100 points to obtain a domain score .
the
final score is obtained by adding each domain score and dividing the sum by 5 to obtain a
total score7 . to measure gait function
, pa , usa ) ,
which consists of an electronic walkway ( 90 460 cm ) . data were sampled from the walkway at
a frequency of 80 hz .
the spatiotemporal characteristics of the gaitrite system have shown
excellent reliability with an intraclass correlation coefficient of between 0.82 and
0.929 . the procedure was explained and
demonstrated to the child . the child was then instructed to walk barefoot at a self - selected
walking speed , with the head facing forward and the eyes looking straight ahead10 .
data on spatial ( step length and stride
length ) and temporal gait parameters ( walking speed and cadence ) were obtained by using
gaitritever 3.2b9 , 11 .
velocity was calculated by dividing the distance by the ambulation
time and was expressed in centimeters per second .
stride length was defined and measured on
the line of progression between the heel points of two consecutive footfalls of the same
foot and was expressed in centimeters .
the faps is integrated within the gaitrite walkway and is considered to be the gold
standard for spatiotemporal gait parameter analysis .
the measurement was performed 3 times , and to eliminate the fatigue of walking ,
the subjects were allowed a 30-second rest after walking once12 .
all measurements were performed twice , before intervention and after 4 weeks of
intervention .
the spatiotemporal gait parameters and the faps for gait function were
measured three times , and the average of the three measurements was used in the analysis .
all of the data were codified and analyzed with spss statistics for windows version
17.0 .
the gross motor function of case a as assessed by gmfm increased from 87.2% at pretest to
94.37% at post - test ; for case b , gmfm increased from 71.17% at pretest to 73.0% at
post - test .
the gait velocity of case a increased from 78.57 cm / s at pretest to 80.705 cm / s
at post - test , while the cadence increased from 135.63 to 136.97 steps / min , the right - sided
step length decreased from 39.01 to 37.03 cm , the left - sided step length increased from
31.76 to 37.27 cm , the right - sided stride length increased from 70.08 to 77.52 cm , the
left - sided stride length increased from 70.08 to 76.85 cm , and the faps increased from 81 to
84 ( table 1table 1.gross motor function and gait function scoresparametercase acase bpretestposttestpretestposttestgmfm ( % ) 87.294.471.273.0velocity ( cm / s)78.680.771.362.3cadence ( step / min)135.6137.0152.9157.6step length ( cm ) right side39.037.028.728.6left side31.837.323.522.1stride length ( cm)right side70.177.552.251.3left side70.176.952.352.9faps ( score)81847584values represent means .
gmfm : gross motor function measure , faps : functional
ambulation performance score ) .
gmfm : gross motor function measure , faps : functional
ambulation performance score the gait velocity of case b decreased from 71.26 cm / s at pretest to 62.27 cm / s at
post - test , while the cadence increased from 152.9 to 157.6 steps / min , the right - sided step
length decreased from 28.70 to 28.55 cm , the left - sided step length decreased from 23.50 to
22.11 cm , the right - sided stride length decreased from 52.18 to 51.25 cm , the left - sided
stride length increased from 52.27 to 52.94 cm , and the faps increased from 75 to 84 ( table 1 ) .
the current neurodevelopmental treatment of cp focuses on maintaining or improving function
in patients with cp .
various treatments may help children with cp to hold an upright body
alignment , strengthen muscles , prevent complications , and improve their daily functions .
although several treatments of cp exist , adjustments to existing and new treatments are
still needed .
many
families and clinicians are interested in the outcomes of treatment using the adeli suit ,
but the rehabilitation community does not have adequate scientific support for its use as a
generally accepted treatment of cp1 .
this study focused on children with cp who received ast for a period of 4 weeks , in order
to examine changes in gmfm , spatiotemporal parameters , and the faps .
an important finding is
improvement in gross motor function , as assessed by gmfm , in both cases .
the applied ast
involved multiple movements based on the children s abilities and included standing up ,
sitting , playing with balls while standing , jumping on the trampoline , walking on different
geographical features , and climbing stairs or ladders .
proprioceptive stimulation by
multiple movements has a profound effect in the treatment of cp because proprioception
stimulates the nervous system at the onset of damage , thus creating a better basis for
successful rehabilitation .
for this reason , gross motor function was increased in both
cases . regarding gait function , the cadence and faps improved after the intervention in both
cases .
step length increased on the left side and decreased on the right side in case a ,
resulting in a decreasing tendency of asymmetries in step length after the intervention .
the results of previous studies on ast suggest that the
subject relies on changes in proprioceptive information from muscles , tendons , and joints .
an increase in the input of proprioception and significant information from the vestibular
organs can affect body position , balance , and posture tension . through active movement
therapy with ast ,
the brain is stimulated and , thus , is retrained to recognize , and
eventually initiate , the correct movement of the muscles , resulting in the improvement of
body alignment through trunk stability , along with improved walking patterns1 , 5 .
body deformation due to a disability prevents patients from maintaining good posture or
weight support in comfortable standing positions .
the location of the elastic bands in the
adeli suit is similar to the position of antagonistic muscle pairs .
motor improvement of the
joints in the lower extremities in antigravity situations was associated with resistance to
the muscles that occurred when patients moved each part of their body that is connected to a
bungee cord .
ast controls each movement in response to the constantly acting gravitational
force , particularly for the head , trunk , and locomotor functions of the legs .
a limitation of this study is that only two children with cp participated in the study .
however , the results show that ast improved gross motor function and functional ambulation
performance in these subjects .
these findings suggest that ast should be recommended for
treatment of cp , as a properly executed intervention would bring more benefits , particularly
for functional improvements in cp . | [ purpose ] the purpose of this study was to determine the effects of adeli suit therapy
( ast ) on gross motor function and gait function in children with cerebral palsy .
[ subjects
and methods ] two participants with spastic cerebral palsy were recruited to undergo ast .
ast was applied in 60-minute sessions , five times per week , with 20 sessions total over 4
weeks .
assessments of gross motor function , spatiotemporal parameters , and functional
ambulation performance for gait were conducted .
[ results ] gross motor function , cadence ,
and functional ambulation performance improved after the intervention in both cases .
[ conclusion ] although additional follow - up studies are required , the results demonstrated
improved gross motor function and functional ambulation performance in the children with
cerebral palsy .
these findings suggest a variety of applications for conservative
therapeutic methods that require future clinical trials in children with cerebral
palsy . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |
PMC5108480 | the lifetime prevalence of low back pain ( lbp ) is high ; 70% of adults have had lbp at some time.1 moreover , the number of patients with lbp in developed countries is increasing in line with the proportion of elderly.2,3 conservative and postoperative treatments for lbp include the use of class 1 medical devices such as a lumbosacral orthosis ( lso).4 cholewicki et al observed that one of the causes of lbp is excessive erector spinae muscle activity , which could be reduced with an lso.5 any decrease in the compressive force exerted on the vertebral body by reducing such activity with an lso would benefit those with osteoporosis and vertebral compression fracture , conditions to which elderly people are vulnerable .
however , a review of data held in the cochrane database found no evidence for the efficacy of lumbar supports alone in preventing and treating lbp.6 although pfeifer et al reported that their newly designed spinal orthosis had several positive effects on muscle strength , body balance , kyphosis angle , and vital capacity in elderly patients with osteoporosis,7 to our knowledge , no previous studies have reported any significant effects of wearing a trunk orthosis to specifically decrease erector spinae activity and low back load in elderly people . to address this issue ,
we previously designed a trunk orthosis to improve trunk and pelvic stability and alignment by means of resistive force provided by joints with springs ( figure 1).8 this orthosis with joints providing resistive force ( orf ) creates resistive force to produce a resistive moment that rotates the trunk backward and pelvis forward . in our previous study , we reported the effect of this orf in modifying trunk alignment and decreasing activity of the erector spinae during static standing in elderly subjects.9 a previous study reported that an lso was effective for decreasing erector spinae activity in an unstable sitting position , where adjustment was needed to balance the upper body,5 while another study showed no positive effects of an lso on decreasing low back load by decreasing low back muscle activity and increasing intra - abdominal pressure.10 to date , there have been no studies on the efficacy of a typical lso or spinal orthosis for reducing low back load during level walking , where the demands for adjusting balance are high .
we previously reported that our orf prototype increased superficial abdominal muscle activity and decreased erector spinae activity during level walking in healthy young adults,11 and here we sought to explore whether our findings can be extended to the trunk muscular activities and the low back joint moment ( lbm ) during level walking in healthy elderly subjects .
this biomechanics study examined the effects of the orf on the gait of healthy elderly people during level walking and compared the effects with those obtained without an orthosis and with an lso .
we hypothesized that the orf and lso would both effectively decrease low back load measured by joint moment and trunk muscular activities during level walking , but that the orf , with its biomechanical function of decreasing low back load , would show a superior effect .
this follow - up to our previous study9 was performed to confirm the hypothesis that orf might decrease low back load not only in static standing but also in level walking , using a new biomechanical method .
from 31 community - dwelling elderly subjects who were candidates for this study , 15 were enrolled ( all males ; mean age , 67.76.1 years ; mean height , 162.45.7 cm ; mean weight , 62.37.8 kg ) after excluding those with neurological disease , pain , history of an orthopedic surgical procedure , history of orthopedic treatment within the past 5 years , and history of lbp within the past 1 year .
the study subjects were the same as those of our previous study.9 the study was approved by the ethics committee of the international university of health and welfare ( 11191 ) .
the orf is shown in figure 1 and its features are described in our previous study.8 briefly , pelvic and upper supports are positioned on the ileum and sternum , respectively .
stainless steel joints , connected to the upper support with a nylon pad and to the pelvic support , produce resistive force through the use of extension springs .
a link mechanism translates the spring - generated tension into a resistive moment on the chest and a reaction moment on the posterior pelvis .
the orf has a release mechanism that releases the resistive force by pulling tension levers downward .
the orf is currently an investigational product that has not been approved by the food and drug administration or by a corresponding national agency for the indication described herein .
the subjects walked 10 m on a level surface at a self - selected speed in a laboratory setting under three conditions : without an orthosis , with the orf , and with an lso ( damen corset , pacific supply , osaka , japan ) .
the damen corset was selected as it is frequently prescribed for patients with lbp . after completing three walking trials without any orthosis
the subjects were allowed 5 minutes to accustom themselves to wearing the orf and the lso .
resistive force on the chest provided by the joints was measured in real time using a strain gauge ( kyowa , tokyo , japan ) and the force data were transferred to a laptop computer by bluetooth ( figure 2 ) and the force was set to a magnitude of 2025 n during static standing .
the pressure between the corset and abdomen was set to 10 mmhg in all measurement conditions.12 gait was recorded with a three - dimensional motion capture system ( vicon 612 , vicon , oxford , uk ) consisting of six force plates ( four from amti , watertown , ma , usa ; and two from kistler , winterthur , switzerland ) and 12 infrared ( ir ) cameras with a sampling rate of 120 hz . referring to a study by seay et al,13 41 ir - reflective markers ( diameter , 14 mm ) were attached to each subject s body .
additionally , three markers were attached over a strain gauge and on bilateral joints of the orf . to measure muscle activity during level walking , electromyograms ( emgs )
were obtained ( biometrics , newport , uk ) at a sampling rate of 1,080 hz for bilateral erector spinae ( 2 cm to the side between l4l5 vertebrae).14 maximum voluntary contraction was measured while one physical therapist manually applied resistant force to the midpoints of the bilateral scapulae , with the subject lying in the prone position on a bed . during acquisition , we performed full - wave rectification feeding into a band pass filter ( 20420 hz ) to decrease noise and used visual 3d analytical software ( c - motion , germantown , md , usa ) .
the obtained emgs were normalized using maximal voluntary contraction during isometric contraction ( as a percent ) , and the root mean square was calculated for a 50 ms window .
subjects performed isometric contractions in prone against gravity with maximum resistance applied by the experimenter to obtain maximal voluntary contraction of the erector spinae.15 visual 3d was used to perform kinetic and kinematic data analysis .
the obtained physical coordinates and ground reaction force data were low - pass filtered with a second - order recursive butterworth filter , with a cutoff frequency of 6 and 18 hz , respectively , according to winter s technique.16 the link segment model consisted of 13 segments : head , trunk , pelvis , bilateral upper arms , forearms , thighs , shanks , and feet .
briefly , the low back extension and flexion moments were calculated using the ground reaction force data obtained from the force plates , the reaction force on the chest obtained from the strain gauge , and the coordinates of the ir - reflective markers on the bodies of the subjects and an orf .
moment exerted by an orf was calculated by multiplying the force measured by a strain gate and moment arm from joint of an orf to the force . in our previous study analyzing the orf effect during static standing,9 we were not able to calculate lbm .
the novelty of the present study lies in applying a new technique to calculate lbm during level walking while wearing an orf .
the moment was subtracted from the lbm calculated by using the ground reaction force data and the coordinates of the ir - reflective markers on the bodies of the subjects because the moment created by orf joints equally gives forward rotation moment on the pelvis because of action reaction law . in the analysis
, segments were regarded as rigid and the joint moments were calculated using a link segment model in which segments were connected together at nodal points . to compute the joint moments ,
joints coordinate data were added to the ground reaction force data , in which the position of the center of mass , the weight portion , and the moment of inertia of each segment were used as parameters .
the measurement data reported by winter16 were used as the body parameters necessary for calculating the lbm .
three - dimensional trunk and pelvic angles were calculated by the eulerian method using coordinate systems as determined by markers on the trunk and pelvis , respectively .
in this study , we defined lbm and bilateral erector spinae activities among these parameters as low back load because lbm and es activities indicate the rotation force around the low back joint and the action of the low back muscles , respectively .
peak values of kinetic and kinematic data acquired during level walking were extracted from the phase between mid - stance and terminal stance ( mts ) , and the pre - swing phase in one gait cycle of the right limb because it was not possible to calculate the lbm when a subject s posterior foot did not contact the force plates ( figure 3 ) .
mean peak values of lbm , three - dimensional trunk and pelvic angles , and integral emgs were calculated from the data obtained in three trials and were selected as representative values for analysis .
comparison was performed using repeated measures analysis of variance ( anova ) after confirming non - deviation of the data and performing the kolmogorov
smirnov test , and variables showing a significant difference were subjected to multiple comparisons with bonferroni correction .
from 31 community - dwelling elderly subjects who were candidates for this study , 15 were enrolled ( all males ; mean age , 67.76.1 years ; mean height , 162.45.7 cm ; mean weight , 62.37.8 kg ) after excluding those with neurological disease , pain , history of an orthopedic surgical procedure , history of orthopedic treatment within the past 5 years , and history of lbp within the past 1 year .
the study subjects were the same as those of our previous study.9 the study was approved by the ethics committee of the international university of health and welfare ( 11191 ) .
the orf is shown in figure 1 and its features are described in our previous study.8 briefly , pelvic and upper supports are positioned on the ileum and sternum , respectively .
stainless steel joints , connected to the upper support with a nylon pad and to the pelvic support , produce resistive force through the use of extension springs .
a link mechanism translates the spring - generated tension into a resistive moment on the chest and a reaction moment on the posterior pelvis .
the orf has a release mechanism that releases the resistive force by pulling tension levers downward .
the orf is currently an investigational product that has not been approved by the food and drug administration or by a corresponding national agency for the indication described herein .
the subjects walked 10 m on a level surface at a self - selected speed in a laboratory setting under three conditions : without an orthosis , with the orf , and with an lso ( damen corset , pacific supply , osaka , japan ) .
the damen corset was selected as it is frequently prescribed for patients with lbp . after completing three walking trials without any orthosis
the subjects were allowed 5 minutes to accustom themselves to wearing the orf and the lso .
resistive force on the chest provided by the joints was measured in real time using a strain gauge ( kyowa , tokyo , japan ) and the force data were transferred to a laptop computer by bluetooth ( figure 2 ) and the force was set to a magnitude of 2025 n during static standing .
the pressure between the corset and abdomen was set to 10 mmhg in all measurement conditions.12
gait was recorded with a three - dimensional motion capture system ( vicon 612 , vicon , oxford , uk ) consisting of six force plates ( four from amti , watertown , ma , usa ; and two from kistler , winterthur , switzerland ) and 12 infrared ( ir ) cameras with a sampling rate of 120 hz . referring to a study by seay et al,13 41 ir - reflective markers ( diameter , 14 mm ) were attached to each subject s body .
additionally , three markers were attached over a strain gauge and on bilateral joints of the orf . to measure muscle activity during level walking , electromyograms ( emgs )
were obtained ( biometrics , newport , uk ) at a sampling rate of 1,080 hz for bilateral erector spinae ( 2 cm to the side between l4l5 vertebrae).14 maximum voluntary contraction was measured while one physical therapist manually applied resistant force to the midpoints of the bilateral scapulae , with the subject lying in the prone position on a bed .
during acquisition , we performed full - wave rectification feeding into a band pass filter ( 20420 hz ) to decrease noise and used visual 3d analytical software ( c - motion , germantown , md , usa ) .
the obtained emgs were normalized using maximal voluntary contraction during isometric contraction ( as a percent ) , and the root mean square was calculated for a 50 ms window .
subjects performed isometric contractions in prone against gravity with maximum resistance applied by the experimenter to obtain maximal voluntary contraction of the erector spinae.15 visual 3d was used to perform kinetic and kinematic data analysis .
the obtained physical coordinates and ground reaction force data were low - pass filtered with a second - order recursive butterworth filter , with a cutoff frequency of 6 and 18 hz , respectively , according to winter s technique.16 the link segment model consisted of 13 segments : head , trunk , pelvis , bilateral upper arms , forearms , thighs , shanks , and feet . briefly , the low back extension and flexion moments were calculated using the ground reaction force data obtained from the force plates , the reaction force on the chest obtained from the strain gauge , and the coordinates of the ir - reflective markers on the bodies of the subjects and an orf .
moment exerted by an orf was calculated by multiplying the force measured by a strain gate and moment arm from joint of an orf to the force . in our previous study analyzing the orf effect during static standing,9 we were not able to calculate lbm .
the novelty of the present study lies in applying a new technique to calculate lbm during level walking while wearing an orf .
the moment was subtracted from the lbm calculated by using the ground reaction force data and the coordinates of the ir - reflective markers on the bodies of the subjects because the moment created by orf joints equally gives forward rotation moment on the pelvis because of action reaction law . in the analysis
, segments were regarded as rigid and the joint moments were calculated using a link segment model in which segments were connected together at nodal points . to compute the joint moments ,
joints coordinate data were added to the ground reaction force data , in which the position of the center of mass , the weight portion , and the moment of inertia of each segment were used as parameters .
the measurement data reported by winter16 were used as the body parameters necessary for calculating the lbm . three - dimensional trunk and pelvic angles were calculated by the eulerian method using coordinate systems as determined by markers on the trunk and pelvis , respectively . in this study
, we defined lbm and bilateral erector spinae activities among these parameters as low back load because lbm and es activities indicate the rotation force around the low back joint and the action of the low back muscles , respectively .
peak values of kinetic and kinematic data acquired during level walking were extracted from the phase between mid - stance and terminal stance ( mts ) , and the pre - swing phase in one gait cycle of the right limb because it was not possible to calculate the lbm when a subject s posterior foot did not contact the force plates ( figure 3 ) .
mean peak values of lbm , three - dimensional trunk and pelvic angles , and integral emgs were calculated from the data obtained in three trials and were selected as representative values for analysis .
comparison was performed using repeated measures analysis of variance ( anova ) after confirming non - deviation of the data and performing the kolmogorov
smirnov test , and variables showing a significant difference were subjected to multiple comparisons with bonferroni correction .
as shown in tables 1 and 2 , all kinematic and kinetic parameters differed significantly among the three conditions . averaged lbm waveforms for all subjects in the three conditions are shown in figure 3 .
the peak flexion moment was observed at the beginning of pre - swing and the peak extension moment at the end of pre - swing . averaged waveforms of lbm and resistive force on the chest while wearing the orf are shown in figure 4 .
force exerted on the chest was 3437 n , which occurred during mts and pre - swing .
significant main effects of an orthosis were observed for the peak flexion and extension moments not only in pre - swing but also in mts ( table 1 ) .
peak extension moments in mts and pre - swing were significantly smaller with the orf than in the other two conditions .
the peak flexion moment in mts was significantly larger with the orf than with the lso ; and in pre - swing was significantly larger with the orf than in the other two conditions .
averaged waveforms of bilateral erector spinae activity for all subjects in the three conditions are shown in figure 5 .
significant main effects of an orthosis were observed in the integral of bilateral erector spinae activity during stance ( table 1 ) .
significant main effects of an orthosis were observed in peak pelvic forward tilt angles in mts and pre - swing , and in the peak pelvic leftward rotation angle in pre - swing ( table 2 ) .
peak pelvic forward tilt angles in mts and pre - swing were significantly larger with the orf than in the other two conditions .
the peak pelvic leftward rotation angle was significantly smaller with the orf than with the lso .
significant main effects of an orthosis were observed in peak thoracic extension angles in mts and pre - swing , and in peak right and left lateral flexion angles in pre - swing .
peak extension angles in mts and pre - swing were significantly larger with the orf than in the other two conditions .
the peak right lateral bending angle in pre - swing was significantly larger with the orf than with the lso .
the peak left lateral bending angle in pre - swing was significantly smaller with the orf than in the other two conditions .
walking velocity was 1.090.10 m / sec without an orthosis , 1.130.12 m / sec with the orf , and 1.180.11 m / sec with the lso .
the lso was significantly faster than without an orthosis ( p=0.017 ) but was not significantly different from with the orf .
averaged lbm waveforms for all subjects in the three conditions are shown in figure 3 .
the peak flexion moment was observed at the beginning of pre - swing and the peak extension moment at the end of pre - swing .
averaged waveforms of lbm and resistive force on the chest while wearing the orf are shown in figure 4 .
force exerted on the chest was 3437 n , which occurred during mts and pre - swing .
significant main effects of an orthosis were observed for the peak flexion and extension moments not only in pre - swing but also in mts ( table 1 ) .
peak extension moments in mts and pre - swing were significantly smaller with the orf than in the other two conditions .
the peak flexion moment in mts was significantly larger with the orf than with the lso ; and in pre - swing was significantly larger with the orf than in the other two conditions .
averaged waveforms of bilateral erector spinae activity for all subjects in the three conditions are shown in figure 5 .
significant main effects of an orthosis were observed in the integral of bilateral erector spinae activity during stance ( table 1 ) .
significant main effects of an orthosis were observed in peak pelvic forward tilt angles in mts and pre - swing , and in the peak pelvic leftward rotation angle in pre - swing ( table 2 ) .
peak pelvic forward tilt angles in mts and pre - swing were significantly larger with the orf than in the other two conditions .
the peak pelvic leftward rotation angle was significantly smaller with the orf than with the lso .
significant main effects of an orthosis were observed in peak thoracic extension angles in mts and pre - swing , and in peak right and left lateral flexion angles in pre - swing .
peak extension angles in mts and pre - swing were significantly larger with the orf than in the other two conditions .
the peak right lateral bending angle in pre - swing was significantly larger with the orf than with the lso .
the peak left lateral bending angle in pre - swing was significantly smaller with the orf than in the other two conditions .
walking velocity was 1.090.10 m / sec without an orthosis , 1.130.12 m / sec with the orf , and 1.180.11 m / sec with the lso .
the lso was significantly faster than without an orthosis ( p=0.017 ) but was not significantly different from with the orf .
we hypothesized that both the lso and orf would effectively decrease low back load during level walking in healthy elderly subjects but the orf would have a superior effect .
there were no significant differences between without an orthosis and with the lso in the peak lbm and integral emg of the erector spinae , or in the pelvic and thoracic angles .
however , significant differences in all these parameters were observed with the orf compared with no orthosis .
moreover , the low back extension moment and emg of the erector spinae were significantly decreased with the orf . collectively , these results suggest that wearing the orf during level walking should help to decrease the low back load in elderly people .
several studies have suggested that using an lso could stabilize the lumbosacral region but not decrease the low back load during static standing or lifting.10 our results indicate that the same applies during level walking also .
interestingly , the orf not only decreased the activity of the erector spinae ( which has higher fatigability in patients with lbp17 ) and the low back extension moment , but also increased the low back flexion moment created by the abdominal muscles .
the biomechanical function of the orf can be explained by a simple model ( figure 6 ) .
the orf can produce an extension moment for the upper trunk that decreases the low back extension moment .
this extension moment also produces a resistive moment on the posterior pelvis ; together , these moments could improve the malalignment commonly seen in elderly people ( ie , lumbar kyphosis with pelvic backward tilt ) .
such malalignment increases both lbp and fall risks.18,19 the extension moment served to extend the upper trunk , and the reaction moment acted as a forward rotation moment for the pelvis . in this way
, the orf increased the peak pelvic forward tilt angle and peak thoracic extension angle through mts and pre - swing .
this function , which can modify alignment in the elderly , would also be beneficial for decreasing the peak lbm , because modifications to the positioning of the pelvis and thorax are directly linked to a decrease in the lever arm from the low back joint to the head , arm , and trunk center of gravity ( figure 6 ) .
a systematic review investigating the activation pattern of trunk muscles during walking in subjects with and without lbp indicated that those with lbp exhibit higher es activity compared with asymptomatic subjects.20 the magnitude of the decreases in erector spinae activity and the low back extension moment while wearing an orf is relatively small .
however , in daily life , people walk a large number of steps , so even though only a small decrease in low back load was evident with the orf , the cumulative difference might have a distinct effect that can help treat and prevent lbp.21 the spinal bones of elderly people , and particularly those of patients with osteoporosis , are more fragile than the middle - aged or young .
typical orthoses would not show a biomechanical effect of decreasing low back load during level walking because most correct only the abdominal region with compressive force or support the pelvis , thorax , and lower back with small resistive force .
only one eccentric type of orthosis , the rucksack - type orthosis , was found to decrease es activity in elderly people during level walking in a previous study.22 the rucksack - type orthosis controls the magnitude of force using weights to move the center of gravity of the upper body , thereby decreasing es activity .
this function is similar to that of the orf , because both orthoses can control a relatively large magnitude of force applied to the upper trunk ; however , the rucksack - type orthosis has the disadvantage of increasing low back compressive force in proportion to the amount of weight , which directly increases the gravitational force on the upper trunk .
however , the orf can apply resistive force horizontally to the chest , thereby avoiding an increase of low back compressive force .
an interesting feature of the orf compared with other orthoses , including the rucksack - type , is its ability to increase the low back flexion moment produced by the abdominal muscles ; the horizontally applied resistive force on the chest can not only decrease activity of the low back extension muscles using the support force , but also activate the abdominal muscles .
the peak moment was larger with the orf in mts than with the lso and in pre - swing than in the two other conditions .
rostami et al reported that using an lso for 4 and 8 weeks decreased deep abdominal muscle thickness.23 although we did not examine deep abdominal muscle activity in this study , these muscles might contribute to increasing the low back flexion moment .
this effect of the orf would be beneficial for exercises aimed at improving abdominal muscle function during level walking in elderly subjects . as we previously found a positive training effect of the orf on such function in hemiparetic patients,8
a potentially negative feature of the orf is that it limits pelvic rotation , which is one of the aspects of gait that increases step length and walking velocity.24 although walking velocity with the orf was not faster than that without an orthosis , it was significantly faster with the lso than without an orthosis .
thus , the stabilization of the pelvis and thorax achieved with an lso might improve gait performance in elderly subjects , and the limited pelvic rotation with the orf might decrease its overall positive effect for improving gait performance .
the orf during level walking served to decrease low back muscle activities and joint moment , and this might be effective in the prevention and treatment of lbp .
only low back extension and flexion moments in the three axial moments were calculated when wearing the orf and therefore low back compressive force , which is a strong indicator of low back load , could not be calculated because the strain gauge measured the orthogonal resistive force on the chest produced by the orf s joints .
finally , only healthy elderly male subjects participated in this study , and only a within - subject trial was conducted .
future studies should include healthy elderly female subjects and subjects who have lbp , and randomized controlled trials should be conducted .
this study has several limitations . wearing the orf during level walking served to decrease low back muscle activities and joint moment , and this might be effective in the prevention and treatment of lbp .
only low back extension and flexion moments in the three axial moments were calculated when wearing the orf and therefore low back compressive force , which is a strong indicator of low back load , could not be calculated because the strain gauge measured the orthogonal resistive force on the chest produced by the orf s joints .
finally , only healthy elderly male subjects participated in this study , and only a within - subject trial was conducted .
future studies should include healthy elderly female subjects and subjects who have lbp , and randomized controlled trials should be conducted .
in conclusion , we demonstrated that the orf can decrease low back load during level walking in healthy elderly people by significantly decreasing lbm and increasing the abdominal moment .
the orf is a promising device for the prevention and treatment of lbp , and we plan to conduct randomized controlled trials with people who have lbp in the future . | purposethe effects of lumbosacral and spinal orthoses on low back pain and gait are not exactly clear .
we previously developed a trunk orthosis with joints providing resistive force on low back load to decrease such load , and confirmed its positive effects during level walking in healthy young adults .
therefore , we aimed to determine the efficacy of this trunk orthosis during level walking in healthy elderly subjects.methodsfifteen community - dwelling elderly subjects performed level walking at a self - selected speed without an orthosis , with our orthosis , and with a lumbosacral orthosis .
kinematic and kinetic data were recorded using a three - dimensional motion analysis system , and erector spinae activity was recorded by electromyography.resultswhen comparing the three conditions , our orthosis showed the following effects : it decreased the peak extension moment , increased the peak flexion moment , decreased the lateral bending angle , increased the peak thoracic extension angle , and had significantly lower erector spinae activity and significantly larger peak pelvic forward tilt angles.conclusionour orthosis with joints providing resistive force decreased low back load and modified trunk and pelvis alignments during level walking in healthy elderly people . | Background
Materials and methods
Subjects
Features of the ORF
Experimental conditions
Experimental setup
Data analysis
Statistical analysis
Results
Low back load
Pelvic and thoracic angles
Gait performance
Discussion
Limitations
Conclusion |
PMC4283739 | various factors affect bond failure including poor operator technique , enamel surface texture , salivary contamination of the prepared enamel surface , bracket properties , masticatory forces and patient s behavior [ 58 ] .
there are also a number of factors affecting bracket retention during orthodontic treatment such as the condition of enamel surface .
soft drinks are commonly used by the adolescents and this habit often continues into adulthood .
soft drinks can be carbonated or noncarbonated ; both types are harmful due to their high sugar content and low level of ph ; which is below the critical level required for enamel demineralization ( ph 5.5 ) .
it is obvious that fermentable carbohydrates can cause caries , which would be even more severe in presence of fixed orthodontic appliances due to increased plaque accumulation .
the most important factors influencing erosion during orthodontic treatment include oral hygiene , diet and orthodontic bonding procedure .
more erosion develops under the influence of carbonated soft drinks rather than noncarbonated ones , due to the presence of carbonic acid . in developed countries ,
coca - cola is the most consumed carbonated drink ( 50% ) , followed by lemon flavored ( 22% ) and orange flavored drinks ( 7% ) .
dough , a yogurt - based , salty drink and non - alcoholic beer are two popular drinks in the iranian diet .
soft drink consumption during orthodontic treatment reduces bracket retention due to enamel softening around brackets [ 10 , 11 ] or degradation of adhesive /composite interface [ 9 , 12 ] ; it may also increase the microleakage beneath the brackets and compromise the bond strength .
microleakage around brackets may contribute to the development of white spot lesions under the brackets [ 19 , 20 ] .
in addition , during fixed orthodontic treatment residual adhesives around the brackets result in more plaque accumulation and increase the risk of decalcification ; therefore , orthodontic patients are advised against consumption of soft drinks during treatment .
omid khoda et al . compared the effects of yogurt - based drinks , 7 up and pepsi on the shear bond strength of orthodontic brackets .
however , information is lacking on the impact of non - alcoholic beer on the bond strength of orthodontic brackets .
the purpose of this study was to determine the effects of two types of soft drinks , coca - cola and a non - alcoholic beer ( istak ) on the shear bond strength and ari of orthodontic metal brackets in vitro .
eighty extracted non - carious human premolars of orthodontic patients were used in this study .
the teeth were cleaned with fluoride - free pumice and the buccal surface was etched with 37% phosphoric acid for 30 seconds , rinsed with water for 15 seconds and air dried with oil - free compressed air until the frosty appearance of enamel appeared . a layer of transbond xt primer ( 3 m , unitek , monrovia , ca . ,
usa ) was applied to each tooth and then light cured ( kerr , le demetron ii , usa ) for 10 seconds with an intensity of 800 mw / cm .
transbond xt adhesive paste was then applied to the base of the metal bracket ( standard edge wise.018 , dentaurum germany ) .
the bracket was centered on the crown of the tooth mesiodistally and along the long axis of the tooth , and was then pressed firmly on the tooth surface .
excess adhesive was removed with a sickle scaler and the adhesive was light cured from four sides of the bracket edge , each for 10 seconds .
all samples were kept in artificial saliva at 37 c for 24 hours to allow complete polymerization of resin .
the specimens were then randomly divided into three groups :
-group 1 ( coca - cola ) : included 30 teeth , which were submerged in coca - cola for 5 minutes twice a day , with equal intervals .
samples were kept in artificial saliva at 37 c .- group 2 [ a non - alcoholic beer , istak ( arianoosh , rasht , iran ) ] : included 30 teeth , which were immersed in istak following the same procedures as for group 1 .
istak is a soft beverage made of malt extract , hops , sugar and carbonated water.coca-cola and istak were stored in a refrigerator .- group 3 ( control ) : included 20 teeth , which were kept in artificial saliva [ 400 mg nacl , 1.21 mg kcl , 780 mg nah2po42h2o , 5 mg na2s.9h2o , 1000 mg co(nh2)2 and 1000 ml of distilled water ] during the 30 days .
-group 1 ( coca - cola ) : included 30 teeth , which were submerged in coca - cola for 5 minutes twice a day , with equal intervals .
after each session the solution was replenished . for the remainder of the time , samples were kept in artificial saliva at 37 c. -group 2 [ a non - alcoholic beer , istak ( arianoosh , rasht , iran ) ] : included 30 teeth , which were immersed in istak following the same procedures as for group 1 .
istak is a soft beverage made of malt extract , hops , sugar and carbonated water .
