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traumatic acute subdural hematoma ( asdh ) is a neurosurgical emergency that requires immediate therapeutic intervention . hematoma evacuation via a craniotomy is indicated in most cases , except for the inoperable patient with irreversible brain stem injuries or if the patient is in a poor general condition . careful monitoring of the neurological status is mandatory even for selected asdh patients with intact consciousness and no brain shift because of the possibility of the unexpected worsening . we report here on a case of spontaneous rapid reduction of a large amount of asdh and we discuss the mechanisms related to the rapid resolution / reduction of asdh . a 61-yr - old man was found unconsciousness following a high speed motor vehicle accident and he was immediately transferred to our institution . on admission , the emergency computed tomography ( ct ) scan revealed a subdural hematoma with a maximal thickness of approximately 2.59 cm in the right fronto - temporo - parietal region with a severe midline shift of 2.42 cm , and this implied cerebral herniation ( fig . 1 ) . the decision was made to treat him conservatively because of his poor clinical condition . with conservative management , an unexpected improvement in his neurologic status was noted within 14 hr of the trauma ( gcs score ; e1vtm3 ) . a repeated ct scan showed a remarkable reduction in the amount of the hematoma and improvement of the midline shift from 2.42 cm to 1.10 cm ( fig . it also demonstrated the new appearance of a high density lesion in the subdural space adjacent to the falx and tentorium , and a low - density area at the previous hematoma site , and especially over the left frontal convexity . thirty days after his admission , a ct scan revealed chronic sdh and an operation was done ( fig . the ct scan 7 days after the operation revealed brain atrophy and improvement of the effacement of the ipsilateral ventricle ( fig . 3b ) . the patient is currently able to obey simply commands and he still remains in a bed - ridden state . . rapid spontaneous resolution of asdh has rarely been reported because these patients usually undergo an operation on an emergency basis . the underlying pathophysiology is not well understood , but several possible mechanisms for the spontaneous resolution have been proposed . two mechanisms are dilution of the hematoma by cerebrospinal fluid ( csf ) due to tearing of the arachnoid membrane and this is followed by wash out ( 1 - 6 ) and compression of the hamatoma by acute brain swelling and this is followed by redistribution of the asdh ( 5 ) . the other theories are extracranial redistribution of the asdh through a skull fracture ( 7 ) , the presence of brain atrophy ( 3 , 8 - 11 ) , and development of subacute spinal sdh ( 12 ) . however , the true mechanisms for spontaneous resolution of asdh still remain unknown . in our case , follow - up ct scan showed a dramatic reduction of the asdh and a new high density region in the subdural space near the falx and tentorium . a low - density area at the anterior portion of the hematoma was also recognized , which implied involvement of csf in the subdural space . sequential follow - up ct scanning revealed brain atrophy after evacuation of the chronic sdh . ( 8) reported rapid spontaneous resolution of an acute subdural hematoma in a patient with cerebral atrophy and they described the aids related brain atrophy as the cause of this phenomenon . they postulated brain atrophy facilitated the redistribution and csf washing out of the asdh . under these conditions , we suggest that the combined effect of a smaller brain and the increased amount of csf in the subarachnoid space due to brain atrophy may favor both the redistribution and dilution of the asdh . however , in our case , the amount of subdural blood was too large to be resolved spontaneously , although there was evidence of brain atrophy on the ct scan . eventually , ct scan showed chronic sdh and an operation was done . based on these observations , we think spontaneous resolution of a considerable amount of asdh is possible , but there is an apparent limit of the amount of spontaneous resolution . wu et al . ( 13 ) , proposed that asdhs might spontaneously resolve when their volume is less than 30 ml , when they are located in the fronto - temporal or tempro - parietal areas near the sylvian fissure , when there is an isodence or low dense space between the hematoma and the inner wall of the skull on ct scan , and when they are associated with cerebral tumefaction and/or cerebral contusion and laceration in neurologically stable patients of a young age . ( 4 ) reported complete disappearance of an asdh with a thickness of 1.5 cm and they proposed that the absence of cerebral contusion is optimal for spontaneous resolution of asdh . however , in our case , the thickness of the hematoma was 2.59 cm and there were cerebral contusions . consequently , we suggest that these findings , including a huge amount of subdural blood ( the thickness of hematoma above 2.5 cm ) is an obstacle to complete spontaneous resolution of asdh . we experienced an unusual case in which the patient 's neurologic condition was improved due to rapid spontaneous reduction of asdh via conservative management , although the initial neurologic status was poor . however , in a real clinical setting , it is very difficult to distinguish which patients should be treated conservatively or surgically . the patient 's age , their premorbid state and the arrival time to the hospital may also be considered . we selected conservative management in this case , but there is no confidence that we should apply the same management principles to other cases with a similar condition . accordingly , it is mandatory to find clinical clues to help determine which patients should be treated with conservatively to manage a large amount of asdh . in conclusion , spontaneous rapid resolution / reduction of asdh may occur in some patients , and even in patients with a large amount of asdh . the mechanisms that are responsible for this are dilution by csf and redistribution of the hematoma in patients with brain atrophy . | the majority of acute post - traumatic subdural hematomas ( asdh ) require urgent surgical evacuation .
spontaneous resolution of asdh has been reported in some cases .
we report here on a case of a patient with a large amount of asdh that was rapidly reduced .
a 61-yr - old man was found unconscious following a high speed motor vehicle accident . on initial examination ,
his glasgow coma score scale was 4/15 .
his pupils were fully dilated and non - reactive to bright light .
brain computed tomography ( ct ) showed a massive right - sided asdh .
the decision was made to treat him conservatively because of his poor clinical condition .
another brain ct approximately 14 hr after the initial scan demonstrated a remarkable reduction of the previous asdh and there was the new appearance of high density in the subdural space adjacent to the falx and the tentorium .
thirty days after his admission , brain ct revealed chronic sdh and the patient underwent surgery .
the patient is currently able to obey simple commands . in conclusion ,
spontaneous rapid resolution / reduction of asdh may occur in some patients .
the mechanisms are most likely the result of dilution by cerebrospinal fluid and the redistribution of hematoma especially in patients with brain atrophy . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
adsorption properties strongly influence the performance of catalytic materials , hence precise knowledge of these properties can assist in characterization , design and optimization of such materials . however , the adsorption properties are generally judged from single - component adsorption measurements often at room temperatures or even under liquid nitrogen conditions , and therefore an extension to practical situations may lead to a severe deviation from the real behavior . in situ adsorption measurements on catalytic materials , especially at high temperature and high pressure conditions , still remain a great challenge . an adsorption measurement device based on a quartz crystal microbalance ( qcm ) is advantageous over the commercialized volumetric and gravimetric methods in a way that it is highly accurate for mass sorption applications , satisfactorily stable in a controlled environment , and more affordable . however , the conventional qcm analysis is limited to temperatures below 80 c . in order to overcome this limitation , we developed an adsorption measurement device based on a high - temperature high - frequency oscillating microbalance ( langatate crystal microbalance , lcm ) , which can realize the adsorption measurements in principle at temperatures as high as 200 - 300 c , owing to the absence of crystalline - phase transitions up to its melting point ( 1,470 c ) . the lcms used in this work have an at - cut ( i.e. , the plate of the crystal microbalance contains the x axis of the crystal and is inclined by 3515 ' from the z axis ) and a resonant frequency of 5 mhz . this device was applied to the adsorption measurements of co2 , h2o , methanol , and dimethyl ether ( dme ) , each in gaseous state , on zeolite h - zsm-5 in the temperature range of 50 - 150 c and pressure range of 0 - 18 bar , aiming at the validation of simulation models for the optimization of bifunctional core - shell catalysts for one - stage production of dme from the synthesis gas . how to operate this device for gas adsorption measurements prior to the adsorption measurements , zeolite h - zsm-5 crystallites ( 0.502 mg ) were synthesized on the gold electrode in the center of the lcm by the steam - assisted crystallization ( sac ) method according to de la iglesia et al . , in such a way that the zeolite crystallites remain attached to the oscillating microbalance . as shown in figure 1 , the lcm used in the adsorption measurement device has polished gold electrodes on both sides , which help to connect the lcm to an oscillator . since the zeolite crystals on the connection points of the gold electrodes to the oscillator would significantly reduce the electroconductivity ( as indicated in figure 1 ) and thus the measurement sensitivity of the lcm , the zeolite h - zsm-5 crystals were deposited on the lcm via the sac method not covering these connection points . the details about the synthesis of zeolite h - zsm-5 on lcm are briefly summarized in the following protocol section and shown in the video protocol in detail . caution : please consult all relevant material safety data sheets ( msds ) before use . several of the chemicals used in the synthesis of zeolite h - zsm-5 are acutely toxic and carcinogenic . please use all appropriate safety practices when performing a nanocrystal reaction including the use of engineering controls ( fume hood , glovebox ) and personal protective equipment ( safety glasses , gloves , lab coat , full length pants , closed - toe shoes ) . moreover , pay special attention when , performing the adsorption measurements with methanol and dme , since both are flammable and explosive dangerous materials . preparation of a zeolite synthesis mixture note : the final synthesis mixture had the following molar composition as adapted from de la iglesia et al . : 1 sio2 : 50 h2o : 0.07 na2o : 0.024 tpa2o : 0.005 al2o3 . thus , the theoretical si / al molar ratio of the synthesized zeolite h - zsm-5 is 100 . dissolve 0.14 g sodium hydroxide in 20.30 g de - ionized water via stirring . alternatively , mix 3.64 g of 1 m naoh with 16.8 g de - ionized water.add 1.16 g tetrapropylammonium hydroxide ( tpaoh ) solution , and stir the solution until it appears clear.add 5.0 g tetraethyl orthosilicate ( teos ) solution dropwise , and stir the solution until it appears clear.keep stirring , while adding 0.09 g aluminum nitrate nonahydrate ( al(no3)39h2o , solid ) into the solution.keep stirring until the solid aluminum nitrate nonahydrate is dissolved . note that the prepared zeolite synthesis mixture should be used within 5 hr due to its aging . alternatively , mix 3.64 g of 1 m naoh with 16.8 g de - ionized water . add 1.16 g tetrapropylammonium hydroxide ( tpaoh ) solution , and stir the solution until it appears clear . add 5.0 g tetraethyl orthosilicate ( teos ) solution dropwise , and stir the solution until it appears clear . keep stirring , while adding 0.09 g aluminum nitrate nonahydrate ( al(no3)39h2o , solid ) into the solution . note that the prepared zeolite synthesis mixture should be used within 5 hr due to its aging . synthesis of zeolite zsm-5 on the lcm via sac
clean the lcm before the zeolite synthesis
wash the lcm thoroughly with the de - ionized water.put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath.dry the lcm at 80 c in an oven . zeolite synthesis
carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes.dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it.add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis.put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis.keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method.right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr.remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min.dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm.put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals.obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . clean the lcm before the zeolite synthesis
wash the lcm thoroughly with the de - ionized water.put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath.dry the lcm at 80 c in an oven . put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath . zeolite synthesis
carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes.dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it.add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis.put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis.keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method.right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr.remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min.dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm.put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals.obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes . dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it . add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis . put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis . keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method . right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr . remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min . dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm . put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals . obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . note : in this work , the lcm without coating and the one coated with h - zsm-5 ( prepared in the last section ) is termed " reference lcm " and " sample lcm " , respectively . moreover , the sample lcm before zeolite deposition is termed " unloaded sample lcm " . in a previous publication in journal of physical chemistry c , a detailed description of the lcm - based adsorption measurement device can be found . in this work , the operation of the device for gas adsorption measurements is presented in this short protocol and in the video protocol in detail . preparation before adsorption measurements
tests on the effects of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath.carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.change the pressure in the sample chamber via dosing pure n2.control the temperature inside the sample chamber by a temperature controller.measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite . activation of sample lcm
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath.carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 .
tests on the effects of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath.carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.change the pressure in the sample chamber via dosing pure n2.control the temperature inside the sample chamber by a temperature controller.measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite .
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air . put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath . carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables . pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully . close the sample chamber , and evacuate it by a vacuum pump . change the pressure in the sample chamber via dosing pure n2 . measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite . activation of sample lcm
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath.carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 .
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air . put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath . carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables . pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully . activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 . adsorption measurementsnote : in this work , the adsorption measurement of co2 at 50 c is presented to give an example . the obtained data from the measurement ( e.g. , resonant frequencies ) and the calculated masses of adsorbed co2 on the h - zsm-5 can be found in table s1 of the supporting information of our previous publication . adjust the temperature inside the sample chamber at the desired temperature of the adsorption measurements ( i.e. , 50 0.1 c ) by a temperature controller , under vacuum conditions , i.e. , only with a negligible amount of adsorbed gas.connect the oscillator to the sample lcm , and measure its resonant frequency by the supporting software of the oscillator via fitting the experimental data with a butterworth - van dyke equivalent circuit model.switch the connection of the oscillator to the reference lcm , and measure its resonant frequency.use the measured resonant frequencies of the sample and reference lcms under vacuum conditions to determine the mass of h - zsm-5 deposited on the sample lcm ( without adsorbed gas ) according to the sauerbrey equation : where is the difference in mass in g , is the number of the harmonic at which the crystal is driven ( in this study , ) , is the difference in resonant frequencies of reference and sample lcms in hz , is the difference of the resonant frequencies between the reference and unloaded sample lcm in hz , is the density of the langatate crystal ( 6.13 g cm ) , is the effective piezoelectrically stiffened shear modulus of the langatate crystal ( 1.910 g cm sec ) , is the resonant frequency of the reference lcm , i.e. , the unloaded lcm , , the area of the lcm ( 1.539 cm ) . note : in this work , the mass of h - zsm-5 deposited on the gold electrode in the center of the lcm is 0.502 mg , which causes a resonant frequency shift of 14,100 hz at 50 c.control the gas pressure of co2 inside the sample chamber by dosing pure gas from the gas cylinder via a mass flow controller ( for methanol and dme , from the evaporator manually via a dosing valve into the chamber ) , or by evacuation via a vacuum pump . here , use a pressure range of co2 adsorption measurements of 0 - 16 bar as shown in figure 2.wait until equilibrium conditions and a stable temperature have been reached , e.g. , the temperature varies within 50 0.1 c.connect the oscillator to the sample lcm , and measure its resonant frequency after exposure to the gas at a given pressure.switch the connection of the oscillator to the reference lcm , and measure its resonant frequency under the same conditions.according to the sauerbrey equation shown above , calculate the total mass of h - zsm-5 deposited on the sample lcm and gas adsorbed on the h - zsm-5 under this gas pressure . by subtracting the mass of h - zsm-5 ( without adsorbed gas ) determined in step 2.2.4 , the mass of co2 adsorbed on the h - zsm-5 under this gas pressure is obtained.repeat the resonant frequency measurements for sample and reference lcms for varying pressures , in order to obtain all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures.finally , obtain the gas adsorption isotherm at 50 c in the studied pressure range of 0 - 16 bar via calculating all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures according to step 2.2.9.for adsorption isotherms at other temperatures , change the stable temperature using the temperature controller , and repeat steps 2.2.1 to 2.2.11.fit the adsorption isotherms with adsorption models like langmuir models via the least squares method to determine the adsorption parameters like adsorption capacities , adsorption enthalpies , and adsorption entropies ( see previous publication and its supporting information ) . adjust the temperature inside the sample chamber at the desired temperature of the adsorption measurements ( i.e. , 50 0.1 c ) by a temperature controller , under vacuum conditions , i.e. , only with a negligible amount of adsorbed gas . connect the oscillator to the sample lcm , and measure its resonant frequency by the supporting software of the oscillator via fitting the experimental data with a butterworth - van dyke equivalent circuit model . switch the connection of the oscillator to the reference lcm , and measure its resonant frequency . use the measured resonant frequencies of the sample and reference lcms under vacuum conditions to determine the mass of h - zsm-5 deposited on the sample lcm ( without adsorbed gas ) according to the sauerbrey equation : where is the difference in mass in g , is the number of the harmonic at which the crystal is driven ( in this study , ) , is the difference in resonant frequencies of reference and sample lcms in hz , is the difference of the resonant frequencies between the reference and unloaded sample lcm in hz , is the density of the langatate crystal ( 6.13 g cm ) , is the effective piezoelectrically stiffened shear modulus of the langatate crystal ( 1.910 g cm sec ) , is the resonant frequency of the reference lcm , i.e. , the unloaded lcm , , the area of the lcm ( 1.539 cm ) . note : in this work , the mass of h - zsm-5 deposited on the gold electrode in the center of the lcm is 0.502 mg , which causes a resonant frequency shift of 14,100 hz at 50 c . control the gas pressure of co2 inside the sample chamber by dosing pure gas from the gas cylinder via a mass flow controller ( for methanol and dme , from the evaporator manually via a dosing valve into the chamber ) , or by evacuation via a vacuum pump . here , use a pressure range of co2 adsorption measurements of 0 - 16 bar as shown in figure 2 . wait until equilibrium conditions and a stable temperature have been reached , e.g. , the temperature varies within 50 0.1 c . connect the oscillator to the sample lcm , and measure its resonant frequency after exposure to the gas at a given pressure . switch the connection of the oscillator to the reference lcm , and measure its resonant frequency under the same conditions . according to the sauerbrey equation shown above , calculate the total mass of h - zsm-5 deposited on the sample lcm and gas adsorbed on the h - zsm-5 under this gas pressure . by subtracting the mass of h - zsm-5 ( without adsorbed gas ) determined in step 2.2.4 , the mass of co2 adsorbed on the h - zsm-5 under this gas pressure is obtained . repeat the resonant frequency measurements for sample and reference lcms for varying pressures , in order to obtain all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures . finally , obtain the gas adsorption isotherm at 50 c in the studied pressure range of 0 - 16 bar via calculating all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures according to step 2.2.9 . for adsorption isotherms at other temperatures , change the stable temperature using the temperature controller , and repeat steps 2.2.1 to 2.2.11 . fit the adsorption isotherms with adsorption models like langmuir models via the least squares method to determine the adsorption parameters like adsorption capacities , adsorption enthalpies , and adsorption entropies ( see previous publication and its supporting information ) . preparation of a zeolite synthesis mixture note : the final synthesis mixture had the following molar composition as adapted from de la iglesia et al . : 1 sio2 : 50 h2o : 0.07 na2o : 0.024 tpa2o : 0.005 al2o3 . thus , the theoretical si / al molar ratio of the synthesized zeolite h - zsm-5 is 100 . alternatively , mix 3.64 g of 1 m naoh with 16.8 g de - ionized water.add 1.16 g tetrapropylammonium hydroxide ( tpaoh ) solution , and stir the solution until it appears clear.add 5.0 g tetraethyl orthosilicate ( teos ) solution dropwise , and stir the solution until it appears clear.keep stirring , while adding 0.09 g aluminum nitrate nonahydrate ( al(no3)39h2o , solid ) into the solution.keep stirring until the solid aluminum nitrate nonahydrate is dissolved . note that the prepared zeolite synthesis mixture should be used within 5 hr due to its aging . alternatively , mix 3.64 g of 1 m naoh with 16.8 g de - ionized water . add 1.16 g tetrapropylammonium hydroxide ( tpaoh ) solution , and stir the solution until it appears clear . add 5.0 g tetraethyl orthosilicate ( teos ) solution dropwise , and stir the solution until it appears clear . keep stirring , while adding 0.09 g aluminum nitrate nonahydrate ( al(no3)39h2o , solid ) into the solution . that the prepared zeolite synthesis mixture should be used within 5 hr due to its aging . synthesis of zeolite zsm-5 on the lcm via sac
clean the lcm before the zeolite synthesis
wash the lcm thoroughly with the de - ionized water.put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath.dry the lcm at 80 c in an oven . zeolite synthesis
carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes.dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it.add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis.put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis.keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method.right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr.remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min.dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm.put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals.obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . clean the lcm before the zeolite synthesis
wash the lcm thoroughly with the de - ionized water.put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath.dry the lcm at 80 c in an oven .
wash the lcm thoroughly with the de - ionized water . put the lcm in a beaker with the de - ionized water , and clean it in an ultrasound bath . zeolite synthesis
carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes.dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it.add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis.put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis.keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method.right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr.remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min.dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm.put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals.obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . carefully place several drops of the prepared zeolite synthesis mixture on the electrode in the center of the lcm as shown in figure 1 using a pipette , since only the zeolite deposited on the gold electrode can cause the resonant frequency shift of the lcm . moreover , avoid spreading the synthesis mixture on the connection points of the gold electrodes to the oscillator , since the zeolite on the connection points would significantly reduce the electroconductivity and thus the measurement sensitivity of the lcm . in addition , removing the zeolite crystals on the connection points after the deposition will destroy the electrodes . dry the lcm with the synthesis mixture at 80 c for 2 hr to obtain a highly viscous gel - like phase on it . add a small amount of de - ionized water ( about 10 ml ) in a teflon - lined autoclave ( 80 ml ) in order to produce the steam during the zeolite synthesis . put the teflon holder in the autoclave , which supports the lcm horizontally above the liquid water at the bottom of the autoclave during the zeolite synthesis . keep the autoclave in an oven at 150 c for 48 hr to synthesize the zeolite on the lcm via the sac method . right after the sac , wash the coated lcm with the de - ionized water and dry it at 80 c for 2 hr . remove the organic template in the zeolite crystals by calcination in a high - temperature oven under an oxidative atmosphere . program the oven as follows : a ) increase the temperature from ambient to 450 c at a rate of 3 c min ; b ) keep the temperature at 450 c for 4 hr ; c ) decrease the temperature from 450 c to room temperature at a rate of 3 c min . dissolve 26.75 g ammonium chloride ( nh4cl , solid ) in 0.4 l de - ionized water . add more de - ionized water in the solution so that the final nh4cl solution is 0.5 l and has the concentration of 1 mol dm . put the coated lcm into the nh4cl solution ( 0.2 l ) in a beaker , and ion - exchange the na - zsm-5 crystals coated on the lcm at 20 c for 2 hr . repeat the ion - exchange using 0.2 l fresh nh4cl solution to get the nh4-zsm-5 crystals . obtain the h - zsm-5 by final calcination using the same parameters as mentioned in step 1.2.2.7 . note : in this work , the lcm without coating and the one coated with h - zsm-5 ( prepared in the last section ) is termed " reference lcm " and " sample lcm " , respectively . moreover , the sample lcm before zeolite deposition is termed " unloaded sample lcm " . in a previous publication in journal of physical chemistry c , a detailed description of the lcm - based adsorption measurement device can be found . in this work , the operation of the device for gas adsorption measurements is presented in this short protocol and in the video protocol in detail . preparation before adsorption measurements
tests on the effects of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath.carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.change the pressure in the sample chamber via dosing pure n2.control the temperature inside the sample chamber by a temperature controller.measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite . activation of sample lcm
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath.carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 .
tests on the effects of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath.carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.change the pressure in the sample chamber via dosing pure n2.control the temperature inside the sample chamber by a temperature controller.measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite .
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air . put the reference and unloaded sample lcms in a beaker with de - ionized water and clean them in an ultrasound bath . carefully place the clean reference and unloaded sample lcms on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables . pretest the installed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully . close the sample chamber , and evacuate it by a vacuum pump . change the pressure in the sample chamber via dosing pure n2 . measure the resonant frequencies of the reference and unloaded sample lcms in the studied temperature and pressure ranges , i.e. , 50 - 150 c and 0 - 16 bar , in order to know the effect of temperature and pressure on the difference in resonant frequencies of the reference and unloaded sample lcms ( in step 2.2.4 ) . the tests show that is significantly affected by the temperature ( 1,200 to 3,000 hz at 50 - 150 c ) , whereas the gas pressure has no significant effect ( change of smaller than 300 hz in the pressure range of 0 - 16 bar ) . use the determined values of in the sauerbrey equation in step 2.2.4 to calculate the adsorbed amount of gases on the zeolite . activation of sample lcm
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air.put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath.carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables.pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully.close the sample chamber , and evacuate it by a vacuum pump.activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 .
clean the o - ring , the lcm holder , and the sample chamber with acetone and pressed air . put the reference lcm in a beaker with de - ionized water , and clean it in an ultrasound bath . carefully place the clean reference lcm and sample lcm on the lcm holder , which is connected to the oscillator via high temperature resistant electric cables . pretest the placed lcms using the oscillator to ensure that the resonant frequencies can be detected successfully . activate the sample lcm at high temperatures ( at least 50 c higher than the temperatures of the adsorption measurements , 200 c in this work ) in vacuum condition overnight to ensure that only a negligible gas amount is adsorbed on the h - zsm-5 . adsorption measurementsnote : in this work , the adsorption measurement of co2 at 50 c is presented to give an example . the obtained data from the measurement ( e.g. , resonant frequencies ) and the calculated masses of adsorbed co2 on the h - zsm-5 can be found in table s1 of the supporting information of our previous publication . adjust the temperature inside the sample chamber at the desired temperature of the adsorption measurements ( i.e. , 50 0.1 c ) by a temperature controller , under vacuum conditions , i.e. , only with a negligible amount of adsorbed gas.connect the oscillator to the sample lcm , and measure its resonant frequency by the supporting software of the oscillator via fitting the experimental data with a butterworth - van dyke equivalent circuit model.switch the connection of the oscillator to the reference lcm , and measure its resonant frequency.use the measured resonant frequencies of the sample and reference lcms under vacuum conditions to determine the mass of h - zsm-5 deposited on the sample lcm ( without adsorbed gas ) according to the sauerbrey equation : where is the difference in mass in g , is the number of the harmonic at which the crystal is driven ( in this study , ) , is the difference in resonant frequencies of reference and sample lcms in hz , is the difference of the resonant frequencies between the reference and unloaded sample lcm in hz , is the density of the langatate crystal ( 6.13 g cm ) , is the effective piezoelectrically stiffened shear modulus of the langatate crystal ( 1.910 g cm sec ) , is the resonant frequency of the reference lcm , i.e. , the unloaded lcm , , the area of the lcm ( 1.539 cm ) . note : in this work , the mass of h - zsm-5 deposited on the gold electrode in the center of the lcm is 0.502 mg , which causes a resonant frequency shift of 14,100 hz at 50 c.control the gas pressure of co2 inside the sample chamber by dosing pure gas from the gas cylinder via a mass flow controller ( for methanol and dme , from the evaporator manually via a dosing valve into the chamber ) , or by evacuation via a vacuum pump . here , use a pressure range of co2 adsorption measurements of 0 - 16 bar as shown in figure 2.wait until equilibrium