-group 3 ( control ) : included 20 teeth , which were kept in artificial saliva [ 400 mg nacl , 1.21 mg kcl , 780 mg nah2po42h2o , 5 mg na2s.9h2o , 1000 mg co(nh2)2 and 1000 ml of distilled water ] during the 30 days .
after 30 days , all teeth were mounted vertically in acrylic blocks measuring 202020 mm ( figure 1 ) so that the load could be applied to the bracket - tooth interface parallel to the buccal tooth surface .
the shear test with a crosshead speed of 0.5 mm / min was performed on the samples with the universal testing machine ( z050 , zwick / roell , ulm , germany ) .
the load at bond failure was recorded using a pc connected to the testing machine .
the load at failure was recorded in newton ( n ) and the stress was calculated in mega pascal ( 1mpa= n / mm ) by dividing the force in newton by the area of the bracket base .
the area of the bracket base was calculated by measuring the width and length of ten bracket bases with a digital caliper accurate to 0.01 mm , which was found to be 10.23 mm .
after debonding , each tooth was examined under a stereomicroscope at 12 magnification and the fraction of the remained adhesive on the tooth surface was scored ( figure 2 ) .
the possible values for the ari were categorized as follows :
-score 1 : 0 to less than 1/6 of adhesive left on the tooth ,- score 2 : 1/6 to less than 2/6 of adhesive left on the tooth ,- score 3 : 2/6 to less than 3/6 of adhesive left on the tooth ,- score 4 : 3/6 to less than 4/6 of adhesive left on the tooth ,- score 5 : 4/6 to less than 5/6 of adhesive left on the tooth ,-
-score 1 : 0 to less than 1/6 of adhesive left on the tooth , -score 2 : 1/6 to less than 2/6 of adhesive left on the tooth , -score 3 : 2/6 to less than 3/6 of adhesive left on the tooth , -score 4 : 3/6 to less than 4/6 of adhesive left on the tooth , -score 5 : 4/6 to less than 5/6 of adhesive left on the tooth , -score 6 : 5/6 to 6/6 of adhesive left on the tooth . during preparation ,
8 samples were lost and there were 26 , 28 and 18 teeth remaining in coca - cola , istak and control groups , respectively .
the data of shear bond strength were statistically analyzed by one - way anova and tukey s post - hoc test and the data of ari scores were analyzed by kruskal - wallis test .
in this study , the shear bond strength and ari of orthodontic brackets in the three groups of coca - cola , non - alcoholic beer ( istak ) and control were evaluated . the mean shear bond strength ( sbs ) and standard deviation of the three groups are shown in table 1 .
the mean sbs was 19.27 6.53 mpa in coca - cola group , 25.36 9.77 mpa in istak group and 25.47 10.02 mpa in the control group .
one - way anova showed a statistically significant difference in sbs of the three groups ( p=0.022 ) .
post - hoc test was used to evaluate the significance of the difference in sbs of each group with the other groups .
this test revealed that coca - cola had significantly lower sbs than istak and the control group .
however , there was no statistically significant difference in sbs of istak and the control group .
the location of bond failure for each sample was evaluated with the ari score and a summary of the data is shown in table 2 .
all three groups showed a similar bond failure mode ( adhesive ) at the enamel - adhesive interface being the most common site of bond failure in the three groups ( 01/6 of adhesive was left on the tooth surface ) .
kruskal - wallis test indicated that the distribution of ari score was similar in the three groups ( p 0.552 ) .
patients diet and the acidity of foods and drinks can impact on shear bond strength of orthodontic brackets [ 912 ] .
recently , consumption of soft drinks has increased in children and adolescents ; these drinks have a negative impact on tooth structure .
the current study was conducted to evaluate the effects of two soft drinks , coca - cola and istak , on the shear bond strength and adhesive remnant index of orthodontic metal brackets , in vitro .
the shear bond strength of brackets was tested using a universal testing machine at a crosshead speed of 0.5 mm / min , similar to what was performed in the studies of gillis and redlich , mascia and chan and oncag .
the present study showed a significant reduction in the shear bond strength of orthodontic brackets in the coca - cola group compared to istak and control groups ; whereas there was no significant difference in the sbs of istak and the control groups .
some previous studies also showed a significant reduction in sbs of brackets exposed to coca - cola .
reported a decrease in the sbs of brackets in the coca - cola and sprite groups in both in vitro and in vivo conditions .
the study of ulusoy et al . revealed a decrease in bracket sbs following the use of coca - cola and rosehip fruit tea , which was suggested to be due to their low ph . in the present study , istak - a soft beverage made of malt extract , hops , sugar and carbonated water -
the reason might be the absence of phosphoric acid in this drink in contrast to coca - cola ; which contains phosphoric acid .
the similarity between our study and those of oncag and ulusoy was that all three experimented on human premolars .
although there were differences in the frequency and duration of exposure to drinks , they all reported a significant reduction in sbs in the coca - cola group . in studies by oncag and ulusoy samples were immersed in soft drinks 3 times a day for 5 minutes during 90 days . in our study , the teeth were immersed in soft drinks twice a day for 5 minutes during 30 days . according to oncag et al .
demineralization around brackets was caused by the acidic soft drinks negatively affecting the bracket - enamel bonding .
on the other hand , softening or degradation of the adhesive/ composite resin interface impairs the interlock at the bracket - adhesive resin - enamel interfaces and may also cause bond failure .
it has been reported that the absorption of acids and acidic drinks may degrade the structure of bisphenol a glycidyl methacrylate or bis - gma based composite resins [ 24 , 9 ] ; the resin matrix is softened and fillers leach out , decreasing the bracket bond strength . according to arikan et al , the presence of microleakage at enamel - adhesive interface is critical for the appearance of white spot lesions and for the formation of caries , while microleakage at the adhesive - bracket interface is related to bond failure .
van eygen et al , in 2005 showed that even short periods of drinking soft drinks ( coca - cola ) reduces enamel microhardness .
hunter et al . evaluated the effect of the frequency of exposure to soft drinks , twice in contrast to four times , and reported that the amount of tissue loss was not proportional to the frequency of exposure to low ph drinks .
owen et al . dipped the samples in drinks for 24 hours for a total of 14 days and observed enamel surface modifications ; which appeared as irregularities in enamel morphology visualized under light and scanning electron microscopes .
even short duration of exposure to soft drinks has adverse effects on the structure and microhardness of enamel .
the results of navarro and omid khoda were different from those of oncag , ulusoy and ours .
navarro did not indicate a significant difference in sbs and ari of coca - cola , schweppes lemon and control groups .
this difference , at least in part , may be due to the use of bovine teeth instead of human teeth . another reason may be the fact that their specimens were not thermocycled ; thermocycling has been proven by some researchers to reduce bond strength between 2070% [ 29 , 30 ] .
although the samples in the present study and ulusoy s were not thermocycled , sbs reduction in the coca - cola group was reported to be significant ; therefore , thermocycling might not be a critical factor in sbs change .
evaluated and compared the effects of acidic soft drinks with a phosphoric acid base such as pepsi , with a citric acid base like 7up , and lactic acid base like carbonated and noncarbonated yoghurt drinks on the shear bond strength of orthodontic brackets .
such different findings may be attributed to the type of composite used in the mentioned studies . the composite used in oncag s ,
ulusoy s and our studies were transbond xt ( light cure ) , but in omid khoda s study , unite ( self cure ) was used ; thus , it may be concluded that unite composite is more resistant to acid than transbond xt .
the mean shear bond strength in our study was much higher than that of previous studies and it was also higher than the suggested clinically acceptable bond level by reynolds and von fraunhofer ; which might probably be due to the lower frequency and duration of exposure to soft drinks compared to other studies or in - vitro condition of the study . according to littlewood et al , bond failure can occur within the bracket , at the bracket - adhesive interface , within the adhesive or at the tooth surface - adhesive interface .
in our study , ari was utilized to determine the extent of residual adhesive on the enamel surface following bracket debonding .
there was no significant difference in ari among the three groups ( coca - cola , istak and control ) .
in fact , in all three groups , a high fraction of samples had less than 1/6 of the adhesive remained on their surfaces .
there was no significant correlation between the sbs and ari in any of the three groups .
although the sbs of the coca - cola group was less than that of the two other groups , the dominant mode of ari in all three groups was similar .
navarro et al . expressed an absence of correlation between ari and sbs in tested groups .
ulusoy et al , in evaluation of the effects of herbal teas reported a significant difference in ari of coca - cola group ( positive control ) with that of other groups .
samples of coca - cola group produced the most consistent separation at the enamel - adhesive interface , leaving the enamel surface intact ( ari score 0 ) .
for all other groups , the majority of bond failures were ari score 1 that is at the enamel - adhesive interface .
ari score 1 means that more adhesive materials are adhered to the bracket base and less adhesive materials remain on the tooth surface .
it was surprising that although both coca - cola and rosehip fruit tea reduced sbs , the mode of bond failure in coca - cola and rosehip fruit tea groups was different .
ari scores were 0 and 1 in coca - cola and rosehip fruit tea groups , respectively .
therefore , the absence of correlation between the sbs and ari in ulusoy s study was consistent with the present study .
presence of low residual adhesive on tooth surface reduces the clean up time , is less troublesome for patients and less harmful to structural integrity of the enamel .
the present study was designed to simulate oral environment as closely as possible ; however , there were still differences left , including varying oral temperature due to diet and absence of bacterial plaque .
evaluated the effects of acidic soft drinks on sbs both in vivo and in vitro and observed that there was no significant difference in sbs of brackets of the same group in vivo and in vitro .
although the difference was not significant , more extensive enamel defects were observed in the in vivo groups compared to the in vitro groups under the scanning electron microscopy .
steffen stated that bacteria of the oral cavity accelerate enamel erosion following exposure to acidic soft drinks . according to the results of the present study ,
istak did not decrease the sbs of orthodontic metal brackets in vitro , in contrast to coca - cola .
further investigations are suggested to evaluate the effects of istak , a kind of non - alcoholic beer , on enamel structure and bonded brackets in vitro and in the oral environment .
this investigation revealed that :
coca - cola significantly decreased the shear bond strength of orthodontic brackets.there was no significant difference in the shear bond strength of brackets between the istak and the control group.adhesive remnant index was not affected by different soft drinks.there was no correlation between the shear bond strength and the adhesive remnant index in any of the three groups .
there was no significant difference in the shear bond strength of brackets between the istak and the control group .
there was no correlation between the shear bond strength and the adhesive remnant index in any of the three groups . | objective : bond failure of brackets during orthodontic treatment is a common problem ; which results in treatment interference , increased treatment time and prolonged clinical time for rebonding of failed brackets . the purpose of this study was to evaluate the effects of coca - cola and a non - alcoholic beer on the shear bond strength and adhesive remnant index ( ari ) of orthodontic metal brackets in vitro.materials and methods : eighty intact human premolars were divided into two experimental groups of coca - cola and non - alcoholic beer ( istak ) , and a control group of artificial saliva . over a period of thirty days , the test groups were immersed in the respective soft drinks for 5 minutes , twice a day . for the remainder of the time , they were kept in artificial saliva at 37c .
the control group was stored in artificial saliva during the experiment .
all samples were subjected to shearing forces using universal testing machine .
ari was determined with a stereomicroscope at 12 magnification .
the data of shear bond strength were statistically analyzed by one - way anova and tukey s post - hoc test and the data of ari scores were analyzed by kruskal - wallis test.results:no significant difference was observed in aris of the three groups ( p 0.552 )
. the shear bond strength of coke group was significantly lower than that of the two other groups ( p 0.035 ) ; but there was no significant difference between the shear bond strength of istak and the control group ( p 0.999).conclusion : coca - cola decreased the shear bond strength of orthodontic brackets . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
PMC3303734 | ppi networks have been widely used to understand biology at the system level [ 13 ] .
network module , a group of proteins that are connected with each other to carry out a function , will be more accurate because a loss or gain of interaction will not break down the module structure .
modules have been applied to predict protein function and disease genes and trace the evolutionary history of networks [ 810 ] . to perform complex biochemical or developmental functions
for example , three modules in s. cerevisiae the set3c complex , protein phosphatase type 2a ( pp2a ) complex , and cell polarity budding share a protein : zds1 .
zds1 can bind pp2a to control mitotic progression , and it also participates in set3c complex during budding processes and repress meiotic process , so zds1 may serve as a bridge between mitosis and meiosis . here
we define these three modules as overlapping modules and define the shared protein as the overlapping nodes .
construction of such network can be helpful for understanding the coordinated relationship between different biological processes .
the problem of identifying modules has been studied by bioinformatics , applied mathematics , and physics .
many methods have been developed to identify modules within a network , and they have been reviewed and evaluated [ 1519 ] .
( 1 ) local seed - based methods which start from a node or clique ( fully connected subgraph ) and follow by an expanding search strategy .
mcode is the first method for module detecting , and it expands highly scoring seed nodes by a local search procedure . but
cfinder is the first algorithm for overlapping communities detection , and it develops a clique percolation method ( cpm ) where k - cliques are explored by rotating about its component ( k-1)-cliques .
cfinder is too slow when applied to dense ppi networks , and particularly it can not detect spoken - like module ( noncliques ) . to overcome this problem , zhang et al .
combines the line graph transformation ( lgt ) and cpm to detect overlapping network modules and builds the overlapping modules network .
wu et al . proposes coach ( core - attachment based method ) to predict complexes by detecting protein - complex core and then adding attachments .
the local protein community finder , lpcf for short , uses two local clustering algorithms to find a community close to a queried protein .
combine a neighbour - sharing score with hierarchical agglomerative clustering to identify both dense network and dense bipartite network structures in a single approach .
reichardt and bornholdt propose a method to detect overlapping ( fuzzy ) communities that maps the graph onto a zero - temperature q - potts model with nearest - neighbor interactions .
combine the idea of modularity function q , spectral relaxation , and fuzzy c - means clustering method for detecting overlapping community structure .
propose a bcd ( betweenness - commonality decomposition ) algorithm which uses edge commonality and edge - betweenness .
other methods such as nonnegative matrix factorization ( nmf ) technique were also used for uncovering overlapping ( fuzzy ) communities [ 27 , 28 ] . besides these topological - based methods , chen and yuan
integrate 265 microarray datasets to detect functional modules in yeast protein - protein interaction network . here
we describe mofinder , an alternative method we have developed that can effectively identify functional modules , especially overlapping modules , from a ppi network .
we used all the overlapping modules detected from human ppi network to generate a graph of module - module communication , and we analyzed the functional properties of the overlapping modules .
cancer genes ( tumor suppressor and oncogene ) and immunome [ 33 , 34 ] were used to annotate cancer- and immune - related proteins .
clustering coefficient of node n is defined as cc(n ) = 2sn / kn(kn 1 ) , where kn is the degree of n and sn is the number of connected links between all neighbors of n. given a predicted module , the p - value of it with respect to a go term is computed by the hypergeometric distribution in ( 1 ) and corrected by bonferroni correction . the functional module is defined as a module enriched in at least one go term ( bonferroni p - value < 0.01 ) :
( 1)p - value=(mk)(nmnk)(nn ) ,
where a predicted complex with size m , k proteins share a go term , and in a total of n proteins , n of them have the same go term . assuming go1 = { go11 , go12 , , go1 m } and go2 = { go21 , go22 ,
, go2n } are two sets of go terms that annotate modules a and b , respectively , the following jaccard index was used to calculate the functional similarity between modules a and b :
( 2)sim(a , b)=|go1go2||go1go2| .
mofinder is based on an amd ( approximate minimum degree ordering ) algorithm [ 35 , 36 ] which has been used for network clustering from electrical engineering .
amd algorithm is usually used in ordering a sparse matrix prior to cholesky factorization ( or for lu factorization with diagonal pivoting ) , and it can transform the sparse matrix to make the nonzero elements close to the diagonal . the approach used by mofinder is summarized in figure 1 .
mofinder first converts the ppi file into a sparse matrix , where a nonzero element represents a protein - protein interaction .
it then performs a global amd of the sparse matrix in which the densely connected elements ( module ) will be clustered along the diagonal .
besides the global amd , which produces the global ordering , a local amd is performed to give the approximate minimum degree ordering .
mofinder uses a sliding window along the diagonal to fetch the local sparse matrix and make the local amd .
the clustering coefficient ( cc ) value of the submatrix in the sliding window is calculated ; if the cc value is not less than the cut - off , mofinder will save the submatrix as a module .
then the sliding window moves one step along the diagonal to find new modules , and the iteration process is repeated until the sliding window reaches the end .
lastly , mofinder removes redundant modules ( if module a belongs to module b , a is removed ) and saves results .
mofinder contains two adjustable parameters : the cc cut - off value and the size of sliding window .
the performance was assessed in term of accuracy of identified modules with respect to annotated function .
mofinder was tested over a broad range of parameters for cc cut - off value ( 0.21 ) and sliding window ( 20450 ) using ppi data from yeast and human .
first , the percentage of functional modules was plotted against a range of cc cut - off values , and for each cc cut - off value , all sizes of sliding window ( 20450 , step = 10 ) were tested and the resulting percentages of functional modules were plotted as a group of points . as shown in figure 2 , the percentage of functional modules increases with the increase of cc cut - off value , and it is observed to have 4 distinct and stable ranges for values of cc cut - off , [ 0.2,0.5 ) , [ 0.5,0.67 ) , [ 0.67,0.84 ) , and [ 0.84,1 ] , respectively . although the highest percentage of functional modules is achieved in the last range ( cc cut - off value [0.84,1 ] ) , using cc cut - off value of this range will identify densely connected complex and ignore other modules .
additionally in this range , mofinder only generates a small number of modules ( e.g. , it predicts , on average , 36 modules from human ppi network when cc cut - off = 0.84 ) .
since the purpose is to detect modules instead of complex , we recommended that the suitable setting of threshold would be in the third range ( cc cut - off value [0.67,0.84 ) ) .
the best choice for cc cut - off value is 0.67 because the number of predicted modules decrease with cc cut - off value ( data not shown ) .
figure 3 shows the number of functional modules matched for the 0.67 cut - off value over all tried sizes of sliding window ( 20450 , step = 10 ) .
so the sliding window should be set to 350 which maximized the number of functional modules .
to achieve best performance , we recommended that the parameter set was cc cut - off value = 0.67 and size of sliding window = 350 .
mofinder was tested using ppi data from yeast and human and compared with the performance of other five software available algorithms : mcode ( default parameters ) , cfinder ( k = 4 , as suggested ) , coach ( default parameters ) nemo ( default parameters ) , and lpcf ( community size was set to 311 which was comparable to mofinder ) .
the percentage of functional modules was used to indicate accuracy , and mofinder was the top performing algorithm with respect to accuracy in yeast ( 93.9% ) ( figure 4(a ) ) and human ( 81.5% ) ( figure 4(b ) ) . also , we compared the major module size of six methods in yeast ( table 1 ) and in human ( see supplementary table 1 in supplementary material available online at doi:10./155/2011/103702 ) .
most of the modules detected by mcode are of size 3 , size 4 for cfinder , size 3 for coach , size 4 for nemo , size 10 for lpcf , and size 5 for mofinder . although the number of modules and the number of proteins assigned to modules were smaller for mofinder than some of these methods , the percentage of functional modules was highest for mofinder .
we applied mofinder to the yeast and human ppi network with default parameters ( cc cut - off = 0.67 , sliding window = 350 ) .
as shown in figure 5 , the overlapping size distribution is different between yeast and human .
most of the modules in yeast ppi network share one protein ( figure 5(a ) ) , but in human ppi network the most common overlapping size is 4 ( figure 5(b ) ) .
for example , three modules ( a , b , and c ) share a protein d , so protein d is counted 3 times ( a - b , a - c , b - c ) . to avoid the overcount problem , we deleted the repeats , so protein d is only counted once .
figure 6(a ) shows that the resulting distribution of overlapping size in yeast is obviously changed , and the most common overlapping size changes into 4 which is similar to human ( figure 6(b ) ) .
these observations suggest that although modules in yeast tend to share less proteins than modules in human , the small overlapping parts ( size 1 and size 2 ) are more repeatedly used in yeast than human , and thus the distribution of overlapping size becomes similar in yeast and human after removing repeats .
since proteins in one module work together to perform functions , a similar function is expected to appear if two modules are overlapping with each other . and the larger the overlapping size , the more likely the same function .
to verify this , we used the go annotation similarity to represent the functional similarity .
figure 7 shows that the average functional similarity is increased with the increase of overlapping size .
such a trend has been observed in both yeast ( figure 7(a ) ) and human ( figure 7(b ) ) .
mofinder identified 221 modules , of which 152 were overlapped with at least one other module .
these overlapped modules were used to construct a module - module communication network ( figure 8(a ) ) . in the communication network , each node is a module , two modules being connected if they share at least one protein . to explore the functional of this network , we used david 6.7 [ 40 , 41 ] to search for enrichment of gene ontology ( go ) terms and the kegg pathways .
we found that go terms and pathways related to cancer and immune response were enriched in the network proteins , so we mapped the cancer and immune - related proteins to the modules .
as shown in figure 8(a ) , of the 47 modules containing immune - related proteins , 33 included cancer - related proteins , and the ratio ( 33/47 ) was greater than expected by chance ( 62 of 152 modules have cancer - related proteins , binomial test , p < 0.01 ) .
therefore , the modules containing immune - related proteins always included cancer - related proteins and vice versa ( 33/62 was greater than expected 47/152 , binomial test , p < 0.01 ) . to explore the communication between functional modules , we map the functional annotation to each module and evaluate the functional similarity between two overlapping modules . the functional similarity is shown as edge color in figure 8 : the values between 0 and 1 are painted with a pink / blue color gradient , and modules without go annotation have gray edges .
figure 8(b ) gives the functional annotation of modules from the largest cluster in figure 8(a ) .
some overlapping modules have the same function , such as the three modules involve in the acetylation of peptidyl - lysine , while several overlapping modules have distinct function , for instance , a module involved in the change of mast cell is overlapping with another module which takes part in the reactions mediated by protein kinases .
one module function is in b - cell activation processes and it contains five proteins : q15464 , o75791 , o43561 , q13094 , and p08575 .
the other module ( p08575 , p20963 , p06729 , and p06127 ) involves in t - cell activation .
these two modules share a protein : p08575 ( receptor - type tyrosine - protein phosphatase c , cd45 ) , which plays a critical role in receptor - mediated signalling in both b and t - cells [ 42 , 43 ] .
consistent with this hypothesis , several studies have illustrated t - cell - dependent b - cell activation .
the module - module communication network included 341 overlapping nodes ( nodes belonging to two or more modules ) .
several studies showed that modular overlaps are potential drug targets because they are key determinants of cooperation between network modules .
so we investigated the potential druggability of overlapping nodes : 56 of them were established drug targets and another 43 proteins were from druggable family , which were 99 druggable proteins in all .
the ratio of druggable proteins ( 99/341 ) was significantly higher than expected ( 20003000 druggable proteins in human , binomial test , p < 0.01 ) .
for both yeast and human interactomes , mofinder surpasses the other five methods in accuracy . furthermore , mofinder is fast in practice for large networks .
for example , when applied to a yeast network including nearly 40,000 interactions ( from i2d ) , the running time of mofinder was only 15 seconds .
since the size of biological networks continues to grow , mofinder is likely to meet the needs of biological analysis .
one is that mofinder specifically detects small - sized modules ( less than 12 ) , but the major module size ( 5 ) is close to the average size of mips complexes ( 6 ) .
mofinder detects 125 modules from the yeast ppi network , which is less than coach and lpcf . from the perspective of the covered proteins of predicted modules ,
these observations suggest another limitation : mofinder is of too strict to detect loosely connected modules , partly because the cc cut - off value is set to 0.67 .
we suppose that setting the cc cut - off value to a small value can increase the number of detected modules especially loosely connected modules ( including pathways ) .
but what is the biological significance of the different clustering coefficient thresholds is still an open question .
yeast is a simple single - celled eukaryote , so the overlapping modules in yeast generally use one protein for communication . on the contrary , human , a multicellular organism , employs more complex system , and thus the overlapping size of human is larger than that of yeast .
we also found the overall distribution of overlapping size is similar between yeast and human after removing repeats .
and in figure 2 the functional steps occur at similar places between yeast and human .
although overlaps may lead to redundant modules which overlap with each other heavily , excluding the overlapping size 4 ( a heavily overlap because the major module size is 5 ) from figures 5 , 6 , and 7 does not change the overall pattern of results .
overlapping modules will work together to carry out several complicated jobs , such as signal transduction .
so constructing a module - module communication network to explore how these modules communicate with each other can help to understand biological complexity .
although we just built such a network in human , similar approach can be applied to other species .
we found that the immune- and cancer - related proteins are always in the same modules .
the association between immune cells and cancer has been discussed , and several clinical studies and experiment have proven that the immune system is a new weapon against cancer .
antitumor adaptive immune responses can suppress tumor growth , and several immunotherapy drugs could cure cancers .
in this paper , we describe a novel algorithm for the identification of overlapping modules in ppi networks .
mofinder performs competitively with other methods and uses two adjustable parameters that enable it to identify modules flexibly .
mofinder is a cross - platform package which is implemented as a c / c++ script , and it can be downloaded and installed free of charge ( http://bsb.kiz.ac.cn/mofinder/ ) . the application of mofinder to human ppi gives clues for fighting against cancer using immune system . and
the overlapping nodes , which are in charge of intermodule crosstalk , could help to identify potential drug targets . | since organism development and many critical cell biology processes are organized in modular patterns , many algorithms have been proposed to detect modules . in this study ,
a new method , mofinder , was developed to detect overlapping modules in a protein - protein interaction ( ppi ) network .
we demonstrate that our method is more accurate than other 5 methods .
then , we applied mofinder to yeast and human ppi network and explored the overlapping information . using the overlapping modules of human ppi network
, we constructed the module - module communication network .
functional annotation showed that the immune - related and cancer - related proteins were always together and present in the same modules , which offer some clues for immune therapy for cancer .
our study around overlapping modules suggests a new perspective on the analysis of ppi network and improves our understanding of disease . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
PMC55326 | the complete genomic sequences of four eukaryotic organisms have been reported ( drosophila melanogaster , caenorhabditis elegans , saccharomyces cerevisiae and arabidopsis thaliana ) .
these genome sequences have been used to demonstrate the utility of comparative genomics in discerning evolutionary as well as possible functional relationships within protein superfamilies [ 1 , 2 ] . with the recent addition of the complete human genome sequence available within the human genome project database ( > 94% sequence available as of february 2001 )
nrs appear to be restricted to metazoans , in which they have key roles in integrating the complexities of homeostasis and development .
in general , nrs function as transcriptional regulators , working in concert with coactivators and corepressors to activate and suppress target gene expression .
the transcriptional activities of about half of the nrs studied in humans are regulated by small lipophilic ligands whereas the other , so - called orphan , receptors await ligand identification .
because of their potential for modulation by exogenous compounds and their central roles in metabolism , these receptors are extremely important targets in human disease .
additionally , nrs represent possible new targets for the control of invertebrate pests in agriculture . the latter approach would be especially promising if certain classes of nrs are shown to be specific to selected invertebrate subgroups .
comparison of complete sets of nrs from evolutionarily divergent organisms should tell us more about the feasibility of these approaches as well as shed light on general phylogenetic relationships among nrs and among species . for the past year
when the c. elegans genome sequence was reported , over 220 nr members were found , and subsequent sequence releases have brought the number of predicted c. elegans nr genes to 270 ( a.s . , unpublished data ) .
this dramatic increase over the number of currently published human nrs ( 48 ) led to speculation that the human nr set could also expand dramatically .
surprisingly , only 21 total nrs were found in the recently reported drosophila genome sequence .
an intriguing question now is whether the total set of human nrs will reflect the diversity seen in c. elegans or instead will parallel that found in drosophila .
we have developed a genomic sequence analysis pipeline utilizing blast searches followed by hmmer domain analysis to identify nr sequences within the human genome .
domain analysis was facilitated by the knowledge that the nr superfamily is unified by a common modular structure .
one hallmark structure that characterizes the family is a dna - binding domain ( dbd ) characterized by two c4-type zinc fingers contained in the amino - terminal half of the proteins .
a second characteristic feature , the ligand - binding domain ( lbd ) , is found at the distal carboxyl terminus and contains a highly conserved transcriptional transactivation function ( af2 ) .
the complete known complement of human nrs was used as a query set to identify candidate novel nr sequences from public human genome databases .
identified candidate sequences were followed up with more detailed bioinformatic and , when warranted , molecular biology analysis . using this approach , we identified two novel nr sequences .
the closest homologs of these sequences were represented by fxr ( nr1h4 ) and hnf4 ( nr2a2 ) .
the fxr - related gene sequence ( fxr - r ) was mapped in silico to chromosomal position 1p13.1 - 1p13.3 , distinct from the chromosomal location of fxr ( 12q23.1 - 20 ) .
interestingly , the positions of the introns within fxr - r were at the same relative positions within the coding sequence as in fxr , suggesting a close evolutionary relationship between these two sequences .
the predicted coding sequence of fxr - r displayed similarity to fxr across nearly the entire length ( 48% sequence identity at the amino acid level ) but contained multiple stop codons ( figure 1a ) .
the sequences of multiple stop codons were confirmed by pcr amplification and subsequent sequencing of fxr - r genomic dna fragments ( see materials and methods ) .
sequence analysis thus indicated that this gene does not code for a functional nr and is likely to be a pseudogene .
surprisingly , real - time quantitative pcr ( rtq - pcr ) detected relatively high levels of expression of fxr - r mrna in testis ( data not shown ) indicating that this gene is a transcribed pseudogene .
the nucleotide sequences from complete and ordered clone al358372 contained eight fragments with amino acid sequence similarity with fxr ( from 5'-3 ' relative to fxr - r , nucleotide positions 13144 - 13480 ; 15053 - 15199 ; 21541 - 21672 ; 21781 - 21879 ; 22015 - 22115 ; 23681 - 23795 ; 24486 - 23795 ; 27041 - 27262 ) .
seven frameshifts ( indicated by asterisks ) were required to preserve the reading frame of hnf4-r relative to hnf4. intron locations in the hnf4 gene are boxed .
the second novel nr gene ( hnf4-r ) was mapped in silico to chromosome position 13q14.11 - 13q14.3 , unlinked to the known hnf4 gene at position 12q12 .
the hnf4-r sequence showed sequence similarity across nearly the entire length of the coding region of hnf4 ( 71.4% sequence identity at the amino acid level ) .
like fxr - r , hnf4-r coding sequence contained multiple stop codons ( figure 1b ) and thus also appears to represent a pseudogene .
nine frame - shifts were necessary to maintain the amino acid reading frame relative to hnf4. the predicted hnf4-r sequence was confirmed by sequence analysis of human genomic dna ( see materials and methods ) .
the predicted coding sequence of hnf4-r was contiguous within the genome , consistent with possible retrotransposition into the genome .
unlike fxr - r , no expression of hnf4-r mrna was detected in any of the tissues examined ( data not shown ) .
only one other nr pseudogene has been reported to date , a pseudogene related to the err receptor .
the identification of fxr - r and hnf4-r brings the total human nr pseudogene number to three .
further evidence that these genes are pseudogenes includes the fact that no homologs of the hnf4-r and fxr - r genes could be found in available mouse , rat , fugu , or drosophila genome sequences .
pseudogene sequences would not be expected to be conserved between genomes even as closely related as human and mouse .
in addition , and in contrast to their closest functional gene homologs hnf4 and fxr , hnf4-r or fxr - r did not display conservation of their amino acid sequences relative to their nucleic acid sequences .
fxr - r and hnf4-r were found using searches that utilized other known human nrs as query sequences .
it remained possible , then , that nr led sequences existed in the human genome that represented homologs of c. elegans nrs but were not identified using mammalian nrs as a query set . to address this question
, we scanned the human genome with the complete sets of c. elegans and drosophila nrs .
extensive analysis using these receptors as query sequences did not reveal a single novel mammalian homolog of these sequences ( no blast hits with p value < 10 ) . from our analysis
, we conclude that the human nr set will not be expanded by orthologs resembling the large number of nrs found in c. elegans .
phylogenetic analysis of vertebrate nrs has defined six ancestral nr subfamilies [ 13 , 14 , 15 ] .
five of the subfamilies are also represented among both the c. elegans and drosophila nrs ( figure 2 ) , consistent with the proposed ancient metazoan origin of these subfamilies .
the earlier analysis identified no arthropod or nematode members of the nr3 subfamily , suggesting that this subfamily might be more recently derived and specific to the deuterostome lineage .
more recently , however , the drosophila genome sequence has revealed a previously unknown nr sequence ( cg7407 ) that falls into the nr3 subfamily ( figures 2,3 ) .
the observation that nr3 is represented in both protostomes and deuterostomes indicates that nr3 , like the other major nr subfamilies , is of an ancient metazoan origin .
. it will be of interest to learn if nr3 is represented in any nematode species , as absence of the nr3 subfamily from nematodes in general would suggest that arthropods and vertebrates may share an evolutionary history that occurred after separation of the nematode lineage .
such an early divergence of the nematode evolutionary lineage would be in disagreement with a recent hypothesis placing nematodes and arthropods in a common evolutionary clade of molting invertebrates and would be more consistent with the traditional placement of nematodes in a lineage that diverged from other metazoans before separation of the major protostome and deuterostome lineages .
relationships within the completed sets of nr superfamily members from humans , c. elegans and drosophila .
a neighbor - joining tree of nr dna - binding domain sequences was generated using the paupsearch feature of the gcg 10.1 program package ( 1,000 bootstrap replicates , midpoint rooting ) ; analysis methods are described in detail in sluder et al . .
significant bootstrap support values are indicated by slashes on the appropriate branches : ( / ) 50 - 79% ; ( // ) 80 - 94% ; ( /// ) 95% ; branches also supported by parsimony analysis are marked by dots ; subfamilies are boxed .
all human ( hs ) and drosophila ( dm ) nrs are included , except for the two human nrs that lack a canonical dbd ( dax-1 ( nr0b1 ) and shp ( nr0b2 ) ) .
the c. elegans ( ce ) sequences include all members of the six major metazoan nr subfamilies as well as selected representatives of the major groupings of divergent c. elegans nrs evident in a larger tree containing all the nematode sequences .
amino acid alignment of drosophila cg7404 with the sequences of human err , err , and err. dmcg7404 exhibits similarity to the human errs in both dna- and ligand - binding domain sequences .
block boxes indicate residues identical in at least two of the four sequences ; gray boxes indicate similar residues .
expression of cg7407 as mrna and the splicing pattern has been confirmed by the recovery and sequencing of cdna ( k.k .
et al . , unpublished data ) . clearly , dramatically divergent evolutionary pathways have shaped the nr sets in separate phylogenetic lineages .
within the six major nr subfamilies , four groups of nrs are currently known only in vertebrates ( thyroid hormone receptors ( tr ) , peroxisome proliferator - activated receptor ( ppar ) , retinoic acid receptors ( rar ) and the steroid receptor group containing glucocorticoid receptor ( gr ) , mineralocorticoid receptors ( mr ) , progesterone receptor ( pr ) and androgen receptors ( ar ) ) .
in addition , both invertebrate genomes encode nrs that are not clearly placed in one of the six defined nr subfamilies ( figure 2 ) .
as previously noted , the three drosophila members of the knirps group define an unusual class of nrs that lack similarity to the lbds of the vertebrate nrs .
most of the c. elegans nrs ( 255 of 270 ) are diverged from those found in humans and flies ( figure 2 ) .
it is unclear whether these divergent nematode nrs represent new subfamilies or are highly diverged members of one or more of the six recognized subfamilies .
in contrast to the situation with the insect knirps group , analyses of potential structures indicates that the majority of the divergent c. elegans receptors are predicted to contain the canonical antiparallel -helix sandwich structure characteristic of ligand - regulated nrs ( a. bogan , c. maina , j .-
chandonia , f. cohen , k. yamamoto , and a. sluder , unpublished data ) .
thus , despite the extensive diversity in sequence of many c. elegans lbd sequences , they are unified by a common structural fold , possibly reflecting the requirement for interaction with a core set of nr cofactors .
since the known structures of nr lbds contain a hydrophobic cleft in which their endogenous hormone ligands are bound , it is likely that most , if not all , orphan receptors will be amenable to modulation by small molecules . in sum , we have found a striking difference between humans , drosophila and c. elegans with respect to their nr sets . there is a finite possibility that the last 5% of the human genome sequence could harbor an additional novel nr sequence , but this is unlikely
such a finding would not change the general conclusion that there are striking differences between the three genomes .
knowledge of all the members of each nr set defines the unique landscape for nr modulation and provides a basis for more detailed phylogenetic studies .
furthermore , such a whole - genome comparison of the types and numbers of genes only reflects one level of nr complexity in an organism .
the impact of transcriptional and post - transcriptional processing events on total nr functional diversity in each proteome will be a subject for future studies .
protein homology searches were performed using individual members of the nr family compared against the human genome database ( htg section from genbank ) using tblastn .
homologous sequences ( those with a blast expectation value of 0.1 or lower ) were then compared with the entire query nr data set using blastn . by choosing the criteria on the basis of automated assessment for 95% sequence identity over 200 base pairs ( bp ) at the nucleotide level , we could map these hits to individual members of the protein family ( ' bins ' ) .
those sequence hits that could not automatically be placed in bins on the basis of the selected criteria were either mapped to other protein families ( if their sequences matched a known gene in genbank ) , considered as potentially novel nr sequences ( when key domains were present ) or deemed irrelevant ( no match to anything ) .
we then applied hidden markov models ( hmms ) built on the dbd and lbd domains using the hmmer package to these potential novel sequences .
the hmm models were used for further substantiation or for ruling out potential novel hits .
first , the hmm model for nr dbd ( zf-c4.hmm from pfam ) domain was used to search the c. elegans protein database ( assembled in house from genbank ) using the hmmer package , and identified 252 c. elegans sequences .
these 252 c. elegans sequences were compared to the human genomic sequence database by the search strategy described above .
second , the above process was repeated for the drosophila genome and again no novel human nr sequences were found .
third , the majority of the c. elegans nrs were grouped into ten subfamilies based on comparative sequence analysis of the dna - binding domain sequences ( a.s . ,
these hmms were then used to search the database of predicted drosophila proteins ( established in - house by extracting from genbank ) with the hmmer package .
the significant hits ( score < 0.1 ) were compared with the 21 known drosophila nrs .
no novel drosophila nr sequences were found . with each new update of the human genomic database ,
the system initiated a new round of searching and filtering , capturing all sequences that were homologous and using a master bin list to filter out sequences that had been annotated in a previous cycle .
this eliminated the need to separate out all new sequences in the database at each update .
a java applet was used as the user 's main interface to the analysis engine and the underlying oracle database .
it was embedded in an html page from which users could view the sequence record by linking to the in - house and public databases as well as all the sequence and domain alignments .
as validation of our bioinformatic search tools , 48 of the 49 known nr sequences in the genome ( including the err pseudogene ) were found .
the gene representing the car nr ( nr1i3 ) is still absent from high - throughput genomic sequence databases .
confirmation of in silico nucleotide sequence was performed by sequencing fxr - r and hnf4-r genomic dna fragments amplified by pcr .
pcr primers were designed using genbank accession numbers al358372 ( fxr - r ) and al138997 ( hnf4-r ) .
fxr - r : 5'-gtt gag tgg aaa tgt gag ag -3 ' and 5'-gta gga atg ttg gca gaa tg -3 ' , product size 566 bp , hnf4-r ; 5'-agc ttg att tgt agc tgt tc -3 ' and 5'-tcc tct ctg ggg cag aa-3 ' product size 1,205 bp .
these primers were used to amplify 100 ng clontech genomic dna using amplitaq gold ( pe biosystems ) using standard pcr amplification conditions .
human tissues were derived from snap frozen surgical specimens ( duke university , durham , nc ) .
some total rnas were obtained from commercial vendors such as clontech ( palo alto , ca ) and zen - bio ( research triangle park , nc ) . to maximize the sensitivity of the quantitation of transcripts
effectiveness of the dnase treatment was confirmed by running pcr with human -actin primers in the absence of reverse transcriptase , to ensure no signal was detected .
the rna was then quantitated using the rna - specific ribogreen dye ( molecular probes ) , and 25 ng aliquoted into a 96-well plate format .
to quantitate mrna expression , rtq - pcr was performed using an abi prism 7700 sequence detection system instrument and software ( pe applied biosystems ) as described [ 20 , 21 ] .
gene - specific primers were synthesized ( keystone , camarillo , ca ) for the sequences fxr - r ( accession number al358372 ) and hnf4-r ( accession number al138997 ) .
protein homology searches were performed using individual members of the nr family compared against the human genome database ( htg section from genbank ) using tblastn .
homologous sequences ( those with a blast expectation value of 0.1 or lower ) were then compared with the entire query nr data set using blastn . by choosing the criteria on the basis of automated assessment for 95% sequence identity over 200 base pairs ( bp ) at the nucleotide level , we could map these hits to individual members of the protein family ( ' bins ' ) .
those sequence hits that could not automatically be placed in bins on the basis of the selected criteria were either mapped to other protein families ( if their sequences matched a known gene in genbank ) , considered as potentially novel nr sequences ( when key domains were present ) or deemed irrelevant ( no match to anything ) .
we then applied hidden markov models ( hmms ) built on the dbd and lbd domains using the hmmer package to these potential novel sequences .
the hmm models were used for further substantiation or for ruling out potential novel hits .
first , the hmm model for nr dbd ( zf-c4.hmm from pfam ) domain was used to search the c. elegans protein database ( assembled in house from genbank ) using the hmmer package , and identified 252 c. elegans sequences .
these 252 c. elegans sequences were compared to the human genomic sequence database by the search strategy described above .
second , the above process was repeated for the drosophila genome and again no novel human nr sequences were found .
third , the majority of the c. elegans nrs were grouped into ten subfamilies based on comparative sequence analysis of the dna - binding domain sequences ( a.s . ,
these hmms were then used to search the database of predicted drosophila proteins ( established in - house by extracting from genbank ) with the hmmer package .
the significant hits ( score < 0.1 ) were compared with the 21 known drosophila nrs .
no novel drosophila nr sequences were found . with each new update of the human genomic database ,
the system initiated a new round of searching and filtering , capturing all sequences that were homologous and using a master bin list to filter out sequences that had been annotated in a previous cycle .
this eliminated the need to separate out all new sequences in the database at each update .
a java applet was used as the user 's main interface to the analysis engine and the underlying oracle database .
it was embedded in an html page from which users could view the sequence record by linking to the in - house and public databases as well as all the sequence and domain alignments .
as validation of our bioinformatic search tools , 48 of the 49 known nr sequences in the genome ( including the err pseudogene ) were found .
the gene representing the car nr ( nr1i3 ) is still absent from high - throughput genomic sequence databases .
confirmation of in silico nucleotide sequence was performed by sequencing fxr - r and hnf4-r genomic dna fragments amplified by pcr .
pcr primers were designed using genbank accession numbers al358372 ( fxr - r ) and al138997 ( hnf4-r ) .
fxr - r : 5'-gtt gag tgg aaa tgt gag ag -3 ' and 5'-gta gga atg ttg gca gaa tg -3 ' , product size 566 bp , hnf4-r ; 5'-agc ttg att tgt agc tgt tc -3 ' and 5'-tcc tct ctg ggg cag aa-3 ' product size 1,205 bp .
these primers were used to amplify 100 ng clontech genomic dna using amplitaq gold ( pe biosystems ) using standard pcr amplification conditions .
human tissues were derived from snap frozen surgical specimens ( duke university , durham , nc ) .
some total rnas were obtained from commercial vendors such as clontech ( palo alto , ca ) and zen - bio ( research triangle park , nc ) . to maximize the sensitivity of the quantitation of transcripts
effectiveness of the dnase treatment was confirmed by running pcr with human -actin primers in the absence of reverse transcriptase , to ensure no signal was detected .
the rna was then quantitated using the rna - specific ribogreen dye ( molecular probes ) , and 25 ng aliquoted into a 96-well plate format .
to quantitate mrna expression , rtq - pcr was performed using an abi prism 7700 sequence detection system instrument and software ( pe applied biosystems ) as described [ 20 , 21 ] .
gene - specific primers were synthesized ( keystone , camarillo , ca ) for the sequences fxr - r ( accession number al358372 ) and hnf4-r ( accession number al138997 ) . | backgroundthe availability of complete genome sequences enables all the members of a gene family to be identified without limitations imposed by temporal , spatial or quantitative aspects of mrna expression . using the nearly completed human genome sequence
, we combined in silico and experimental approaches to define the complete human nuclear receptor ( nr ) set .
this information was used to carry out a comparative genomic study of the nr superfamily.resultsour analysis of the human genome identified two novel nr sequences .
both these contained stop codons within the coding regions , indicating that both are pseudogenes .
one ( hnf4 -related ) contained no introns and expressed no detectable mrna , whereas the other ( fxr - related ) produced mrna at relatively high levels in testis .
if translated , the latter is predicted to encode a short , non - functional protein .
our analysis indicates that there are fewer than 50 functional human nrs , dramatically fewer than in caenorhabditis elegans and about twice as many as in drosophila . using the complete human nr set we made comparisons with the nr sets of c. elegans and drosophila .
searches for the > 200 nrs unique to c. elegans revealed no human homologs .
the comparative analysis also revealed a drosophila member of nr subfamily nr3 , confirming an ancient metazoan origin for this subfamily.conclusionsthis work provides the basis for new insights into the evolution and functional relationships of nr superfamily members . | Background
Results and discussion
Materials and methods
Computational identification of human NR sequences
Genomic DNA analysis of novel human NR sequences
mRNA expression analysis |
PMC4156265 | exposure to ionizing radiation has dramatically
increased in modern
society , raising serious health concerns .
ionizing radiation is widely used in medicine for research , diagnosis ,
and therapy ; it is also used in manufacturing and construction .
in addition , accidental exposure to ionizing radiation occurs during
air travel and space missions .
the deleterious consequences of ionizing
radiation exposure can result in cancer and non - cancer - related diseases , including cardiovascular diseases and cognitive decline .
it has therefore
become a priority to develop new tools that can effectively and rapidly
screen populations for radiation exposures .
methods for rapid
and efficient biological dosimetry could find
applications not only for estimating the risk of disease induced by
medical , occupational , or accidental exposure to radiation but also for countermeasures .
radiological incidents
like the recent fukushima explosion on december 2013 underscore the
need for a quick and reliable way to identify exposed individuals
in the days immediately following a radiological accident so that
medical management can begin as soon as possible thereafter .
while
cytogenetics remains the gold standard for assessing radiation exposure ,
such a technique might not be sensitive enough to detect subtle molecular
changes evoked by radiation exposure before the occurrence of cellular
and organ damage , nor does it give insight into signaling pathways
triggered by ionizing radiation .
radiation biology has traditionally
focused on dna damage and repair ,
mutation induction , and chromosomal rearrangements . until recently
,
comparatively little attention was paid to other molecular consequences
of irradiation that may underlie the risk of pathology .
in addition
to interacting with genomic dna , however , ionizing radiation may alter
the structure and function of key cellular components such as proteins
and lipids , activating a pro - inflammatory response and ultimately
affecting cell signaling .
recent radiation research
is , therefore , beginning to adopt a systems biology approach to investigate
the pleiotropic effects of exposure to ionizing radiation on multiple
cellular components .
metabolic phenotyping is a modern analytical approach that
uses
state - of - the - art instrumentation such as mass spectrometry to characterize
the molecular composition ( i.e. , the phenotype ) of biofluids . to date ,
metabolic phenotyping investigations in radiation research have mainly
focused on biomarker discovery in urine .
the capability to phenotype blood ,
however , provides novel opportunities to investigate the molecular
response to radiation , thus yielding mechanistic insights as well
as discovering novel biomarkers .
evidence indeed suggests that blood
factors induced by irradiation may reflect and contribute to an ongoing
inflammatory response to the initial radiation - induced injury . here
, we applied a multiplatform mass
spectrometry - based metabolic
phenotyping strategy to investigate in mouse serum the molecular response
to acute -radiation exposure and describe a lipidomic biosignature
that has the potential to be used as an indicator of radiation exposure
and as a potential target for therapeutic interventions .
all chemicals were purchased from sigma - aldrich
( seelze , germany ) and were of analytical - grade purity or higher .
lipid
standards were purchased from avanti polar lipids ( alabaster , al ,
usa ) , cayman chemical ( ann arbor , mi , usa ) , biomol ( plymouth meeting ,
pa , usa ) , and larodan fine chemicals ( malm , sweden ) . for solid - phase
extraction , 96-well , waters oasis hlb plates ( 60 mg ) were obtained
from waters corporation ( milford , ma , usa ) .
n = 5 ) were irradiated
at georgetown university with 8 gy of rays ( cs source , 1.67 gy / min ) , corresponding to an exposure to radiation
that is expected to kill half of the exposed population within 30
days ( ld50/30 ) .
sham control male c57bl/6 mice ( 810
weeks old ; n = 5 ) were treated the same way as irradiated
mice .
in particular , they were transported to the irradiator at the
same time , introduced into the irradiation pie , and spun in the irradiator ,
minus the radiation exposure .
serum was collected using serum separators ( bd biosciences , ca ,
usa ) .
all experimental conditions and
animal handling were carried out in accordance with animal protocols
approved by the georgetown university animal care and use committee
( guacuc ) .
total metabolites were extracted
from serum samples by 1:40 dilution in 66% acetonitrile containing
2 m debrisoquine sulfate and 30 m 4-nitrobenzoic acid
as internal controls . following centrifugation at 13 000 g , the supernatant was stored at 80 c for further
analysis .
metabolites were separated using an acquity uplc system
( waters corporation , milford , ma , usa ) fitted with a beh hilic 2.1
100 mm , 1.7 m column maintained at 30 c .
mobile phase a consisted of 95:5 acetonitrile / water
( v / v ) containing 10 mm ammonium acetate ( ph 8.0 ) ; mobile phase b consisted
of 50:50 acetonitrile / water ( v / v ) containing 10 mm ammonium acetate
( ph 8.0 ) . a 10 min linear gradient from 100 to 80% a with a 3 min
re - equilibration time was applied .
ms analyses were performed on a
synapt g2-s mass spectrometer ( waters corporation , wilmslow , uk ) acquiring
from 50 to 1500 m / z in positive
electrospray ionization mode .
data were
collected in two channels all of the time : low collision energy ( 6.0
v ) , for the molecular ions , and high collision energy ( 1540
v ) , for product ions .
the ion mobility spectrometry ( ims ) gas was
nitrogen , and the ims t - wave velocity and height were 900 m / s and
40 v , respectively .
lipids were extracted from 25 l of
serum following protein precipitation using 100 l of 2:1 chloroform / methanol
( v / v ) .
samples were vortex - mixed and centrifuged for 10 min at 10 000 g and 4 c .
the bottom phase was collected , dried ,
and resuspended in 100 l of 50:50 acetonitrile / isopropanol
( v / v ) for further ms analysis .
total lipid analysis was performed
on an ionkey / ms system composed of an acquity uplc m - class , the ionkey
source , and an ikey csh c18 , 130 , 1.7 m ( particle size ) ,
150 m 100 mm column ( waters corporation , milford , ma ,
usa ) coupled to a synapt g2-si mass spectrometer ( waters corporation ,
wilmslow , uk ) .
the capillary voltage was 2.8 kv , and the source temperature
was 110 c .
injections were 0.5 l using the partial - loop
mode , and the column temperature and flow rate were 55 c and
3 l / min , respectively . mobile phase a consisted of 60:40 acetonitrile / water
( v / v ) with 10 mm ammonium formate + 0.1% formic acid .
mobile phase
b consisted of 90:10 isopropanol / acetonitrile ( v / v ) with 10 mm ammonium
formate + 0.1% formic acid .
the gradient was programmed as follows :
0.02.0 min from 40% b to 43% b , 2.02.1 min to 50%
b , 2.112.0 min to 99% b , 12.012.1 min to 40% b , and
12.114.0 min at 40% b. a 3 min re - equilibration time was applied .
ms analyses were performed , acquiring from 50 to 1500 m / z in positive electrospray ionization mode with
a capillary voltage of 2.8 kv .
data were collected in two channels
all of the time : low collision energy ( 6.0 v ) , for the molecular ions ,
and high collision energy ( 1540 v ) , for product ions .
the
ims gas was nitrogen , and the ims t - wave velocity and height were
900 m / s and 40 v , respectively .
metabolites were extracted
from mice sera using a specific 96-well plate system for protein removal ,
internal standard normalization , and derivatization using the absolute
idq p180 kit ( biocrates life sciences ag , innsbruck , aut ) .
the methods
have been described in detail , and the preparation
was performed according to the user manual included with the kit .
briefly , 10 samples ( n = 5 sham - irradiated group
and n = 5 irradiated group ) were added to the center
of the filter on the upper 96-well kit plate , at 10 l per well ,
and then dried using a nitrogen evaporator .
subsequently , 50 l
of a 5% solution of phenylisothiocyanate was added for derivatization
of the amino acids and biogenic amines .
the metabolites
were extracted using 300 l of a 5 mm ammonium acetate solution
in methanol and transferred by centrifugation into the lower 96-well
plate .
the extracts were diluted with 600 l of the ms
running solvent for further ms analysis using uplc coupled with the
xevo tq - s mass spectrometer ( waters corporation , wilmslow , uk ) .
one
blank sample ( i.e. , no internal standards and no sample added ) , three
water - based zero samples ( phosphate - buffered saline ) , and three quality - control
samples were also added to the kit plate .
the quality control samples ,
composed of human serum containing metabolites at several concentration
levels , were used to verify the performance of the assay and mass
spectrometer .
a seven - point serial dilution of calibrators was added
to the kit s 96-well plate to generate calibration curves used
to quantify biogenic amines and amino acids .
the kit included a mixture
of internal standards for quantifying the natural metabolites : chemical
homologous internal standards were used to quantify glycerophospholipid
and sphingomyelin species , whereas stable isotope - labeled internal
standards were used to quantify the other compound classes .
the amount
of internal standards was identical in each well , and the internal - standard
intensities of zero sample and sample wells were compared to allow
conclusions on ion - suppression effects to be made .
acylcarnitines ,
glycerophospholipids , and sphingolipids were subjected to flow - injection
analysis ( fia ) using uplc - xevo tq - s in positive mode .
hexose was analyzed
using a subsequent fia acquisition in negative electrospray ionization
mode .
amino acids and biogenic amines were analyzed using an acquity
uplc system connected to a xevo tq - s mass spectrometer ( waters corporation ,
wilmslow , uk ) in positive electrospray ionization mode ( table s1 ) .
extraction of oxylipins
was performed as previously described . briefly , after thawing on ice , 50 l of serum samples was
treated immediately with antioxidants ( 0.2 mg butylated hydroxytoluene
( bht / edta ) ) and spiked with the following internal standards : 6k - pgf1-d4 , txb2-d4 , pgf2-d4 , pge2-d4 , pgd2-d4 , lte4-d3 , ltb4-d4 , 12,13
dihome - d4 , 9,10-dihome - d4 , 14,15-dihetre - d11 , 15-deoxy--12,14-pgj2-d4 , 20-hete - d6 , 9s - hode - d4 , 12s - hete - d8 , 5s - hete - d8 , 5-oxo - ete - d7 .
serum samples were loaded onto the solid - phase
extraction plate and then eluted using 1.5 ml of ethyl acetate after
wetting the plate wells with 0.5 ml of methanol .
the eluent was reduced
under nitrogen and subsequently reconstituted in a 50 l solution
of 50:50 methanol / acetonitrile ( v / v ) containing 100 nm 1-cyclohexyluriedo-3-dodecanoic
acid , a quality marker for the analysis . oxylipins were analyzed
using uplc ( waters corporation , milford , ma , usa ) coupled to electrospray
ionization on a xevo tq - s mass spectrometer ( waters corporation , wilmslow ,
uk ) .
the autosampler was cooled to 10 c . for each analysis ,
3 l of sample was injected onto a beh c18 1.7 m , 2.1
100 mm column ( waters corporation , milford , ma , usa ) maintained
at 40 c .
mobile phases were composed
as follows : a = 0.1% acetic acid and b = 90:10 ( v / v ) acetonitrile / isopropanol
( 0.001.00 min from 25% b to 33% b , 1.018.00 min b
to 95% , 8.018.50 min to 95% b , 8.5110.00 min to 25%
b ) .
electrospray ionization was performed in the negative - ion mode
applying a capillary voltage of 2 kv , a source temperature of 120
c , a desolvation gas temperature of 350 c , a desolvation
gas flow of 650 l / h , a cone - gas flow of 150 l / h , a collision voltage
of 15 v , and a cone voltage of 35 v. oxylipins were detected by means
of multiple reaction monitoring ( mrm ) transitions optimized using
synthetic standards ( table s2 ) .
for global molecular profiling ,
samples were acquired as ion maps ( retention time versus m / z ) using data - independent analysis ( ms ) , which provided information for both the intact precursor ions
( at low collision energy ) and the fragment ions ( high collision energy ) .
after retention - time alignment , an aggregate run representing the
compounds in all samples was generated .
this aggregate run was used
for peak picking and then propagated to all runs to detect the same
ions in each .
a combination of analysis of the variance ( anova ) including
principal component analysis ( pca ) and partial , least - squares discriminant
analysis ( pls - da ) identified metabolites most responsible for differences
between sample groups .
these searches were performed by the human metabolome database ( hmdb ) and lipidmaps and by fragmentation patterns ,
retention times , and ion - mobility - derived collision cross sections
versus commercially available reference standards , when available .
for quantification purposes , mean concentrations and sem values for
each group were calculated using mrm transition and appropriate internal
standards to normalize for variations in sample preparation and ms
detection .
univariate analyses ( student s t - test ) were conducted to assess for significance ( p value < 0.05 was considered significant ) , and false - discovery
rate ( fdr ) was used to control for multiple comparison .
data
processing and analysis was conducted using progenesis qi ( nonlinear
dynamics , newcastle , uk ) , metabolyzer , and metaboanalyst 2.0 .
data quantification
was performed using targetlynx software ( waters corporation , milford ,
ma , usa ) and metidq software ( biocrates life sciences ag , innsbruck ,
aut ) .
to characterize the molecular response associated
with exposure
to radiation , we phenotyped the metabolomes of irradiated mice ( 8
gy , ld50/30 ) and sham control mice using a multiplatform
mass spectrometry - based approach ( figure 1 ) .
serum metabolomes from sham control and irradiated mice were subjected
to global metabolic profiling to screen for major molecular alterations .
identification and quantification of bioactive and low - abundance metabolites
followed ( figure 1 ) .
the overall effects on
blood cell levels in combination with various symptoms associated
with the acute radiation syndrome following exposure to a ld50/30 have been described extensively in the literature .
cb57bl/6
mice were irradiated ( 8 gy ; n = 5 ) or subjected to
the same treatment minus irradiation ( sham control group ; n = 5 ) .
blood was collected , and serum samples were prepared
and divided into two aliquots . before extraction , a mixture of internal
standards was added to the serum to normalize for variations in sample
preparation or ms detection .
the
results were integrated for the generation of a unique metabolic biosignature ,
which indicates exposure to radiation . to determine the overall effect of radiation on the molecular
phenotype
,
we compared the serum metabolome of sham control and irradiated mice
using a microfluidic device coupled with an electrospray - tof detector ,
detecting thousands of molecular features in serum samples ( figure s1 ) .
pca showed that the -irradiated
group was well - separated from the sham control group , underscoring
that radiation induces a profound molecular response in blood .
significant
changes were filtered using anova , highlighting the molecular features
that contributed most to the variance between the two groups of mice
( figure s1 ) . among the dysregulated molecular
components
, we tentatively identified metabolites belonging to the
classes of amino acids , carnitines , sphingomyelins , and phosphatidylcholines
as discriminators of the sham control and irradiated groups ( table 1 ) .
lipids are ranked according to
their anova p values . to further investigate these initial findings , in
an independent
set of experiments
, we applied a multiplexed metabolic profiling approach .
using stable - isotope - dilution tandem ms , we identified and quantified
more than 180 endogenous metabolites from six biochemical classes :
biogenic amines , amino acids , glycerophospholipids , sphingolipids ,
sugars , and acylcarnitines ( table s3 ) .
multivariate statistical analyses showed that exposure to
radiation induced significant molecular changes in mouse sera ( figure 2a , b and figure s2a ) .
in particular , we observed a decrease in the levels of diacyl phosphatidylcholines
( pcs ) containing polyunsaturated fatty acids ( pufas ) ( figure 2c and table s3 ) but a
significant increase in the levels of ether pcs in mice exposed to
radiation compared with that in their sham - irradiated littermates
( figure 2d and table s3 ) .
moreover , in the irradiated animals , the levels of lysophosphatidylcholines
and sphingomyelins ( sm ) were also increased ( table
s3 ) .
such differences were found to be significant after multiple - comparison
correction ( fdr < 0.01 ) .
other significant differences between
sham control and irradiated mice included the levels of serine and
carnitines , which increased after irradiation ( figure 2b and table s3 ) . targeted metabolic profiling .
( a ) pls - da analysis showed a marked
separation of serum metabolites belonging to irradiated and sham control
mice , highlighting the features that contributed most to the variance
between the two groups .
the colored boxes on the right indicate the relative concentrations
of the corresponding metabolite in each group under study .
the variable
importance in projection ( vip ) , a weighted sum of squares of the pls
loadings , takes into account the amount of explained y variation in each dimension .
( c , d ) levels of diacyl pcs ( c ) and
ether pcs ( d ) in irradiated and sham control ( n =
5 , student s t test ; * * * , p < 0.001 ) .
pc species can be hydrolyzed to release pufas that
can then be
converted into oxylipins by means of enzymatic and nonenzymatic reactions .
to measure the levels of such , usually
, low - abundance metabolites ,
we used a tandem ms with multiple reaction monitoring for over 100
oxylipins ( figure 1 ) .
the overall oxylipins
composition was sufficient to discriminate between irradiated and
sham control animals ( figure s2b and figure 3a , b ) .
we observed a marked increase in the levels
of arachidonic acid - derived metabolites ( figure 3b and table 2 ) . in particular , a significant
increase after multiple comparison correction was found for hydroxyeicosatetraenoic
acids ( 12-hete , 11-hete , 8-hete ) , 14,15 dihydroxy - eicosatrienoic acid
( 14,15-dihetre ) , and 12-hydroxy - heptadecatrienoic acid ( 12-hhtre )
in mice exposed to radiation versus that in the sham control animals
( figure 3c and table 2 ) .
our results also highlighted a marked decrease in the levels of
diols of epa including 17,18-dihete and 14,15-dihete in the serum
of mice exposed to radiation versus that in the serum of sham - treated
mice ( figure 3d and table 2 ) .
minor decreases were also observed for other ala- or dha - derived
metabolites ( figure 3b and table 2 )
( a ) pls - da analysis showed a marked
separation of serum oxylipins belonging to irradiated and sham control
mice , highlighting the features that contributed most to the variance
between the two groups .
the colored boxes on the right indicate the relative concentrations
of the corresponding metabolite in each group under study .
variable
importance in projection ( vip ) , a weighted sum of squares of the pls
loadings , takes into account the amount of explained y variation in each dimension .
( c , d ) levels of omega-6 oxylipins
( c ) and omega-3 oxylipins ( d ) in irradiated and sham control mice
( n = 5 , student s t test ;
* , p < 0.05 ; * * * , p < 0.001 ) .
fdr , false discovery rate . to obtain a metabolic biosignature
of mice that would indicate
radiation exposure , we combined the quantitative results derived from
the multiplexed metabolic profiling analyses , underscoring a significant
molecular contrast between sham control and irradiated mice ( figure 4a , b ) .
the sham control molecular composition exhibits
a decidedly distinct
pattern and structure compared with that of the irradiated one , suggesting
that radiation elicits a very distinct metabolic response ( figure s3 ) .
( a ) correlation analysis
was used to visualize the overall relationships between different
features and the irradiated phenotype .
( b ) clustering result shown
as a heatmap ( distance measure using pearson ; clustering algorithm
using ward ) providing an intuitive visualization of the characteristic
lipidomic biosignature found in irradiated mice versus sham control
mice . each colored cell on the map corresponds to a concentration
value , with samples in rows and features / compounds in columns .
in this study , we investigated
the molecular response to radiation
exposure in mice using a multiplatform mass spectrometry - based strategy .
we phenotyped the serum of irradiated and sham control mice , reporting
novel metabolic pathways altered after radiation exposure .
most of
the study s novelty resides in the finding that radiation induces
a differential regulation of diacyl pcs and ether pcs as well as omega-6
and omega-3 lipid mediators .
such radiation - induced changes in lipid
composition could have repercussions on ( 1 ) the physicochemical properties
of cellular membranes and lipoproteins , ( 2 ) the cellular antioxidant
buffering capacity , and ( 3 ) cell signaling .
many of these molecular
changes may not be specific to the radiation exposure but act instead
as general makers of cellular / organism stress and inflammation .
limited studies have investigated the effects of ionizing radiation
on the blood level of metabolites and lipids in particular .
it is reported that treatment of lipid membranes
with ionizing radiation leads to lipid degradation , which is mediated
by both oxidation and fragmentation processes .
such studies , however , aimed to measure the content of
a limited number of lipid metabolites in reductionistic models such
as synthetic lipid membranes and cellular models . by using a multiplatform
mass spectrometry - based approach , we were able to describe in a more
comprehensive fashion the alterations in the molecular phenotype of
mouse serum following irradiation . in our study , we analyzed metabolites
with a wide dynamic range of concentrations : from low - abundance oxylipins ,
in the picomolar range , to the higher abundance precursor phospholipids ,
in micromolar amounts .
the observed phospholipids rearrangement ,
a decrease in the levels
of diacyl pc species and an increase in ether pcs ( plasmalogens ) ,
might ultimately have repercussions on the physicochemical properties
of the cellular membranes and lipoproteins .
the phospholipid
class of plasmalogens is indeed ubiquitously found in considerable
amounts as a constituent of mammalian cell membranes and of serum
lipoproteins .
plasmalogens are more susceptible than diacyl pcs to
oxidative reactions because of the reactivity of their enol - ether
function ; indeed , it is reported that they are effective as endogenous
antioxidants .
oxidation of the vinyl ether markedly prevents the oxidation of
highly polyunsaturated fatty acids , and products of plasmalogen degradation
do not propagate lipid oxidation . on the other hand
, the oxidation
of the vinyl ether bond of plasmalogens leads to the release of fatty
aldehydes , which are toxic to cells .
the pathological
role of plasmalogens and the biochemical pathways underlying their
upregulation after radiation exposure remain to be elucidated .
the most intriguing observation of our
study is that acute exposure
to radiation induced a specific mobilization of bioactive
oxylipins ( figure 5a , b ) .
these lipid mediators
are currently the focus of considerable interest , for they are also
key messengers for cellular homeostasis , inflammation , platelet aggregation ,
and vascularization .
oxylipins are produced via enzymatic or nonenzymatic oxygenation
of both omega-6 and omega-3 pufas .
three major enzymatic pathways
are involved in their generation : cyclooxygenase ( cox ) , lipoxygenase
( lox ) , and cytochrome p450 ( cyp450 ) ( figure 5a , b ) . pathway analysis .
diet - derived omega-6 linoleic acid ( la ) and omega-3
alpha - linolenic acid ( ala ) are transformed into longer chains pufas
by the sequential action of desaturases and elongases .
pufas can be
found in blood as unesterified fatty acids , esterified to phospholipids ,
or converted into the oxygenated metabolites oxylipins .