conditions and a stable temperature have been reached , e.g. , the temperature varies within 50 0.1 c.connect the oscillator to the sample lcm , and measure its resonant frequency after exposure to the gas at a given pressure.switch the connection of the oscillator to the reference lcm , and measure its resonant frequency under the same conditions.according to the sauerbrey equation shown above , calculate the total mass of h - zsm-5 deposited on the sample lcm and gas adsorbed on the h - zsm-5 under this gas pressure . by subtracting the mass of h - zsm-5 ( without adsorbed gas ) determined in step 2.2.4 , the mass of co2 adsorbed on the h - zsm-5 under this gas pressure is obtained.repeat the resonant frequency measurements for sample and reference lcms for varying pressures , in order to obtain all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures.finally , obtain the gas adsorption isotherm at 50 c in the studied pressure range of 0 - 16 bar via calculating all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures according to step 2.2.9.for adsorption isotherms at other temperatures , change the stable temperature using the temperature controller , and repeat steps 2.2.1 to 2.2.11.fit the adsorption isotherms with adsorption models like langmuir models via the least squares method to determine the adsorption parameters like adsorption capacities , adsorption enthalpies , and adsorption entropies ( see previous publication and its supporting information ) . adjust the temperature inside the sample chamber at the desired temperature of the adsorption measurements ( i.e. , 50 0.1 c ) by a temperature controller , under vacuum conditions , i.e. , only with a negligible amount of adsorbed gas . connect the oscillator to the sample lcm , and measure its resonant frequency by the supporting software of the oscillator via fitting the experimental data with a butterworth - van dyke equivalent circuit model . switch the connection of the oscillator to the reference lcm , and measure its resonant frequency . use the measured resonant frequencies of the sample and reference lcms under vacuum conditions to determine the mass of h - zsm-5 deposited on the sample lcm ( without adsorbed gas ) according to the sauerbrey equation : where is the difference in mass in g , is the number of the harmonic at which the crystal is driven ( in this study , ) , is the difference in resonant frequencies of reference and sample lcms in hz , is the difference of the resonant frequencies between the reference and unloaded sample lcm in hz , is the density of the langatate crystal ( 6.13 g cm ) , is the effective piezoelectrically stiffened shear modulus of the langatate crystal ( 1.910 g cm sec ) , is the resonant frequency of the reference lcm , i.e. , the unloaded lcm , , the area of the lcm ( 1.539 cm ) . note : in this work , the mass of h - zsm-5 deposited on the gold electrode in the center of the lcm is 0.502 mg , which causes a resonant frequency shift of 14,100 hz at 50 c . control the gas pressure of co2 inside the sample chamber by dosing pure gas from the gas cylinder via a mass flow controller ( for methanol and dme , from the evaporator manually via a dosing valve into the chamber ) , or by evacuation via a vacuum pump . here , use a pressure range of co2 adsorption measurements of 0 - 16 bar as shown in figure 2 . wait until equilibrium conditions and a stable temperature have been reached , e.g. , the temperature varies within 50 0.1 c . connect the oscillator to the sample lcm , and measure its resonant frequency after exposure to the gas at a given pressure . switch the connection of the oscillator to the reference lcm , and measure its resonant frequency under the same conditions . according to the sauerbrey equation shown above , calculate the total mass of h - zsm-5 deposited on the sample lcm and gas adsorbed on the h - zsm-5 under this gas pressure . by subtracting the mass of h - zsm-5 ( without adsorbed gas ) determined in step 2.2.4 , the mass of co2 adsorbed on the h - zsm-5 under this gas pressure is obtained . repeat the resonant frequency measurements for sample and reference lcms for varying pressures , in order to obtain all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures . finally , obtain the gas adsorption isotherm at 50 c in the studied pressure range of 0 - 16 bar via calculating all the masses of co2 adsorbed on the h - zsm-5 sample under different gas pressures according to step 2.2.9 . for adsorption isotherms at other temperatures , change the stable temperature using the temperature controller , and repeat steps 2.2.1 to 2.2.11 . fit the adsorption isotherms with adsorption models like langmuir models via the least squares method to determine the adsorption parameters like adsorption capacities , adsorption enthalpies , and adsorption entropies ( see previous publication and its supporting information ) . figure 1 shows the photographs , light microscopy and scanning electron microscopy ( sem ) images of the coated and uncoated lcm sensor ( left ) , as well as their x - ray diffraction ( xrd ) patterns ( right ) . from both , light and scanning electron microscopy ( figure 1b and c ) , the connection points of the gold electrodes to the oscillator most of the zeolite crystals on top of the lcm - sensor are isolated and show characteristic rounded - boat morphology , with the ( 010)-plane predominantly facing up . besides , some crystals additionally show the typical intergrowth behavior ( " twinned crystals " ) . moreover , the loaded h - zsm-5 ( si / al molar ratio of 100 according to the composition of the synthesis mixture ) on the langatate crystal has been investigated by xrd and wavelength - dispersive x - ray ( wdx ) spectroscopy . in figure 2 , co2 adsorption isotherms for the h - zsm-5 zeolite obtained with the lcm device in the temperature range of 50 - 150 c and pressure range of 0 - 16 bar , as well as the fit of the single site langmuir isotherm model to the experimental data , are shown to give a representative example . as shown in figure 2 , the determined adsorption isotherms of co2 were fitted with a single site langmuir isotherm well . figure 3 shows the diagram of ln(k'i ) vs. 1,000/t for co2 as derived from the adsorption isotherms , i.e. , the temperature dependence of the adsorption constants determined from the fit of the adsorption isotherms . the adsorption enthalpies and entropies of co2 were determined by fitting with the van't hoff equation ( see the supporting information of the previous publication ) . the results of the model fitting show that the adsorption capacity , adsorption enthalpy and adsorption entropy for co2 in h - zsm-5 are 4.0 0.2 mmol g , 15.3 0.5 kj mol and 56.3 1.5 j mol k , respectively . the high quality of the fit of the single site langmuir isotherm and the van't hoff equation as shown in figures 2 and 3 supports the assumption of a constant adsorption capacity ( i.e. , saturation loading ) and enthalpy ( i.e. , heat of adsorption ) to be valid at least for the range of conditions used . moreover , the adsorption parameters of co2 determined by the lcm - based adsorption measurement device in this work compare well to values reported in literature , i.e. , the adsorption capacity , adsorption enthalpy and adsorption entropy reported for co2 in mfi - type zeolites vary in the range of 2.1 - 3.8 mmol g , 19 - 28.7 kj mol , and 43.7 - 82.7 j mol k , respectively , in the temperature range of 30 - 200 c and pressure range of 0 - 5 bar . ( a ) photographs of the coated and uncoated sensor ( right ) , ( b ) light microscopy and ( c ) scanning electron microscopy images . the x - ray diffraction patterns of coated and uncoated lcm sensor ( right ) . adsorption isotherms for co2 in h - zsm-5 at 50 ( ) , 75 ( ) , 100 ( ) , and 150 c ( ) . the symbols represent the experimental data , the error bars indicate the measurement uncertainty of the resonant frequencies caused by , e.g. , the temperature instability , and calculated according to the sauerbrey equation as described in step 2.2.4 , and the lines represent the fit of the single site langmuir isotherm model to the experimental data . in this work , the successful synthesis of the zeolite h - zsm-5 crystals on the gold electrode in the center of the lcm sensor by sac is demonstrated , i.e. , the zeolite is successfully loaded on the lcm sensor without covering the connection points of the gold electrodes to the oscillator . thus , the zeolite can oscillate together with the lcm sensor , while the lcm sensor keeps its good electroconductivity and measurement sensitivity . compared to the conventional qcm devices which are limited below 80 c , the lcm device presented in this work is successfully used for the adsorption measurements at temperatures as high as 150 c , i.e. , at or close to the temperature of reactions in industry . higher than 200 c , the measurement uncertainty may exceed the mass of the adsorbed gas , since , with the increasing temperature above 150 c , the mass of the adsorbed gas has a significant decrease , whereas the measurement uncertainty increases significantly due to the decreasing temperature control precision . thus , in future experiments , a new method should be developed to deposit more zeolite on the lcm , which causes more gas to adsorb , and moreover compensates the effect of the temperature and pressure on . this could help to extend the application range of the lcm device to higher temperatures . during the experiment , the critical steps in the zeolite synthesis are steps 1.2.2.1 , 1.2.2.4 , 1.2.2.5 and 1.2.2.7 , while those in the adsorption measurements are steps 2.1.1.3 , 2.1.1.4 , 2.2.1 , 2.2.5 and 2.2.6 . in step 1.2.2.1 , avoid placing too much of the synthesis mixture on the lcm , which would spread on the connection points of the gold electrodes . in step 1.2.2.4 , carefully put the teflon holder with the lcm in the autoclave to ensure that the lcm is horizontal and does not contact the liquid water at the bottom . in steps 1.2.2.5 and 1.2.2.7 , do not use a higher temperature in the zeolite synthesis and calcination , since our previous experiments show that it leads to degradation of the lcm . in the adsorption measurements , the position of the lcm sensors has a significant effect on the connectivity of the lcm sensors to the oscillator , and therefore on the quality of the resonant frequency signals . therefore , pay special attention to steps 2.1.1.3 and 2.1.1.4 , in which the lcms are loaded on the holder and pretested . the lcms should be in the position that they are connected with the oscillator via the connection points of the electrodes ( indicated in figure 1 ) . in addition , in steps 2.2.1 and 2.2.6 , ensure that a stable temperature is achieved before the measurements , since this also increases the measurement accuracy . moreover , in step 2.2.5 , feed the gas slowly , in order to have a small change of the temperature inside . this helps the temperature to become stable again after a short time . since the sac synthesis method for zeolite h - zsm-5 on the lcm sensor could be extended to other zeolites easily , the lcm - based adsorption measurement device is expected to be used for them as well . moreover , due to its high accuracy and low cost , this device is expected to be applicable to any material , which could be coated on the lcm , in order to investigate its adsorption properties at high temperatures . | we present a high - temperature and high - pressure gas adsorption measurement device based on a high - frequency oscillating microbalance ( 5 mhz langatate crystal microbalance , lcm ) and its use for gas adsorption measurements in zeolite h - zsm-5 .
prior to the adsorption measurements , zeolite h - zsm-5 crystals were synthesized on the gold electrode in the center of the lcm , without covering the connection points of the gold electrodes to the oscillator , by the steam - assisted crystallization ( sac ) method , so that the zeolite crystals remain attached to the oscillating microbalance while keeping good electroconductivity of the lcm during the adsorption measurements .
compared to a conventional quartz crystal microbalance ( qcm ) which is limited to temperatures below 80 c , the lcm can realize the adsorption measurements in principle at temperatures as high as 200 - 300 c ( i.e. , at or close to the reaction temperature of the target application of one - stage dme synthesis from the synthesis gas ) , owing to the absence of crystalline - phase transitions up to its melting point ( 1,470 c ) .
the system was applied to investigate the adsorption of co2 , h2o , methanol and dimethyl ether ( dme ) , each in the gas phase , on zeolite h - zsm-5 in the temperature and pressure range of 50 - 150 c and 0 - 18 bar , respectively .
the results showed that the adsorption isotherms of these gases in h - zsm-5 can be well fitted by langmuir - type adsorption isotherms .
furthermore , the determined adsorption parameters , i.e. , adsorption capacities , adsorption enthalpies , and adsorption entropies , compare well to literature data . in this work
, the results for co2 are shown as an example . |
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the intestinal microbiota is composed of ~100 trillion commensal bacteria and has co - evolved with the host by participating in many essential physiologic and metabolic functions . immunological effects of the microbiota are not simply due to the presence of innocuous bacteria , but to the biological activities of the gut microbiota consortium . in many cases , regulation of development and/or effector functions of different immune cell populations ( such as treg cells vs th17 cells ) the relative abundance of these different immunomodulatory members can direct the general nature of host mucosal and systemic immunity . previously , we referred to such immunomodulatory members of the microbiota as autobionts . we and other groups have shown that segmented filamentous bacteria can potently induce interleukin-17-producing cd4 t cells ( th17 cells ) in the small intestine of mice . we have also shown that a combination of 46 strains of clostridia indigenous to conventionally reared mice can induce treg cells in the mouse colonic lamina propria and thereby contribute to protecting mice against colitis and allergic responses . in the most recent publication by our group , we identified 17 strains of human - derived clostridia as potent inducers of treg cells . starting from a complete healthy human fecal sample , a sequence of selection steps was applied to obtain treg cell - inducing human - derived bacterial strains using gnotobiotic techniques . we first observed full treg cell induction in the colon of ex - germ - free mice orally inoculated with a chloroform - treated human fecal sample . then the cecal contents from these mice were treated with chloroform , diluted , and serially transplanted into other germ - free mice , while monitoring treg induction capability . we succeeded in obtaining colonized mice in which the complexity of the gut microbiota was greatly decreased without sacrificing treg - inducing potency . from these mice , we cultured and selected 17 strains which , when mixed together and orally administered to gem - free mice and rats , were able to induce a significant accumulation of cd4foxp3 treg cells in the colon . furthermore , repeated oral ingestion of the mixture of 17 strains rendered specific - pathogen - free mice resistant to experimental allergic diarrhea and trinitrobenzene sulfonic acid ( tnbs)-induced colitis . therefore , the 17 strains have at least a prophylactic effect in mouse colitis models . the 17 treg - inducing strains isolated in our study all belong to the class clostridia . . some clostridia species , including clostridium tetani , c. botulinum , and c. perfringens , are well - known pathogens that are often isolated as a singular cause of infectious disease , and c. difficile is responsible for antibiotic - associated diarrhea and colitis . on the other hand , clostridia species are extremely heterogeneous and many of them inhabit the large intestine of human and animals as predominant symbiotic microbes . clostridia species can be classified into 19 clusters ( i to xix ) , and many of the pathogenic species listed above belong to cluster i. the 17 treg - inducing strains fall within clusters iv , xiva , and xviii of clostridia ( fig . 1 ) . it has been reported that species within clusters xiva and iv are indispensable for various physiological host functions . for instance , colonization with clostridia clusters xiva and iv normalizes the enlarged cecum found in germ - free mice and supports epithelial growth and turnover . it was also shown that colonization with clostridia cluster xiva strains renders mice resistant to c. difficile colonization . antibiotic sensitivities are categorized as susceptible ( white ) , intermediate ( light orange ) or resistant ( dark orange ) . antimicrobial susceptibility testing was performed using the broth microdilution method with dry plates ( eiken chemical , japan ) according to the clinical and laboratory standards institute ( clsi ) guidelines m11-a8 and m100-s23 . briefly , each strain grown on eggerth - gagnon agar was harvested and suspended in abcm broth ( eiken chemical , tokyo , japan ) . the number of colony - forming units ( cfu ) was adjusted to 1 10 cfu / ml and an aliquot ( 100 l ) of the suspension was inoculated into each well of the plates . after incubation at 37 c for 48 h , bacterial growth was visually assessed to determine the minimal inhibitory concentrations ( mics ) . the etest ( sysmex - biomrieux , japan ) was also employed to test for susceptibility to metronidazole and vancomycin . it should be noted that there have been several reports of clostridia clusters iv , xiva , and xviii species ( clostridium clostridioforme , clostridium innocuum , and clostridium ramosum , in particular ) in clinical specimens of opportunistic infections . however they are rarely single isolates , but rather a fraction of the multiple organisms in these clinical samples . considering that commensal clostridia species colonize in close proximity to the gut epithelial surface , they may translocate when the barrier is compromised , not necessarily because of specific pathogenic properties , but simply because of their local abundance . sequencing of the genomes of the 17 strains revealed that they lack known toxins and virulence factors . some of the 17 strains possess genes encoding putative sialidase , hyaluronidase , flagella - related protein , and fibronectin binding protein , but with low similarity to genes found in pathogenic clostridia species . furthermore , ingestion of a mixture of the 17 strains of clostridia was effective in preventing colitis induced by tnbs and even by dextran sulfate sodium ( dss ) ( data not shown ) , which chemically disrupts the epithelial barrier . given the lack of major virulence factors and toxins in their genomes and the lack of any toxicity upon dosing to animals , the clostridia strains isolated in our study appear to be safe for clinical use . to further confirm that the isolated clostridia strains are not harmful all strains were susceptible to ampicillin - sulbactam , piperacillin - tazobactam , amoxicillin - clavulanate , metronidazole , and chloramphenicole , although some strains were resistant to penicillin g and ampicillin alone , and strains 1 , 3 , 8 , and 18 showed low level resistance to vancomycin with mic values of 4 to 8 g / ml ( fig . 