( a , b ) the
activities of cox , lox , and cyp450 enzymes catalyze the formation
of hundreds of oxylipins species with different biological activities
starting from the omega-6 pufas precursors ( a ) and the omega-3 pufas
( b ) .
the irradiated mice had marked alterations in the lox , cox , and
cyp450 pathways , resulting in the increase of omega-6 oxylipins ( red )
and decrease of omega-3 oxylipins ( green ) .
significantly , it is reported that ionizing radiation activates
cox and lox pathways and that nonsteroidal anti - inflammatory drug
treatment reduces radiation - induced expression of pro - inflammatory
cytokines ( e.g. , tnf- , tgf- , il-6 , and il-1 / ) . in agreement with these observations ,
our results further suggest
that oxylipins - mediated inflammation might be a critical signaling
step for the organism response to radiation .
macrophage activation
leading to a persistent inflammatory response has been described in
radiation therapy for cancer in animals following irradiation and
in atomic bomb survivors . like other inflammatory
cytokines ,
, they can be rapidly activated after tissue irradiation
as part of the immune response to stress factors and thus they might
disrupt cellular homeostasis over time , causing detrimental health
effects .
it is therefore reasonable to
speculate that oxylipins - mediated inflammation could be involved in
the bystander and abscopal effects associated with clinical exposures
to ionizing radiation in a radiotherapy - type situation .
our study
indicates that 24 h following irradiation omega-6 pro - inflammatory
lipid mediators derived from the cox and lox pathways ( e.g. , hetes ,
pgf2 , tbx2 ) are upregulated , while the levels of omega-3 anti - inflammatory
mediators ( e.g. , 5-hepe , 9-hotre ) are downregulated .
notably , some
of the most significant oxylipins alterations in the serum of irradiated
mice were related to the metabolism of the cyp450 pathway , including
14,15-dihetre , 14,15-dihete , and 17,18-dihete ( figure 5 ) .
the cyp450 family of enzymes can produce epoxides from
pufas , which are subsequently metabolized by the soluble epoxide hydrolase
( seh ) to the corresponding vicinal diols , dihydroxyeicosatrienoic
acids .
aa - derived cyp metabolites are known to
play an important role in renal and cardiovascular function and are
linked to the development of hypertension and related disease . omega-3
pufa - derived cyp metabolites are described as possessing
anti - inflammatory and analgesic properties , as inhibitors of platelet aggregation , and as pulmonary smooth - muscle relaxants .
our results support the hypothesis that cyp450-mediated
oxylipins metabolism might represent a major biochemical pathway in
response to acute exposure to ionizing radiation .
most notably , in
human and animal studies , the cyp - dependent metabolite profiles were
generally reflective of the pufa intake , suggesting the use therapeutic interventions designed to modulate
the levels of oxylipins as a preventive measure or as a countermeasure
to cope with the debilitating effects of radiation exposure .
oxylipins levels might greatly affect
intrinsic cellular radiosensitivity ,
the incidence and type of radiation - induced tissue complications ,
and diseases . the balance between omega-6 pro - inflammatory
and omega-3 anti - inflammatory lipid mediators may play a key role
in the different phases of the response to radiation : a pro - inflammatory
phase followed by a pro - resolution phase to restore cellular homeostasis .
notably , it is reported that
an unbalanced inflammatory response resulting as a consequence of
radiation exposure might contribute to both cancer and non - cancer - related
diseases , including eye and brain diseases and
cardiovascular disease .
therefore , the ability to control oxylipin pathways with dietary
interventions ( i.e. , omega-3 supplementation ) might alleviate and
eventually offset many of the side effects linked to either radiation
therapy or accidental exposure .
notably , the most significant molecular alterations induced
by
exposure to radiation affected lipid metabolism .
our study , however ,
also reports a dysregulation of the metabolism of amino acids ( e.g. ,
serine ) and carnitines ( figure 4 and table s3 ) in mice exposed to radiation .
such
findings , together with more modest alterations of intermediates of
the urea cycle ( e.g. , citrulline , fdr = 0.05 ) , were previously reported
and linked to radiation - induced gastrointestinal and liver damage . by offering a bird s eye view of the molecular
response to radiation exposure ,
our study highlights the interplay
between lipid metabolism and the network of biochemical pathways for
more polar metabolites , which is an object of continued and renewed
investigation by the scientific community .
using
a metabolic phenotyping approach , we identified a novel molecular
response in mouse serum following exposure to radiation .
the
integration of metabolic profiling information provided a detailed
molecular biosignature that might be used as an indicator of radiation
exposure and , potentially , as a predictor of radiosensitivity .
most
important , the molecular components identified in the present study
( i.e. , plasmalogens and oxylipins ) would enable us to modulate their
levels using currently available pharmacological treatments or nutritional
intervention .
if validated in humans , our findings could find
applications in
personalized medicine .
numerous attempts have been made to develop
dna - based diagnostic assays to identify cancer patients who might
develop severe adverse healthy tissue reactions in response to radiotherapy .
we speculate that baseline levels of oxylipins ( e.g. , omega-6/omega-3
ratio ) might serve as a companion diagnostic tool for radiation therapy
to help differentiate cancer patients who would respond best to radiotherapy
treatment from radiosensitive patients who may be unable to tolerate
the additional inflammatory response induced by radiotherapy . | exposure
to ionizing radiation has dramatically increased in modern
society , raising serious health concerns . the molecular response to
ionizing radiation , however , is still not completely understood . here ,
we screened mouse serum for metabolic alterations following an acute
exposure to radiation using a multiplatform mass - spectrometry - based
strategy . a global , molecular profiling revealed that mouse serum
undergoes a series of significant molecular alterations following
radiation exposure .
we identified and quantified bioactive metabolites
belonging to key biochemical pathways and low - abundance , oxygenated ,
polyunsaturated fatty acids ( pufas ) in the two groups of animals .
exposure to radiation induced a significant increase in the
serum levels of ether phosphatidylcholines ( pcs ) while decreasing
the levels of diacyl pcs carrying pufas . in exposed mice , levels of
pro - inflammatory , oxygenated metabolites of arachidonic acid increased ,
whereas levels of anti - inflammatory metabolites of omega-3 pufas decreased .
our results indicate a specific serum lipidomic biosignature that
could be utilized as an indicator of radiation exposure and as novel
target for therapeutic intervention .
monitoring such a molecular response
to radiation exposure might have implications not only for radiation
pathology but also for countermeasures and personalized medicine . | Introduction
Materials and
Methods
Results
Discussion
Conclusions |
PMC3015817 | since the initial description by walsh and donker in 1982 regarding the anatomic basis for erectile function , urologists have continuously explored technical advances that would improve both the oncologic and quality of life outcomes following radical prostatectomy .
anatomical nerve - sparing methods have become mainstream practice with resultant improvements in erectile function rates following what was once considered a morbid procedure .
although some interval modifications have been achieved in the radical retropubic prostatectomy operation , the next most significant advancement in surgical technique has been the introduction of laparoscopy . in the late 1990s , guillonneau and vallancien introduced laparoscopic radical prostatectomy as a viable surgical alternative for treating prostate cancer .
subsequently , several centers have gone on to incorporate laparoscopic prostatectomy into their surgical armamentarium with successful outcomes .
having set the stage for laparoscopy in the surgical treatment of prostate cancer , urologists then sought to improve upon such techniques with the use of robotics and the implementation of robotic radical prostatectomy . throughout the evolution of technology and treatment strategies ,
the urologic community has stringently emphasized the need to demonstrate both oncologic and therapeutic efficacy of procedures .
the acceptance of changes in surgical technique has always been predated by demonstrable evidence from multiple institutions , regarding both the clinical and pathologic criteria that are essential for the successful treatment of prostate cancer . as such ,
we have entered the robotic age in the treatment of prostate cancer with encouraging reports from a few centers of excellence . here
then , we hope to further diversify the experience in robotic prostatectomy and enrich the confidence of treating urologists by reporting our operative technique , oncologic and therapeutic outcomes , and learning curve from our initial series of over 140 consecutive patients treated by robotic radical prostatectomy .
between january 2003 and may 2005 , 174 consecutive patients with clinically localized prostate cancer underwent a robotic radical prostatectomy performed by 2 surgeons ( dss and ds ) at the new york - presbyterian hospital .
thirty - one patients with a follow - up period of less than 3 months were excluded from analysis in this study .
all patients had biopsy proven adenocarcinoma of the prostate and were staged according to the 2002 american joint committee on cancer ( ajcc ) classification .
all patients underwent a robotic radical prostatectomy with the da vinci surgical system ( intuitive surgical inc . ,
briefly , following induction of general endotracheal anesthesia , the patient was placed in the lithotomy position with a steep trendelenberg .
the abdominal and groin areas were prepped and draped , intravenous antibiotics were administered before skin incision , and a 20-f , 30-cc urethral catheter was placed .
a 1.5-cm incision was made to the left of the umbilicus , and a veress needle was used to insufflate the abdomen with co2 to a pressure of 15 cm h0 .
a 10/12-mm trocar for the camera was placed at the umbilicus , two 8-mm trocars for the robotic arms were placed just lateral to the medial umbilical ligaments , a 5-mm suction port was introduced to the right of the umbilicus , and 10/12-mm and 5-mm trocars were introduced 2 fingerbreadths above the iliac crest on the patient 's right and left side , respectively .
overall , 6 ports were used , and the da vinci robot was docked . starting with a 30 angle - up lens ,
the medial umbilical ligaments were incised up the anterior abdominal wall allowing cephalad reflection of the bladder and entry into the space of retzius .
the lens was changed to a 0 angle , and the endopelvic fascia was opened in a cautery - free manner to expose the apex of the prostate .
the levator muscles were swept posterolaterally , and the puboprostatic ligaments were sharply divided to gain further length beyond the apex of the prostate .
the superficial and deep dorsal veins were ligated with a 0-vicryl stitch , and a second 0-vicryl stitch was then placed for back bleeding over the anterior surface of the prostate .
the anterior bladder neck was divided , and the ureteral orifice position and presence of a median lobe were assessed , and then the posterior bladder neck was divided .
the vasa were divided and the seminal vesicles were dissected in a cautery - free manner to avoid potential injury to the neurovascular bundles . the posterior layer of denonvillier 's fascia was divided allowing for identification of perirectal fat , which served as a guide between the prostate and rectum . to optimize nerve sparing , all prostatic pedicles were clipped and sharply divided without using electrocautery .
the lateral prostatic fascia was incised on each side allowing the neurovascular bundles to fall posterolaterally , and a bilateral nerve - sparing procedure was performed when possible .
the neurovascular bundle was released distally to the level of the urethra and prostatic apex . at this point ,
the only remaining attachments of the prostate were the dorsal vein complex ( dvc ) and the urethra .
the dvc and urethra were then divided rendering the prostate free , and the specimen was placed in a 10-mm endocatch bag .
intraoperative frozen sections was used judiciously to help decrease the incidence of positive surgical margins .
the urethrovesical anastomosis was performed in a running fashion by using a double - arm 2 0 monocryl suture .
an 18-fr , 10-ml urethral catheter was placed and irrigated to ensure a water - tight anastomosis .
a bilateral pelvic lymph node dissection was performed following completion of the urethrovesical anastomosis in patients with a greater than 2% chance of positive lymph nodes as obtained from the partin predictive tables .
borders of the node dissection were the external iliac vein anteriorly , obturator nerve posteriorly , bifurcation of common iliac artery superiorly , and cooper 's ligament inferiorly .
a # 10 flat jp drain was placed into the pelvis and brought out through the left - side 5-mm port .
the da vinci robot was then undocked , all trocars were removed under direct vision , and the specimen was removed through the camera port .
the fascia at the extraction site was closed with a 2 0 vicryl suture , and all skin incisions were closed with a 4 0 biosyn stitch .
postoperative psa values , urinary continence , and erectile function were evaluated 6 weeks postoperatively , every 3 months for the first year , every 6 months for the second year , and yearly thereafter . before surgery , all men were evaluated with the following data recorded : age , bmi , clinical t stage , biopsy gleason score , and prostate - specific antigen ( psa ) .
operative outcome measures included operative time , estimated blood loss ( ebl ) , and complications .
postoperative outcomes were length of hospital stay , catheter time , pathology , margin status , biochemical recurrence , and return of continence . the chi - square ( ) log - rank test with the yates correction factor and the unpaired 2-tailed student t test were used to compare variables between the first and second 70 patients in our series .
between january 2003 and may 2005 , 174 consecutive patients with clinically localized prostate cancer underwent a robotic radical prostatectomy performed by 2 surgeons ( dss and ds ) at the new york - presbyterian hospital .
thirty - one patients with a follow - up period of less than 3 months were excluded from analysis in this study .
all patients had biopsy proven adenocarcinoma of the prostate and were staged according to the 2002 american joint committee on cancer ( ajcc ) classification .
all patients underwent a robotic radical prostatectomy with the da vinci surgical system ( intuitive surgical inc . , sunnyvale , ca ) .
briefly , following induction of general endotracheal anesthesia , the patient was placed in the lithotomy position with a steep trendelenberg .
the abdominal and groin areas were prepped and draped , intravenous antibiotics were administered before skin incision , and a 20-f , 30-cc urethral catheter was placed .
a 1.5-cm incision was made to the left of the umbilicus , and a veress needle was used to insufflate the abdomen with co2 to a pressure of 15 cm h0 .
a 10/12-mm trocar for the camera was placed at the umbilicus , two 8-mm trocars for the robotic arms were placed just lateral to the medial umbilical ligaments , a 5-mm suction port was introduced to the right of the umbilicus , and 10/12-mm and 5-mm trocars were introduced 2 fingerbreadths above the iliac crest on the patient 's right and left side , respectively .
overall , 6 ports were used , and the da vinci robot was docked . starting with a 30 angle - up lens ,
the medial umbilical ligaments were incised up the anterior abdominal wall allowing cephalad reflection of the bladder and entry into the space of retzius .
the lens was changed to a 0 angle , and the endopelvic fascia was opened in a cautery - free manner to expose the apex of the prostate .
the levator muscles were swept posterolaterally , and the puboprostatic ligaments were sharply divided to gain further length beyond the apex of the prostate .
the superficial and deep dorsal veins were ligated with a 0-vicryl stitch , and a second 0-vicryl stitch was then placed for back bleeding over the anterior surface of the prostate .
the anterior bladder neck was divided , and the ureteral orifice position and presence of a median lobe were assessed , and then the posterior bladder neck was divided .
the vasa were divided and the seminal vesicles were dissected in a cautery - free manner to avoid potential injury to the neurovascular bundles .
the posterior layer of denonvillier 's fascia was divided allowing for identification of perirectal fat , which served as a guide between the prostate and rectum . to optimize nerve sparing ,
the lateral prostatic fascia was incised on each side allowing the neurovascular bundles to fall posterolaterally , and a bilateral nerve - sparing procedure was performed when possible .
the neurovascular bundle was released distally to the level of the urethra and prostatic apex . at this point ,
the only remaining attachments of the prostate were the dorsal vein complex ( dvc ) and the urethra .
the dvc and urethra were then divided rendering the prostate free , and the specimen was placed in a 10-mm endocatch bag .
intraoperative frozen sections was used judiciously to help decrease the incidence of positive surgical margins .
the urethrovesical anastomosis was performed in a running fashion by using a double - arm 2 0 monocryl suture .
an 18-fr , 10-ml urethral catheter was placed and irrigated to ensure a water - tight anastomosis .
a bilateral pelvic lymph node dissection was performed following completion of the urethrovesical anastomosis in patients with a greater than 2% chance of positive lymph nodes as obtained from the partin predictive tables .
borders of the node dissection were the external iliac vein anteriorly , obturator nerve posteriorly , bifurcation of common iliac artery superiorly , and cooper 's ligament inferiorly . a # 10
flat jp drain was placed into the pelvis and brought out through the left - side 5-mm port .
the da vinci robot was then undocked , all trocars were removed under direct vision , and the specimen was removed through the camera port .
the fascia at the extraction site was closed with a 2 0 vicryl suture , and all skin incisions were closed with a 4 0 biosyn stitch .
postoperative psa values , urinary continence , and erectile function were evaluated 6 weeks postoperatively , every 3 months for the first year , every 6 months for the second year , and yearly thereafter .
before surgery , all men were evaluated with the following data recorded : age , bmi , clinical t stage , biopsy gleason score , and prostate - specific antigen ( psa ) .
operative outcome measures included operative time , estimated blood loss ( ebl ) , and complications .
postoperative outcomes were length of hospital stay , catheter time , pathology , margin status , biochemical recurrence , and return of continence . the chi - square ( ) log - rank test with the yates correction factor and the unpaired 2-tailed student t test were used to compare variables between the first and second 70 patients in our series .
the mean operative time was 241 minutes ( range , 120 to 540 ) , with a decrease of over 100 minutes when comparing our initial series of 70 with our more recent 70 patients ( 318 vs 209 , p<0.001 ) .
the mean estimated blood loss ( ebl ) was 274 ml ( range , 10 to 800 ) , with a significant decrease in our more recent series of patients ( 387 vs 155 , p<0.001 ) . in 127 men , data regarding nerve sparing were available .
one hundred and nine of 127 men ( 86% ) had a bilateral nerve - sparing operation , whereas 13/127(10% ) had unilateral nerve - sparing , and 5/127(4% ) had bilateral nerve resections .
sixty - six of the last 70 patients ( 94% ) had bilateral nerve - sparing operations .
five patients ( 3% ) had a conversion to open surgery for bleeding , large intravesical median lobes , adherence to the rectum , and robot malfunction .
of our most recent 70 patients , 65 ( 93% ) were discharged within 24 hours of surgery .
the urethral catheter was removed at a mean time of 8.9 days ( range , 1 to 29 ) . at a mean follow - up of 11 months
, 96% of patients ( 137/143 ) had a psa<0.2 ng / ml . four patients had postoperative complications including an ileus , small bowel obstruction1 requiring reoperation , and a urethral stricture requiring repeat dilatation .
two patients ( 1% ) did not have any tumor identifiable in the final pathology specimen , 1 of whom received preoperative hormonal deprivation therapy .
organ - confined ( oc ) disease ( pt2 ) was present in 122/141 patients ( 87% ) and extracapsular extension ( ece ) was present in the final pathology analysis ( pt3/t4 ) of 19/141 ( 13% ) patients .
twenty - four of 141 men ( 17% ) in our series had a positive margin on examination of the specimen , with a decrease from 23% ( 16/70 ) to 11% ( 8/70 ) when comparing our more recent 70 patients with the initial 70 ( p=0.07 ) .
patients with ece had a significantly higher rate of positive surgical margins than did those with oc disease ( 47% vs 12% , p<0.001 ) .
when exclusively considering pt3 disease , we further noted a decrease in positive margins when comparing our first 70 patients ( 5/9 ; 56% ) with our latter 70 ( 2/8 ; 25% ) .
no significant difference existed in positive margin rates in the gleason 7 cohort ( 19/122 , 16% ) and the gleason > 7 group ( 4/19 , 21% ) ( p=0.55 ) . over 40% ( 14/33 ) of the positive margins
the number of positive apical , anterior , and posterior positive margins remained unchanged as experience matured in this series ; however , margin positivity at the bladder neck decreased from 7 in the first cohort of patients to 2 in our latter operative experience .
patients requiring no urinary pads were considered continent , and the use of even one pad per day for occasional stress urinary incontinence was not considered continent for the purposes of our analysis .
the median time to complete continence was 3.5 months , and over 95% of patients were fully continent at 1-year follow - up .
because our data are awaiting maturity , we will hold off on reporting potency data in this analysis .
as robotic surgery becomes a more accepted treatment modality for clinically localized prostate cancer , many urologists are struggling to incorporate this technique into their therapeutic armamentarium .
increasingly , surgeons are foregoing open retropubic approaches with a technically challenging robotic - assisted technique for which efficacy data are currently limited but growing .
it is the intent of our analysis to objectively analyze the robotic approach from an oncologic standpoint , and in doing so , to characterize the oncologic and therapeutic efficacy of this relatively new and growing technology .
although the ultimate measure of any intervention is the ability to prolong long - term survival , modifications in surgical technique can be assessed in the short - term by analyzing pertinent oncological principles .
one such variable is pathology margin status . it is generally agreed that a positive margin is indicative of incomplete tumor resection and bears significant prognostic importance .
several institutions have demonstrated the independent prognostic significance of positive surgical margins across all stages of disease .
han et al further point out that the rate of positive surgical margins has declined dramatically from around 40% during the early 1980s to less than 10% more recently .
although these observations are likely multifactorial , the lower positive margin rates over the past decade are largely attributable to the stage migration that has occurred in the era of psa screening .
it is imperative , however , to also consider contemporary refinements in preoperative planning , biopsy strategies , and surgical technique .
as we embark on the robotic age for the treatment of prostate cancer , it is essential that we adhere to surgical principles learned over the last 3 decades . in this study
, we report an overall positive margin rate of 17% . during the first half of our experience
, we noted a 23% positive margin rate , which declined to 11% over our last 70 consecutive patients .
further , no difference existed in the pathologic distribution of tumors between our initial 70 patients and our latter 70 ( 9 vs 10 cases of ece ) . collectively , these data suggest that the decline in the positive margin rate is most attributable to an improved surgical skill set in the robotic technique rather than a decrease in the number of biologically aggressive tumors
. subset analysis of the data also notes a significantly lower incidence of positive margins when comparing specimens with organ - confined disease with those with extracapsular extension ( 12% vs. 47% , p<0.001 ) .
noteworthy , however , is the decrease in positive margin rates from 56% to 25% in the pt3 subgroup of tumors during the evolution of our experience .
this underscores that robotic prostatectomy is a reasonable surgical alternative for organ - confined , as well as pt3 disease .
thus , even in our initial robotic experience , the results compare favorably with those of other reported robotic series , as well as with the current oncologic gold standard open retropubic approach .
the specific location of positive margins is explainable both by surgical experience and technique . as depicted in table 4 ,
bladder neck positive margins declined over 3-fold when comparing the first half of our robotic experience and our most recent surgical series .
the antegrade approach utilized during a robotic prostatectomy can be quite challenging and choosing the appropriate location for incision of the bladder neck can be difficult . as our experience in robotic surgery has matured , we have learned to take a wider bladder neck and robotically reconstruct the bladder as necessary . this modification has probably resulted in decreased positive bladder neck margins as our experience increased . in terms of the anterior and apical margins , we noted that 15% ( 5/33 ) of all positive margin sites were at each of these 2 anatomic locations , respectively .
this compares favorably with other published reports in which the apical positive margin rates range from 5% to 28% .
we believe that the anterior and apical dissection is enhanced with the robotic approach likely due to the use of an angled lens resulting in improved visualization of the dorsal vein / urethral complex . at 1-year follow - up , 97% ( 139/143 ) of men were fully continent without the requirement of any pads .
our 3-month follow - up data , however , indicate that less than 50% of patients are fully continent at this time point .
anecdotally , this is lower than the continence rates we have observed for our contemporary open retropubic prostatectomy series .
one possible explanation is that the enhanced robotic visualization may result in excessive dissection of the striated sphincter complex beyond the apex of the prostate . to improve the early continence rates , we have made several modifications in our current technique including tacking the urethra to the periosteum of the pubis and avoiding excessive dissection beyond the apex of the prostate . with such modifications ,
our short - term continence rates have improved in a more contemporary group of patients ( data not shown ) .
robotic - assisted radical prostatectomy is an effective modality for the treatment of clinically localized prostate cancer .
we acknowledge that a learning curve needs to be overcome ; however , with a rigorous , focused robotic educational program , early oncologic functional results compare favorably with results of the open retropubic approach . | objective : to report the operative technique , oncologic and therapeutic outcomes , and learning curve from our initial series of over 140 patients treated by robotic radical prostatectomy.methods:between january 2003 and may 2005 , 143 patients with clinically localized prostate cancer underwent a robotic radical prostatectomy .
prospective data collection included patient age , body mass index ( bmi ) , clinical t stage , biopsy gleason score , and prostate - specific antigen ( psa ) .
operative outcome measures included operative time , estimated blood loss ( ebl ) , and complications .
postoperative outcomes were length of hospital stay , catheter duration , pathology , margin status , biochemical recurrence , and return of continence.results:mean operative time was 241 minutes with an ebl of 274 ml .
five patients ( 3% ) required conversion to open surgery .
the average hospitalization was 1.8 days , and foley catheters were removed after 8.9 days .
twenty - four of 141 men ( 17% ) had a positive surgical margin , with a decrease from 23% in the first half of our experience to 11% in the latter half .
patients with an extracapsular extension had a significantly higher positive surgical margin rate than did those with organ - confined disease ( 47% vs 15% ) . over 40% of the positive margins
were located posteriorly . at a mean follow - up of 11 months
, 96% of patients had a psa<0.2 ng / ml . the median time to complete continence was 3.5 months , and over 95% of patients were fully continent at 1 year.conclusion:robotic radical prostatectomy is an effective treatment modality for clinically localized prostate cancer .
although a learning curve needs to be overcome , patients experienced benefits in convalescence with early oncologic and functional outcomes comparable to those of the open approach .
longer - term results are needed ; however , patient outcomes in our series are encouraging . | INTRODUCTION
METHODS
Patient Selection
Operative Technique
Follow-up
Data collection, Outcomes, and Statistical Analysis
RESULTS
DISCUSSION
CONCLUSION |
PMC4601462 | livedoid vasculopathy ( lv ) is a rare cutaneous disease which manifests mainly as recurrent leg ulcers .
these ulcers heal with atrophic , porcelain white scars called atrophie blanche , which is an alternative named used for the disease .
inflammation , autoimmunity and hypercoagulability are the main pathogenetic factors with the latter being the most predominant among them .
the recurrent nature and controversial pathogenesis makes this disorder a very challenging one to treat .
we herein report a case of lv associated with hyperhomocysteinemia which responded well to hyperbaric oxygen therapy ( hbot ) , thus providing a novel , efficient and safe option for management .
this 30-year - old male presented with complaints of multiple ulcers over both legs of 6 years duration .
the patient gave history of initially developing few ulcers on the left foot after prolonged wearing of boots for duration of 7 days ( 18 hours / day ) .
he progressively developed similar multiple ulcers over both lower legs and dorsum of feet over a duration of 2 weeks .
they were associated with moderate intensity pain and would increase on walking or running and reduce with rest .
the ulcers had a waxing and waning course and would generally heal with course of antibiotics and rest .
dermatological examination revealed multiple ulcers distributed in a reticular pattern over medial and lateral aspects of both lower legs , extensor aspect of both ankles and dorsum of both feet [ figure 1 ] .
multiple atrophic porcelain white scars were also present in the interspersed and adjoining regions along with post inflammatory hyperpigmentation .
ulcers had well - defined margins , sloping edges and the floor had granulation tissue with no discharge [ figure 2 ] .
distribution of ulcers on : ( a ) the medial aspect of both legs and feet .
( b ) lateral aspect of dorsum of left feet and leg close up images revealing ( a ) livedoid ulcers on leg long with atrophie blanche and ( b ) reticulate pattern of ulcers on dorsum of foot investigations revealed normal hemogram , liver and renal function tests .
antinuclear antibodies , hepatitis b surface antigen ( hbsag ) , anti - hepatitis c , vdrl and hiv testing were all negative . antineutrophil cytoplasmic antibodies ( anca ) , antiphospholipid antibodies ( apla ) , protein c and s , antithrombin iii , factor v leiden , prothrombin gene mutation and methylene tetrahydrofolate reductase ( mthfr ) gene polymorphisms were all negative .
a skin biopsy from the ulcers revealed ulcerated epidermis with intervening orthokeratotic epidermis , mild acanthosis and spongiosis .
most of the vessels showed thickened walls with fibrin deposition and thrombus formation [ figure 3 ] . with these features a diagnosis of lv with associated
histopathology revealing extensive fibrin deposition and thrombus formation in the dermal vessels with absent inflammation [ hematoxylin and eosin stain
10 ] he was prescribed oral prednisolone 60 mg along with , pentoxyphylline , low - dose aspirin , folic acid and local cleaning of ulcers with normal saline .
patient was given a course of hbot for the ulcers along with continuation of pentoxyphylline , aspirin and folic acid .
the patient was administered hbot in a multiplace chamber at pressure of 2.5 atmospheres for 90 min daily , 6 days a week . at the end of 10 sittings of hbot ,
ulcers in the feet and ankles showed signs of healing in the form of appearance of granulation tissue in the base , shrinking margins , reduced inflammation and reduced tenderness . by the end of 15 sittings ,
the patient was followed up for 1 year during which no recurrence of ulcers was noted .
lv is presently described as a condition manifesting as recurrent leg ulcers with thrombosis of the microcirculation as the main pathogenesis .
the important procoagulant causes include hyperhomocysteinemia , proteins c and s deficiency , factor v leiden mutation , prothrombin gene mutation , antithrombin iii deficiency and antiphospholipid antibody syndrome .
the genetic causes of homocystenemia are mainly due to enzyme defects such as cystathione--synthase , methylenetetrahydrofolate reductase and methionine synthase deficiencies .
acquired causes include nutritional deficiencies , renal failure , use of folic acid and vitamin b6 antagonists , cardiovascular diseases and peripheral arterial occlusive disease .
hbot is a recognized modality of treatment of various problem wounds and non - healing ulcers due to various etiologies .
the non - healing ulcers form the single largest group of indications for which hbot is being administered .
this group includes various indications like diabetic foot ulcers , arterial insufficiency ulcers , ulcers due to venous stasis , decubitus ulcers and fungal infections .
these disorders have a common pathology in having ischemia and infection as the most common causes of non - healing .
hyperbaric oxygen acts by delivering 100% oxygen at high pressures ( 2 - 3 atmospheres pressure ) wherein oxygen dissolves 20 times more in plasma and tissue fluids than at room air pressure . therefore
, a high oxygen gradient is produced across the wound site negating the damaging effects of tissue ischemia .
it also increases the release of fibrinolytics from endothelial cells , promotes angiogenesis of ischemic tissue and facilitates neovascularization , thereby accelerating healing in lv .
other proposed mechanisms include increased collagen formation , inhibition of bacterial overgrowth and reduced tissue reperfusion injury .
contraindications for hbot include previous chest or ear surgery , pregnancy , seizures , malignancy , pneumothorax , emphysema and upper respiratory tract infections .
so chest x - ray , pulmonary function tests , and eardrum examination are a must before starting therapy .
severe adverse effects are very rare and middle ear barotraumas are the most common reported adverse effects .
our patient had lv associated with homocystenemia and he showed very good response to hbot .
lv itself has been very rarely reported from india and few reports globally have reported use of hbot for lv .
hbot therapy is therefore a novel , relatively cost - effective , opd treatment option in lv .
it is probably not only a means of therapy for healing the ulcers , but also to get longer recurrence free periods .
hyperbaric oxygen therapy is a novel , safe and beneficial therapy for livedoid vasculopathyhyperbaric oxygen therapy can not only help in healing ulcers , it can also help in prolonging remission periods in such cases .
hyperbaric oxygen therapy is a novel , safe and beneficial therapy for livedoid vasculopathy hyperbaric oxygen therapy can not only help in healing ulcers , it can also help in prolonging remission periods in such cases . | a 30-year - old male presented to the dermatology department with complaints of multiple ulcers over both legs of 6 years duration .
the ulcers had a waxing and waning course with present exacerbation of lesions since 1 month .
dermatological examination revealed multiple ulcers distributed in a reticular pattern over medial and lateral aspects of both lower legs , extensor aspect of both ankles and dorsum of both feet .
multiple interspersed atrophic porcelain white scars were also present
. investigations revealed raised serum homocysteine levels .
a skin biopsy from the ulcers showed features of livedoid vasculopathy . following recurrence of lesions after oral corticosteroid therapy , the patient
was given a course of hyperbaric oxygen therapy for the ulcers to which he responded very well .
this case is being presented for the novel option of hyperbaric oxygen therapy in livedoid vasculopathy , which by itself is rarely reported in this part of the world . | Introduction
Case Report
Discussion |
PMC3710328 | signal transducer and activator of transcription ( stat ) family proteins are requisite molecules in the development of t helper subsets .
stat1 and stat4 are required for the development of th1 cells that secrete ifn and mediate immunity to intracellular pathogens .
stat3 promotes th17 development and the secretion of il-17 , a cytokine required for immunity to extracellular bacterial and fungal infections .
stat6 is required for the differentiation of th2 and th9 cells , which contribute to extracellular parasite immunity .
the in vivo function of stat6 has been defined largely following the generation of gene - deficient mice .
stat6 mice develop normally , breed as homozygotes , but have altered immune responses in vivo . in the absence of stat6 , mice are resistant to the development of allergic inflammation , and have exaggerated responses to inflammatory insults resulting in greater immunity to intracellular pathogens , but greater susceptibility to autoimmune inflammation , with differences in phenotype attributed to different mouse lines with targeted stat6 alleles . some of the studies that defined these functions utilized experiments involving adoptive transfer of lymphocytes , or where cells or tumors were adoptively transferred into stat6 hosts . whether cells behave normally in these transfer experiments
has not been extensively examined . in experiments to test if deficiency in stat4 or stat6 affected development of ctl specific for non - classical mhc molecules , we observed autoreactivity of stat6 ctl for stat6-expressing cells .
the development of stat6-specific reactivity by stat6 cd8 t cells might have important implications for the interpretation of experiments where stat6 cd8 t cells are exposed to stat6 expressing cells in vivo .
to examine the responses of balb / c , stat4 and stat6 mice ( h2 , qa1 and mta ) as responders against nzb / b1nj ( nzb ) ( h2 , qa1 and mta ) stimulator cells , we generated ctl lines from each strain .
we found no consistent differences in the ability of the stat4 or stat6 cd8 t cells to develop responder ctl against qa1 or mta antigens , compared with balb / c t cells ( fig .
however , we did observe that stat6 anti - nzb ctl could lyse target cells from stat4 and balb / c mice ( fig .
in contrast , balb / c and stat4 responders did not lyse balb / c , stat4 or stat6 cells ( fig .
the ability of stat6 ctl to lyse these target cells was unexpected because stat4 , stat6 and balb / c are syngeneic , differing only in the regions adjacent to the targeted stat4 or stat6 alleles .
ctl lines generated from secondary mlcs were assayed against stat6 and stat6 con a blast ( cab ) target cells in a standard cr release assay .
( a ) balb / c , ( b ) stat4 and ( c ) stat6 anti - nzb responder ctl were assayed against nzb , balb / c , stat4 or stat6 cab target cells .
lysis of nzb cells in ( a ) and ( b ) represents mta and/or qa1 restricted responses .
stat6 anti - nzb ctl ( c ) lyse nzb , stat4 and balb / c , but not stat6 target cells , indicating lysis of stat6 cells .
( d ) stat6 anti - balb / c , ( e ) stat6 anti - stat4 ctl and ( f ) stat4 anti - stat6 mlc were assayed against balb / c , stat4 , stat6 , h2 , b6.tla or h2 ( b10.riii ) cab target cells .
all data are the mean sd of triplicate assays and are representative of three or more experiments . to confirm these data and to rule out problems due to the use of one specific mouse strain ( nzb ) , we examined responses of balb / c , stat4 and stat6 as stimulators and responders of ctl in similar assays . stat6
anti - balb / c and stat6 anti - stat4 responder ctl also lyse stat4 and balb / c target cells ( fig .
1d and e ) , whereas stat4 anti - stat6 ctl do not lyse stat4 , stat6 or balb / c target cells ( fig .
to determine the restricting mhc molecule involved in these responses , we examined mice expressing different mhc haplotypes and found the responses were restricted to h2 .
stat6 anti - stat4 or anti - balb / c ctl lysed target cells from h2 ( balb / c , stat4 and nzb ) mouse strains ( figs . 1d and e and 2 ) , but not cells expressing h2 ( b6.tla ) , h2 ( b10.riii ) , or h2 ( c3h / hej and b10.br ) ( figs .
1d and e and 2c ) , suggesting that the ctl were not alloreactive . to determine whether the restricting element(s ) are h2-k , -d and/or -l molecules , we further compared stat6 ctl responses against a / j ( h2-k , -d and -l ) , balb / c ( h2-k , -d and -l ) and balb.dm2 ( h2-k , -d and l null ) target cells using two different stat6 ctl lines .
2a and b ) are recognized by the polyclonal responder ctl . since h2 cells ( c3h.hej or b10.br )
2c ) , recognition of cells from a / j ( h2-k , -d and -l ) by the 241.44 line shows restriction to h2-d and/or -l ( fig .
in contrast , the lack of response of the 240.43 line demonstrates the failure of these cells to recognize h2-d , suggesting the reactivity against another h2 class i molecule , most likely h2-k ( fig .
restricted ctl were also able to lyse balb.dm2 cells , which express h2-k and h2-d , but not h2-l , further suggesting that h2-l restriction is not a prominent reactivity in these ctl lines ( fig .
thus , stat6 ctl can develop with specificities restricted to h2-k , h2-d or h2-l .
stat6 anti - balb / c ctl lines ( a ) 241.44 , ( b d ) 240.43 were incubated with target cells from h2 , or h2 expressing mouse strains in a standard cr release assay . ctl line 241.44 ( a ) ( h2-d and -l - restricted ) lyses a / j ( h2-k , -d and -l ) target cells , whereas ctl line 240.43 ( b ) ( h2k - restricted ) did not lyse a / j or ( d ) balb.dm2 ( h2kd ) . neither line lysed h2 target cells ( c , data not shown ) .
data are the mean sd of triplicate assays and are representative of two or more experiments . confirming these data , in figure 3 we show that pre - incubating a / j or stat4 target cells with monoclonal antibodies reactive with h2-k , -d and h2-l molecules blocks target cell recognition and lysis by these ctl .
lysis of stat4 or a / j target cells is blocked by monoclonal antibody 30 - 5 - 7s that reacts with h2-d or -l but not h2-k or h2-k , confirming restriction of this line to h2-l ( fig . 3a and b ) . in figure 3c ,
lysis of stat4 targets by the stat6 anti - balb 240.43 line is not blocked by 30 - 5 - 7s , but rather is blocked by the two antibodies which bind h2-k molecules , 34 - 1 - 2s and 34 - 7 - 23s , demonstrating restriction to h2-k . taken together , the data clearly demonstrate that stat6 anti - balb / c or anti - stat4 ctl are restricted to either h2-k ,- d or -l .
figure 3.stat6 anti - balb / c or stat4 ctl are h2-k , -d or l restricted .
( a c ) stat6 anti - balb / c ctl lines 241.44 ( a and b ) , and 240.43 ( c ) were incubated with cab target cells from stat4 ( a and c ) or a / j ( b ) mice and cytotoxicity was measured in a standard cr release assay . the ability of anti - mhc i antibodies to block lysis was tested .
data are the mean sd of triplicate assays and are representative of two or more experiments . since the primary difference between balb / c or stat4 and stat6 mice is the stat6 gene product , we tested whether stat6 ctl recognized peptides derived from the stat6 protein .
we tested a stat6 peptide previously shown to be presented by tumor cells and observed the h2-k restricted ctl recognize stat6 target cells incubated with the sywsdrlil peptide ( fig .
4 ) , which fits the h2-k consensus motif of xyx6(i / l / v)l .
we additionally tried several stat6 peptides that fit the h2-d or h2-l consensus motifs , ( xgpx(k / r)x3(l / i / f ) and ( xpx6(l / m / f ) , respectively , but none conferred reactivity .
stat6 anti - balb / c ctl 240.43 ( left ) or stat6 anti - stat4 ctl 268 ( middle ) were incubated with stat6 cab target cells at an effector : target cell ratio of 12:1 in a standard cr release assay .
cytotoxicity was assessed in the absence or presence of 9-mer peptides from h-2k binding peptides from hiv gp160 ( rgpgrafvti , vgnpiilp ) , or stat6 ( sywsdrlil ) .
the alloreactive b10 ( h2 ) anti - balb / c ( h2 ) ctl 262 ( right ) did not require peptide for lysis and was not affected by target cell incubation with peptides .
data are the mean sd of triplicate assays and are representative of three or more experiments .
these data demonstrate responses by stat6 ctl against h2 cells from stat6-expressing mouse strains including balb / c , nzb and stat4 .
the polyclonal ctl populations generated from the stat6 responder spleen cells initially recognized k , -d and -l restricted antigens , although with weekly passage in vitro some populations eventually self - selected for a predominantly h2-k , -d or -l restricted phenotype .
use of target cells from recombinant inbred mouse strains and blocking by antisera specific for h2 class i molecules confirms their mhc restriction . the h2-k restricted ctl recognized the stat6-derived peptide sywsdrlil .
these data clearly illustrate the importance of running negative control experiments , particularly when working with genetically altered strains of mice . in some situations
for example , in evaluations of immune responses within stat6 mice against exogenous antigens or allergens , or in studies of inflammation or hypersensitivity , the results may show no effects easily attributable to altered peptide presentation by mhc class i molecules on stat6 cells .
however , it is possible that results could be influenced by subtle changes in reactivity of cells maturing in the stat6 environment and/or by the unusual peptide repertoire transferred on h2 molecules with stat6 cells .
such changes in immune responses due to these altered peptide expression might be negligible or difficult to identify and quantitate , but should nonetheless be considered . however , the situation could be more complicated in adoptive transfer experiments .
although most adoptive transfer experiments involving stat6 mice use transferred purified cd4 t cells , in experiments where stat6 splenocytes or cd8 t cells are transferred to wild type recipients , it is possible that stat6-specific cd8 t cells could mediate a graft vs. host response , resulting in background inflammation , apart from the specific responses being studied .
conversely , when stat6-expressing cells are transferred into stat6 recipients , it is possible that stat6 cd8 t cells reactive against stat6 peptides might eliminate transferred cells , masking some functional activity of the transferred cells .
the results from most of these experiments are consistent with a phenotype arising from stat6-deficiency , suggesting that the contribution of anti - stat6 immunity may be modest
. the contribution of anti - stat6 ctl responses may be more important in studies of graft rejection .
two reports demonstrated that stat6 mice demonstrated shorter graft survival times in models of cardiac transplant with minor histocompatibility differences , and where ctla4-ig was used to block graft rejection . although it is possible that some of this increase is due to the increased propensity of stat6 t cells to develop into inflammatory t cells , it is likely that anti - stat6 immunity may also be contributing to the observed phenotype .
the anti - stat6 immunity that develops in mice lacking endogenous stat6 clearly contributes to the increased anti - tumor immunity in stat6 mice .
have shown delayed and reduced primary mammary carcinoma growth in stat6 mice using 4t1 , a balb / c mammary carcinoma that is usually malignant , non - immunogenic and metastatic .
the authors hypothesize that the deletion of the stat6 gene facilitates development of potent anti - tumor immunity via a cd4-independent pathway .
our data suggest that immunization with the h2 stat6-expressing tumor might simply stimulate cd8 ctl specific for h2 restricted antigens from stat6-expressing cells , which lyse the cells regardless of expression of tumor antigens . indeed ,
jensen et al . demonstrate that enhanced immunity to 4t1 cells in stat6 mice is dependent upon the stat6 peptide used in our studies . similarly , kacha et al .
show that effector cells from stat6 mice have increased lytic activity and produce increased levels of ifn in response to the h2 tumor p1.htr .
thus , it is not clear that these studies actually demonstrate an effect of stat6 function , but rather may reflect immune responses that are not tolerant to stat6 peptides presented in the context of mhc i. it is important to note that autoreactivity to a missing self - protein is not a ubiquitous phenomenon .
although there is clearly an anti - stat6 ctl response in stat6 mice , there is no corresponding anti - stat4 ctl response in stat4 mice .
it is possible that stat4 peptides do not compete effectively for binding to mhc class i such that no reactive cells are positively selected in the thymus . alternatively
, mhc class i binding peptides from stat4 might be homologous enough to other endogenous peptides that reactive t cells are negatively selected in the thymus .
the differences between the phenotypes of stat4 and stat6 ctl illustrate that the autoreactive phenomenon described here must be tested empirically for each mutant strain being studied . in summary ,
our data illustrate the importance of demonstrating maintenance of self - tolerance in mice made defective in a particular gene by homologous recombination . particularly in genes that have immune function , where common techniques involve transplantation and adoptive transfer
, it is critical to define the ability of t cells to recognize peptides from the targeted proteins as foreign .
failure to do so could result in misinterpretation of how a gene of interest contributes to immune responses .
stat4 and stat6 mice were generated and backcrossed for at least 10 generations to balb / c mice as described previously .
b10.br , c3h / hej breeder mice and balb / c.dm2 spleen cells were the kind gift of dr james forman .
balb / c , c57bl/6j , nzb / b1nj , c3h / hej , b10.br and a / j breeder mice were purchased from the jackson laboratory .
all animals were bred and housed in the iusm - evansville animal facility and procedures were approved by the iusm iacuc . where indicated , mice were immunized intraperitoneally with 25 10 spleen cells in 0.5 ml balanced salt solution , rested for at least 7 d , euthanized , and their spleens removed for the in vitro generation of ctl . using specific strain combinations as described , ctl were generated in secondary mlc as described .
briefly , 5 10 responder and 5 10 irradiated ( 3000rad ) stimulator spleen cells were incubated for 67 d in rpmi 1640 supplemented with 710% fetal bovine serum , 1% l - glutamine and 50 ng / ml 2-mercaptoethanol at 37c in 7% co2 .
some ctl lines were re - stimulated weekly with irradiated stimulator cells in supplemented mishell - dutton medium that also contained t cell growth factors from ( nh4)2so4-precipitated supernatants of phorbol myristic acetate - stimulated el4.il2 cells .
generation of concanavalin a ( cona ) blast cells ( cab ) , cr labeling of target cells , the standard 46 h cr release assay , controls , and the method for determination of the % specific lysis of target cells have been described .
standard error was computed by propagation of errors as previously described.cr release assays for analysis of peptide binding , peptide blocking and antibody blocking were performed using pre - incubations as previously described .
rma - s - d , t2-d , e3 and ltkd cell lines were provided by dr d. marguiles .
using anti - cd4 antibody gk1.4 ( pharmingen ) and anti - cd8 antibody yts169.4 ( kindly provided by dr .
stat4 and stat6 mice were generated and backcrossed for at least 10 generations to balb / c mice as described previously .
b10.br , c3h / hej breeder mice and balb / c.dm2 spleen cells were the kind gift of dr james forman .
balb / c , c57bl/6j , nzb / b1nj , c3h / hej , b10.br and a / j breeder mice were purchased from the jackson laboratory .
all animals were bred and housed in the iusm - evansville animal facility and procedures were approved by the iusm iacuc . where indicated , mice were immunized intraperitoneally with 25 10 spleen cells in 0.5 ml balanced salt solution , rested for at least 7 d , euthanized , and their spleens removed for the in vitro generation of ctl .
using specific strain combinations as described , ctl were generated in secondary mlc as described . briefly
, 5 10 responder and 5 10 irradiated ( 3000rad ) stimulator spleen cells were incubated for 67 d in rpmi 1640 supplemented with 710% fetal bovine serum , 1% l - glutamine and 50 ng / ml 2-mercaptoethanol at 37c in 7% co2 .
some ctl lines were re - stimulated weekly with irradiated stimulator cells in supplemented mishell - dutton medium that also contained t cell growth factors from ( nh4)2so4-precipitated supernatants of phorbol myristic acetate - stimulated el4.il2 cells .
generation of concanavalin a ( cona ) blast cells ( cab ) , cr labeling of target cells , the standard 46 h cr release assay , controls , and the method for determination of the % specific lysis of target cells have been described .
standard error was computed by propagation of errors as previously described.cr release assays for analysis of peptide binding , peptide blocking and antibody blocking were performed using pre - incubations as previously described .
rma - s - d , t2-d , e3 and ltkd cell lines were provided by dr d. marguiles .
flow cytometry analyses of ctl lines was performed as previously described . using anti - cd4 antibody gk1.4 ( pharmingen ) and anti - cd8 antibody yts169.4 ( kindly provided by dr . | the generation of germline gene mutations in mice has been an invaluable tool for experimental biology .
however , studying immune responses that develop in the absence of a specific protein that could alter thymic selection complicates experimental interpretations .
we observed that cd8 + t cells from stat6/ mice displayed autoreactivity to stat6-expressing cells , associated with specific stat6 peptides binding to mhc class i molecules .
these results suggest caution in interpreting experiments where stat6-expressing cells are transferred into stat6/ mice , or where adoptive transfer of stat6/ lymphocytes is performed .
our results further highlight additional considerations when studying immune responses involving cell transfer into gene - deficient mice . | Introduction
Results
Discussion
Material and Methods
Mice
Cell lines and assays |
PMC4908610 | midgut volvulus occurs when there is a rotation of small bowel around its mesenteric vascular pedicle .
it is more commonly seen in the pediatric population , with approximately 85% of malrotation cases presenting in the first 2 weeks of life , and more than 90% of patients will present by their 1st birthday , . in adults ,
midgut volvulus is a rare surgical disease , with reported incidences between 0.00001% to 0.19% . despite its rarity
, midgut volvulus remains an important cause of small bowel obstruction and more importantly it can compromise blood supply to the small intestine resulting in mesenteric ischemia and infarction . as such , it requires a high index of clinical suspicion and a low threshold for operative intervention to prevent or treat the development of intestinal ischemia and/or gangrene , as this scenario carries a high morbidity and mortality rate .
those without any underlying or identifiable cause can be classified as primary cases , and are even less commonly seen . in this case report , we present a rare case of adult primary midgut volvulus mimicking acute appendicitis .
a 35-year - old chinese male presents with 1-day history of intermittent and worsening right iliac fossa pain .
past medical history includes renal stones previously treated in japan with shock wave lithotripsy a few years ago .
he denies any previous surgeries . on examination , he was afebrile , and haemodynamically normal ( blood pressure 123/79 mm hg , heart rate 68/min , oxygen saturation 98% on room air ) .
chest radiograph did not reveal any free air under diaphragm , excluding a possibility of a perforated viscus .
the patient was clinically diagnosed to have acute appendicitis , and appendectomy was promptly arranged .
after an incision was made in the mcburney s area , a moderate amount of serous fluid and dilated small bowel loops were seen on entry into the peritoneal cavity .
the appendix only appeared mildly inflammed , and upon running the bowel a jejunal volvulus was noted ( fig . 1 , fig .
this appeared secondary to extensive adhesions between the small bowel loops , and extensive adhesiolysis was performed .
the frequency and mode of presentation for midgut volvulus in adults differ by geography . in western countries ,
the annual incidence of midgut volvulus has been estimated to be about 1.75.7 per 100,000 population , and 36% of patients present with intestinal obstruction ( io ) . in africa and asia , about 2460 per 100,000 population suffer from midgut volvulus annually , with 2050% of these presenting with io . to understand intestinal rotation and its anomalies
the embryo s gut is formed as a straight tube in the fourth week of fetal life . during the fifth week , a vascular pedicle develops .
the superior mesenteric vessel supplies the midgut , and is defined by a rapidly enlarging loop with the superior mesenteric artery running out to its apex .
the cephalad portion of the loop gives rise to the first 20 feet of small bowel , and the remainder of the loop forms the distal small bowel and colon up to the splenic flexure .
intestinal rotation primarily involves the midgut . during the development of the intestine , rotation of the intestine
stage i occurs in weeks 510 , involving midgut extrusion into the extra - embryonic cavity , a 90 counter - clockwise rotation , and return of the midgut into the fetal abdomen .
stage ii occurs in week 11 and involves further counterclockwise rotation within the abdominal cavity completing a 270 rotation , bringing the duodenal
loop behind the superior mesenteric artery with the ascending colon to the right , the transverse colon above , and descending colon to the left .
stage iii involves fusion and anchoring of the mesentery , where the cecum descends , and the ascending and descending colon attach to the retroperitoneum , .
stage i anomalies include omphaloceles caused by failure of the gut to return to the abdomen .
stage iii anomalies include an unattached duodenum , mobile cecum , and an unattached small bowel mesentery .
primary midgut volvulus is more common in children and young adults , and can not be attributed to any predisposing anomaly found intra - operatively . in a previous study of midgut volvulus , the small bowel in high - risk populations were shown to have a corresponding longer mesentery , with deficient mesenteric fat , and has a narrower insertion .
a fiber - rich diet has also been implicated , particularly a large amount , with resultant forceful small bowel peristalsis and eventual volvulus , . in this case report
, our patient did not have any of these known predisposing factors , with the midgut volvulus likely secondary to congenital adhesion bands .
other predisposing conditions that could increase the likelihood of small bowel volvulus include a narrow mesenteric root , malrotation , internal hernias , diverticulosis , congenital bands , jejunal lipomatosis , colostomy , fistula , meckel s diverticulum , calcified mesenteric lymph nodes , mesenteric lipomas , pregnancy , endometriosis , abscess , mycobacterial disease , aneurysms , and hematomas , and tumors such as mesenteric lymphangiomas or jejunal adenocarcinomas , , .
midgut volvulus in adults can present with acute colicky abdominal pain usually in the peri - umbilical or epigastric regions .
they may then develop small bowel obstruction and present with nausea , vomiting and/or abdominal distension . in patients without prior history of abdominal surgery or other obvious causes like hernias , there should be a high index of suspicion of midgut volvulus as a cause of the intestinal obstruction .
patients at risk of midgut volvulus such as those with congenital midgut malrotation are usually asymptomatic .
many remain so , with the anomaly discovered only at autopsy or incidentally during ct scans performed for another indication . however , some may have chronic and unexplained abdominal discomfort , and even fewer may report acute episodes of severe abdominal pain , .
localization of small intestinal loops predominantly in the right side or absence of cecal gas shadow should arouse suspicion of malrotation ; this was not present in our patient .
ultrasonography has been reported to be helpful in diagnosis of intestinal malrotation in the literature , with the classically described whirpool sign .
standard upper gastrointestinal series may show a vertical duodenum not crossing the midline , with the entire small bowel found in the right half of the abdomen .
the gold standard of diagnosis is a computed topography ( ct ) scan of the abdomen .
malrotation can be diagnosed with ct with the anatomic location of a right - sided small bowel , a left - sided colon , an abnormal relationship of the superior mesenteric vessels , and aplasia of the uncinate process .
whirpool sign is also visible on ct imaging in patients with or without malrotation signifying volvulus .
diagnostic imaging should not be considered in a distressed patient with a rigid abdomen , as further delay will increase the likelihood or worsen any developing bowel ischemia , causing unnecessary morbidity . in our case , we elected to operate on the patient immediately with a clinical diagnosis of appendicitis . to our surprise
a delay in operative intervention by performing a ct imaging might have compromised blood flow to bowel and could have resulted in bowel ischemia necessitating bowel resection .
the classic treatment for incomplete intestinal malrotation is the ladd s procedure , which entails division of ladd s bands and mobilization of the duodenum , division of adhesions around the sma to broaden the mesenteric base , placing the small bowel primarily to the right and the large bowel to the left of the midline , and an appendectomy .
these principles have remained the same since ladd s address to the new hampshire medical society in 1936 .
recently reported a series of malrotation patients managed laparosopically . in patients with volvulus secondary to other causes ,
the principles of management include early surgical intervention to decompress bowel , resection of non - viable bowel and addressing the underlying cause , e.g. adhesiolysis for adhesions .
the frequency and mode of presentation for midgut volvulus in adults differ by geography . in western countries ,
the annual incidence of midgut volvulus has been estimated to be about 1.75.7 per 100,000 population , and 36% of patients present with intestinal obstruction ( io ) . in africa and asia , about 2460 per 100,000 population suffer from midgut volvulus annually , with 2050% of these presenting with io .
the embryo s gut is formed as a straight tube in the fourth week of fetal life . during the fifth week , a vascular pedicle develops .
the superior mesenteric vessel supplies the midgut , and is defined by a rapidly enlarging loop with the superior mesenteric artery running out to its apex .
the cephalad portion of the loop gives rise to the first 20 feet of small bowel , and the remainder of the loop forms the distal small bowel and colon up to the splenic flexure .
intestinal rotation primarily involves the midgut . during the development of the intestine , rotation of the intestine
stage i occurs in weeks 510 , involving midgut extrusion into the extra - embryonic cavity , a 90 counter - clockwise rotation , and return of the midgut into the fetal abdomen .
stage ii occurs in week 11 and involves further counterclockwise rotation within the abdominal cavity completing a 270 rotation , bringing the duodenal
loop behind the superior mesenteric artery with the ascending colon to the right , the transverse colon above , and descending colon to the left .
stage iii involves fusion and anchoring of the mesentery , where the cecum descends , and the ascending and descending colon attach to the retroperitoneum , .
stage i anomalies include omphaloceles caused by failure of the gut to return to the abdomen .
stage iii anomalies include an unattached duodenum , mobile cecum , and an unattached small bowel mesentery .
primary midgut volvulus is more common in children and young adults , and can not be attributed to any predisposing anomaly found intra - operatively . in a previous study of midgut volvulus , the small bowel in high - risk populations were shown to have a corresponding longer mesentery , with deficient mesenteric fat , and has a narrower insertion .
a fiber - rich diet has also been implicated , particularly a large amount , with resultant forceful small bowel peristalsis and eventual volvulus , . in this case report
, our patient did not have any of these known predisposing factors , with the midgut volvulus likely secondary to congenital adhesion bands .
other predisposing conditions that could increase the likelihood of small bowel volvulus include a narrow mesenteric root , malrotation , internal hernias , diverticulosis , congenital bands , jejunal lipomatosis , colostomy , fistula , meckel s diverticulum , calcified mesenteric lymph nodes , mesenteric lipomas , pregnancy , endometriosis , abscess , mycobacterial disease , aneurysms , and hematomas , and tumors such as mesenteric lymphangiomas or jejunal adenocarcinomas , , .
midgut volvulus in adults can present with acute colicky abdominal pain usually in the peri - umbilical or epigastric regions .
they may then develop small bowel obstruction and present with nausea , vomiting and/or abdominal distension . in patients without prior history of abdominal surgery or other obvious causes like hernias , there should be a high index of suspicion of midgut volvulus as a cause of the intestinal obstruction .
patients at risk of midgut volvulus such as those with congenital midgut malrotation are usually asymptomatic .
many remain so , with the anomaly discovered only at autopsy or incidentally during ct scans performed for another indication
. however , some may have chronic and unexplained abdominal discomfort , and even fewer may report acute episodes of severe abdominal pain , .
localization of small intestinal loops predominantly in the right side or absence of cecal gas shadow should arouse suspicion of malrotation ; this was not present in our patient .
ultrasonography has been reported to be helpful in diagnosis of intestinal malrotation in the literature , with the classically described whirpool sign .
standard upper gastrointestinal series may show a vertical duodenum not crossing the midline , with the entire small bowel found in the right half of the abdomen .
the gold standard of diagnosis is a computed topography ( ct ) scan of the abdomen .
malrotation can be diagnosed with ct with the anatomic location of a right - sided small bowel , a left - sided colon , an abnormal relationship of the superior mesenteric vessels , and aplasia of the uncinate process .
whirpool sign is also visible on ct imaging in patients with or without malrotation signifying volvulus .
diagnostic imaging should not be considered in a distressed patient with a rigid abdomen , as further delay will increase the likelihood or worsen any developing bowel ischemia , causing unnecessary morbidity . in our case
, we elected to operate on the patient immediately with a clinical diagnosis of appendicitis . to our surprise
a delay in operative intervention by performing a ct imaging might have compromised blood flow to bowel and could have resulted in bowel ischemia necessitating bowel resection .
the classic treatment for incomplete intestinal malrotation is the ladd s procedure , which entails division of ladd s bands and mobilization of the duodenum , division of adhesions around the sma to broaden the mesenteric base , placing the small bowel primarily to the right and the large bowel to the left of the midline , and an appendectomy .
these principles have remained the same since ladd s address to the new hampshire medical society in 1936 . generally , symptomatic patients with malrotation should have surgical intervention .
recently reported a series of malrotation patients managed laparosopically . in patients with volvulus secondary to other causes ,
the principles of management include early surgical intervention to decompress bowel , resection of non - viable bowel and addressing the underlying cause , e.g. adhesiolysis for adhesions .
adult midgut volvulus is a rare cause of small bowel obstruction but has the potential for morbidity and mortality if not diagnosed early with intestinal ischaemia and related sequlae . as such
, clinicians should maintain a reasonable index of suspicion and in diagnostic dilemmas , we recommend performing a contrast ct scan early .
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request . | highlightsadult midgut volvulus is a rare but important cause of small bowel obstruction.our patient , 35 year old male , was clinically suspected to have acute appendicitis.intra-operatively , we found a normal appendix and jejunal volvulus with adhesions.if in doubt , early contrast ct scan should be performed.delay to diagnosis and surgical intervention may result in bowel ischemia . | Introduction
Presentation of case
Discussion
Epidemiology
Embryology
Etiology
Clinical presentation
Radiological diagnosis
Management
Conclusion
Conflicts of interest
Funding
Ethical approval
Consent
Author contribution
Guarantor |
PMC3385641 | acute myocardial infarction ( mi ) remains a leading cause of morbidity and mortality worldwide . according to the latest report of the american heart association , every 25 seconds , an american will have a coronary event , and approximately every minute , someone will die of a coronary event . in 2010 , 785,000 americans experienced an mi , and approximately 470,000 had a recurring mi .
heart failure can result from adverse remodeling of the collagenous scar that replaces the damaged myocardium in the left ventricle ( lv ) after mi .
lv remodeling is mediated by cell survival , inflammation , angiogenesis , and turnover of the extracellular matrix ( ecm ) .
markers of lv remodeling can be either determined in the circulation ( e.g. , serum or plasma ) or detected in the heart by imaging technologies or biopsy .
post - mi , levels of specific matrix metalloproteinases ( mmps ) increase and mediate left ventricular remodeling .
mmp-9 has been reported as a prognostic indicator of cardiac dysfunction in mi patients [ 2 , 3 ] .
mmp-9 deletion has also been shown to improve remodeling of the lv in mice [ 4 , 5 ] .
we hypothesized that the analysis of plasma proteins post - mi in wild - type ( wt ) and mmp-9 null mice will identify prospective markers of early mi that are mmp-9 dependent .
termed as the most complex proteome , plasma is an intricate body fluid , containing a wide diversity of proteins .
plasma has been investigated using targeted evaluations , to measure markers that detect mi or predict outcomes following mi . for examples , the muscle form of creatine kinase ( ck - mb ) , troponins , and c - reactive protein are used clinically to determine both presence of mi and extent of myocardial damage [ 7 , 8 ] .
mmp-9 , galectin-3 , and brain natriuretic peptide have been used to evaluate lv responses to mi [ 911 ] .
plasma has also been investigated using proteomic approaches , but this has been fraught with technical issues , primarily because the range of protein levels in the plasma is 10 , and the ten most abundant proteins account for 90% of the total protein concentration [ 12 , 13 ] .
serum albumin is a high abundant protein in plasma , and it is the leading candidate for selective removal prior to proteomics analysis of less abundant proteins in plasma .
albumin can also be removed by ligand chromatography [ 14 , 15 ] , and isoelectric trapping .
nonetheless , the use of depletion methods may also result in specific removal of low abundant cytokines , lipoproteins , and peptide hormones of interest .
accordingly , we hypothesized that using a fractionation protocol for the analysis of plasma proteins post - mi in wild - type ( wt ) and mmp-9 null mice would identify prospective markers of early mi that are mmp-9 dependent .
in our study , we performed protein fractionation prior to protein separation by 1d - page and ms analysis . by doing so , we avoided using depletion methods and concomitantly reduced the presence of albumin and enriched for lower abundance proteins .
all animal procedures were conducted according to the guide for the care and use of laboratory animals ( nih notice number : not - od-12 - 020 ) and were approved by the institutional animal care and use committee at the university of texas at san antonio .
male 48 months old c57bl6/j wild - type ( wt ) ( n = 3 ) and mmp-9 null mice ( n = 3 ) were used in this study .
animals were housed at constant temperature ( 22 2c ) on a 12 h light / dark cycle .
they were fed ad libitum on standard laboratory mice chow and had free access to tap water .
mi was made by permanent ligation of the left anterior descending coronary artery as described previously .
animals without mi ( day 0 ) were used as controls ( n = 3/genotype ) . at one day post - mi ,
mice were anesthetized with 5% isoflurane , plasma was collected , the coronary vasculature was flushed with 0.9 m saline , and the hearts were excised .
the hearts were separated between right and left ventricles and were stained with 1% 2,3,5-triphenyltetrazolium chloride ( sigma ) and photographed for measurement of infarct area .
plasma was collected at days 0 and 1 post - mi , snap frozen and stored at 80c . at sacrifice , heparin ( 100 l of 1000 usp units / ml ) was injected intraperitoneally , and 5 min after heparin injection , blood was collected from the carotid artery of the mouse .
plasma was fractionated using the gellfree 8100 fractionation system ( protein discovery , inc . ) .
five hundred micrograms of total protein were reduced for 10 min at 50c with 1x acetate sample buffer ( protein discovery , inc . ) and 0.053 m dithiothreitol ( dtt ) .
after samples being cooled down to room temperature , 15 mm iodoacetamide was added , and samples were alkylated in the dark for 10 min . for protocol optimization , we used six different protocols where samples were either run in an 8% , 10% , or 12% tris - acetate cartridge combined with one of three fractionation programs .
we tested three different fractionation programs shown in figures 2 , 3 , and 4 .
for all of the programs , mes was used as the running buffer ( 0.05 m mes , 0.05 m tris , 0.1% sds ph 7.9 ) . for each of the six protocols
tested , twelve fractions ( 150 l / fraction ) were collected per sample and proteins were visualized on a 12% bis - tris gel by sds - page .
the proteins in fraction 1 were separated in a 1020% tricine / peptide gel .
the gel region containing visually detectable proteins from the lane for fraction 12 ( the highest molecular weight fraction ) on the bis - tris gel was excised into three slices .
each slice was separately destained and dehydrated and the proteins digested in situ with trypsin ( promega ) .
the digests were analyzed by capillary hplc - electrospray ionization tandem mass spectrometry ( hplc - esi - ms / ms ) on a thermo fisher ltq orbitrap velos mass spectrometer fitted with a new objective digital picoview 550 nanoesi source .
online hplc separation of the digests was accomplished with an eksigent / ab sciex nanolc - ultra 2-d hplc system : column , picofrit ( new objective ; 75 m i.d . )
packed to 15 cm with c18 adsorbent ( vydac ; 218ms 5 m , 300 ) .
precursor ions were acquired in the orbitrap in profile mode at 60,000 resolution ( m / z 400 ) ; data - dependent collision - induced dissociation ( cid ) spectra of the six most intense ions in the precursor scan above a set threshold were acquired at the same time in the linear trap .
mascot ( versions 2.3.02 ; matrix science ) was used to search the uninterpreted cid spectra against a combination of the mouse subset of the ncbinr database ( mus .