1 ) . since c. ramosum and c. innocuum are reported to have intrinsic resistance to glycopeptides and lipopeptides ( vancomycin ) , and genes identical to vanb2 ligase of enterococcus spp . can be found in c. hathewayi , c. boltae , and c. inocuum - like bacteria , it is not surprising that strains 1 , 3 , 8 and 18 , which have 16s sequence similarities with those species , showed low level resistance to vancomycin . overall , none of the 17 strains have high - level resistance to antibiotics tested ; rather they show a benign safety profile . further careful characterization of the strains and pre - clinical studies will be required before clinical translation . the precise mechanism underlying how the 17 strains of clostridia stimulate the induction of colonic treg cells remains to be further elucidated . one suggested mechanism is the production of short chain fatty acids ( scfas ) , which have multiple metabolic and immune functions . in the context of treg induction , scfas can elicit a tgf-1 response in epithelial cells , which can contribute to de novo induction of peripheral treg cells ( ptreg ) . scfas , particularly butyrate , can suppress dendritic cell activation through suppression of expression of the nfb component relb . it has also been shown that butyrate activates signaling pathways through gpr109a to induce anti - inflammatory genes in dendritic cells . in addition to its effects on dendritic cells , butyrate can directly stimulate thymic treg cell ( ttreg ) proliferation through activation of gpr43 and the differentiation of nave cd4 t cells into ptreg cells through histone h3 acetylation of the foxp3 gene intronic enhancer by inhibition of histone deacetylase ( hdac ) . the genomes of the 17 strains contain abundant genes predicted to be involved in the biosynthesis of acetate and butyrate ( fig . 2 ) . scfa production by each of the 17 strains when cultured in vitro in glucose - supplemented medium was analyzed by a validated liquid chromatography - electrospray ionization - tandem mass spectrometry ( lc - esi - ms / ms ) system ( fig . 3 ) . among scfas , acetate and butyrate were detected at high concentrations in most of the culture supernatants except those of strains 1 , 3 , 8 , and 18 . notably , strains 9 , 13 , 16 , 27 , and 29 showed very high butyrate production ( fig . 3 ) . these findings are consistent with previous reports showing that bacterial strains belonging to clostridia cluster iv and xiva were positive for butyryl - coa : acetate coa transferase , an enzyme responsible for butyrate production . importantly , mono - colonization of gf mice with one of each of the 17 strains was insufficient to induce treg cells in vivo ; however , the mixture of 17 strains is effective , suggesting synergistic effects in a microbial community - dependent manner . potential metabolic pathways leading to the production of acetate and butyrate from acetyl - coa are shown . the thickness of the arrow and the number on the left of the arrow indicates the copy number of genes identified in the genomes of the 17 strains . liquid chromatography - electrospray ionization - tandem mass spectrometry ( lc esi - ms / ms ) analysis of short chain fatty acid metabolites was performed on 48 h cultures of peptone - yeast extract - fildes solution supplemented with glucose at a concentration of 1% ( w / v ) . fatty acids in the culture supernatant were derivatized with 2-nitrophenylhydrazine and purified by liquid / liquid extraction . short chain fatty acid peaks were identified by both their specific ms / ms ion - transitions and comparison of the retention times with those of known short chain fatty acids in a standard solution . we also examined enzymatic properties of the 17 strains using api zym , rapid i d 32a , and api 20a systems in vitro ( fig . enzyme activities related to virulence such as trypsin , -chymotrypsin , and gelatin hydrolase , which have been implicated in infective endocarditis , and -glucuronidase , which may release toxic substances by cleaving glucuronic acid - conjugation in the intestine , were very low or absent in the 17 strains . of note some strains were positive for the activity of principal mucin degrading enzymes , such as -galactosidase and n - acetyl--glucosaminidase , and many strains exhibited strong activity of -glucosidase , which is an intestinal enzyme that breaks down a variety of carbohydrates . strain 9 and 13 could convert tryptophan into indole , which is reported to enhance epithelial barrier function and contribute to suppression of colitis . together with scfa production profiles , the 17 strains are diverse and heterogeneous in terms of enzymatic and metabolic properties . considering that the dysbiotic microbiota associated with inflammatory bowel disease ( ibd ) is frequently accompanied by significantly decreased diversity , the use of a functionally diverse mixture like the 17 strains of clostridia described here may be reasonable for clinical applications to correct dysbiosis . apizym and rapid api 32a tests ( sysmex biomrieux ) were used for determination of enzymatic activities of each of the 17 strains . cells from cultures grown on eggerth - gagnon blood agar plates for 48 h at 37 c in an anaerobic chamber were suspended in saline and the turbidity was adjusted to 56 in the mcfarland scale ( approx . aliquots of 65 l of the suspensions were inoculated into cupules in the strip . in the case of rapid api 32a , the turbidity was matched to 4 in the mcfarland scale and aliquots of 55 l were applied to the cupules . the strips were incubated at 37 c for 4 h and the reactions were determined according to the manufacturer s instructions . imbalance in the gut microbiota , termed dysbiosis , significantly contributes to various human diseases . several studies have reported decreased levels of clostridia and bacteroidetes in fecal samples from ibd patients . marked decreases in the prevalence of clostridia have been associated with increased risk of airway hypersensitivity and atopic dermatitis . animal studies have also shown that bacterial dysbiosis helps perpetuate the cycle of chronic inflammation characteristic of ibd . therefore , manipulation of the gut microbiota as a therapeutic strategy holds great promise for immuno - inflammatory diseases including ibd and allergy . alteration of the gut microbiome via fecal microbiota transplantation ( fmt ) , which involves placing stool from a healthy donor via duodenal tubing , colonoscopy , or enema , has been shown effective in patients with pseudomembranous colitis induced by clostridium difficile infection , and researchers and patients are interested in testing the potential of fmt for the treatment of other disease associated with disruption of the intestinal microbiota , including insulin resistance , multiple sclerosis , and ibd . while fmt has been established as a proof of principle for the feasibility of manipulating the human microbiome as a therapeutic strategy , the development of commercial products based on fecal transplants faces a number of hurdles from manufacturing , quality assurance , pathogen contamination risk , donor selection , and patient acceptance perspectives . therefore , treatment with a composite of well - characterized benign microorganisms is more desirable from many perspectives for medical purposes . there are many probiotics with a history of safe use in humans for medicinal purposes . however , probiotics currently in use have generally been selected based on properties such as ease of culture and tolerance to acid and oxygen and are not among the major colonizers of the human gut . in other words , they have not been isolated based on their ability to correct microbiome dysbiosis associated with human disease or to boost specific arms of the host immune system . presumably as a result , the dysbiotic microbiota are refractory to treatment with currently available individual probiotic strains , and most probiotics tested to date have demonstrated , at best , mediocre effects in the clinic . thus , there is a compelling need to identify more robust therapeutic organism compositions that are compatible and symbiotic to the host and , ideally , able to induce broader changes to the microbial ecosystem to correct dysbiosis and drive the immune system to normal homeostasis . in this context , we believe that the 17 strains of clostridia isolated in our study can form the basis for a future live biotherapeutic product to treat certain forms of ibd , allergy , and other immune - inflammatory diseases . compositions based on these strains could ( 1 ) help correct microbiome imbalances ; ( 2 ) be easily administered orally and not necessarily frequently , providing patients with a convenient therapeutic option ; ( 3 ) be safe for human use , since they are commensal strains that are long - term colonizers of the healthy human gut , do not have prominent virulence factors , and are sensitive to antibiotics ; ( 4 ) be viewed favorably by patients given that they are natural compositions ; and ( 5 ) be manufactured with traditional fermentation methods and developed under the live biotherapeutics route outlined by the fda , thus circumventing the manufacturing , regulatory , and patient acceptance hurdles that hamper commercialization of fecal transplants . our next step should be optimization of the therapeutic mixture by elimination of nonessential components of the 17 strains for treg cell induction . in addition to this , we need to identify and isolate other bacterial strains from human intestine that can regulate differentiation and activation of other immune cell subsets , such as th17 cells . our long - term goal is to offer a diverse but minimal consortium of microbes that can colonize the human gut and restore and maintain immune homeostasis to prevent and cure immune - inflammatory diseases . | the gut microbiota plays important roles in the development of the host immune system .
we have previously shown that a combination of 46 strains of commensal clostridia isolated from conventionally reared mice can induce the accumulation of cd4+foxp3 + regulatory t ( treg ) cells in the mouse colonic lamina propria .
subsequently , we succeeded in isolating and selecting 17 strains of clostridia from a healthy human fecal sample that can significantly increase the number and function of colonic treg cells in colonized rodents , thereby attenuating symptoms of experimental allergic diarrhea and colitis . here
we characterize each of the 17 strains of human - derived clostridia in terms of sensitivity to antibiotics and ability to produce short chain fatty acids and other metabolites , and discuss their potential as biotherapeutics to correct dysbiosis and treat immune - inflammatory diseases . |
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liver transplantation is a major surgical procedure that is associated with a high incidence of postoperative complications . acute lung injury ( ali ) is a common complication after orthotopic liver transplantation ( olt ) that occurs in 34.2%77.8% of patients receiving olt and in 7.8%23.6% of patients with post - olt ali aggravated by acute respiratory distress syndrome ( ards ) , which itself carries a mortality rate of 76.5% [ 24 ] . overwhelming inflammation has been shown to play an important role in the progress of ali induced by olt , but the initiative mechanism of inflammation remains unclear . toll - like receptor 4 ( tlr4 ) is an important member of the tlr family and can be activated by noxious stimuli to initiate nuclear factor-b ( nf-b ) signaling and a downstream inflammatory response [ 6 , 7 ] . overexpression of tlr4 , which is induced by different factors , including ischemia - reperfusion , endotoxin , trauma , and mechanical ventilation , has been shown to play a critical role in ali [ 811 ] . tlr4 has also been shown to be involved in ischemia - reperfusion injury pathophysiology in certain important organs such as the liver and kidney as well as in the heart [ 12 , 13 ] . at present , there is no evidence to support the important role of tlr4 in ali after olt . therefore , a clarification of the role played by tlr4 may help to explain the mechanism of ali induced by liver transplantation and may also contribute to the development of effective intervention measures for ali . the purposes of the current study are ( 1 ) to observe the neutrophil tlr4 expression in patients to provide clues for tlr4 involved in ali after olt ; ( 2 ) to dynamically investigate the changes in lung pathology , tlr4 expression , and inflammatory mediators in lung tissue during orthotopic autologous liver transplantation ( oalt ) in a rat model ; ( 3 ) to evaluate the relative importance of tlr4 in the development of ali during olt using rna interference ( rnai ) , which is an established method for inhibiting target gene expression in a rat model [ 14 , 15 ] . this study was approved by the research ethics board of the third affiliated hospital , sun yat - sen university , china ( number chictr - och-12002255 ) . twenty - two asa iii or iv patients of both sexes ( 19 males , 3 females ) , 3359 years of age , weighing 4974 kg , and undergoing olt , were studied . all of the patients received modified piggyback liver transplantation , a liver transplantation technique of side - to - side caval anastomosis during orthotopic hepatic transplantation without inferior vena caval occlusion . diagnosis of acute lung injury ( ali ) was based on clinical findings , and diagnosis criteria were as follows : acute onset , pao2/fio2 300 , bilateral pulmonary infiltrates detected by x - ray , and pulmonary wedge pressure ( pcwp ) 18 mmhg or no clinical evidence of congestive heart failure . the peripheral arterial blood samples ( 4 ml ) were collected for measurement of tlr4 expression and concentration of tnf- and il-1 at t1 ( after induction of anesthesia ) , t2 ( 1 hr after anhepatic phase ) , t3 ( 4 hrs after graft reperfusion ) , and t4 ( 24 hrs after graft reperfusion ) . 2 ml of the blood sample was stored in the edta anticoagulant tube for flow cytometry for detection of tlr4 expression on the surface of pmns cells and the other 2 ml was centrifuged at 3000 r / min for 10 min and then stored in 30c for assay of serum cytokines tnf- and il-1. male sprague - dawley ( sd ) rats , aged 810 weeks and weighing 220250 g , were purchased from medical experimental animal center of guangdong province . all the animal research protocols used in this study were approved by the institutional animal care and use committee of sun yat - sen university in guangzhou , china . three 21-nt sirna duplexes targeting rat tlr4 gene ( number nm 019178 ) were designed using the sirna target finder and design tool available at http://www.ambion.com and were commercially obtained from ribobio ( guangzhou , china ) . green fluorescent protein ( gfp ) sirna , with no homology to tlr4 gene , was taken as sirna control . tlr4 sirna was found to have no toxic effect on hepatic stellate cells ( hsc ) using cck-8 assay ( data not showed ) . after transfection and incubation for 48 hrs , hepatic stellate cells ( hsc ) were harvested and expression of tlr4 at both mrna and protein levels was detected by real - time pcr ( rt - pcr ) and western blot , respectively . tlr4 sirna with maximum inhibition rate was selected for in vivo experiments : tlr4 sirna1 , target sequence 1 : gcatagaggtacttcctaa . 3-dtdt ggaucuuguacaccuagaa-5 ( antisense ) . after anesthesia with pentobarbital sodium ( 30 mg / kg , i.p . ) , rats received intratracheal injection of 500 l of normal saline ( ns group ) , 500 l ( 50 nmol ) of sirna vector or tlr4 sirna ( nc and sirna group ) . 