( 145,083 sequences ) ) and a database of common contaminants ( 179 sequences ) .
methionine oxidation was considered as a variable modification ; trypsin was specified as the proteolytic enzyme , with one missed cleavage allowed . a secondary search of the cid spectra using x !
tandem and mascot results , and determination of protein and peptide identity probabilities were accomplished by scaffold ( version 3 ; proteome software ) .
the thresholds for acceptance of peptide and protein assignments in scaffold were 95% and 99.9% , respectively .
total proteins ( 10 g ) were loaded onto either 412% bis - tris gels ( proteins > 50 kda ) or 1020% tricine gels ( proteins < 50 kda ) and run by sds - page .
proteins were transferred to a nitrocellulose membrane which was hybridized overnight at 4c with primary antibody .
primary antibodies used were antiserum amyloid a1 ( number af2948 , r&d ) , anti - alpha-2 macroglobulin ( number ab52651 , abcam ) and antineutrophil - associated gelatinase lipocalin ( ngal , aka lipocalin 2 ; number ab63929 , abcam ) ( number ab63929 , abcam ) .
immunoblot intensities ( arbitrary units ) were assessed using a one - way anova with newman - keuls multiple comparison test .
infarct areas were similar between wt and mmp-9 null mice ( p = 0.85 ; figure 1 ) , indicating that both groups received a similar injury stimulus .
the different fractionation programs are shown in the figures . when using fractionation program number 1 and an 8% acetate cartridge , proteins were only observed in fractions 10 to 12 ( figure 2 ) . by changing voltage intensities and step duration
the protein ' profiles differed depending of the type of cartridge used ( figure 3 ) . since serum albumin is approximately 66 kda , we focused on protocols that provided fractions with reduced albumin content .
the combination of program number 3 with the 8% acetate cartridge yielded fractions with these characteristics , where most of the albumin was seen in fractions 2 to 11 ( figure 4 ) .
these conditions were considered optimal for our examination , and fractions 1 and 12 , which showed reduced levels of albumin , were further analyzed by hplc - esi - ms / ms on an orbitrap velos .
supplemental tables 1 and 2 list the proteins identified in both fractions ( see supplementary material available online at doi:10.1155/2012/397103 ) , per genotype and time point . of the 145 proteins identified in the wt mice , 12 proteins were present only at day 0 , and 45 proteins were just observed 1 day post - mi ( figure 5(a ) ) . in the mmp-9
null mice , 195 proteins were identified ; of which 19 were unique to day 0 and 61 proteins were observed only post - mi ( figure 5(b ) ) . the molecular weight of proteins observed in fraction 1 ranged from 7 kda to 69 kda , although fragments of higher molecular weight proteins ( e.g. , c - terminus of alpha-2 macroglobulin ) were also present .
the majority of proteins observed in fraction 12 had molecular weights ranging from 45 kda to 263 kda ; nevertheless , lower molecular weight proteins such as transthyretin ( 16 kda ) were also observed .
the unweighted spectrum counts were used to provide measure of relative abundance ( figure 6 ) .
two percent of the proteins identified in wt animals were ecm proteins , while ecm proteins accounted for 3% of the total identified proteins in mmp-9 null mice .
serum amyloid a ( saa ) , a marker of inflammation , was observed at both time points .
saa was identified in fraction 1 of both genotypes and levels at day 0 were significantly different ( p < 0.001 ) from levels at day 1 post - mi ( figure 7(a ) ) .
ngal levels post - mi were significantly higher than at day 0 ( p < 0.05 ) but no differences were observed between genotypes ( figure 7(b ) ) .
alpha-2 macroglobulin , a generic mmp inhibitor and an mmp-9 substrate , was observed only post - mi in the wt group .
nevertheless , alpha-2 macroglobulin precursor was observed in both groups at days 0 and 1 post - mi .
quantification of alpha-2 macroglobulin showed a significant difference between genotypes 1 day post - mi ( figure 7(c ) ) .
the discovery of plasma markers remains challenging due to the complexity of the samples and the wide range of protein concentrations .
in addition , the analysis of proteomics data is a complex multistep process . therefore , to overcome these problems , effective sample preparation is of outmost importance .
efficient sample preparation will reduce component complexity and enrich for lower abundance proteins while depleting or reducing the most abundant ones .
we used a novel fractionation technique to interrogate plasma from wt and mmp-9 null mice at day 1 post - mi .
this novel technique provides all of the advantages of 1-d gel electrophoresis , with the additional benefits of increased loading capacity and high yield liquid phase recovery .
the most significant findings of this study were that ( 1 ) multiple proteins were identified in post - mi plasma , compared with day 0 control plasma ; ( 2 ) serum amyloid a is good marker of early mi but it is not mmp-9 dependent ; ( 3 ) alpha-2 macroglobulin may be an mmp-9 dependent marker . these results combined indicate that a fractionation followed by 1-d gel / lc / ms analysis strategy is effective to isolate and identify plasma proteins changes in response to mi .
we identified a total of 145 unique proteins in the wt samples and 195 unique proteins in the mmp-9 null samples . known markers of inflammation , such as haptoglobin and saa , were among the proteins identified .
studies from the malm preventive study , sweden , have shown that elevated plasma levels of haptoglobin are a risk factor for mi .
recently , devaux 's group identified haptoglobin as a potential biomarker of prognosis of heart failure in patients with acute mi .
interestingly , they state that low levels of haptoglobin early post - mi favor heart failure .
we identified haptoglobin as a potentially increased post - mi marker ; nevertheless , studies with longer time points post - mi will have to be developed to confirm the role of haptoglobin in progression to heart failure .
saa is a known marker of inflammation , and saa levels inversely correlate with cardiac function .
one - day post - mi the levels of saa were significantly higher , in both genotypes , confirming an association with mi .
we plan to investigate if changes in plasma saa are correlated with progression to heart failure post - mi . from the proteins present only post - mi
ngal is a marker of kidney injury , as well as matrix degradation and inflammation .
this protein has previously been reported to be associated with mi and heart failure [ 24 , 25 ] .
a recent paper by akcay and colleagues shows that 1-year mortality rates were significantly higher in patients with high levels of ngal .
our results were in accordance with the previous reports , showing a robust increase in ngal levels post - mi .
although the association between mmp-9 and alpha-2 macroglobulin has been previously reported , this is the first time that alpha-2 macroglobulin is associated with mmp-9 in the myocardial infarction setting .
the higher levels of alpha-2 macroglobulin observed in the mmp-9 mice post - mi suggest that this protein may be an mi biomarker that is mmp-9 dependent .
the protocols developed in this study can be used for other biological samples besides plasma .
we are currently developing a fractionation method to investigate secreted proteins in cell culture media . like in plasma , albumin is highly abundant in the commercially available serums used to supplement culture media . in vitro ,
the levels of secreted proteins are very low compared to the values observed in culture serum , making it very difficult to identify and quantify the proteins produced by the cells .
fractionation of samples is an easy and reproducible technique that can be used in a variety of models and biological samples .
mouse models of mi are very useful and important given the unique ability to genetically manipulate these animals .
however , it is important to remember that the mi mouse model does not fully mimic the human disease .
thus , postinfarct remodeling of the lv likely has differences between the mouse and human that will need to be taken into account before full translation can occur .
thus , the discovery and development of biomarkers has high potential for providing a real benefit for screening , diagnosis , prognosis , prediction of recurrence , and therapeutic monitoring of mi patients . in conclusion , by performing plasma fractionation prior to proteomics analysis , we were able to reduce the presence of high abundant proteins , such as albumin , and enrich samples for the detection of lower abundance proteins .
we compared plasma samples from wild - type and mmp-9 null mice post - mi , and identified alpha-2 macroglobulin as a prospective mi marker which may be mmp-9 dependent .
this technical report revealed that a fractionation approach is a useful technique to evaluate plasma samples . | following myocardial infarction ( mi ) , matrix metalloproteinase-9 ( mmp-9 ) levels increase , and mmp-9 deletion improves post - mi remodeling of the left ventricle ( lv ) .
we provide here a technical report on plasma - analysis from wild type ( wt ) and mmp-9 null mice using fractionation and mass - spectrometry - based proteomics .
mi was induced by coronary artery ligation in male wt and mmp-9 null mice ( 48 months old ; n = 3/genotype ) .
plasma was collected on days 0 ( pre- ) and 1 post - mi .
plasma proteins were fractionated and proteins in the lowest ( fraction 1 ) and highest ( fraction 12 ) molecular weight fractions were separated by 1-d sds - page , digested in - gel with trypsin and analyzed by hplc - esi - ms / ms on an orbitrap velos .
we tried five different fractionation protocols , before reaching an optimized protocol that allowed us to identify over 100 proteins .
serum amyloid a substantially increased post - mi in both genotypes , while alpha-2 macroglobulin increased only in the null samples . in fraction 12
, extracellular matrix proteins were observed only post - mi .
interestingly , fibronectin-1 , a substrate of mmp-9 , was identified at both day 0 and day 1 post - mi in the mmp-9 null mice but was only identified post - mi in the wt mice . in conclusion , plasma fractionation offers an improved depletion - free method to evaluate plasma changes following mi . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion |
PMC2686467 | understanding and predicting protein rna interactions at the atomic level is crucial for our ability to interfere with such processes as gene expression and regulation .
rna interactions based on the sequences and folds of the corresponding protein ( 13 ) or rna molecules ( 46 ) .
however , these do not always capture the similarity in the local regions which are responsible for protein rna recognition .
these regions are important since even proteins of the same family can form different interactions with rna nucleotides ( 7,8 ) . by analyzing the amino acid composition in rna binding sites ,
several successful methods for prediction of rna binding regions were developed ( 912 ) . however , there are only few methods ( 5 ) that distinguish between two main interaction types formed across protein rna interfaces : ( i ) interactions with the backbone of double - stranded rna molecules ; ( ii ) interactions with single - stranded rna bases that are buried in the protein binding pockets ( 14 ) . here , we focus on the classification and prediction of protein interactions formed with single - stranded nucleotide bases .
sequences of such nucleotides , which are not involved in local base pairs and are extruded from the surrounding double - stranded helix were also termed extruded helical single strands and described as structural classification of rna ( scor ) motifs ( 6,15 ) . as estimated by the recent study of ellis and jones ( 16 ) , the flexibility in the protein binding sites
is not significant and should allow structural prediction of protein rna interaction . in our recent work ( 17 )
we observed that most of the protein interacting nucleotides are part of a consecutive fragment of at least two nucleotides , whose rings have significant interactions with the protein .
many such pairs were observed to share the same protein binding cavity and > 30% of these pairs are -stacked .
we showed that the classification of the nucleotide and dinucleotide binding sites reveals similarities in patterns important in protein rna recognition .
we further showed that searching for known binding patterns on the surface of a target protein allows the prediction of its dinucleotide binding sites with a high success rate ( 17 ) . here , we present the rna binding site data base ( rsitedb ) , which details and classifies the nucleotide and dinucleotide binding pockets from all known protein rna complexes .
the database contains the 3d physico - chemical patterns that describe the main types of the interactions .
the on - line search engine of rsitedb allows to search these patterns in a query protein .
this predicts the binding sites and the binding modes of rna dinucleotide . the on - line structural search against the entire data set of 3d patterns takes only several minutes and provides an atomic level prediction of protein rna interactions . as illustrated in figure 1
, rsitedb contains the nucleotide binding sites extracted from all known protein rna complexes .
these binding sites are classified into clusters according to the spatial arrangement of the protein physico - chemical and geometrical properties .
the created clusters provide a set of 3d consensus binding patterns , which represent the main types of protein nucleotide interactions .
this classification is useful both for the analysis of existing interactions and for prediction of unknown ones .
rsitedb is based on the classification of the 3d binding patterns extracted from all protein rna complexes .
first , given a protein rna complex , rsitedb analyzes and classifies its protein nucleotide interactions .
second , given an unbound protein structure , rsitedb predicts the regions that can function as dinucleotide binding sites .
rsitedb is based on the classification of the 3d binding patterns extracted from all protein rna complexes .
first , given a protein rna complex , rsitedb analyzes and classifies its protein nucleotide interactions .
second , given an unbound protein structure , rsitedb predicts the regions that can function as dinucleotide binding sites .
the first option allows the analysis of existing complexes , we refer to it as rsitedb analysis subsequently .
this retrieves the properties and the similarities of nucleotide and dinucleotide binding sites stored in the database .
this is a different type of search algorithm which performs an online structural search of the query protein against the database of 3d - consensus binding patterns .
the search algorithm is based on an efficient geometric hashing algorithm ( 18 ) , which allows a simultaneous comparison to all of the database patterns ( 17 ) .
regions that are structurally and physico - chemically similar to any of these patterns are predicted to serve as binding sites .
the rna nucleotides , bound to the top ranking patterns from the database , predict the binding modes of nucleotides to the query protein .
below we detail the information provided by rsitedb and the different ways to query it .
the sections are organized according to the screens presented to the user at the different stages of the analysis ( see figures 24 ) .
the top left figure illustrates the basic analysis of the input protein rna complex .
for each pair of interacting chains it presents the number of nucleotide and dinucleotide binding sites .
making the selection marked in light blue allows the user to explore the table of dinucleotide binding sites presented at the bottom .
the top right figure illustrates one of the dinucleotide binding sites , which is marked in light blue .
the extruded rna nucleotides are purple sticks and the surface of the protein binding pocket is represented by green dots .
blue , acceptors red , donors / acceptors green , hydrophobic aliphatic orange and aromatic white / gray .
figure 3.rsitedb classification .
rsitedb details the properties of the binding sites in the cluster ( top table ) and provides the transformations that can align them in 3d space .
each binding site in a cluster is described by its pdb code , chain identifies and nucleotide identities ( e.g. 1sj3pru51g52 ) .
it has three columns which provide the following details of its matched pseudocenters : ( i ) chain identifier and residue number ; ( ii ) residue type and ( iii ) pseudocenter type . although the pseudocenters are not required to have the same amino acid identity or origin ( backbone or side chain )
, we indicate the conservation of these ( * or b / s , respectively ) .
it describes the complex which initiated the cluster and was used for the pattern construction .
these provide the prediction of the interactions of the query protein with the nucleotide bases .
the query protein is cyan and the amino acids involved in the predicted interactions are represented by sticks .
the protein which represents the pattern is magenta ribbons and its binding site surface is green dots .
the top left figure illustrates the basic analysis of the input protein rna complex . for each pair of interacting chains
making the selection marked in light blue allows the user to explore the table of dinucleotide binding sites presented at the bottom .
the top right figure illustrates one of the dinucleotide binding sites , which is marked in light blue .
the extruded rna nucleotides are purple sticks and the surface of the protein binding pocket is represented by green dots .
blue , acceptors red , donors / acceptors green , hydrophobic aliphatic orange and aromatic white / gray .
rsitedb details the properties of the binding sites in the cluster ( top table ) and provides the transformations that can align them in 3d space .
each binding site in a cluster is described by its pdb code , chain identifies and nucleotide identities ( e.g. 1sj3pru51g52 ) .
it has three columns which provide the following details of its matched pseudocenters : ( i ) chain identifier and residue number ; ( ii ) residue type and ( iii ) pseudocenter type .
although the pseudocenters are not required to have the same amino acid identity or origin ( backbone or side chain ) , we indicate the conservation of these ( * or b / s , respectively ) .
it describes the complex which initiated the cluster and was used for the pattern construction .
these provide the prediction of the interactions of the query protein with the nucleotide bases . as shown in the right figure , rsitedb visualizes the constructed complex . the query protein is cyan and the amino acids involved in the predicted interactions
the protein which represents the pattern is magenta ribbons and its binding site surface is green dots .
the pseudocenters and the rna dinucleotides are colored as in figure 1 . given the structure of a protein rna complex ( specified by its pdb code ) , rsitedb details its interacting protein - rna chains .
for each pair of chains , rsitedb details the number of atomic contacts , which are defined by atoms within a distance of 5 .
a pair of protein rna chains is considered to be interacting if there are at least 10 atomic contacts between them .
we further analyze the protein interacting nucleotides and dinucleotides , their geometries and the properties of the corresponding protein binding sites .
we define a nucleotide binding site by the protein connolly solvent accessible surface area ( 19 ) within 2 from the surface of the rna base .
nucleotides with a protein binding site area larger than 3 are defined as protein interacting .
given a pair of extruded consecutive nucleotides that interact with the protein , a dinucleotide binding site is defined by a pair of corresponding nucleotide binding sites .
figure 1 presents the details provided by rsitedb and illustrates one binding pocket accommodating a pair of consecutive -stacked nucleotides .
rsitedb presents such parameters as the distance and angle between consecutive nucleotides as well as the binding site surface area .
rsitedb considers the protein binding sites represented by their surfaces and the physico - chemical properties termed pseudocenters ( 20 ) .
these are points in 3d space extracted from the protein amino acids that represent groups of atoms according to the interactions in which they may participate : hydrogen - bond donor ( don ) , hydrogen - bond acceptor ( acc ) , mixed donor / acceptor ( dac ) , hydrophobic aliphatic ( ali ) and aromatic contacts ( pi ) .
rna complex is either assigned to a cluster of similar binding sites or described as unique .
members of the same cluster share a similar spatial arrangement of pseudocenters , which we term a 3d consensus pattern .
the multiple alignment of nucleotide and dinucleotide binding sites is performed with the multibind and rnabind methods , respectively ( 17 ) .
figure 3 illustrates the analysis of the 3d consensus pattern described by the pseudocenters matched by the alignment .
for each matched pseudocenter , we present its property as well as the details of the amino acid that originated it . as illustrated in figure 3 , rsitedb provides a jmol visualization of the multiple alignment and of the common pattern shared by the cluster members .
first , it accounts for the spatial physico - chemical properties of dinucleotide binding sites .
second , it is based on a classification methodology , which performs multiple binding sites alignment and validates the spatial superimposition of the cluster members .
this overcomes the problem that objects similar in pairs may not be similar as a whole group .
specifically , by using multiple alignment , we assess the quality of the constructed 3d consensus pattern , which is required to be shared by all cluster members and to constitute at least 30% of each binding site ( 17 ) .
the classification algorithm was applied to a non - redundant data set of protein rna complexes , which is provided at the rsitedb web site .
this data set was constructed by considering all high - resolution x - ray structures of protein rna complexes [ ndb release may 2008 ( 21 ) , resolution better than 3 ] .
we extracted all pairs of interacting chains and removed protein and rna chains with sequence identity above 25% and 60% in both chains , respectively .
the classification of this non - redundant data set created 61 clusters of binding sites with more than one member .
approximately 44% of these clusters involve proteins with different sequences [ < 25% similarity and different pfam annotations ( 25 ) ] .
complexes that were removed due to redundancy , were added at the later stages of classification , and were assigned to the cluster of the closest homologue that fulfills the classification requirement ( i.e. the constructed 3d consensus pattern is at least 30% of each of the cluster members ) . using this procedure , 60% of all the dinucleotide binding sites and 45% of the single nucleotide binding sites
the same procedure was applied to the available nmr structures , which are assigned to the created clusters and are analyzed by rsitedb . here , we use the created clusters to predict the rna binding sites that accommodate unpaired extruded nucleotides . specifically , given a potentially unbound protein structure ,
these are defined by the above described clusters . due to the low number of significant clusters of single nucleotide binding sites , currently only the dinucleotide patterns are used for the prediction .
the search is performed with the rnapred algorithm ( 17 ) , which outputs a list of alignments to different protein regions that are recognized to contain some of the constructed 3d patterns . for each alignment , we detail the rigid transformation that can superimpose the pattern upon the protein in 3d space .
we apply the transformation and provide a pdb complex of the solution which includes the query protein , the superimposed 3d patterns , its binding site surface and rna dinucleotides .
figure 4 presents an example of output page which details the matched pseudocenters and visualizes the predicted complex .
alternatively , the user can download all of the alignments with the corresponding pdb files of their superimposition .
we provide scripts for the rasmol software , allowing an off - line visualization of the results . using leave - one - out tests ,
interestingly , 32% of the correct predictions were made based on proteins with different sequences ( < 25% identity and no common pfam domain ) and could not be obtained based on recognition of sequence motifs .
the main contribution of our knowledge - based predictions is that they describe the protein physico - chemical patterns that may be involved in interactions and predict the spatial orientation of the rna nucleotides in the protein binding site independent of the protein overall sequences or folds .
as illustrated in figure 1 , rsitedb contains the nucleotide binding sites extracted from all known protein rna complexes .
these binding sites are classified into clusters according to the spatial arrangement of the protein physico - chemical and geometrical properties .
the created clusters provide a set of 3d consensus binding patterns , which represent the main types of protein nucleotide interactions .
this classification is useful both for the analysis of existing interactions and for prediction of unknown ones .
rsitedb is based on the classification of the 3d binding patterns extracted from all protein rna complexes .
first , given a protein rna complex , rsitedb analyzes and classifies its protein nucleotide interactions .
second , given an unbound protein structure , rsitedb predicts the regions that can function as dinucleotide binding sites .
rsitedb is based on the classification of the 3d binding patterns extracted from all protein rna complexes .
first , given a protein rna complex , rsitedb analyzes and classifies its protein nucleotide interactions .
second , given an unbound protein structure , rsitedb predicts the regions that can function as dinucleotide binding sites .
the first option allows the analysis of existing complexes , we refer to it as rsitedb analysis subsequently .
this retrieves the properties and the similarities of nucleotide and dinucleotide binding sites stored in the database .
this is a different type of search algorithm which performs an online structural search of the query protein against the database of 3d - consensus binding patterns .
the search algorithm is based on an efficient geometric hashing algorithm ( 18 ) , which allows a simultaneous comparison to all of the database patterns ( 17 ) .
regions that are structurally and physico - chemically similar to any of these patterns are predicted to serve as binding sites .
the rna nucleotides , bound to the top ranking patterns from the database , predict the binding modes of nucleotides to the query protein .
below we detail the information provided by rsitedb and the different ways to query it .
the sections are organized according to the screens presented to the user at the different stages of the analysis ( see figures 24 ) .
the top left figure illustrates the basic analysis of the input protein rna complex . for each pair of interacting chains
making the selection marked in light blue allows the user to explore the table of dinucleotide binding sites presented at the bottom .
the top right figure illustrates one of the dinucleotide binding sites , which is marked in light blue .
the extruded rna nucleotides are purple sticks and the surface of the protein binding pocket is represented by green dots .
blue , acceptors red , donors / acceptors green , hydrophobic aliphatic orange and aromatic white / gray .
rsitedb details the properties of the binding sites in the cluster ( top table ) and provides the transformations that can align them in 3d space .
each binding site in a cluster is described by its pdb code , chain identifies and nucleotide identities ( e.g. 1sj3pru51g52 ) .
it has three columns which provide the following details of its matched pseudocenters : ( i ) chain identifier and residue number ; ( ii ) residue type and ( iii ) pseudocenter type . although the pseudocenters are not required to have the same amino acid identity or origin ( backbone or side chain ) , we indicate the conservation of these ( * or b / s , respectively ) . the rna dinucleotides are represented as sticks , colored by their atoms .
figure 4.prediction of dinucleotide binding sites . the results of searching rsitedb with a query protein .
it describes the complex which initiated the cluster and was used for the pattern construction .
these provide the prediction of the interactions of the query protein with the nucleotide bases .
the query protein is cyan and the amino acids involved in the predicted interactions are represented by sticks .
the protein which represents the pattern is magenta ribbons and its binding site surface is green dots .
the top left figure illustrates the basic analysis of the input protein rna complex . for each pair of interacting chains
making the selection marked in light blue allows the user to explore the table of dinucleotide binding sites presented at the bottom .
the top right figure illustrates one of the dinucleotide binding sites , which is marked in light blue .
the extruded rna nucleotides are purple sticks and the surface of the protein binding pocket is represented by green dots .
blue , acceptors red , donors / acceptors green , hydrophobic aliphatic orange and aromatic white / gray .
rsitedb details the properties of the binding sites in the cluster ( top table ) and provides the transformations that can align them in 3d space .
each binding site in a cluster is described by its pdb code , chain identifies and nucleotide identities ( e.g. 1sj3pru51g52 ) .
it has three columns which provide the following details of its matched pseudocenters : ( i ) chain identifier and residue number ; ( ii ) residue type and ( iii ) pseudocenter type .
although the pseudocenters are not required to have the same amino acid identity or origin ( backbone or side chain ) , we indicate the conservation of these ( * or b / s , respectively ) .
it describes the complex which initiated the cluster and was used for the pattern construction .
these provide the prediction of the interactions of the query protein with the nucleotide bases .
the query protein is cyan and the amino acids involved in the predicted interactions are represented by sticks .
the protein which represents the pattern is magenta ribbons and its binding site surface is green dots .
given the structure of a protein rna complex ( specified by its pdb code ) , rsitedb details its interacting protein - rna chains .
for each pair of chains , rsitedb details the number of atomic contacts , which are defined by atoms within a distance of 5 .
a pair of protein rna chains is considered to be interacting if there are at least 10 atomic contacts between them .
we further analyze the protein interacting nucleotides and dinucleotides , their geometries and the properties of the corresponding protein binding sites .
we define a nucleotide binding site by the protein connolly solvent accessible surface area ( 19 ) within 2 from the surface of the rna base .
nucleotides with a protein binding site area larger than 3 are defined as protein interacting .
given a pair of extruded consecutive nucleotides that interact with the protein , a dinucleotide binding site is defined by a pair of corresponding nucleotide binding sites .
figure 1 presents the details provided by rsitedb and illustrates one binding pocket accommodating a pair of consecutive -stacked nucleotides .
rsitedb presents such parameters as the distance and angle between consecutive nucleotides as well as the binding site surface area .
rsitedb considers the protein binding sites represented by their surfaces and the physico - chemical properties termed pseudocenters ( 20 ) .
these are points in 3d space extracted from the protein amino acids that represent groups of atoms according to the interactions in which they may participate : hydrogen - bond donor ( don ) , hydrogen - bond acceptor ( acc ) , mixed donor / acceptor ( dac ) , hydrophobic aliphatic ( ali ) and aromatic contacts ( pi ) .
rna complex is either assigned to a cluster of similar binding sites or described as unique .
members of the same cluster share a similar spatial arrangement of pseudocenters , which we term a 3d consensus pattern . for each cluster ,
the multiple alignment of nucleotide and dinucleotide binding sites is performed with the multibind and rnabind methods , respectively ( 17 ) .
figure 3 illustrates the analysis of the 3d consensus pattern described by the pseudocenters matched by the alignment . for each matched pseudocenter ,
we present its property as well as the details of the amino acid that originated it . as illustrated in figure 3
, rsitedb provides a jmol visualization of the multiple alignment and of the common pattern shared by the cluster members .
first , it accounts for the spatial physico - chemical properties of dinucleotide binding sites .
second , it is based on a classification methodology , which performs multiple binding sites alignment and validates the spatial superimposition of the cluster members .
this overcomes the problem that objects similar in pairs may not be similar as a whole group .
specifically , by using multiple alignment , we assess the quality of the constructed 3d consensus pattern , which is required to be shared by all cluster members and to constitute at least 30% of each binding site ( 17 ) .
the classification algorithm was applied to a non - redundant data set of protein rna complexes , which is provided at the rsitedb web site .
this data set was constructed by considering all high - resolution x - ray structures of protein rna complexes [ ndb release may 2008 ( 21 ) , resolution better than 3 ] .
we extracted all pairs of interacting chains and removed protein and rna chains with sequence identity above 25% and 60% in both chains , respectively .
the classification of this non - redundant data set created 61 clusters of binding sites with more than one member .
approximately 44% of these clusters involve proteins with different sequences [ < 25% similarity and different pfam annotations ( 25 ) ] .
complexes that were removed due to redundancy , were added at the later stages of classification , and were assigned to the cluster of the closest homologue that fulfills the classification requirement ( i.e. the constructed 3d consensus pattern is at least 30% of each of the cluster members ) . using this procedure , 60% of all the dinucleotide binding sites and 45% of the single nucleotide binding sites
the same procedure was applied to the available nmr structures , which are assigned to the created clusters and are analyzed by rsitedb .
here , we use the created clusters to predict the rna binding sites that accommodate unpaired extruded nucleotides . specifically , given a potentially unbound protein structure , we search its surface for regions similar to the 3d consensus binding patterns .
these are defined by the above described clusters . due to the low number of significant clusters of single nucleotide binding sites , currently only the dinucleotide patterns are used for the prediction .
the search is performed with the rnapred algorithm ( 17 ) , which outputs a list of alignments to different protein regions that are recognized to contain some of the constructed 3d patterns . for each alignment , we detail the rigid transformation that can superimpose the pattern upon the protein in 3d space .
we apply the transformation and provide a pdb complex of the solution which includes the query protein , the superimposed 3d patterns , its binding site surface and rna dinucleotides .
figure 4 presents an example of output page which details the matched pseudocenters and visualizes the predicted complex .
alternatively , the user can download all of the alignments with the corresponding pdb files of their superimposition .
we provide scripts for the rasmol software , allowing an off - line visualization of the results . using leave - one - out tests ,
interestingly , 32% of the correct predictions were made based on proteins with different sequences ( < 25% identity and no common pfam domain ) and could not be obtained based on recognition of sequence motifs .
the main contribution of our knowledge - based predictions is that they describe the protein physico - chemical patterns that may be involved in interactions and predict the spatial orientation of the rna nucleotides in the protein binding site independent of the protein overall sequences or folds .
all of the files that describe the classification and its data sets are provided at the website .
the prediction algorithm , which screens the protein of interest against the database of 3d consensus patterns , is extremely fast with an average running time of 3 min . in the case of longer running times , caused by the large size of the query protein or the server overload
, the user can provide an email to which a link to the output page will be sent .
the visualization of the results is based on jmol , which requires a web browser that supports java applets .
clore phd fellowship ( a.s .- p . ) ; israel science foundation ( 281/05 to h.j.w . ) ; national institute of allergy and infectious diseases ( niaid ) ; national institute of health ( 1uc1ai067231 ) ; binational us - israel science foundation ( bsf ) ; hermann minkowski - minerva center for geometry at tau ; national cancer institute ; national institutes of health ( contract noi - co-12400 ) ; intramural research program of the nih , national cancer institute , center for cancer research . | we present a new database and an on - line search engine , which store and query the protein binding pockets that interact with single - stranded rna nucleotide bases .
the database consists of a classification of binding sites derived from protein rna complexes .
each binding site is assigned to a cluster of similar binding sites in other protein rna complexes .
cluster members share similar spatial arrangements of physico chemical properties , thus can reveal novel similarity between proteins and rnas with different sequences and folds .
the clusters provide 3d consensus binding patterns important for protein nucleotide recognition .
the database search engine allows two types of useful queries : first , given a pdb code of a protein rna complex , rsitedb can detail and classify the properties of the protein binding pockets accommodating extruded rna nucleotides not involved in local rna base pairing .
second , given an unbound protein structure , rsitedb can perform an on - line structural search against the constructed database of 3d consensus binding patterns .
regions similar to known patterns are predicted to serve as binding sites .
alignment of the query to these patterns with their corresponding rna nucleotides allows making unique predictions of the protein rna interactions at the atomic level of detail .
this database is accessable at http://bioinfo3d.cs.tau.ac.il/rsitedb . | INTRODUCTION
RsiteDB overview
RsiteDB analysis
RsiteDB classification
RsiteDB prediction
PERFORMANCE AND AVAILABILITY
FUNDING |
PMC4515906 | gaseous microemboli are a concern during surgery with cardiopulmonary bypass as potential cause of neurocognitive deficit since these microbubbles were verified by ultrasonic microbubble counters operating with blood flow rates up to 6 l / min [ 1 , 2 ] . in haemodialysis ( hd ) with much lower blood flow rates ( up to 0.6 l / min ) , microbubbles have also been a concern , but proper measurement was difficult .
microemboli were detected in the subclavian vein downstream from the arteriovenous fistula of hd patients a decade ago [ 3 , 4 ] .
it was speculated that microbubbles lodge in the capillary bed of various organs , mainly the lungs , and that they obstruct the blood flow in the capillaries , causing tissue ischaemia and micro - infarct , followed by inflammatory reaction , complement activation and tissue damage .
patients who undergo surgery with cardiopulmonary bypass are usually treated only once , whereas hd patients are repeatedly treated , in total > 600 h each year .
any infusion of microbubbles during hd may become significant for the patient 's health due to the cumulative exposure time .
when it became technically possible to measure microbubbles in hd , it was demonstrated that they may pass the venous bubble catcher and the safety system against air infusion without triggering an alarm , in vitro and during treatments .
they observed a significantly higher number of patients with elevated embolic counts on the arterial side than would be expected if an open foramen ovale was the only option , and concluded that a considerable number of microemboli seem to pass the lung .
two years later , a case report of a chronic hd patient was published : he suffered from cardiac arrest and died .
the autopsy revealed a closed foramen ovale ; the microscopic investigation verified microembolies of air that were surrounded by fibrin in the lungs , the heart and the brain .
the investigators concluded that microbubbles were present before death , passed the cardiopulmonary circulation , entered the left heart and were distributed throughout the body .
autopsies of five patients ( died either during or up to 2.3 days after the hd session ) revealed pulmonary fibrosis and pulmonary microemboli ( fibrin around air bubbles ) .
a study by british investigators confirmed microemboli in the arteriovenous fistulae of hd patients during their treatment , but found no evidence of microembolization in the middle cerebral artery .
microbubbles were determined at the site of the arteriovenous access for different blood levels in the venous bubble catcher in a randomized , double - blinded study : the lowest number of microbubbles was observed during high blood levels , confirmed in a second study .
recently , an australian group published a computational analysis of microbubbles in hd : most bubbles with a diameter of < 50 m , and many bubbles in the range of 50200 m pass through the venous bubble catcher .
it seems that microbubbles generally occur in hd treatments . up to now , the available information on the potentially harmful microbubbles in hd is not consistent .
the aim of our investigation was to detect possible sources of the generated microbubbles and to increase the knowledge on microbubble formation .
the study was designed as purely observational , non - interventional investigation in a single centre , without documentation of patient data .
twenty - nine treatments of 21 patients were observed , covering a broad range of patient and treatment conditions ( types of blood access , treatment modes , blood flow rates and arterial pressures ) .
the applied diagnostic medical device was the non - invasive , bubble counter bcc200 ( gampt gmbh , merseburg , germany ) , a pulsed ultrasonic doppler system with a transmission frequency of 2 mhz , measuring microbubbles in the range of 10500 m .
the applied dialysis machines were 5008 haemodialysis systems ( fresenius medical care , germany ) .
two types of dialysers were applied : xevonta hi23 ( bbraun , germany ) and fx1000 ( fresenius medical care , germany ) .
the standard position of the two sensor heads of the microbubble counter was on the arterial line ( between arterial access and blood pump ) and on the venous line ( between venous bubble catcher and venous access ) . for a consistency check ,
the arterial and the venous sensor heads of the bcc200 were positioned during a stable treatment at the same blood line in a row .
the number of measured microbubbles differed by 8% in the normal and in reversed orders ; thus , the two sensor heads give identical information with a deviation below 10% .
some air reservoirs in the blood tubing were obvious , such as in the venous bubble catcher .
other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size .
. the usually high level of blood was not changed due to the purely observational character of our study .
bubbles were found
at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments .
measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m .
it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case
, we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter .
obviously , the visible volume had become the source of many microbubbles . in a single case ,
the two sensor heads were positioned up- and downstream of the arterial luer lock connector . the downstream sensor head measured 46 microbubbles and the upstream sensor head only 2 , indicating that small amounts of air were entering the blood line at this connection . at this part of the tubing system ,
as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter .
arterial pressures were observed in the range between 230 and 80 mmhg . in total , measurements with complete data sets were obtained from 29 treatments , including 21 patients , with an observed treatment time summed up to 7871 min ( 131 h ) .
we observed a correlation between the count of microbubbles and the blood flow rate and the arterial pressure , see figures 1 and 2 .
the microbubble rate increased with the blood flow rate and the negative arterial pressure .