48 hrs later , animals in the ns , nc , and sirna group received orthotopic autologous liver transplantation ( oalt ) while rats in sham group only received laparotomy and vessels separation without oalt . in first part of the experiment , twenty - two patients were divided into ali group and non - ali group according to their postoperative diagnosis after olt surgery . in second part of the experiment , rats in group s ( sham operated , n = 16 ) received abdominal operation and liver dissection under anesthesia , without cold perfusion and reperfusion . rats in oalt model group ( group m , n = 16 ) all received oalt using the above - described method [ 17 , 18 ] . in groups s and m , rats were randomly killed at 4 , 8 , 16 , and 24 hrs after liver reperfusion ( i.e. , groups m1 , m2 , m3 , m4 , resp . ) , and rats in group s were killed at corresponding times after liver liberation ( groups s1 , s2 , s3 , and s4 , resp . ; in the third part of the experiment , eighty - eight animals were randomly allocated to one of the four groups : the sham group ( group s ) , the oalt group in which rats were administrated saline as a control ( group ns ) , the oalt group in which rats were administrated sirna expression vector also as a control ( group nc ) , and the oalt group in which rats were administrated tlr4 sirna ( group sirna ) . the rats ( n = 14 per group ) were used to assess survival rates . then the remaining rats ( n = 8 per group ) were killed and samples were collected at 8 hrs . all rats were anesthetized with pentobarbital ( 30 mg / kg body weight , i.p . ) . tracheal intubation was performed with inserting a 14-gauge intravenous catheter which was then connected to small animal ventilators ( alc - v9a ; shanghai alcott biotech co. , ltd . , rats were mechanically ventilated with a standard tidal volume ( 10 ml / kg ) ventilation protocol ( inspiratory / expiratory ratio of 1 : 1 ; respiratory rate 50 breaths / min ; 100% oxygen ) . all the animals were killed with deep anesthesia and lung tissues and blood samples were collected for dynamic analysis of pulmonary function , histopathology , tlr4 gene and protein expression , and cytokines . the partial pressure of oxygen ( pao2 ) was measured using an istat portable blood gas analyzer ( g7 + chips , abbott laboratories , usa ) according to the provided manufacturer 's protocol . for assessing the severity of alveolar edema , the wet weight of the superior lobe of the right lung was accurately measured and baked to constant weight ( 80c , 24 hrs ) to calculate the water content of lung as follows : water content = ( lung wet weight lung dry weight)/lung wet weight 100 . for histologic evaluation , fresh lung sections were fixed in 10% buffered formalin and embedded in paraffin ; sections ( 4 m ) were stained with hematoxylin and eosin . a pathologist who was blinded to research group assignment analyzed the samples and determined the levels of lung injury according to a scoring system as described by derks and jacobovitz derks . total rna was extracted from lung samples by trizol ( invitrogen ) according to the provided manufacturer 's protocol . reverse transcription ( rt ) of the lung samples was performed according to the introduction of taqman microrna reverse transcriptase kit ( life technologies , gaithersburg , md , usa ) . quantitative real - time pcr ( qrt - pcr ) was performed using the taqman universal pcr master mix , noamperase ung ( life technologies ) , and the light cycler 480 system ( roche , switzerland ) . the relative expression of the target genes ( tlr4 and -actin ) was calculated by the 2 method . the qpcr primers were as follows : tlr4 forward primer : 5-cctaggcaccagggtgtgat-3. tlr4 reverse primer : 5-ttggtgacaatgccgtgttc-3. -actin forward primer : 5-tgcttgtggtagccactgta-3. -actin reverse primer : 5-cctcattctggctcgagtag-3. tlr4 forward primer : 5-cctaggcaccagggtgtgat-3. tlr4 reverse primer : 5-ttggtgacaatgccgtgttc-3. -actin forward primer : 5-tgcttgtggtagccactgta-3. -actin reverse primer : 5-cctcattctggctcgagtag-3. expression of tlr4 was assayed using western blotting according to our previous study . the primary antibodies used were monoclonal rabbit anti - mouse tlr4 ( 1 : 500 dilution , santa cruz biotechnology , usa ) . the secondary antibody used was goat anti - rabbit hrp - conjugated igg ( 1 : 2000 dilution , cell signaling technology , usa ) . protein band density was measured using uvp labworks imaging analysis software ( uvp labworks , upland , ca , usa ) . frozen sections of lung tissue were immunofluorescent stained using polyclonal rabbit anti - mouse nf-b p65 ( 1 : 1000 dilution , cell signaling technology , usa ) according to our previous study . flow cytometry was performed using phycoerythrin - cy5-labelled monoclonal antibody for tlr4 ( ebioscience , san diego , usa ) of human according to the provided manufacturer 's protocol . the contents of tnf- and il-1 in human serum were measured by elisa kits ( rapidbio lab , california , usa ) according to the provided manufacturer 's protocol of the kits . lung tissues harvested from rats were homogenized with cold normal saline and then centrifuged at 4000 r / min for 15 minutes . the contents of tnf- and il-1 in lung tissue and serum were measured by respective elisa kits ( r&d systems , minneapolis , mn , usa ) . the levels of tnf- and il-1 were expressed as pg / g protein in lung tissue and pg / ml in serum sample . mpo activity in lungs was used as an indicator of peripheral blood polymorphonuclear leukocytes ( pmns ) infiltration . using a method previously described , mpo activity was detected and defined as the quantity of enzyme required degrading 1 mmol of h2o2 at 37c and was expressed in mu/100 mg wet tissue . to observe whether or not tlr4 gene silencing improves the prognosis of liver transplantation , we observed survival rates . the rats ( n = 14 per group ) receiving the same protocols were used to assess survival rates . from the onset of reperfusion , animals were monitored via video recording for 24 hrs . two hours after reperfusion , the survivors were transferred to their individual cages and allowed free access to food and water . all data were analyzed using spss 12.0 software ( spss , chicago , illinois ) . the differences among the groups were evaluated by using one - way analysis of variance ( anova ) followed by the tukey test . pmns migration in circulation system has been shown to be involved in lung inflammatory injury induced by lps . we observed tlr4 expression on pmns from peripheral blood of patients received olt in order to find out the relation between tlr4 and ali induced by olt . we first confirmed the different levels of tlr4 on pmns and the associated inflammatory cytokines in patients ( basic information in table 1 ) who received olt surgery with or without ali . as shown in table 1 , patients with ali exhibited more ascites ( p = 0.01 ) and longer anhepatic phase ( p < 0.032 ) . moreover , severe fluctuating of circulatory volume is assumed as more blood loss volume ( p = 0.034 ) and total output ( p = 0.003 ) which lead to more infusion of red blood cell ( p = 0.021 ) existing in patients with ali compared to the non - ali ones . expression of tlr4 on pmns was increased gradually following olt , which was significantly higher in ali group than that in non - ali group ( p < 0.05 ) at 24 hrs after neohepatic phase . moreover , the tlr4 downstream cytokines tnf- and il-1 were synchronously increased after olt , and ali group exhibited significantly increased tnf- and il-1 level ( p < 0.05 ) at 24 hrs after neohepatic phase ( table 2 ) . these results indicated that inflammatory response especially tlr4 associated pathway was activated in patients with ali following olt . in order to investigate the underlying mechanism of ali following olt , the oalt model was involved in our present study . the model could well mimic the hemodynamic change during liver transplantation ( table 3 ) . furthermore , we detected pathological and functional change in the injured lung at 4 hrs , 8 hrs , 16 hrs , and 24 hrs following oalt . table 4 shows no statistical significance in body weight and anhepatic phase duration time of the animals . as a result , we found collapse of pulmonary architecture ; severe infiltration of polymorphonuclear and mononuclear cells into intra - alveolar , interstitial , and edematous interstitial space existed ( figure 1(a ) ) in rats that received oalt , accompanied with severe lung edema and dysfunctional pulmonary blood gas barrier which reflected in higher water content of lung ( p < 0.05 , figure 1(c ) ) and lower pao2 ( p < 0.05 , figure 1(d ) ) , which were correlated with increased pathological injury score ( p < 0.05 , figure 1(b ) ) . and these changes peaked at 8 hrs after reperfusion while they gradually recovered after 24 hrs . these above results showed that ali occurred rapidly after oalt and maximized at 8 hrs after liver transplantation . to investigate the underlying mechanism that accounted for ali induced by oalt , we detected the tlr4 and its downstream cytokines which play important roles in mediating inflammatory cascades . as shown in figures 2(a)2(c ) , the mrna and protein level of tlr4 in lung tissue were simultaneously elevated from 4 hrs after reperfusion and peaked at the timepoint of 8 hrs while they gradually decreased from 16 hrs after reperfusion . furthermore , oalt obviously increased the level of tnf- and il-1 in lung tissue or serum from oalt groups , as illustrated in figures 2(d)2(h ) . interestingly , we also found that the level of tnf- and il-1 in lung tissue reached maximum at 8 hrs . by contrast , the increase of serum cytokines seemed to be earlier evident as tnf- reached maximum at 4 hrs while concentration of il-1 peaked at 8 hrs . these results indicated that tlr4 and its associated downstream cytokines might act as the mediator of ali progress which was obviously injured at 8 hrs after reperfusion , and this representative timepoint was chosen for further studies . sirna administration , as a gradually mature technology , was used in our current study to knock down tlr4 induction . first , in hsc cell line , we tested three tlr4 sirna sequences and determined that sequence 1 was the most effective in inhibiting tlr4 protein and mrna expression ( figures 3(a ) and 3(c ) ) . then , we confirmed effective knockdown of oalt - induced tlr4 protein expression in rat lung after intranasal sirna administration . tlr4 sirna significantly inhibited oalt - induced tlr4 protein ( figures 3(b ) and 3(f ) ) and mrna ( figure 3(e ) ) expression in rat lung lysates whereas nonspecific sirna administration had no effects ( figure 3(b ) ) and intranasal sirna administration had no effects on liver tlr4 expression ( figures 3(b ) and 3(d ) ) . next , nf-b p65 , as a downstream transcription factor of tlr4 pathway , was involved in verifying the effects of tlr4 sirna on signaling transduction . as shown in figures 4(a ) and 4(b ) , nf-b p65 nuclear translocation was significantly increased in nc group ( p < 0.05 versus sham ) while it significantly decreased with tlr4 sirna treatment ( p < 0.05 versus group nc ) ; by contrast , the expression of nf-b p65 in cytoplasm was opposite . levels of lung and serum tnf- and il-1 , important makers of inflammation during oalt and oalt - induced ali , were also decreased in rats administrated tlr4 sirna compared with nonspecific sirna ( figures 4(d)-4(e ) ) . rats administrated tlr4 sirna exhibited lower concentration of myeloperoxidase in injured lung tissue than rats administrated nonspecific sirna after oalt ( figure 4(c ) ) . this result demonstrated that pmns migrated to injured lung following oalt . to determine the functional role of tlr4 expression on ali induced by oalt as shown in figure 5(b ) , we found that knockdown of tlr4 significantly decreased mortality compared with rats given nonspecific sirna in the oalt model ( p < 0.05 ) . these differences in mortality correlated with decreased lung injury parameters after oalt , evident by significantly decreased pathological scores ( figures 5(a ) and 5(c ) ) and water content of lung ( figure 5(d ) ) with increased arterial pao2 ( figure 5(e ) ) compared with group nc ( p < 0.01 ) . furthermore , tlr4 protein levels were positively correlated with lung pathological scores ( r = 0.8523 ; p < 0.001 ) , which indicated the important role of tlr4 activation in the pathophysiologic process of ali induced by oalt . these results indicated that tlr4 induction served as an important response to early stage of ali induced by oalt in vivo . clinical outcome in the present study showed that high level of tlr4 expression in circulating pmns existed in patients with ali after olt , accompanied with high level of cytokines , providing clues of tlr4 overexpression involved in ali induced by olt . lung inflammatory injury was induced by oalt in a rat model , evidenced by the increase of pathological scores and the release of cytokines along with decreases in pao2 and the high mrna and protein expression of lung tissue tlr4 . silencing tlr4 expression reduced the nuclear transfer of subunit p65 and attenuated ali , after oalt . these clinical and experiment results indicate that tlr4 may contribute to ali induced by liver transplantation and that tlr4 might be a potential intervention target . liver transplantation causes severe trauma and complex pathophysiological alterations , such as hemodynamic instability , liver and intestinal ischemia - reperfusion ( i / r ) injury , and the subsequent bacterial or endotoxin translocation and neutrophil infiltration due to i / r injury , leading to large amount of cytokines production and multiple inflammatory pathway activation [ 24 , 25 ] . lung is considered an early and vulnerable remote organ affected by olt , and ali occurs after olt in a high incidence rate . ali has been characterized as an acute inflammation with sequestration of neutrophils and the subsequent enzymatic products in lung , resulting in increased microvascular permeability and then interstitial and pulmonary edema . in our study , proinflammatory cytokines tnf- and il-1 significantly increased in the circulation and lungs in rats with ali after liver transplantation . the degree of lung injury was proportional to the tnf- and il-1 levels in rat lung tissue . these results suggested that proinflammatory cytokines flooding , which may be due to neutrophil infiltration and multiple inflammatory pathway activation , played critical roles in amplifying lung inflammation and injury . as an important member of the tlr family , tlr4 is expressed on multiple cell types . tlr4 is especially enriched in monocytes - macrophages , neutrophils , and endothelial cells that are closely related to the inflammatory response . tlr4 overexpression has been shown in injured lung induced by trauma or lps [ 2931 ] . in the current study , we found higher expression of tlr4 on circulating pmns and higher levels of cytokines existed in patients with ali than those in non - ali patients . interestingly , level of tlr4 expression on circulating pmns was increased persistently even at 24 hours after neohepatic phase in ali patients , indicating that tlr4 expression on circulating pmns was closely related to prognosis of patients at the early stage of reperfusion . also , the tlr4 level was raised transiently in non - ali patients , which may be due to an acute phase response . on the other hand , the migration and activation of neutrophil have been demonstrated to initiate and aggravate the lung inflammatory response induced by traumatic factors or endotoxin [ 32 , 33 ] . neutrophils infiltration can be reflected by mpo activity which was released by the intravascular degranulation of neutrophils . we also found high level of mpo activity in injured lung tissue following oalt in the animal experiments , indicating that neutrophils infiltration might play a critical role in ali induced by liver transplantation . furthermore , tlr4 mrna and protein expression were found to be dynamically upregulated in lung tissues , which coincided with significant increases in cytokines and enhanced mpo activity in rats . all these data are consistent with the interpretation that neutrophils infiltration and upregulation of tlr4 expression participate in the development of ali during olt . furthermore , recently , tlr4 in endothelial cell was showed to be a key determinant of lps - induced pmns infiltration in lungs [ 35 , 36 ] . tlr4 stimulation in endothelial cells induced rapid expression of cell surface adhesion proteins , resulting in increased neutrophils rolling and firm adhesion . massive numbers of pmns accumulated in lungs in response to activation of tlr4 . however , study showed impaired pmns recruitment into the alveolar space in tlr4 null mice . moreover , pmns tlr4 has also been reported to regulate interaction of pmns with activation of endothelial cells . another study using chimeric mice proved that endothelial cell tlr4 rather than pmn tlr4 plays the dominant role in lps - induced pmns infiltration in lungs . however , our study differs in that we examined the role of tlr4 in a model of lung injury induced by multiple factors from liver transplantation which was more in line with clinical practice and both endothelial cell tlr4 and pmn tlr4 might be involved in the progress of ali induced by oalt . studies have confirmed that tlr4 could activate transcription factors nuclear factor b ( nf-b ) , amplify inflammatory responses , and lead to the injury ( including lung injury ) of multiple tissues and end - organs induced by endotoxin , massive transfusion , and trauma [ 40 , 41 ] . the tlr4/nf-b pathway activation has an effect on the production of inflammatory cytokines and chemokines , resulting in pmns infiltration and the subsequent exaggerated inflammatory response . excess production of proinflammatory cytokines such as tnf- and il- can deteriorate the inflammatory process . in our current study , tnf- and although there are various pathways involved in cytokines synthesis , tlr4/nf-b pathway has been proven to be playing an important role in cytokines ( tnf- and il- ) productions ; our current study also demonstrated that the level of cytokines could reflect effects on treatment of tlr4 sirna , indicating the close relation between tlr4/nf-b pathway activation and downstream cytokines ( tnf- and il- ) production . interestingly , a recent study reported that tlr4 was a receptor for hmgb1-mediated ali after liver partial ischemia / reperfusion injury . various studies have shown that the high expression of tlr4 on separated lung cells plays a role in ali [ 4346 ] . although we found a high expression of tlr4 in ali , we did not investigate the cell type positioning of tlr4 sirna , which will be involved in our study in the future . in addition , study has shown that activation of tlr4 alone or in conjunction with tlr2 could attenuate lung injury , associated with promotion of prosurvival effects on alveolar epithelium cell . similarly , tlr3-deficient mice were also showed to be protected from lung damage after hyperoxic exposure in vivo , accompanied with increased survival rate . collectively , the above finding implies that the role of tlrs in ali is highly contextual . and , in the present study , our data support the idea that tlr4 is the major pathway that mediates the oalt - induced ali . however , the role of tlr2 and tlr3 in the oalt - induced ali needs to be explored in future studies to make the mechanism of tlrs acting on ali more clear . in conclusion , our study provides compelling evidence that the upregulation of tlr4 was involved in the ali that occurred in rats after liver transplantation and downregulation of tlr4 by sirna was proven to be an important therapy for ali induced by liver transplantation . thus , the potential manipulation of tlr4 activity to control lung injury may permit advances in liver transplantation . furthermore , various tlr4 antagonists are under development for indications like septic shock , suggesting that such strategies may effectively reduce ali early in the course of the disease and tlr4 may also act as an important target in the development of clinical drug for ali treatment . | acute lung injury ( ali ) is a severe complication of orthotopic liver transplantation ( olt ) with unclear underline mechanism .