1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
fig .
2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
an overview of our observational results concerning microbubble rate is shown in figure 3 .
the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure .
3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm .
the number of patients ( n ) as well as the number of treatments ( n ) per group is noted .
for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below .
microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm .
the number of patients ( n ) as well as the number of treatments ( n ) per group is noted .
for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below .
generally , a large difference between the groups was seen with a spread in microbubble rate up to a factor of 5 .
microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter ,
blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) .
this observation was possibly due to the substitution fluid applied during online haemodiafiltration , prepared from degassed dialysate .
the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg )
was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) .
these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate .
a large spread was observed also concerning the number and volume of the injected microbubbles .
the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l .
the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l .
some air reservoirs in the blood tubing were obvious , such as in the venous bubble catcher .
other potential air reservoirs were magnitudes smaller ; in rare cases , air bubbles with a diameter of a few millimetres were seen , and in most cases , air bubbles were found only after attentive search due to their extremely small size .
. the usually high level of blood was not changed due to the purely observational character of our study .
bubbles were found
at the upper part of the dialyser on the dialysate side, at the dialyser caps on the blood side of the dialyser, in the blood line segment of the roller pump, in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments . at the upper part of the dialyser on the dialysate side , at the dialyser caps on the blood side of the dialyser , in the blood line segment of the roller pump , in syringes with fluids for injections ( such as heparin or iron ) , partly injected into the blood line during the treatments .
measurements with the bubble counter usually revealed a broad range of sizes , with bubble diameters between 10 and 300 m , mostly between 10 and 200 m .
it was repeatedly observed that the start of the blood pump was accompanied by an increase in the bubble count , higher than the level when the blood pump was running continuously . in a single case , we could follow a small visible air volume moving through the heparin line into the main blood line ; this volume vanished downstream of the dialyser , but instead 33 microbubbles were detected by the microbubble counter .
obviously , the visible volume had become the source of many microbubbles . in a single case
, the two sensor heads were positioned up- and downstream of the arterial luer lock connector .
the downstream sensor head measured 46 microbubbles and the upstream sensor head only 2 , indicating that small amounts of air were entering the blood line at this connection . at this part of the tubing system ,
as planned , a broad range of treatment conditions could be investigated , including the treatment modes online haemodiafiltration and hd , one single - needle treatment , access via cimino fistula , teflon shunt or catheter .
arterial pressures were observed in the range between 230 and 80 mmhg . in total , measurements with complete data sets were obtained from 29 treatments , including 21 patients , with an observed treatment time summed up to 7871 min ( 131 h ) . we observed a correlation between the count of microbubbles and
the blood flow rate and the arterial pressure , see figures 1 and 2 .
the microbubble rate increased with the blood flow rate and the negative arterial pressure .
1.microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
fig .
2.microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
microbubble rates as function of the blood flow rate , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
microbubble rates as function of the negative arterial pressure , measured during 29 treatments ( haemodialysis and online haemodiafiltration ) in 21 patients .
an overview of our observational results concerning microbubble rate is shown in figure 3 .
the data were grouped first according to the treatment mode , hd or online haemodiafiltration , and then according to blood access and cannula diameter . additionally , the largest group of 14 treatments with identical blood access , cannula diameter and treatment mode was divided into three groups by arterial pressure .
3.microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm .
the number of patients ( n ) as well as the number of treatments ( n ) per group is noted .
for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below .
microbubble rates for different treatments : haemodialysis ( hd ) and online haemodiafiltration ( ohdf ) , blood access by catheter [ single needle ( sn ) catheter or double lumen ( dl ) catheter ] , gore tex shunt or cimino fistula , with cannula diameters of 1.8 mm or 2.0 mm .
the number of patients ( n ) as well as the number of treatments ( n ) per group is noted .
for all groups the blood flow ( qblood ) and the arterial pressure ( part ) are listed below .
generally , a large difference between the groups was seen with a spread in microbubble rate up to a factor of 5 .
microbubble rates below 40 per minute were observed for hd treatments with catheters at blood flow rates of 200 ml / min ( 1st bar ) , and online haemodiafiltration treatments in patients with goretex shunts ( 3rd bar ) or cimino fistulas with mean blood flow rates < 360 ml / min ( 5th and 6th bars ) . at similar conditions concerning blood access , cannula diameter , blood flow rate and arterial pressure , a much higher microbubble rate was observed during hd than during online haemodiafiltration ( 2nd bar versus 6th bar ) .
this observation was possibly due to the substitution fluid applied during online haemodiafiltration , prepared from degassed dialysate .
the highest microbubble rates were observed for high blood flow rates and low arterial pressures ( 4th and 7th bars ) . a low arterial pressure ( 210 mmhg )
was observed ( although a large cannula diameter was applied : 2.0 mm ) at the very high blood flow rate ( 550 ml / min ) . at a lower blood flow rate ( 376 ml / min ) with a lower cannula diameter ( 1.8 mm ) , the arterial pressure was also low ( 198 mmhg ) .
these observations indicate that a high blood flow rate and a low arterial pressure may lead to a high microbubble rate .
a large spread was observed also concerning the number and volume of the injected microbubbles .
the lowest microbubble rate was observed in patients with goretex shunts , a cannula diameter of 1.8 mm , treated by online haemodiafiltration at a medium blood flow rate ( 339 ml / min ) and a moderate arterial pressure ( 112 mmhg ) , resulting in a mean value of 3723 microbubbles per treatment with a total volume of 17 l .
the highest microbubble rate was observed in patients with cimino fistula , a cannula diameter of 2.0 mm , treated by online haemodiafiltration at a high blood flow rate ( 550 ml / min ) and a low arterial pressure ( 210 mmhg ) , resulting in a mean of 29 889 microbubbles per treatment with a total volume of 232 l .
our observations increased the knowledge on formation kinetic and quantification of microbubbles in hd . in interventional studies , more microbubbles
were observed for a low blood level in the venous bubble catcher than for a high level [ 7 , 12 , 13 ] .
air might be dragged below the blood level , and at low blood levels , the buoyant force might not be sufficient to separate small bubbles from downwards flowing blood . then , small bubbles may be injected into the patient [ 6 , 7 , 1214 ] . due to the non - interventional character of our study , the blood level in the venous bubble catcher remained without change ; we could not observe that the air above the high blood level contributes to the formation of microbubbles .
the persistent air bubbles found on the dialysate side of the dialyser did not vanish possibly due to a slow degradation of large bubbles at the air fluid interface by the degassed dialysate ( the dissolution time for air bubbles in water strongly increases with larger bubble diameter ) .
the observed split of a visible bubble into smaller fragments that were no longer visible gave cause for three concerns concerning patient safety .
first , as already demonstrated [ 6 , 7 , 1214 ] , microbubbles may pass the safety system against air infusion without triggering an alarm .
second , microbubbles may be transported into the patient , and if small enough , pass the lung capillaries , and be distributed throughout the body .
larger gas bubbles have a higher dissolution time and a higher chance to get stuck in a capillary .
moreover , microbubbles may increase or decrease depending on surrounding pressure ( e.g. sparkling water effect ) .
consequently , different scenarios concerning the lifetime of microbubbles inside the human body are possible .
the microbubbles measured upstream of the arterial luer lock connector were possibly a remnant of these microbubbles inside the patient 's body .
third , the total surface area increases . if a bubble is divided into smaller bubbles , then the surface area increases ( e.g. for a split into 64 bubbles , the surface area increases by the factor of 4 ) .
each increase in the surface area may also mean an increase in interaction of air with blood and the endothelium of the capillaries .
microbubbles may originate from the remaining air of an incomplete initial filling with insufficient rinsing of the extracorporeal circuit . if the remaining air was the only source of the microbubbles , then the microbubble count should monotonously decrease . in the beginning of the treatments ,
the mean microbubble count was an order of magnitude larger than during the rest of the treatment .
even in long treatments ( e.g. nocturnal dialysis ) , the number of microbubbles was not decreasing towards the end of the treatment , indicating either other sources of microbubble generation or large
stores of initially not removed air somewhere in the blood tubing . an increase in bubble count
was repeatedly observed at the start of the blood pump , possibly due to a sudden displacement of the remaining bubbles in the blood line , suction of bubbles from a sideline ( e.g. heparin line ) or from the roller pump segment into the main line .
our simultaneous measurement up- and downstream of the arterial luer lock connector indicated that , in the negative pressure region , small amounts of air may enter the blood line .
this single observation at the arterial connector did not allow generalization concerning prevalence of incomplete tightness .
the involved volume was so small that the safety system against air infusion did not react . generally , the tightness of luer lock connectors is under discussion [ 1720 ] .
the substitution fluid is made from degassed dialysis fluid , thus should be counteracting the microbubbles ( e.g. by diluting ) . on the other hand , online haemodiafiltration
is often performed with a high blood flow rate and a low negative arterial pressure .
possibly , a high microbubble rate may not be due to the online haemodiafiltration per se , but due to a low negative arterial pressure ( e.g. by small cannula diameter ) .
a large randomized clinical trial demonstrated the survival benefit of online haemodiafiltration ; a major influence of microbubbles as a confounding factor on the primary outcome parameter all - cause mortality was unlikely , because the blood flow rate was similar in both the study groups ( differences only between 2 and 5% ) .
we did not observe treatments without microbubbles , although the measurements covered different conditions ; thus , it seems that microbubbles are always present during a standard hd treatment .
this observation is in line with other investigations , and microbubbles were observed during treatments with the dialysis machines of all manufacturers [ 4 , 8 , 11 , 12 ] , indicating that microbubbles are a general side effect of hd .
but the range of the microbubble counts was large ; obviously , in the standard hd setting , there are treatments with a low generation of microbubbles .
there are several limitations of this non - interventional study : the study was performed in a single centre with a small number of patients and treatments .
nevertheless , some preventive actions were identified ( partially mentioned in earlier studies ) :
the cannula diameter should be adjusted to the blood flow rate. the arterial luer lock connector should be tightened. careful filling and sufficient rinsing in the preparation phase might reduce remaining gas bubbles. syringes filled with fluids for injections ( such as heparin or iron ) and side lines should be given special care. the blood in the venous bubble catcher should be kept at a high level [ 6 , 7 , 12 , 13]. extreme blood flow rates and arterial pressures should be avoided.from the visible bubbles ( at blood pump segment , dialyser cap , venous chamber and heparin line ) , the total volume was estimated to a few microliters , obviously not enough to be the only source of microbubbles .
careful filling and sufficient rinsing in the preparation phase might reduce remaining gas bubbles .
syringes filled with fluids for injections ( such as heparin or iron ) and side lines should be given special care .
the blood in the venous bubble catcher should be kept at a high level [ 6 , 7 , 12 , 13 ] .
systematic investigations in this field are scarce . considering the clinical observations , one might hypothesize that a degassing or cavitation phenomenon of blood may contribute in generating microbubbles , even if the threshold pressure gradient is not achieved .
the results should be further investigated in sound laboratory experiments , in vitro and clinical studies . generally , during hd
, microbubbles are infused in large numbers into the patients , but the involved volume is extremely small . if there is an impact on the patient 's health , it seems scarcely due to the miniscule volume of an individual microbubble , because this volume is many orders of magnitude below any volume inducing air embolism .
the large surface areas of the microbubbles and their enormous number appear to be more eligible candidates .
during hd , a large number of microbubbles may reach the patients , but the involved volume is extremely small .
origin and pathophysiologic consequences of this phenomenon are not well understood but deserve further study and , if necessary , a new blood line design aiming to minimize blood air interfaces . | backgroundthe infusion of microbubbles as a side effect of haemodialysis was repeatedly demonstrated in recent publications , but the knowledge on the source of microbubbles and on microbubble formation is scarce.methodsmicrobubbles in the range of 10500 m were measured by a non - invasive bubble counter based on a pulsed ultrasonic doppler system in a non - interventional study of a single centre .
totally , 29 measurements were performed in standard treatments covering a broad range of patient and treatment conditions ( types of blood access , treatment modes , blood flow rates and arterial pressures).resultsseveral possible sources of microbubbles could be identified such as an arterial luer lock connector at negative pressure and remnant bubbles from insufficient priming , but some sources of microbubbles remain unknown .
microbubbles were found in all treatments , haemodialysis ( hd ) and online haemodiafiltration .
the lowest average microbubble rates ( 17 16 microbubbles per minute ) were observed in patients treated by online haemodiafiltration at medium blood flow rates and moderate arterial pressures and the highest average microbubble rates ( 117 63 microbubbles per minute ) at high blood flow rates ( 550 ml / min ) and low arterial pressures ( 210 mmhg ) .
generally , the microbubble rate correlated to both blood flow rate ( correlation coefficient r = 0.45 ) and negative arterial pressure ( r = 0.67).conclusionsmicrobubbles are a general side effect of hd ; origin and pathophysiologic consequences of this phenomenon are not well understood , and deserve further study . | Introduction
Materials and methods
Results
Observation of visible bubbles
Detection of microbubbles
Discussion
Conclusion
Conflict of interest statement |
PMC3263266 | soft tissue defects of the extremity that expose underlying critical structures like bone / tendon / joint require a flap cover .
many of these defects , especially if they are moderate to large in size , require free tissue transfer for a successful reconstruction .
a soft tissue defect requiring a free flap which is longer than that provided by the conventional large free flaps like latissimus dorsi ( ld ) and anterolateral thigh ( alt ) is a challenging problem .
we have used the boomerang - shaped extended rectus abdominis myocutaneous ( beram ) flap to reconstruct nine such defects .
this flap has a robust blood supply , consistent anatomy and has caused acceptable donor morbidity .
the purpose of this study was to retrospectively analyse the outcome of nine such reconstructions ( in terms of flap outcome , complications , donor site morbidity and functional outcome ) performed in our unit during the last 3 years .
we also conducted a clinical study on 30 volunteers ( 10 males , 10 females and 10 children ) , where we used standard markings for alt flap and ld flap and did a comparison of the available flap lengths with our design of beram flap .
we present nine patients who underwent coverage of long soft tissue defects with the beram flap in our unit in last 3 years [ table 1 ] . in five cases ,
the flap was used in upper extremity , and in four cases , it was used in lower extremity soft tissue reconstruction .
the age of patients ranged between 8 years to 39 years and all were male patients .
the defect sizes ranged between 25 and 39 cm in length and between 8 and 12 cm in width .
a pattern of the defect was created , with the position of recipient vessels clearly marked .
we believe that the most important measurement in this kind of situation is the distance from the location of the ideal recipient vessels to the distal - most point of the defect .
a line was drawn from the pubic symphysis to the umbilicus , and extended upwards and laterally towards the inferior angle of scapula [ figures 1 and 2 ] .
the proximal ( inferior ) vertical segment of flap extended from pubic symphysis to umbilicus and overlay the rectus abdominis muscle at paramedian position , and the width of the flap was kept limited to allow primary closure of this part of the defect in all cases .
the distal ( superior ) oblique cutaneous segment was designed symmetrically on a line from umbilicus to inferior angle of scapula the distal limit of the flap was kept at the mid - axillary line in seven cases , and extended up to the posterior axillary line in the other two cases ( case nos 4 and 6 ) the other usual flap sites ( ld , alt flap , groin flap , parascapular flap ) were also marked according to standard measurements mentioned by mathes and nahai and maximum possible flap dimensions were noted . in all cases , we found that beram flap provided the longest flap .
diagrammatic representation of design and axis of the flap and local vascular anatomy picture depicting the flap design on patient adrenaline in saline ( 1 in 200 ) was injected along flap margins .
distal cutaneous part of flap was raised from the chest wall starting from the distal tip of the flap and proceeding towards the umbilicus [ figure 3a ] .
dissection proceeded in the subcutaneous plane until the lateral border of the anterior rectus sheath was crossed and the first major paraumbilical perforator(s ) encountered [ figure 3b ] .
( c ) transverse incision at rectus sheath just superior to first paraumbilical perforator a transverse incision was made in the rectus sheath just superior to the perforator(s ) [ figure 3c ] and another vertical incision made in the sheath just lateral to the major perforator(s ) .
the rectus muscle was cut transversely a little above the umbilicus ( preserving the main paraumbilical perforators in the flap .
the medial skin margin of the flap was incised just lateral to the umbilicus , bringing the incision to the midline below the umbilicus .
the flap included the oblique supra - umbilical skin paddle , the lower half of the rectus muscle along with its overlying rectus sheath , fat and skin [ figure 3d ] . as the rectus muscle was lifted off from its bed , the deep inferior epigastric vessels were identified and followed to their origin from the external iliac vessels [ figure 3e and f ] .
the pedicle was divided and the flap transferred [ figure 3 g and h ] . in all our cases ,
skin was closed in layers with a negative suction drain [ figure 3i ] . in one patient ,
in whom the oblique cutaneous portion of the flap was broader , that part of the donor area was skin grafted .
( e ) identifying the deep inferior epigastric artery on the deeper surface of the rectus muscle .
( f ) completion of flap dissection showing comparison with 15 cm ruler and diea pedicle at its origin ( g ) final defect after thorough debridement and harvested beram flap kept aside .
the patient was positioned supine under general anaesthesia . the recipient defect was prepared and healthy recipient vessels were identified .
a pattern of the defect was created , with the position of recipient vessels clearly marked .
we believe that the most important measurement in this kind of situation is the distance from the location of the ideal recipient vessels to the distal - most point of the defect .
a line was drawn from the pubic symphysis to the umbilicus , and extended upwards and laterally towards the inferior angle of scapula [ figures 1 and 2 ] .
the proximal ( inferior ) vertical segment of flap extended from pubic symphysis to umbilicus and overlay the rectus abdominis muscle at paramedian position , and the width of the flap was kept limited to allow primary closure of this part of the defect in all cases .
the distal ( superior ) oblique cutaneous segment was designed symmetrically on a line from umbilicus to inferior angle of scapula the distal limit of the flap was kept at the mid - axillary line in seven cases , and extended up to the posterior axillary line in the other two cases ( case nos 4 and 6 ) the other usual flap sites ( ld , alt flap , groin flap , parascapular flap ) were also marked according to standard measurements mentioned by mathes and nahai and maximum possible flap dimensions were noted . in all cases , we found that beram flap provided the longest flap .
diagrammatic representation of design and axis of the flap and local vascular anatomy picture depicting the flap design on patient
distal cutaneous part of flap was raised from the chest wall starting from the distal tip of the flap and proceeding towards the umbilicus [ figure 3a ] .
dissection proceeded in the subcutaneous plane until the lateral border of the anterior rectus sheath was crossed and the first major paraumbilical perforator(s ) encountered [ figure 3b ] .
( c ) transverse incision at rectus sheath just superior to first paraumbilical perforator a transverse incision was made in the rectus sheath just superior to the perforator(s ) [ figure 3c ] and another vertical incision made in the sheath just lateral to the major perforator(s ) .
the rectus muscle was cut transversely a little above the umbilicus ( preserving the main paraumbilical perforators in the flap .
the medial skin margin of the flap was incised just lateral to the umbilicus , bringing the incision to the midline below the umbilicus .
the flap included the oblique supra - umbilical skin paddle , the lower half of the rectus muscle along with its overlying rectus sheath , fat and skin [ figure 3d ] . as the rectus muscle was lifted off from its bed , the deep inferior epigastric vessels were identified and followed to their origin from the external iliac vessels [ figure 3e and f ] .
the pedicle was divided and the flap transferred [ figure 3 g and h ] . in all our cases ,
skin was closed in layers with a negative suction drain [ figure 3i ] . in one patient ,
in whom the oblique cutaneous portion of the flap was broader , that part of the donor area was skin grafted .
( e ) identifying the deep inferior epigastric artery on the deeper surface of the rectus muscle .
( f ) completion of flap dissection showing comparison with 15 cm ruler and diea pedicle at its origin ( g ) final defect after thorough debridement and harvested beram flap kept aside .
a 40-year - old male presented with type iiib open fracture of radial head , loss of lateral condyle of humerus and composite tissue loss over the extensor aspect of the right forearm and distal arm [ figure 4a ] .
he underwent wound debridement , external fixator application , lateral collateral ligament reconstruction [ figure 4b ] and coverage with a 35 12 cm beram flap [ figure 4c g ] .
a ) side swipe injury with composite tissue loss over the extensor aspect of the right forearm and distal arm .
( h ) after flap debulking at 1 year a 35-year - old industrial worker sustained a major crush injury with type iiib fracture in both bones of right leg , with metatarsal fracture with composite tissue loss ( 47 8 cm ) over anterolateral aspect of right leg and foot .
patient underwent medial gastrocnemius muscle flap cover for proximal one - third of the right leg and free beram flap ( 43 8 cm ) transfer for the critical raw area over the distal two - thirds of anteromedial aspect of the right leg and dorsum of foot .
residual raw area and superolateral part of flap donor area was covered with split skin graft .
he started non - weight - bearing walking in 3 weeks and walking without support in 12 weeks .
this was a 30-year - old male with severe crush injury of the left forearm following a vehicular accident .
after debridement , we were left with a soft tissue defect of 35 10 cm .
considering the gross comminution along with bone loss , a decision was taken to create a single bone forearm by plate osteosynthesis between distal radius and proximal ulna .
wound was covered by a free beram flap ( 35 10 cm ) with end - to - side arterial anastomosis to brachial artery and three end - to - end venous anastomoses .
the bones united uneventfully . later on , the patient underwent secondary flap debulking procedure . in his recent follow - up , he was found to be able to perform all his daily activities and was able to drive a bike using his single bone forearm .. an 8-year - old male child had an avulsion injury of the anteromedial aspect of the left leg , extending from the knee to the distal third of leg , exposing subluxated knee joint and most of the tibia .
defect measuring 12 30 cm was covered by free beram flap . in this case , the flap marking had been extended beyond the mid - axillary line .
postoperatively , the distal 5 cm of the flap got necrosed [ figure 5a ] which was debrided and covered with an inferiorly based local transposition skin flap [ figure 5b ] .
he was able to walk with support in 4 weeks time ( a ) 1 week postoperative picture of flap with partial distal necrosis .
( b ) post debridement of necrotic segment of flap and coverage with inferiorly based local transposition flap we also conducted a clinical study with randomly selected 30 volunteers ( 10 children aged 415 years , 10 adult males and 10 adult females ) [ figure 6 ] .
the subject 's height , standard flap marking and measurements were taken for alt and ld flaps according to the standard measurements given by mathes and nahai , and beram flap was marked according to above - described markings .
the comparison of possible flap length in relation to volunteer 's height and comparison of ld and alt flap length to beram flap length was done [ table 2 ] .
representation of clinical study showing standard flap markings and comparison of different flap lengths in centimeters clinical study : children 's measurements adult female measurements adult male measurements
a 40-year - old male presented with type iiib open fracture of radial head , loss of lateral condyle of humerus and composite tissue loss over the extensor aspect of the right forearm and distal arm [ figure 4a ] .
he underwent wound debridement , external fixator application , lateral collateral ligament reconstruction [ figure 4b ] and coverage with a 35 12 cm beram flap [ figure 4c g ] .
a ) side swipe injury with composite tissue loss over the extensor aspect of the right forearm and distal arm .
a 35-year - old industrial worker sustained a major crush injury with type iiib fracture in both bones of right leg , with metatarsal fracture with composite tissue loss ( 47 8 cm ) over anterolateral aspect of right leg and foot .
patient underwent medial gastrocnemius muscle flap cover for proximal one - third of the right leg and free beram flap ( 43 8 cm ) transfer for the critical raw area over the distal two - thirds of anteromedial aspect of the right leg and dorsum of foot .
residual raw area and superolateral part of flap donor area was covered with split skin graft .
he started non - weight - bearing walking in 3 weeks and walking without support in 12 weeks .
this was a 30-year - old male with severe crush injury of the left forearm following a vehicular accident .
after debridement , we were left with a soft tissue defect of 35 10 cm .
considering the gross comminution along with bone loss , a decision was taken to create a single bone forearm by plate osteosynthesis between distal radius and proximal ulna .
wound was covered by a free beram flap ( 35 10 cm ) with end - to - side arterial anastomosis to brachial artery and three end - to - end venous anastomoses .
the bones united uneventfully . later on , the patient underwent secondary flap debulking procedure . in his recent follow - up , he was found to be able to perform all his daily activities and was able to drive a bike using his single bone forearm ..
an 8-year - old male child had an avulsion injury of the anteromedial aspect of the left leg , extending from the knee to the distal third of leg , exposing subluxated knee joint and most of the tibia .
defect measuring 12 30 cm was covered by free beram flap . in this case , the flap marking had been extended beyond the mid - axillary line .
postoperatively , the distal 5 cm of the flap got necrosed [ figure 5a ] which was debrided and covered with an inferiorly based local transposition skin flap [ figure 5b ] .
he was able to walk with support in 4 weeks time ( a ) 1 week postoperative picture of flap with partial distal necrosis .
( b ) post debridement of necrotic segment of flap and coverage with inferiorly based local transposition flap
we also conducted a clinical study with randomly selected 30 volunteers ( 10 children aged 415 years , 10 adult males and 10 adult females ) [ figure 6 ] .
the subject 's height , standard flap marking and measurements were taken for alt and ld flaps according to the standard measurements given by mathes and nahai , and beram flap was marked according to above - described markings .
the comparison of possible flap length in relation to volunteer 's height and comparison of ld and alt flap length to beram flap length was done [ table 2 ] .
representation of clinical study showing standard flap markings and comparison of different flap lengths in centimeters clinical study : children 's measurements adult female measurements adult male measurements
our clinical experience of nine cases combined with results of our clinical study has confirmed that our design of free beram flap consistently provides a flap length which is an average 32.6% longer than the standard ld myocutaneous / skin flap and an average 42.2% longer than the standard alt flap in adults . in children , beram is 71% longer than alt flap and 46.7% longer than ld flap.the free beram flap was consistently reliable in all the seven cases in which the distal end did not extend beyond the mid - axillary line . in the two patients in whom the flap had been extended beyond the mid - axillary line , the portion beyond the mid - axillary line was lost.in our follow - up so far ( minimum 1 year ) ,
no patient has reported a hernia or a weakness of the abdominal wall.in our clinical study , we found that for adult men and women , the available flap length to ( patient ) height ratio for alt , ld and beram flaps was on average 19.79% , 21.31% and 28.09% , respectively .
our clinical experience of nine cases combined with results of our clinical study has confirmed that our design of free beram flap consistently provides a flap length which is an average 32.6% longer than the standard ld myocutaneous / skin flap and an average 42.2% longer than the standard alt flap in adults . in children , beram is 71% longer than alt flap and 46.7% longer than ld flap .
the free beram flap was consistently reliable in all the seven cases in which the distal end did not extend beyond the mid - axillary line .
in the two patients in whom the flap had been extended beyond the mid - axillary line , the portion beyond the mid - axillary line was lost . in our follow - up so far ( minimum 1 year ) , no patient has reported a hernia or a weakness of the abdominal wall . in our clinical study , we found that for adult men and women , the available flap length to ( patient ) height ratio for alt , ld and beram flaps was on average 19.79% , 21.31% and 28.09% , respectively .
pennington and pelly in 1980 reported two successful rectus abdominis myocutaneous free flaps based on deep inferior epigastric artery ( diea ) .
they mentioned a long pedicle with wide vascular lumen , easy harvest and aesthetic and functional acceptability as advantages .
the disadvantages included the need to reconstruct anterior rectus sheath to prevent hernia formation and the bulk of the flap .
the vascular anatomy of the diea and its paraumbilical perforators has been well studied.[46 ] variants of diea - based flaps like vertical rectus abdominis muscle / myocutaneous ( vram ) and transverse rectus abdominis muscle / myocutaneous ( tram ) flaps have been described earlier . while technically our flap qualifies to be an extended deep inferior epigastric free flap , the same name would be applied to most of the above - mentioned variants and modifications .
free boomerang - shaped extended rectus abdominis myocutaneous ( beram ) flap because the phrase boomerang - shaped closely describes the shape of the flap and makes it easy to understand and remember .
he observed that by including the paraumbilical cutaneous perforator(s ) , the flap can be designed with a diagonally placed upper skin island extending beyond the rectus muscle from umbilicus to costal margin , giving a long flap with an equally long vascular pedicle in a free flap and wide arc of rotation in case of a pedicled flap .
this is because these perforators communicate by means of choke vessels to the anterior branches of the lateral intercostal vessels at a 45 angle to the anterior axillary line .
boyd et al . performed cadaveric studies by dye injections , dissections , and barium radiographic studies to delineate the vascular territories of superior epigastric and deep inferior epigastric vessels and they observed that majority of the abdominal wall was stained through injection of the deep inferior epigastric system .
the beram flap has several attributes that make it unique and many interesting comparisons can be made to its cousins , the vram and tram flaps .
first , the distal skin paddle is easy to mark and elevate . on an average , a 1012 cm wide flap
can easily be harvested with direct closure and the length - to - width ratio is around 3:1 in all these flaps .
the beram flap is simpler because of a more limited inclusion of rectus muscle ( only the inferior half ) , while in vram flap the extra muscle dissection adds time to the procedure and probably increases postoperative pain . in obese patients , owing to the characteristic distribution pattern of fat in the abdominal wall ,
the distal skin paddle in vram flap is likely to be bulkier than that of beram flap .
the beram flap distal skin paddle ( beyond the periumbilical area ) is quite thin in most people , and in 67% cases , it has been less than 1 cm in thickness .
the beram flap skin paddle donor site can be closed immediately without compromising the visualisation of the pedicle , unlike the tram flap , where closure of the donor site must be performed after harvest of the flap .
the distal skin paddle which lies beyond the repaired rectus sheath can be made as broad as is required , and if necessary the donor area is grafted .
the skin paddle donor site is closed without additional elevation of skin flaps as seen in the tram flap closure , which requires elevating and mobilising the upper abdominal skin . the rectus muscle and
anterior fascial dissection is the same as a free tram flap , as opposed to the greater muscle dissection of the vram flap .
boyd , taylor and other investigators have shown the pedicled extended rectus abdominis myocutaneous flap to be versatile in its arc of rotation and very reliable , with no reported cases of distal flap necrosis .
no significant long - term donor - site complications were noted in our study or in other studies .
a similar flap was described by edmond and franklyn as periumbilical axial flap which was designed on an axis between the umbilicus and the inferior angle of the scapula running upwards laterally as far as the mid - axillary line or even the posterior axillary line . in our series , the flap had been extended beyond the mid - axillary line in two cases . in both these cases , we lost this portion of the flap .
all the above designs of flaps involve a proximal muscle with diea pedicle and a distal skin paddle of variable length .
our design of beram flap provides the longest continuous skin paddle of any flap in the body .
we feel in certain situations having a skin paddle for the major part of the defect has definite advantages .
the myocutaneous flap is , however , bulkier than a muscle flap asko et al . did a comparative study of 51 patients operated by ld and rectus abdominis free flap and they found that average rectus flap skin island was larger than that used for the ld flap ( maximal length 47 vs. 34 cm ) , which shows ram flap to be 38% longer than ld flap , but in our clinical study we found that beram flap was 32.6% longer than ld flap in adults , using standard landmarks . in our surgical experience of over 200 ld muscle flaps
, we have frequently noticed that if we transfer a complete ld muscle , the distal 34 cm of the flap does not survive , so the final length difference would be even more .
gnter germann and markus hlbauer mentioned ld muscle dimensions : length 35 cm ( range 2142 cm ) and width 20 cm ( range 1426 cm ) .
did a study of anterolateral femoral flap applied anatomy based on a doppler analysis of 50 volunteer adults and cadaver dissections of 42 legs ; vascular injection technique were used in 35 cadavers .