toll - like receptor 4 ( tlr4 ) has been identified as a key receptor mediating inflammation .
we hypothesized that tlr4-mediated pulmonary inflammation may contribute to development of ali during olt .
patients with or without ali were observed for serum cytokines and expression of tlr4 on peripheral blood polymorphonuclear leukocytes ( pmns ) .
next , rats which underwent orthotopic autologous liver transplantation ( oalt ) were divided into sham and model groups . pulmonary function and the level of tlr4 expression and cytokines
were analyzed .
furthermore , the role of tlr4 in oalt - mediated ali was assessed in rats treated with tlr4-sirna before oalt . the pmns tlr4 expression and
the serum tnf- and il- level were higher in patients with ali than those with non - ali .
interestingly , lung tlr4 expression was significantly increased after 8 hours of oalt with increased levels of tnf- and il- , which lead to lung pathological damage and an increase of lung myeloperoxidase content .
moreover , knockdown of tlr4 reduced lung cytokines release and reversed the above pathologic changes after oalt and finally improved rats ' survival rate . in conclusion ,
tlr4 overexpression , potentially by stimulating proinflammatory cytokine overproduction , contributes to the development of ali after olt . |
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in 2013 , there were 382 million people with diabetes worldwide and it is predicted that this will increase to 592 million people by 2035 . diabetic retinopathy ( dr ) is the most serious ocular complication of diabetes and is the leading cause of preventable blindness in working age populations . dr accounted for 5% of global blindness in 2002 , approximately five million people worldwide . it is estimated that up to 50% of people with proliferative dr who do not receive timely treatment will become legally blind within five years . although up to 98% of visual loss secondary to dr can be prevented with early detection and treatment , once it has progressed , vision loss is often permanent . despite this , comprehensive dr screening rates are poorly achieved globally , with less than 50% of australian and american diabetic patients receiving appropriate screening [ 6 , 7 ] . the number of people with diabetes living in rural areas is increasing worldwide and is expected to reach 145 million people by 2035 . patients living in rural and remote areas have poorer access to specialist ophthalmology services [ 8 , 9 ] . indigenous people worldwide are particularly vulnerable to eye disease , with blindness six times higher in indigenous australians than for non - indigenous australians [ 1 , 10 ] . impaired vision affects national economies through loss of productivity and earning capacity as well as having significant negative social impacts on communities worldwide , with vision impaired individuals relying heavily on social support . a review of international rural remote dr screening models by the authors found that the vast majority of published models use ophthalmologists as the primary image graders . given the growing number of diabetic patients worldwide , poor achievement of screening recommendations , and limited access to ophthalmology services in rural and remote communities , there is a recognised need for innovative approaches to the delivery of dr screening . this has led to trials utilising nonophthalmologist graders , with numerous studies demonstrating the efficacy of nonophthalmologist graders in detecting dr [ 4 , 1115 ] . this paper presents the results of the evaluation of an innovative remote outreach dr screening ( rodrs ) service delivered in remote communities in a state of australia . the service aims to improve rates of dr screening for patients in remote settings with previously limited access to screening . in 2012 , the rodrs service was implemented in a hospital district and health service in a state of australia . prior to 2012 the implementation of the service was prompted by concerns that diabetic patients living in the region were not undergoing dr screening , and to utilise visiting ophthalmology services more efficiently . optometrists and ophthalmologists service four rural and three remote communities in the region , with ophthalmologists visiting the district triannually ( figure 1 ) . the rodrs program visits 11 remote communities in the district annually . these remote communities have nurse - led clinic facilities with visiting general practice services and three communities have visiting optometry and ophthalmology services . the documented diabetic population in each community ranges from 3 to 49 people and is listed in table 1 . patient lists are then sent to each primary health care centre ( phc ) and the regional diabetes educator , who adjusts lists adding patients and removing those no longer living in the district . diabetic patients are contacted by community health from the rural hub or by their local phc to invite them for annual screening . posters are displayed in community public areas and local health workers raise awareness of the screening visit . on the day of screening , local health workers visit residents in their homes to remind them of the visiting service and provide transport to clinics if required . a registered nurse and indigenous health worker ( indigenous refers to aboriginal peoples and/or torres strait islanders ) ( ihw ) based in the rural hub travel via four - wheel drive to 11 remote communities with a fundal camera . this forms part of an existing chronic disease network in the district . remote communities are located between 117 km and 693 km ( approximately 1.5 to 7 hours ' drive ) from the rural hub , which itself is located 687 km from the closest major regional hospital and 1176 km from the state capital . dr screening is performed in phcs ( except for one community where it is hospital - based ) . a brief patient history , random blood glucose level ( bgl ) , hba1c , cholesterol level , blood pressure ( bp ) , and body mass index ( bmi ) are collected . a visual acuity using a snellen chart patients then undergo fundal photography by a registered nurse using an automated nonmydriatic camera ( centervue drs ) . one 45 fundal photograph centred on the macula and with view of the optic nerve is captured of each eye . if an adequate image can not be acquired , patients undergo dilation with tropicamide ( mydriacyl ) 0.5% unless contraindicated . a visiting diabetes educator , podiatrist , and dietician also attend most remote clinics to provide a comprehensive diabetes service . clinical information and fundal images are transferred to an urban , regional , or locally based rural general practitioner ( gp ) accredited to perform dr image grading ( four gps involved ) . participating gps completed a four - hour online dr upskilling program through the university of queensland masters of medicine ( general practice ) program followed by an accreditation assessment through the royal australian and new zealand college of ophthalmologists ( ranzco ) queensland faculty [ 17 , 18 ] . gps complete a 50-patient ( 100 eyes ) exam and must achieve at least 75% concordance with an ophthalmologist reviewer for accreditation [ 17 , 18 ] . gp image grading was provided by a distant accredited gp ; however , in 2014 it was performed by a locally based rural accredited gp , in collaboration with the visiting ophthalmologist . the gp grader assesses the adequacy of the image , evaluates the image for the presence of dr or other pathology , grades dr ( if present ) according to the wisconsin system , and nominates an appropriate management plan ( figure 2 ) . an urban - based buddy ophthalmologist provides support to the gp grader and visits the region triannually . if no pathology is detected by the gp grader , screening results are sent to the phc for filing , with a copy sent to the patient . if pathology is identified , results are sent to the phc , the patient , and the patient 's nominated gp to arrange ophthalmology referral . those patients with mild or moderate nonproliferative dr ( npdr ) are referred to the visiting ophthalmologist , to be seen during their triannual visit in the community closest to them ( figure 2 ) . ophthalmologist is notified of any patients with severe npdr or proliferative dr ( pdr ) , and depending on the timing of their next visit to the region , the patient will either be reviewed by the regional ophthalmology team or urgently transferred to a larger centre with permanent ophthalmology services . patients for whom an adequate image can not be obtained are generally referred to the visiting ophthalmologist , as this may indicate another pathology such as cataract . this retrospective , descriptive screening record audit had three aims : to identify the proportion of patients with documented diabetes mellitus ( type 1/type 2 ) residing in 11 remote communities who underwent dr screening with the rodrs service , to compare the proportion of those patients screened by the program who underwent appropriate dr screening prior to and following the implementation of the rodrs service , to identify the proportion of screened patients with mild , moderate , or severe npdr and pdr.a further paper explores the acceptability of the program to patients , health professionals , and other key stakeholders . to identify the proportion of patients with documented diabetes mellitus ( type 1/type 2 ) residing in 11 remote communities who underwent dr screening with the rodrs service , to compare the proportion of those patients screened by the program who underwent appropriate dr screening prior to and following the implementation of the rodrs service , to identify the proportion of screened patients with mild , moderate , or severe npdr and pdr . data was collected at phcs during dr screening visits to 11 remote communities in a state of australia . eligible participants were patients with type 1 or type 2 diabetes mellitus aged 18 years or older , attending dr screening in remote communities between april 2012 and december 2014 . patients were excluded from participation if they had no perception of light in either eye , were terminally ill or deemed too unwell to participate , or had a physical or mental disability that prevented either screening or treatment . all eligible patients attending dr screening clinics were invited to take part in the study and all patients consented to participate ( n = 142 ) . however , one patient was screened with gestational diabetes mellitus and therefore was excluded from analysis . the australian national health and medical research council ( nhmrc ) developed national guidelines for the recommended frequency of dr screening . the guidelines recommend that all patients with diabetes ( type 1 and type 2 ) undergo at least biennial screening . however , patients at high risk of dr , including indigenous australians and patients living in rural and remote communities , should be considered for annual examinations . in this paper the main outcome measures included ( i ) the proportion of the documented diabetic population living in remote communities who underwent dr screening with the program , ( ii ) the proportion of those patients screened by the program who underwent appropriate screening ( in line with the nhmrc guidelines ) prior to and following the intervention , ( iii ) the quality of images captured by the screening team , ( iv ) the proportion of screening episodes with dr detected and the type of dr identified , and ( v ) the proportion of screening episodes which required ophthalmology referral . clinical data on dr screening prior to the intervention was based on self - report . all screening data were entered initially into an excel database , cleaned , and then imported into and analysed using spss ( version 22 ) . summary statistics are presented as frequency ( percentage ) for categorical variables and mean ( standard deviation ) for continuous variables that were normally distributed ; otherwise median and interquartile ranges were reported . where appropriate chi - square tests ( gender , ethnicity , and dr detection in high risk patients ) , mann - whitney u ( age , hba1c ) , and an independent sample t - test ( systolic and diastolic bp ) were used to assess bivariate associations . a mcnemar 's test was performed to determine a p value for proportions screened prior to and following implementation of the model . a p value of 0.05 was considered to be statistically significant . in accordance with advice from the human research ethics committee ( hrec ) , the above project was compliant with the nhmrc guidance ethical considerations in quality assurance and evaluation activities and therefore was not recommended for hrec review ( hrec/15/qrbw/122 ) . a total of 218 screening episodes were recorded across 11 remote communities between april 2012 and december 2014 . the program screened 141 patients with 47 patients ( 33.3% ) screened twice and 15 patients ( 10.6% ) screened three times . of the 11 remote communities visited by the screening team , eight communities were visited three times , two communities were visited twice , and one community was visited once . the proportion of the total documented diabetic population ( residing in 11 remote communities ) screened by the program significantly increased throughout the operation of the service from 33.7% in 2012 to 47.9% in 2014 ( table 1 ; figure 3 ) . the odds ratio for being screened in 2014 compared with 2012 was 2 ( 95% ci 1.492.68 ; p = 0.00003 ) . a total of 141 patients were identified as eligible for participation in this study . of these indigenous patients comprised 23.5% of patients screened , with most indigenous patients identifying as australian aboriginal . patients ranged from 18 to 90 years of age , with a median age of 63 years . the median duration of diabetes was 6 years with 32.1% of patients considered at high risk of dr ( duration of diabetes more than 10 years ) [ 2 , 20 ] . this is similar to the national diabetes profile with diabetes slightly more common in males than females ( 5.1% of males and 4.2% of females ) , although the rate of diabetes is highest amongst those aged 75 to 84 years , slightly older than the population screened in this study . overall , 51.8% of patients had an hba1c 7% indicating poor glycaemic control and 28.5% of patients had an hba1c 8% ( associated with increased risk of dr ) ( table 3 ) [ 2 , 20 , 22 ] . systolic bp ranged from 100 mmhg to 212 mmhg with a mean systolic bp of 140.5 mmhg . diastolic bp ranged from 54 mmhg to 120 mmhg , with an average diastolic bp of 82.9 mmhg . most patients had suboptimal bp control with 69.8% of patients with a systolic bp reading 130 mmhg and 46% of patients with a diastolic bp 85 mmhg . in addition , many patients were considered at high risk of dr with a systolic bp 150 mmhg ( 25.9% ) or a diastolic bp 90 mmhg ( 32.4% ) [ 2 , 20 ] . of the 141 patients screened by the program , 16.3% had received appropriate dr screening prior to the implementation of the service ( screening in line with recommendations made in the nhmrc guidelines ) , but 36.2% of patients had never been screened ( figure 4 ) . following the introduction of the program , 66.3% of eligible patients received appropriate screening ( odds ratio 1.93 ; 95% ci of 1.422.64 ; p = 0.00025 ) . ( note : a total of 92 patients were included in the analysis of appropriate screening following the implementation of the model ( 49 patients excluded ) . a total of 36 patients were excluded as rescreening could not be evaluated for the following reasons : ( i ) screened in a community only visited once by the program ( one community ) , ( ii ) screened for the first time in 2014 , or ( iii ) screened for the first time in 2013 in those communities not visited in 2014 ( two communities ) . a further 13 patients who were deceased or had moved from the community were excluded . ) commonly recorded reasons for not attending community screening included previous screening by an ophthalmologist or optometrist , working or travelling out of town , illness / hospitalisation , or unable to be contacted . there was no significant difference between those patients who underwent appropriate screening with the model based on gender ( 1.216 ; 1 df ; p = 0.27 ) , indigenous status ( 0.007 ; 1 df ; p = 0.93 ) , age ( z = 1.84 ; p = 0.07 ) , hba1c ( z = 0.37 ; p = 0.71 ) , systolic bp ( t = 0.29 ; p = 0.77 ) , or diastolic bp ( t = 0.74 ; p = 0.46 ) . a total of 13.9% of screening episodes required rescreening due to inadequate images ( table 5 ) . further analysis demonstrated that the vast majority of inadequate images occurred in 2013 , with a rate as high as 26.5% of screening episodes ( figure 5 ) and traced to a faulty camera . following camera servicing , table 4 describes the