, we also found that alt flap length varies between 12 and 38 cm ( average 28.4 cm . ) depending on the patient 's height .
in our experience beram flap is the free flap which provides the longest skin paddle in the body . it is easy to harvest , has a long reliable pedicle , and causes minimal donor site morbidity .
based on the findings of this study , it could be argued that beram flap would be the free flap of choice for coverage of long soft tissue defect over extremities in males , and females beyond child - bearing age .
owing to the significant scarring of abdominal wall , we would not recommend this flap for use in females of , or below child - bearing age .
more studies , including dye injection studies , are warranted to more precisely define the safe distal limit of beram flap .
larger prospective clinical studies along with long - term functional and quality of life evaluations are warranted to conclude that one flap is superior to other for reconstruction of extremity . | background : a soft tissue defect requiring flap cover which is longer than that provided by the conventional long free flaps like latissimus dorsi ( ld ) and anterolateral thigh ( alt ) flap is a challenging problem . often , in such a situation , a combination of flaps is required . over the last 3 years
, we have managed nine such defects successfully with a free boomerang - shaped extended rectus abdominis myocutaneous ( beram ) flap .
this flap is the slightly modified and free version of a similar flap described by ian taylor in 1983.materials and methods : this is a retrospective study of patients who underwent free beram flap reconstruction of soft tissue defects of extremity over the last 3 years .
we also did a clinical study on 30 volunteers to compare the length of flap available using our design of beram flap with the maximum available flap length of ld and alt flaps , using standard markings.results:our clinical experience of nine cases combined with the results of our clinical study has confirmed that our design of beram flap consistently provides a flap length which is 32.6% longer than the standard ld flap and 42.2% longer than the standard alt flap in adults .
the difference is even more marked in children .
the beram flap is consistently reliable as long as the distal end is not extended beyond the mid - axillary line.conclusion:beram flap is simple in design , easy to harvest , reliable and provides the longest possible free skin / myocutaneous flap in the body .
it is a useful new alternative for covering long soft tissue defects in the limbs . | INTRODUCTION
MATERIALS AND METHODS
Surgical technique
BERAM flap design and marking
Flap dissection
CASE REPORTS
Case 1
Case 2
Case 3
Case 4
Clinical study
RESULTS
DISCUSSION
CONCLUSIONS |
PMC3364026 | finger vein recognition is a promising biometric recognition technology which verifies identities through finger vein patterns .
medical studies have shown that the finger vein pattern is unique and stable . in detail , the finger veins of an individual are different from the others ' , and even the veins captured from a single individual are quite different from one finger to another . furthermore , the finger veins are also invariant for healthy adults .
compared with fingerprints , finger veins are hard to be forged or stolen as they are hidden inside the fingers .
the contactless captures of finger veins also ensure both convenience and cleanliness , and they are user - friendly .
furthermore , finger veins are less affected by physiology and environment factors such as dry skin and dirt .
thirdly , the features are extracted . finally , the finger vein images are matched based on the extracted features .
the preprocessing procedure includes image enhancement , normalization , and segmentation . for image enhancement , yang and yan incorporated directional decomposition and frangi filtering to enhance the image quality .
yang and yang introduced multi - channel gabor filter to enhance the images and obtained better performance . finger vein segmentation is also a very important step , and there are some typical methods including line tracking , mean curvatures , and region growth - based feature .
however , a summary of these approaches with the typical references is provided in table 1 .
wu and liu extracted the pca features and then trained a neural network for matching , which results in a high recognition rate .
since pca transforms the 2-dimensional image matrix to a 1-dimensional vector , the covariance matrix is always large in size and it is time intensive to obtain the projection matrix which is composed of the covariance matrix 's eigenvectors . yang et al .
proposed 2dpca to reduce the size of the covariance matrix and save time for computing projection matrices . in order to represent the characteristics of the 2-dimensional images more accurately , zhang and zhou introduced ( 2d ) pca which can reflect the information of the image in row and column directions , respectively , use less time to compute the projection matrix , and get better experimental results on face recognition
wu and liu used pca and lda to extract features and train a svm model for recognition . measuring
for example , knn requires a distance metric to find the neighbors of the target instance and then conducts classification or regression based on the distance metric .
typical distance metrics , such as euclidean distance , make significant contribution in some application domains . in some conditions
, these metrics can not satisfy the assumption that the distances between instances from the same class are small while those from different classes are large .
there are two challenges for finger vein recognition : ( 1 ) how to efficiently extract distinguishing features and ( 2 ) how to design a strong classifier with high recognition rate and fast recognition speed to make the system more practical in real - world applications . to overcome these two challenges , in this paper
we apply ( 2d ) pca to extract the features from finger vein images . in order to address the shortcoming of traditional distance - metric - based classifiers
, we build a classifier for each individual based on metric learning . with regard to training samples of each classifier ,
thus , we use smote technology to oversample the positive samples to balance the two classes before training the classifier .
the experimental results show that the proposed method has good performance on finger vein recognition .
pca is a typical linear dimensionality reduction and feature extraction method . due to the transformation from the 2-dimensional image matrix into a 1-dimensional column vector ,
pca often makes the size of the corresponding covariance matrix too large , and computing the eigenvectors and eigenvalues becomes time - consuming . in order to solve this problem , yang et al .
2dpca directly uses the image matrix to compute pca features without transforming the 2-dimensional image matrix into a 1-dimensional column vector .
therefore , it reduces the size of corresponding covariance matrix and obtains the feature projection matrix with less time .
however , 2dpca works only for the row direction of images . to address the problem ,
zhang and zhou proposed ( 2d ) pca which captures the image information from not only the row direction but also the column direction .
the experimental results show that ( 2d ) pca outperforms 2dpca and pca in terms of both recognition rate and running time . the process of ( 2d ) pca is described as follows . considering m finger vein images , which are denoted by a1 , , am , we compute the mean image matrix as a = ( 1/m)jaj and the image covariance matrix gas
( 1)g= 1mj=1m(aja)t(aja ) .
for a random image matrix a , the key of obtaining the new features is to get the projection matrix x r , nd . then the new features are calculated as y = ax .
the total scatter of the projected samples is used to determine a good projection matrix x , where the total scatter of the projected samples can be characterized by the trace of the covariance matrix of the projected feature vectors . from this point of view , we adopt the following criterion :
( 2 ) j(x)=trace{e[(ye(y))(ye(y))t ] } = trace{e[(ax e(ax))(ax e(ax))t ] } = trace{xte[(ae(a))t(ae(a))]x}.
( 3)j(x)=trace{xtgx}.
it has been proven that j(x ) gets the maximum when the projection matrix x is composed by the d orthonormal eigenvectors coupled to the d largest eigenvalues . in so saying , x obtains the optional value , and d can be controlled by setting a threshold as follows :
( 4)i=1dij=1nj ,
where is a user - specific threshold and 1 , 2 , , n is the top - n largest eigenvalues of g. because x only reflects the information in the row direction , zhang and zhou proposed alternative 2dpca which reflects the information in the column direction and combines 2dpca with alternative 2dpca to obtain a new method called ( 2d ) pca . here is the process of alternative 2dpca .
let the image matrix aj = [ ( aj), ,(aj ) ] , and the mean image matrix a=[(a(1))t, ,(a(n))t]t , where aj and a(i ) , denote the ith row vector of aj and a(i ) respectively .
the image covariance matrix can be rewritten as
( 5)g1=1mj=1 m k=1m(aj(k)a(k))t(aj(k)a(k ) ) .
similarly , to achieve the projected matrix x in 2dpca ,
we can obtain the projection matrix z r from ( 2 ) and ( 5 ) .
we can also compute q in the same manner as we compute d in 2dpca . using the projected matrix x , z in 2dpca and alternative 2dpca , respectively
, we can obtain the new feature we can see from ( 6 ) that the new feature c reflects more information of the image than the features obtained by 2dpca and alternative 2dpca .
furthermore , the dimension of c is smaller , and thus ( 2d ) pca costs less time than 2dpca and alternative 2dpca for image processing .
the main task of the metric learning is to find a better distance metric , based on which the distances between the samples from same class become small while those from different classes become large .
this helps to improve the performance of the machine learning methods . to overcome the shortage of the knn classifier using euclidean distance , weinberge et al .
proposed a metric learning method called lmnn ( large margin nearest neighbor ) which learns a distance metric to improve the performance of knn classifiers .
the metric is obtained by learning a linear transformation matrix l. with this distance metric , the distance between the same - class instances becomes smaller , and they are separated from the other instances by a large margin .
( i = 1 , , n ) denote the feature vector of training instances and
the essence of lmnn is to obtain a new distance d(xi , xj ) = ||l(xixj)|| = ( xixj)ll(xi xj ) after learning a linear transformation l matrix . with this distance metric ,
the distance between the instance and its k nearest neighbors will be minimized and the distance between the instances in different classes will be larger .
green circles denote instances from the first class , yellow squares denote instances from the second class , and red squares denote instances from the third class .
consider the instance denoted by the white circle , which is treated as a test instance from the first class , in our following analysis .
based on euclidean distance , we find 4 nearest neighbors , and this test instance is misclassified into the second class .
however , using the lmnn - learned metric ; this instance is separated from the second and the third instances .
the other is to resample the original dataset , either by oversampling the minority class and/or undersampling the majority class .
chawla et al . proposed an oversampling approach called smote where the minority class is oversampled by creating
the minority class is oversampled by taking each minority class sample and introducing synthetic examples along the line segments joining any / all of the k minority class nearest neighbors . depending upon the amount of oversampling required , neighbors from the k nearest neighbors are randomly chosen .
the proposed method includes training process and recognition process . as shown in figure 2 ,
a classifier is built for each individual , and the samples from a certain individual are treated as positive and others are negative . in the verification mode , we input a test sample to corresponding classifier to verify whether the sample comes from this individual based on the classification result . in the identification mode , we input a test sample to every classifier and identify which individual this sample belongs to . in the training process , it is necessary to preprocess the infrared images of the finger veins .
preprocessing includes grayscale , roi selection , and normalization ( e.g. , size normalization and gray normalization ) .
after the preprocessing , we apply ( 2d ) pca to extract the features of the training samples .
then we label the samples as positive and negative class accordingly and oversample the positive samples with smote .
we learn a new distance metric , that is , the transformation matrix l , with lmnn .
the preprocessing and feature extraction in the recognition process are similar to that in the training process .
after that , we input the features of the samples to train classifier to verify the individual based on the classification result .
the preprocessing includes image grayscale , roi selection , size normalization , and gray normalization . the original image ( an example is shown in figure 3(a ) )
is a 24-bit color image with a size of 320 240 . in order to reduce the computational complexity
, we transform the original image to an 8-bit image based on the gray - scale equation y = r 0.299 + g 0.588 + b 0.114 , where r , g , and b denote the value of red , green , and blue .
as the background of finger vein region might include noise , we employ an edge - detection method to segment the finger vein region from the gray - scale image .
a sobel operator with a 3 3 mask [ -101 - 202 - 101 ] is used for detecting the edges of fingers .
the width of the finger region can be obtained based on the maximum and minimum abscissa values of the finger profile , and the height of the finger region is similarly detected .
the size of the selected roi is different from image to image due to personal factors such as different finger size and changing location .
therefore it is necessary to normalize the roi region to the same size before the feature extraction process by ( 2d ) pca .
we use bilinear interpolation for size normalization in this paper , and the size of the normalized roi is set to be 96 64 ( as shown in figure 3(c ) ) . in order to extract efficient features ,
gray normalization is used to obtain a uniform gray distribution ( as shown in figure 3(d ) ) .
formally , we have
( 7)p(i , j)=p(i , j)g1g2g1 ,
where p(i , j ) is the pixel value of the original image , g1 is the min pixel value of original image , g2 is the max pixel value of original image , and p(i , j ) is the pixel value of image after gray normalization . after the preprocessing , we extract the features for each image by ( 2d ) pca and assign labels for them .
a classifier is trained for every individual , where the samples belonging to this individual are treated as positive and others are negative .
we oversample the positive samples by smote to obtain an augmented training set which achieves class balance in general .
lmnn is then used on the augmented training set to obtain a transformation matrix l. with this new distance metric , a knn classifier is built for classification . in the verification mode , we input the feature vector of a test sample to a classifier which represents a certain individual , and then we verify whether the sample belongs to this individual based on the classification result . in the identification mode , we employ all classifiers to classify the test sample .
if only a classifier c classifies it as positive class , this sample belongs to the individual which corresponds to the classifier c. if there are many classifiers classifying the sample as positive class , then we use the training accuracy rate for decision making : the sample belongs to the individual that corresponds to the classifier with the best training accuracy .
the experiments were conducted using our finger vein database which is collected from 80 individuals ' ( including 64 males and 16 females , asian race ) index fingers of right hand , where each index finger contributes 18 finger vein images .
each individual participated in two sessions , separated by two weeks ( 14 days ) .
the age of the participants was between 19 and 60 years , and their occupations included university students , professors , and workers at our school .
the capture device was manufactured by the joint lab for intelligent computing and intelligent system of wuhan university , china , which is illustrated in figure 4 .
after roi extraction and size normalization , the size of the region used for feature extraction is reduced to 96 64 .
therefore , there are 80 classes , where each class contains 18 samples in our database .
all the experiments are implemented with matlab and conducted on a machine with 2.4 g cpu and 4 g memory .
we design three experiments to verify the efficiency of the proposed method . in experiment 1
, we extract features by ( 2d ) pca and then compare the classification performance of the metric - learning - based method and the classic euclidean - distance - based method . in experiment 2 , we compare the classification performance of knn classifier combined with lmnn using different number of training samples . in experiment 3
we select 480 , 720 , 960 , and 1200 images ( i.e. , 6 , 9 , 12 , and 15 images for each individual ) for training , and the rest of 960 , 720 , 480 , and 240 images ( i.e. , 12 , 9 , 6 , and 3 images for each individual ) are left for testing , respectively .
we treat the training samples from each individual as the positive class and construct a center point for each class , where the ith feature of the center point is calculated by averaging the corresponding feature values of the training samples .
as there are 80 individuals , we then obtain 80 center point ci ( i=1,2, ,80 ) . for any testing sample c
, we estimate the euclidean distances from sample c to each center point , d(c , ci)=||c - ci||2 ( i=1,2, ,80 ) .
if ( c , cj)=mind(c , ci)(i = 1,2 , , 80 ) , then c goes to the jth class.the metric - learning - based method works similarly as the euclidean - distance - based method except for the usage of the learned distance metric d(c , ci)=||l(c - ci)||2 ( i=1,2, ,80 ) .
it is clearly seen that the recognition rate of the metric - learning - based method is higher than the euclidean - distance - based method . with distance metric transformation ,
two samples from different classes with small euclidean distance are dragged farther . on the other hand ,
next we are going to provide an intuitive explanation based on the example shown in figures 6 and 7.these two figures show the data distribution of the data set with 480 training samples and 960 testing samples .
we obtain 25 features for each sample using ( 2d ) pca and select 2 features with the largest contribution to euclidean distance metric .
similarly , these two features are transformed to a new metric space using lmnn , as shown in figure 7 .
the samples of the first individual ( including 6 training images and 12 testing images ) are treated as positive , and the rest of them are considered as negative . in figures 6 and 7 , we use red plus to denote positive training samples , green plus for positive testing samples , blue star for negative training samples , and yellow star for negative testing samples .
it is shown from figure 6 that it is difficult to distinguish the first class from the others because the distances between samples in the first class and the other classes are indiscriminating .
however , by using lmnn , the samples in the first class are gathered together , as shown in figure 7 . in detail , the positive samples are located mainly in the area of abscissa value between 0 and 10 . on the contrary ,
this makes it easier to discriminate samples in the first class from samples in the other classes .
in this experiment ,
we select 480 , 720 , 960 , and 1200 images ( i.e. , 6 , 9 , 12 , and 15 images for each individual ) for training , and the rest of 960 , 720 , 480 , and 240 images ( i.e. , 12 , 9 , 6 , and 3 images for each individual ) are left for testing , respectively .
we treat the training samples from each individual as the positive class and construct a center point for each class , where the ith feature of the center point is calculated by averaging the corresponding feature values of the training samples .
as there are 80 individuals , we then obtain 80 center point ci ( i=1,2, ,80 ) . for any testing sample c , we estimate the euclidean distances from sample c to each center point , d(c , ci)=||c - ci||2 ( i=1,2, ,80 ) .
if ( c , cj)=mind(c , ci)(i = 1,2 , , 80 ) , then c goes to the jth class .
the metric - learning - based method works similarly as the euclidean - distance - based method except for the usage of the learned distance metric d(c , ci)=||l(c - ci)||2 ( i=1,2, ,80 ) .
it is clearly seen that the recognition rate of the metric - learning - based method is higher than the euclidean - distance - based method . with distance metric transformation ,
two samples from different classes with small euclidean distance are dragged farther . on the other hand ,
next we are going to provide an intuitive explanation based on the example shown in figures 6 and 7 .
these two figures show the data distribution of the data set with 480 training samples and 960 testing samples .
we obtain 25 features for each sample using ( 2d ) pca and select 2 features with the largest contribution to euclidean distance metric .
similarly , these two features are transformed to a new metric space using lmnn , as shown in figure 7 .
the samples of the first individual ( including 6 training images and 12 testing images ) are treated as positive , and the rest of them are considered as negative . in figures 6 and 7 , we use red plus to denote positive training samples , green plus for positive testing samples , blue star for negative training samples , and yellow star for negative testing samples .
it is shown from figure 6 that it is difficult to distinguish the first class from the others because the distances between samples in the first class and the other classes are indiscriminating .
however , by using lmnn , the samples in the first class are gathered together , as shown in figure 7 . in detail , the positive samples are located mainly in the area of abscissa value between 0 and 10 . on the contrary ,
this makes it easier to discriminate samples in the first class from samples in the other classes .
experiment 2 in this experiment , we select 6 , 9 , 12 , and 15 images from each individual as training samples to build a knn classifier .
here the number of neighbors , that is , k , is empirically set to be 3 in knn .
we obtain different recognition rates with different numbers of training samples , and the experimental results are shown in figure 8.overall , the recognition rate increases with the number of training images increases .
when the number of the training images goes to 15 , the recognition rate reaches 96.67% .
it is also worth noting that , as compared to table 2 , the knn - based method outperforms the above - mentioned metric - learning - based method and the euclidean - distance - based method , by considering the same number of training images .
in this experiment , we select 6 , 9 , 12 , and 15 images from each individual as training samples to build a knn classifier .
the number of neighbors , that is , k , is empirically set to be 3 in knn .
we obtain different recognition rates with different numbers of training samples , and the experimental results are shown in figure 8 .
when the number of the training images goes to 15 , the recognition rate reaches 96.67% .
it is also worth noting that , as compared to table 2 , the knn - based method outperforms the above - mentioned metric - learning - based method and the euclidean - distance - based method , by considering the same number of training images .
we select 1200 images ( 15 images for each individual ) for training and 240 images ( 3 images for each individual ) for testing .
we use smote to oversample the positive samples to be 5 , 10 , 20 , 30 , 40 , and 50 times as large as the original set .
we observe that the recognition rate does not improve by only increasing a small number of synthetic positive samples , as shown in smote-5 and smote-10 .
after that , the recognition rate increases by about 3% , and finally it achieves 99.17% with smote-40 or smote-50 . with a sufficiently large set of synthetic positive samples ,
we select 1200 images ( 15 images for each individual ) for training and 240 images ( 3 images for each individual ) for testing .
we use smote to oversample the positive samples to be 5 , 10 , 20 , 30 , 40 , and 50 times as large as the original set .
we observe that the recognition rate does not improve by only increasing a small number of synthetic positive samples , as shown in smote-5 and smote-10 .
after that , the recognition rate increases by about 3% , and finally it achieves 99.17% with smote-40 or smote-50 . with a sufficiently large set of synthetic positive samples , the recognition performance would not improve any more .
this paper proposes a new finger vein recognition method based on ( 2d ) pca and metric learning .
firstly , we extract features by ( 2d ) pca and then train a binary classifier for each individual based on metric learning .
furthermore , we address the class imbalance problem by using smote oversampling before the classifier is trained .
( 1 ) we apply ( 2d ) pca to extract features of finger vein image , where ( 2d ) pca reflects the information in both the row direction and the column direction , and it is more efficient for feature extraction as compared to pca and 2dpca .
( 2 ) we build the knn classifier based on metric learning using lmnn which changes the sample distribution in the new metric space .
lmnn makes the distance between the samples from the same class smaller and the distance between the samples from different classes larger .
this is incorporated with individually trained classifiers which reflect the characteristics of the corresponding individuals .
( 3 ) we note the class - imbalance problem ; that is , when building the classifier for an individual , the number of the samples from the other individuals is considerably large .
we tackle it by oversampling the positive samples with smote , and the experimental results validate the effectiveness .
promising future work includes the exploration of features with better discrimination as well as the processing finger vein images of low quality . | finger vein recognition is a promising biometric recognition technology , which verifies identities via the vein patterns in the fingers . in this paper , (
2d)2 pca is applied to extract features of finger veins , based on which a new recognition method is proposed in conjunction with metric learning .
it learns a knn classifier for each individual , which is different from the traditional methods where a fixed threshold is employed for all individuals . besides , the smote technology is adopted to solve the class - imbalance problem .
our experiments show that the proposed method is effective by achieving a recognition rate of 99.17% . | 1. Introduction
2. Technical Background
3. The Proposed Method
4. Experimental Result and Analysis
5. Conclusion |
PMC3399359 | they provide end - to - end care with newborn screening , diagnostic assessment of patients , dispensing of hearing aids , and appropriate follow - up to ensure good outcomes are obtained .
historically , audiology services were commonly commissioned from the acute sector and have had a low priority because of the silent and insidious nature of the disability .
in addition , the general public do not see hearing impairment as a dramatic health problem requiring urgent intervention .
first presentation in the uk is usually to the general practitioner ( gp ) .
although the majority ( 80% ) of uk patients access their hearing care through the nhs , there is also an option to use the independent sector without a gp referral .
there are recent initiatives involving screening , for example , by telephone or internet , which may in future lead to self - referral without the need for gp involvement .
the public health challenge of hearing impairment is growing due to the demographics of the population , as age is the major determinant of hearing loss .
hearing impairment in the uk affects one in ten adults aged 5574 years . over the next 15 years hearing impairment
almost one in four ( 22.6% ) over 75-year olds reports moderate or severe worry because of hearing problems .
typically , those who are referred for hearing assessment have had a hearing problem for 10 or more years , are aged in their mid-70s and have a substantial hearing problem .
the older that people are when they present for assessment and intervention , the more difficult they find adaptation to and care of their hearing aids
. the degree of hearing impairment is a major factor that predicts ability to benefit from hearing aids .
our research for the health technology assessment ( hta ) programme shows that a disease marker of an impairment of 35 db hearing level ( hl ) at 3 k hz gives the best outcomes for those who accept interventions .
we found that 14% of the 5574 year age group have bilateral impairment of at least 35 db hl ; only 3% had effective amplification through the use of hearing aids .
the hta study also found a high comorbidity of ent symptoms : hearing , balance and tinnitus . in 5574
year olds , about 40% of those reporting hearing difficulties also report tinnitus , and about 20% of those reporting hearing difficulty also report both tinnitus and dizziness .
there are no research data on the number of patients with hearing loss a gp will see and refer to audiology in a year .
one study in leicester found the numbers referred in a two - year period ranged between 54 referrals from two individual gps ( with a special interest in ent ) and 110 patients each from 14 other individual gps .
gp decision - making about individual referrals is based on clinical knowledge , diagnostic tools available , knowledge of the patient , patient self - reporting , and the gp 's knowledge and experience of local services and pathways .
not all patients who present to their gp with hearing problems are referred for treatment .
the hta report found that of those who have consulted their gp about hearing , only 38% also went to hospital ; only 41% in the age band 5574 years .
the decision not to refer is not in itself a failure and may be soundly based .
anecdotal evidence and information from surveys indicates that gps may have low awareness of the needs of people who are deaf or hard of hearing .
the nhs audiology modernisation programme , begun in 2001 , has resulted in significant quality improvements , and since 2006 , in reduced national waiting times .
the major challenge recently has been high variability of referral to treatment ( rtt ) times , identified in 2009 through national data collection , ranging from 2 weeks direct access rtt in some audiology services to 15 weeks in others .
another relevant policy development in the uk nhs is the introduction , through the new nhs health and social care bill , of any qualified provider ( aqp ) . unlike the previous contractual arrangements , which were restricted to a small group of patients ,
this enables a variety of services to tender for and provide care to nhs patients .
the aims were : significant improvements in diagnosing hearing loss , more timely and effective referrals , better patient outcomes .
significant improvements in diagnosing hearing loss , more timely and effective referrals , better patient outcomes .
the nhs audiology improvement programme was launched in july 2009 , supported by nhs improvement .
the department of health report improving access to audiology services ( 2007 ) included a specific commitment to apply service improvement tools .
the nhs audiology improvement programme recognised that a range of approaches is needed to change people 's behaviour and build an evaluative culture .
audiology services involve a multi - stage , multi - factorial mix of human and technical processes , with often long care pathways ( these are long - term conditions ) and multiple human interaction variables .
using randomised controlled trials ( rcts ) to develop the evidence base is not feasible for these kinds of services .
evaluative case studies , using multiple methods and different sources of evidence , were seen to be the most effective model , as the emphasis is on the real - life setting in which these services are delivered and received .
evidence from evaluative case studies is more likely to sustain improvements after the initial halo effect of pilots has gone .
ham et al . in 2002 found that almost one - third of the nhs national booked admissions programme pilots failed to sustain improvements .
our approach was based on staff deciding which improvements were possible in their local context , not imposing a central template .
consequently , our hypothesis was threefold : evaluative case studies , using a variety of methods , would enable local staff to review their own practice , central and local expertise can together identify what works best .
field testing by practitioners means that it is more acceptable for national rollout , combining pathway redesign and peer support provides a sustainable model to build the evaluative culture needed for long term improvements .
evaluative case studies , using a variety of methods , would enable local staff to review their own practice , central and local expertise can together identify what works best .
field testing by practitioners means that it is more acceptable for national rollout , combining pathway redesign and peer support provides a sustainable model to build the evaluative culture needed for long term improvements . providing evidence of good practice from services and research is an important tool to offer commissioners :
otherwise , funders will always focus on simpler targets like waiting times , numbers seen , and costs , particularly at a time when productivity and cost savings are high on the health agenda .
18 audiology improvement pilot sites across all areas of audiology were set up during 2009/10 to support and develop innovative ways to demonstrate measurable benefits of whole pathway redesign .
the focus was on how and why these innovations succeed or fail , and how the context in which they are delivered would influence the outcome .
the immediate challenge was how could busy staff engage with change and innovation whilst at the same time keeping the everyday business of audiology services running ?
this paper describes two of the 18 pilot projects , both of which involve gp decision making and direct referral to audiology .
these two pilots focused on age - related hearing loss and tinnitus , in particular , improving primary care referrals by supporting greater gp clinical engagement .
these examples focus on those critical interfaces where , historically , the hearing care journey can so easily break down for patients : between primary and secondary care , particularly for patients with tinnitus and complex hearing needs .
the work relies on moderately small numbers , but the results are sufficiently robust to indicate the scale of improvements which may be possible .
findings are likely to become more substantial when greater numbers of patients are treated as the pilots move into mainstream provision .
the traditional way to manage patients who may need a hearing aid is for the gp to refer to the audiology service : the patient is assessed at one appointment and fitted with aid / s at another , after the custom made earmould / s have been manufactured .
however , recent advances in technology , specifically open fit hearing aids mean that for some patients , these two appointments can be combined and suitable patients assessed and fitted ( a&f ) at the same appointment .
open fit hearing aids have been shown to provide increased comfort to patients , and a more natural quality of sound , as well as being more discreet .
one appointment saves time , so that if suitable patients can be identified before they arrive at audiology ( i.e. , if they are triaged in primary care ) , then the process can become very efficient for both patients and service providers .
this project involved devising a new referral form for gps , which together with a very simple hearing screening device , allowed the audiology service to direct patients either to the traditional two - appointment pathway or the new assess and fit pathway .
the overall aim of the leicester pilot , which ran during 2009 and 2010 , was to promote gp clinical engagement locally by enabling gps to triage their patients .
the project also provided gps with up - to - date information about hearing aid technology and hearing services .
the hearing screening device that was used had been developed by siemens and the mrc hearing & communication group .
the hearcheck screener can identify age - related hearing problems and predict people who could benefit from intervention .
it is a simple , low - cost , hand - held device which produces a fixed series of 6 pure tones , ( 75 , 55 , and 35 db hl at 3 khz and 55 , 35 , and 20 db hl at 1 khz ) .
it is unable to identify low frequency or conductive hearing loss , so needed to be combined with providing gps with revised referral criteria and information on management of hearing loss .
this meant that gps were also then able to brief patients on the nature of their condition , telling them that they may get a hearing aid at one appointment if that were suitable , so their patients were clear what to expect at each stage of their pathway . at the start of the project
two of the four original practices used health care assistants to carry out the triage on their behalf .
the engagement , interest , and support of the four gp practices involved in the initial pilot of the technology and questionnaire were integral to its success .
the screening devices , which cost around 100 each , were loaned to the gps in the pilot .
calibration is needed every three years and the devices are expected to last around five years .
the audiology team recorded all details of the triage , and all subsequent measurements and outcomes for every patient , ( including qualitative data ) to enable a full analysis of the triage in terms of the agreed outcome measures . in nottinghamshire ,
patients with tinnitus were being referred via ent ; appropriate patients would then be referred onto audiology for specialist advice and support , which added an extra step and wait into the process .
a local audit found a high rate of non - responders for patients referred from ent , and significant waits and bottlenecks in the referral pathways for tinnitus patients .
this was also an opportunity to provide information about the condition and specialist support available .
all local gps were able to refer into the service to maximise the numbers and allow for a measurable evaluation of the outcomes .
clear referral criteria were drawn up for gps to ensure appropriate patients would be referred .
the clinic was set up on the choose and book system to allow referrals to be accepted electronically .
a press release was also issued to raise public awareness to coincide with national tinnitus week .
data on all waiting times were recorded and a detailed patient satisfaction questionnaire was completed by all directly referred tinnitus patients .
numbers of patients requiring or requesting subsequent ent consultations were recorded to ensure that all aspects of an efficient service were considered .
at the end of the trial , 97 patients were referred who had been triaged in primary care for age , hearing , vision , ability to concentrate and manual dexterity .
53 not suitable for a&f were invited for the traditional pathway ( 23 excluded on age , 39 excluded on hearing , 1 on vision , 1 on cognition , 0 on manual dexterity ) .
26 were fitted on the day , 4 at a later date , and 9 had hearing too good for , or declined , a hearing aid .
the work relies on moderately small numbers , so some caution in interpretation is needed , but the numbers are sufficient to justify some rough estimates of savings which could be made .
for example , in the last 12 months , the department received 2600 direct referrals , so if 27% of them had been a&f , the number of a&f would be 702 patients , giving a time saving per year of 350 clinical hours .
patient - related outcome measurespatient related outcome measures demonstrated that the quality of service had not been compromised by the a&f procedure .
there was a good correlation between the primary care screening test result and the clinic results , indicating that the hearcheck is providing the right information for triage .
importantly , the pilot also found that the patients were more likely to be referred at their first visit .
data from the patient satisfaction questionnaire found that 84% respondents said that their gp referred them without delay , ensuring that the management of their hearing loss could be promptly assessed.by the end of 2011 , a further 51 gp practices had bought hearcheck screeners , a substantial development from the 4 practices who first engaged with the pilot .
they had seen the benefits and were able to secure meetings for the audiology team with other practices .
patient related outcome measures demonstrated that the quality of service had not been compromised by the a&f procedure .
there was a good correlation between the primary care screening test result and the clinic results , indicating that the hearcheck is providing the right information for triage .
importantly , the pilot also found that the patients were more likely to be referred at their first visit .
data from the patient satisfaction questionnaire found that 84% respondents said that their gp referred them without delay , ensuring that the management of their hearing loss could be promptly assessed . by the end of 2011 ,
a further 51 gp practices had bought hearcheck screeners , a substantial development from the 4 practices who first engaged with the pilot .
they had seen the benefits and were able to secure meetings for the audiology team with other practices .
the pilot ran from december 2009 to december 2010 and 100 patients were seen through the new pathway . during the pilot the average waiting time referral to treatment ( rtt ) for tinnitus patients was reduced from 14 weeks ( range = 5 to 53 weeks ) to 2 weeks ( range = 1 to 9 weeks ) .
23% ( 23 out of the 100 patients seen ) were red flagged as requiring / requesting referral to ent and 43% were seen as
the quality of referrals received from gps was high , and few inappropriate referrals were received .
patient - related outcome measuresthe patient satisfaction questionnaire found that 100% of patients felt their first hospital appointment was sooner than they had thought it would be,86% felt more confident to manage their condition better,86% had left their appointment with an understanding of the plan for their care .
the patient satisfaction questionnaire found that 100% of patients felt their first hospital appointment was sooner than they had thought it would be,86% felt more confident to manage their condition better,86% had left their appointment with an understanding of the plan for their care .
100% of patients felt their first hospital appointment was sooner than they had thought it would be , 86% felt more confident to manage their condition better , 86% had left their appointment with an understanding of the plan for their care .
stakeholder and team engagement were sometimes difficult , because of busy workloads and the need to change mindsets . creating opportunities to inform gps about present day audiology services was challenging .
the initial cost of the hearcheck screener could prove a sticking point with some gps but it can be argued that for the long term , savings in the reduction of the patient pathway will cover these concerns .
these pilots demonstrate that engaging gps in service improvement is most successful when they are clinically engaged , by providing information on management of hearing loss , up - to - date information on local pathways , clear referral criteria , and , where available , the use of innovative technology like the hearcheck screening device .
the results show significant improvements in referral patterns and benefits for patients . on - going communication with gps at every opportunity
has been integral to the success and sustainability of these pilots , which were always viewed as longer - term projects . by the end of 2011 , a further 51 gp practices in the leicester pilot area had bought hearcheck screeners .
we now have new tools and pathways to support initial assessment of hearing loss in primary care .
as services improve , and patients who have been referred report good experience and outcomes back to their gp , so gps ' internal map of local services will change and so will their referral patterns .
when audiology staff work with gps to provide tools for referring patients more effectively , the resulting service benefit will help re - draw that internal map more quickly . throwing a spotlight in this way on the pattern of care patients receive in a service which has low priority in the nhs shows how changing the pathway can improve the service , leading in turn to better models for commissioning of audiology services in the future . a national publication , pushing the boundaries , has been produced by nhs improvement to share the learning from the pilot phase of the programme by describing in detail the work in the 18 pilot sites . a second publication
was published in march 2011 . as the nhs moves into a new phase involving new commissioning models and potential new providers through the aqp route
future pathways will need to reflect the development of new technologies , enabling self - assessment , for example , through internet or telephone platforms , which could lead to self - referral to audiology services .
gps have a critically important role in the current hearing care pathway : appropriate and early referral is essential . improved referral criteria based on research and better information on hearing loss for gps , and a simple triage using an audiometric screening device , can lead to earlier identification and management of hearing problems in primary care .
people with hearing impairment are highly likely to have other problems such as tinnitus and balance disorders .
balance disorders contribute in part as risk factors for falls and other accidental injury which are frequent causes of loss of independence , avoidable illness and mortality .
given the comorbidity , there is an added opportunity for gps to improve benefit for their patients , if tinnitus and dizziness could also be identified and managed . | the public health challenge of hearing impairment is growing , as age is the major determinant of hearing loss .
almost one in four ( 22.6% ) over 75-year olds reports moderate or severe worry because of hearing problems .
there is a 40% comorbidity of tinnitus and balance disorders .
good outcomes depend on early presentation and appropriate referral .
this paper describes how the nhs improvement programme in england used service improvement methodologies to identify referral pathways and tools which were most likely to make significant improvements in diagnosing hearing loss , effective referrals and better patient outcomes .
an audiometric screening device was used in gp surgeries to enable thresholds for effective referrals to be measured in the surgery . revised referral criteria , the use of this device , new assess and fit technology in the audiology clinic , and direct access pathways
can transform audiology service delivery so that patient outcomes are measurably better .
this , in turn , changes the experience of gps , so they are more likely to refer patients who can benefit from treatment . at the end of 2011 , 51 gp practices in one of the audiology pilot areas had bought hearcheck screeners , a substantial development from the 4 practices who first engaged with the pilot .
| 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
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