proportion of screening episodes where images were normal , abnormal , or inadequate and required reimaging . a total of 58.9% of screening episodes were normal , 24.3% had dr detected , and 16.8% produced inadequate images . the majority of patients with dr had mild to moderate npdr detected ( 21% ) ; however , in 3.2% of screening episodes sight - threatening dr was detected ( defined by severe npdr or pdr ) . gp graders identified diabetic maculopathy in 5.6% of screening episodes , with all cases detected in 2014 . there was no statistically significant association between detection of dr and the absence or presence of appropriate dr screening prior to the implementation of the service ( 0.525 ; 1 df ; p = 0.47 ) . dr was detected more often in patients with a duration of diabetes more than 10 years and an hba1c 8% , cut - offs previously shown to increase the risk of dr [ 2 , 20 ] . a total of 51.6% of patients with diabetes longer than 10 years had dr detected , compared with 25.2% of patients with in screening episodes where the patient 's hba1c was 8% , dr was detected more often ( 47.1% , compared to 23.4% with no dr ) ( 10.602 ; 1 df ; 0.001 ) . there was no statistically significant association between a diastolic bp 90 mmhg ( 0.363 ; 1 df ; p = 0.547 ) or a systolic bp 150 mmhg ( 3.447 ; 1 df ; p = 0.063 ) and detection of dr . a total of 28.2% of screening episodes were referred to the buddy ophthalmologist for review of dr or secondary to identification of another pathology . this is above the national population average for appropriate dr screening and is a significant achievement in remote populations with minimal to no access to optometry and ophthalmology services . since the introduction of the program , screening of the eligible diabetic population living in remote communities has become more comprehensive , increasing from 33.7% to 47.9% across its three years of operation . international rural and remote dr screening programs reported population coverage ranging from 39% to 85% [ 2331 ] . indeed , achieving high rates of screening is particularly challenging in these study communities given the transient and highly mobile nature of the population , the fact that patients are often employed away from townships , the delivery of screening only once annually , and the lack of a fully coordinated approach to screening with visiting optometry and ophthalmology services . low health literacy and limited patient contact with local health services are also recognised barriers to achieving comprehensive population coverage . it is hoped that with continued service promotion and improved community awareness , patient uptake will continue to improve . despite limited ophthalmology resources , a review of international rural remote dr screening models by the authors found that the majority of programs use ophthalmologists as the primary image graders . most countries are not adequately meeting screening recommendations and the number of people with diabetes continues to rise [ 6 , 7 , 32 , 33 ] . further to this , evidence suggests that on average 70% of fundal images captured show no retinopathy . there is thus a need to explore innovative approaches to dr screening within a range of settings . previous studies have demonstrated the efficacy of gp graders , with an australian pilot of dr grading by general practitioners demonstrating good sensitivity ( 87% ) and specificity ( 95% ) . during the operation of the rodrs service , just 28.2% of patients required ophthalmology referral for dr . this model may thus provide a more efficient solution to managing limited specialist ophthalmology resources in rural and remote areas . many international rural remote dr screening models have identified the successful integration of screen - positive patients with ophthalmology follow - up to be particularly challenging [ 34 , 35 ] . a benefit of the rodrs program is the integration of screen - positive patients with specialist follow - up through the use of a buddy ophthalmologist , who supports the gp grader and provides visiting services to the region . the rodrs program also integrated dr screening with other diabetes care , providing a holistic multidisciplinary diabetes service that enables patients to easily complete their annual cycle of care . the significance of this is demonstrated by data released by the national diabetes strategic advisory group indicating that just 18% of australian diabetics had a claim made by their gp for an annual cycle of care [ 36 , 37 ] . it is hoped that provision of a coordinated approach to diabetes care will increase the proportion of diabetic patients undergoing annual dr screening and completing their annual cycle of care . the rodrs service is inherently unique in its delivery , using local health professionals to screen diabetic patients and a local gp to grade images . this community - based approach enables the service to tailor itself to the needs of the local population and workforce . also notable are the high screening rates amongst the indigenous population , comprising 23.5% of patients screened . providing a service that meets the needs of the local indigenous community is vitally important given that just 20% of indigenous australians undergo appropriate dr screening and rates of blindness are six times higher than for non - indigenous australians . this is consistent with other rural remote australian studies with reported detection rates ranging from 11% to 45% , but with the majority of programs reporting rates of 16 to 18% [ 23 , 24 , 38 , 39 ] . this is significant and exemplifies the benefits of the program in detecting abnormality and avoiding preventable blindness . this could be explained by differing terminology amongst gp graders , with some graders believing they should only report diabetic macula oedema . other australian rural remote models have reported rates of diabetic macula oedema ranging from 0.2% to 2.8% , whilst international studies reported rates of clinically significant macula oedema ranging from 4.4% to 6.1% [ 23 , 28 , 38 , 4042 ] . the recent release of the consultation paper for the development of the australian national diabetes strategy has highlighted improved eye screening as a key challenge for the future . models such as this one provide a successful approach both to screening and comprehensive ongoing management of patients with dr . further research is needed to identify the generalisability of this model in terms of infrastructure , payment models , and incentives for quality . the australian medical services advisory committee ( msac ) , the group that advises government on additional medical services nationally , has recently recommended a medicare benefits schedule ( mbs ) item number for nonmydriatic retinal photography in primary care settings , significantly improving the feasibility of this model of care in australian communities . no data was available on the proportion of screen - positive patients who actually underwent follow - up by an ophthalmologist . given the increasing number of remote australians with diabetes , the development and trial of efficient workforce solutions for dr screening are of growing importance . it utilises existing infrastructure and the local health workforce to develop a community - driven and delivered service that meets the needs of the local population . it integrates dr screening into an already existing multidisciplinary diabetes service , providing comprehensive and holistic diabetes care . | background . up to 98% of visual loss secondary to diabetic retinopathy ( dr ) can be prevented with early detection and treatment . despite this ,
less than 50% of australian and american diabetics receive appropriate screening .
diabetic patients living in rural and remote communities are further disadvantaged by limited access to ophthalmology services .
research design and methods .
dr screening using a nonmydriatic fundal camera was performed as part of a multidisciplinary diabetes service already visiting remote communities .
images were onforwarded to a distant general practitioner who identified and graded retinopathy , with screen - positive patients referred to ophthalmology .
this retrospective , descriptive study aims to compare the proportion of remote diabetic patients receiving appropriate dr screening prior to and following implementation of the service
. results . of the 141 patients in 11 communities who underwent
dr screening , 16.3% had received appropriate dr screening prior to the implementation of the service .
in addition , 36.2% of patients had never been screened . following the introduction of the service ,
66.3% of patients underwent appropriate dr screening ( p = 0.00025 ) . conclusion .
this innovative model has greatly improved accessibility to dr screening in remote communities , thereby reducing preventable blindness .
it provides a holistic , locally appropriate diabetes service and utilises existing infrastructure and health workforce more efficiently . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
paget s disease of bone ( pdb ) is a common skeletal disorder characterized by osteoclast activation , which provokes increased but disorganized bone turnover , leading to pathological fractures , bone deformity , and bone pain . the disease has a strong genetic component , but the genes responsible have not been fully characterized . the most important predisposing gene is sqstm1 , which is mutated in 10% of patients with the disease . the sqstm1 causal mutations increase osteoclastogenesis by enhancing receptor activator of nuclear factor kappa b ( nf-b ) ( rank ) signaling in osteoclasts and their precursors ( cavey et al . linkage studies in families ( lucas et al . , 2008 ) coupled with genome - wide association studies ( gwas ) ( albagha et al . , 2010 , albagha et al . , 2011 ) have identified a strong susceptibility locus for pdb at the optn locus on chromosome 10p13 . the optn gene encodes optineurin , a ubiquitously expressed cytoplasmic protein involved in many cellular processes including regulation of nf-b signaling ( zhu et al . , 2007 ) , autophagy , and innate immunity ( wild et al . , 2011 ) , we investigated the role of optn in regulating bone turnover and evaluated the molecular mechanisms by which variants at the optn locus predispose to pdb . in order to identify disease - causing mutations in the optn locus , we conducted mutation screening of the coding exons of optn in 200 pdb patients , but results showed no mutations in the protein - coding region ( data not shown ) . however , the top gwas hit ( rs1561570 ) was found to be a strong expression quantitative trait locus ( eqtl ) in human monocytes ( zeller et al . , 2010 ) and in peripheral blood mononuclear cells ( westra et al . , 2013 ) with substantially reduced levels of optn mrna expression in carriers of the pdb - predisposing allele with an evidence of an allele - dose effect ( figure 1 ) . these observations indicate that susceptibility to pdb is associated with reduced expression of optn and raise the possibility that optn might act as a negative regulator of osteoclast function . here , we studied the expression of optineurin during osteoclast differentiation and investigated the effects of optn knockdown in primary bone - marrow - derived macrophage ( bmdm ) cultures . expression of optineurin increased considerably during osteoclast differentiation , following stimulation of the cultures with macrophage colony - stimulating factor ( m - csf ) and receptor activator of nf-b ligand ( rankl ) ( figure 2a ) . knockdown of optineurin by small hairpin rna ( shrna ) in m - csf- and rankl - stimulated bmdms significantly increased osteoclast numbers and size when compared with a non - targeting shrna control ( figures 2b and 2c ) . overexpression of optn in raw 264 cells resulted in a substantial reduction in the number and size of multinucleated trap+ cells formed upon stimulation with rankl ( figure s1 ) . these experiments illustrate that optineurin acts as a negative regulator of osteoclast differentiation in vitro . to investigate the effects of optn on osteoclast activity and bone remodelling in vivo , we conducted skeletal phenotyping of mice homozygous for a d - to - n amino acid substitution at codon 477 of the optineurin protein ( optn ) . this is a loss - of - function mutation that encodes a protein that is unable to bind to lys63-linked ubiquitin chains ( gleason et al . , 2011 ) . bone marrow macrophage cultures from optn mice formed significantly more osteoclasts than those from wild - type ( wt ) littermates and the number of hypernucleated osteoclasts was significantly increased ( figure 3a ) , replicating exactly the observations in wt cultures subjected to shrna optn knockdown ( figure 2c ) . to investigate if optn affects osteoblast differentiation and/or function , we performed an in vitro bone nodule assay and osteoblast - osteoclast co - culture assays . there was no difference between optn and wt mice in the ability of calvarial osteoblasts to form bone nodules in vitro ( figure 3b ) . however , osteoblast - osteoclast co - culture assays showed that osteoblasts from optn mice had reduced ability to support osteoclast differentiation when compared to wt osteoblast cultures ( figure 3c ) . in contrast , and consistent with bone marrow culture data presented in figure 3a , osteoclast precursor cells derived from optn mice showed enhanced sensitivity to osteoblast stimulation that appears to compensate for the reduction in the ability of mutant osteoblast to support osteoclast differentiation ( figure 3c ) . quantitative bone histomorphometry showed that optn mice had significantly increased number of osteoclasts per bone surface ( oc.n / bs ) and resorption surfaces ( oc.s / bs ) as compared with wt ( figure 3d ) . indices of bone formation ( osteoid surface per bone surface [ os / bs ] , osteoid volume per bone volume [ ov / bv ] , and mineral apposition rate [ mar ] ) were also significantly higher in optn mice ( figures 3e and 3f ) , and there was a non - significant trend ( p = 0.08 ) for an increase in bone formation rate ( bfr / bs ) . these observations illustrate that optineurin negatively regulates osteoclast activity both in vitro and in vivo . the lack of an effect on osteoblast differentiation in vitro suggests that the increased bone formation we observed in optn in vivo was most probably secondary to the increase in bone resorption , as occurs in humans with pdb ( ralston et al . , 2008 ) . since pdb is also characterized by the development of focal osteolytic lesions that predominantly affect older people , we performed further skeletal phenotyping of optn mice to investigate bone mass and bone structure and to look for evidence of focal osteolytic lesions in aged mice using micro - computed tomography ( micro - ct ) scanning of lower limbs . there was no difference between genotypes in bv / tv , trabecular number , or structure in young mice ( figures s2a and s2b ) . however , analysis of older mice aged between 8 and 18 months revealed evidence of a focal osteolytic lesion in the left femur in one optn mutant mouse aged 15 months ( figure 3 g ; table s1 ) . we then investigated the presence of bone lesions in another loss - of - function mouse model in which the c - terminal polyubiquitin - binding domain of optn has been deleted , resulting in a truncated protein with low expression ( optn ) . analysis of the hindlimbs of optn mice ( n = 8) by micro - ct showed no pagets - disease - like lesions ( table s1 ) . these observations illustrate that while the d477n loss - of - function mutation in optn increases bone turnover , this does not result in net bone loss , presumably because the increase in bone resorption is coupled with that of bone formation . the development of a focal osteolytic lesion in one optn mutant mouse ( 10% of mice aged 15 months ) , but not in optn , supports the hypothesis that loss of function in optineurin can lead to a pdb - like phenotype while illustrating that additional factors must also be present for focal osteolytic lesions to become fully penetrant . controlled rankl - induced nf-b activation is essential for osteoclast differentiation and function and for the maintenance of normal bone turnover . previous studies have suggested optn as a negative regulator of tnf--induced nf-b activation in immune cells ( maruyama et al . , 2010 , nagabhushana et al . , 2011 , sudhakar et al . , 2009 , zhu et al . , 2007 ) , but its role in rankl - induced nf-b activation is yet unknown . in order to investigate the effects of optn on intracellular signaling in osteoclasts , we studied rankl - induced nf-b activation during osteoclast differentiation in cultures from optn mice as well as in optn knockdown cultures . we found no difference in rankl - induced nfb activation , as measured by phosphorylation of ib , in bmdms from d477n mutant mice compared with wt ( figure 4a ) . however , following rankl stimulation , there was a progressive increase in nf-b activation that was significantly greater in cultures from optn as compared with wt mice , with a maximal effect at 3 days ( figure 4b ) . similar findings were observed in optn knockdown cultures in which reduced expression of optn by knockdown resulted in enhanced nf-b activity after rankl stimulation ( figure 2d ) . these findings indicate that the inhibitory effects of optn become most apparent as osteoclast differentiation proceeds and is consistent with the observation that optn levels increase substantially during osteoclast differentiation ( figure 2a ) . a similar mechanism has previously been reported in cyld null mice , which show no abnormalities of rankl - induced nf-b activation in bmdms but show enhanced nf-b activation as osteoclast differentiation proceeds ( jin et al . , 2008 ) . previous studies have shown that the deubiquitinase enzyme cyld plays an important negative regulatory role in nf-b signaling in osteoclasts by interacting with p62 and traf6 ( jin et al . , 2008 ) . it has also been reported that optineurin is required for cyld - dependent inhibition of nf-b activation in immune cells ( nagabhushana et al . , 2011 ) . since the d477n mutation is located in the region that binds to cyld ( nagabhushana et al . , 2011 ) , we studied the effects of the mutant protein on cyld binding by immunoprecipitation in osteoclasts . this confirmed that the d477n optn variant had an impaired ability to bind cyld compared with wt optn ( figure 4c ) , indicating that the inhibitory effect of optn on osteoclast is mediated , in part , by a cyld - dependent pathway . it has previously been shown that the expression of mutant optn protein was higher than that of wt in multiple tissues including bmdms ( gleason et al . , 2011 ) . in line with these findings , we also observed that the expression of the mutant optn protein during osteoclast differentiation was higher than that of the wt ( figures 4c and 4e ) . this difference was more noticeable from day 3 post - rankl stimulation ( figure 4e ) , possibly due to increased nf-b activity , since a putative nf-b binding site has been reported in optn promoter ( sudhakar et al . , 2009 ) . however , the increased levels of expression of the mutant optn protein were clearly not sufficient to compensate for its reduced ability to suppress nf-b activity and osteoclast differentiation . studies have shown that while rankl stimulates osteoclast activity , it also initiates a negative auto - regulatory effect on osteoclasts through induction of interferon beta ( ifn- ) expression ( takayanagi et al . , 2002 , hayashi et al . , 2002 ) , which has an inhibitory effect on osteoclast by interfering with rankl - induced expression of c - fos ( takayanagi et al . , previous studies have shown that optineurin is involved in ifn- signaling in immune cells ( gleason et al . , 2011 , munitic et al . , 2013 ) , we investigated if optn plays a role in this negative regulatory loop in osteoclast by studying the expression of ifn- and c - fos in both wt and optn mutant cells . we found that rankl induced the expression of ifn in both wt and optn mutant cells , but the expression levels were significantly lower in mutant cells compared to those observed in wt ( figure 4d ) . additionally , c - fos expression during later stages of osteoclast differentiation ( 3 days post - rankl stimulation onward ) from optn mice was higher than that observed in wt ( figure 4e ) . these data suggest that in addition to its inhibitory effect on nf-b signaling , optn may also exert an inhibitory role on osteoclast differentiation by modulating the ifn- signaling pathway . coupling between bone formation by osteoblasts and bone resorption by osteoclasts is a complex mechanism , but it is well known that osteoblasts produce many pro - osteoclastogenic cytokines to regulate osteoclast function such as tnfsf11 ( rankl ) and il-6 . expression of these two cytokines in osteoblast cultures derived from optn mice was significantly lower than that observed in wt cultures , which is consistent with the reduced ability of mutant osteoblast to support osteoclast differentiation ( figures 4f and 4 g ) . in conclusion , we have demonstrated that reduced expression or loss of optn function in mice leads to enhanced osteoclast differentiation , identifying optn as a negative regulator of osteoclast differentiation . the underlying mechanisms are complex but involve rankl - induced nf-b activation , an interaction with cyld , and regulation of ifn- signaling with regulatory effects that are cell - type specific , dependent on its expression level and on its ability to bind polyubiquitin . our data suggest that the common genetic variant rs1561570 at the optn locus increases susceptibility to pdb by reducing levels of optn expression , probably leading to enhanced osteoclast differentiation . the generation of optn knockin mice was described previously ( gleason et al . , 2011 ) . to generate optn , the optn mice were first crossed to flpe mice to remove the flpe transgene , and these mice were then crossed to bal-1 cre mice to delete exon 12 . micro - ct analysis was performed using an in vivo skyscan 1076 or an ex vivo skyscan 1172 system . live animals were scanned on the in vivo scanner looking for the development of pdb - like lesions as previously described ( daroszewska et al . , 2011 ) , and those showing evidence of lesions were further analyzed on the ex vivo scanner and then subjected to histological analysis as described below . image reconstruction was performed using the skyscan nrecon package and trabecular bone parameters measured using skyscan ctan software . the hindlimbs were fixed for 24 hr in 4% formalin - buffered saline and stored in 70% ethanol prior to embedding in methyl methacrylate . sections were stained for tartrate - resistant acid phosphatase ( trap ) with aniline blue counterstain to visualize osteoclasts and osteoid analysis was done after von kossa staining with van gieson counterstaining according to standard protocols as previously described ( erben and glsmann , 2012 ) . histomorphometry was performed using custom - built software and followed standardized nomenclature and recommendations ( dempster et al . , 2013 ) . bone marrow cells were isolated from the long bones of 4-month - old mice and cultured for 48 hr in the presence of m - csf ( 100 ng / ml ) to generate bmdms . adherent cells were reseeded and stimulated with m - csf ( 25 ng / ml ) and rankl for 5 days until osteoclasts were formed . cells were fixed with 4% ( v / v ) formaldehyde in pbs and stained for trap . trap - positive multinucleated osteoclasts ( more than three nuclei ) were counted , and numbers were compared to the wt control . for the nf-b activation assay , bmdms were generated as described above and incubated under standard conditions for 24 hr . cells were serum starved for 1 hr followed by stimulation with 100 ng / ml rankl . cell lysates were collected at different time points and assayed by immunoblotting . for the nf-b luciferase reporter assay , bmdms were transduced with lentiviral nf-b luciferase reporter for 2 days followed by selection for 48 hr using puromycin ( 5 g / ml ) . the cells were then plated in 96-well plates and luciferase activity was measured with a steadyglo - luciferase reporter assay system at the indicated time points following m - csf ( 25 ng / ml ) and rankl stimulation ( 100 ng / ml ) using a bio - tek synergy ht plate reader . the medium used was complete minimum essential medium alpha modification ( mem ) , and all cultures were incubated under standard conditions of 5% co2 and 37c in a humidified atmosphere . osteoblasts were isolated from the calvarial bones of 2-day - old mice by sequential collagenase / edta digestion and cultured in complete mem medium . on reaching confluence , cells were detached with trypsin , re - plated in 12-well plates at a density of 1 10 cells / well , and cultured in osteogenic medium ( complete mem supplemented with 50 g / ml vitamin c and 3 mm -glycerophosphate ) . the medium was replaced three times per week , and cultures were continued for up to 21 days . mineralized nodules were detected using alizarin red staining , and bone nodule formation was quantified by destaining the cultures in 10% ( w / v ) cetylpyridinium chloride and dissolving the stain in 10 mm sodium phosphate ( ph 7.0 ) . the absorbance of the extracted stain was then measured at 562 nm and compared to an alizarin red standard curve . alizarin red values were corrected for viable cell number as determined by the alamar blue assay . bone marrow cells from wt mice were isolated and cultured for 48 hr in complete mem supplemented with m - csf ( 100 ng / ml ) . the adherent bmdms were then transduced with either lentiviral particles containing shrna targeted against the optn gene or negative control particles ( non - targeting lentiviral particles ) . transduced cells were selected for 48 hr using puromycin ( 5 g / ml ) . optn - depleted bmdms were then plated in 96-well plates and stimulated with m - csf ( 25 ng / ml ) and rankl ( 25 or 50 ng / ml ) until osteoclasts were formed . trap - positive multinucleated osteoclasts were counted and numbers were compared to the non - targeted negative control . values in the graphs indicate group means sem , as indicated in the legends . | summarypaget s disease of bone ( pdb ) is a common disease characterized by osteoclast activation that leads to various skeletal complications .
susceptibility to pdb is mediated by a common variant at the optineurin ( optn ) locus , which is associated with reduced levels of mrna .
however , it is unclear how this leads to the development of pdb . here
, we show that optn acts as a negative regulator of osteoclast differentiation in vitro and that mice with a loss - of - function mutation in optn have increased osteoclast activity and bone turnover .
osteoclasts derived from optn mutant mice have an increase in nf-b activation and a reduction in interferon beta expression in response to rankl when compared to wild - type mice .
these studies identify optn as a regulator of bone resorption and are consistent with a model whereby genetically determined reductions in optn expression predispose to pdb by enhancing osteoclast differentiation . |
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hepatic cirrhosis is the clinical and pathologic result of a multifactorial chronic liver injury characterized by extensive fibrosis and nodular regeneration replacing the normal liver parenchyma . it is well known that cirrhosis is associated with a markedly increased risk of hepatocellular carcinoma ( hcc ) , the sixth most common malignancy worldwide and third most common cause of cancer related death . the detection of hepatic malignancy in cirrhotic patients is a diagnostic challenge due to distortion of the hepatic architecture . in this article we discuss and illustrate the wide spectrum of intra- and extrahepatic findings on computed tomography ( ct ) in patients with cirrhosis . common pathologic features of cirrhosis include hepatic fibrosis , nodular distortion of hepatic architecture , and perfusion abnormalities . bridging bands usually have variable thickness and may mimic a tumor capsule due to delayed contrast enhancement . focal confluent fibrosis is defined as a peripheral wedge - shaped hypoattenuated area on unenhanced and venous phase ct . on delayed phase , enhancement of overlying capsular retraction with volume loss in areas of focal confluent fibrosis is an important feature to differentiate this entity from malignant conditions ( figure 1 ) . additional early detectable morphologic changes of the liver include widening of the porta hepatis , enlargement of the interlobar fissure , and expansion of pericholecystic space . during advanced stages shrinkage of the liver is seen , especially in alcohol - induced cirrhosis . the medial segment ( iv ) of the left lobe shrinks with concomitant hypertrophy of the lateral segments ( ii , iii ) , giving a tongue - like appearance . these changes lead to a nodular contour and heterogeneity of the liver , which is classically associated with cirrhosis . hepatic steatosis is a nonspecific reversible response of hepatocytes to chronic injury , commonly seen in alcohol - induced cirrhosis . when hepatosteatosis occurs , the average liver attenuation is at least 10 hounsfield units ( hu ) less than the splenic parenchyma on unenhanced ct . the identification of normal course vascular structures in areas of fatty infiltration is crucial to differentiate this abnormality from hepatic tumors . evolving hepatic nodular lesions are another important feature of cirrhosis . in attempt to standardize the terminology , an international working party has suggested terms and definitions of nodular lesions in cirrhotic patients . these are categorized as regenerative nodules , dysplastic nodules , and hcc . a regenerative nodule ( rn ) is a well - defined area of liver parenchyma that has enlarged in response to necrosis and altered circulation . based on gross morphologic features , the nodular regeneration can be classified as micronodular ( <3 mm in diameter ) or macronodular ( > 3 mm in diameter ) . unless a regenerative nodule contains iron , it is rarely seen on a noncontrast ct . if iron deposition is present ( siderotic nodule ) , the nodule appears hyperdense to the surrounding liver on a non - contrast ct ( figure 2 ) . micronodular changes are rarely identified on ct , despite being present in all cirrhotic livers . regenerative nodules do not enhance in the arterial phase ( figure 3 ) and are isodense to the remaining parenchyma on the venous phase , making them indistinguishable from the hepatic background . a combination of micro- and macronodular regeneration is the most common morphologic presentation seen in cirrhotic patients . a dysplastic nodule ( dn ) is defined as a nodular region of dysplastic hepatocytes without histologic features of malignancy . dns commonly measure 510 mm and most of them are undetectable by ct since , even after the administration of contrast , the majority is isoattenuating . dysplastic nodules can be further characterized as low grade or high grade , according to the degree of dysplasia . tumor angiogenesis appears to be a mandatory step in the evolution of dysplastic nodules to hcc . during this process , there is a progressive increase in the arterial supply and a concomitant decrease in the portal venous supply to these lesions . the major shift in angiogenesis typically occurs during the transition from low - grade dns to high - grade dns . new vessels composed of nontriadal arteries become dominant and the absence of portal tracts is noted . the increasingly dedifferentiated nodule appears more markedly enhanced on post - contrast early arterial phase image , occasionally mimicking an hcc ( figure 4 ) . several reports have described the detectability of dysplastic nodules on dynamic ct scans . in a large series of liver transplantation specimens , small dns ( < 5 mm ) the detection rate for dysplastic nodules smaller than 2 cm has been reported , in pretransplant three - phase helical ct study , to be 39% . hcc is a malignant neoplasm composed of cells with hepatocellular differentiation and is almost exclusively seen in patients with cirrhosis . the development of hcc in the cirrhotic liver is described either as de novo hepatocarcinogenesis or as a multistep progression , from low - grade dysplastic nodules to high - grade dysplastic nodule , then to dysplastic nodule with microscopic foci of hcc , then to small hcc , and finally to overt carcinoma . hcc is classified histologically as trabecular , pseudoglandular , compact , and scirrhous , with the trabecular pattern being the most common . the fibrolamellar type of hcc has distinct clinical , histologic , and prognostic features and is commonly seen in young patients with no history of cirrhosis or chronic liver disease . small lesions enhance homogeneously , while large lesions are heterogeneous with a characteristic mosaic pattern , due to intralesional necrosis . approximately 80%90% of hccs are highly vascular lesions demonstrating intense contrast enhancement during the arterial phase . in the venous phase , hcc demonstrates washout and becomes isodense with the liver parenchyma , thereby making its detection difficult . about 10%20% of hccs are hypovascular and show contrast enhancement slightly less than that in the surrounding liver on arterial phase images , making the imaging differentiation with dns difficult . the intranodular vascular changes of these lesions revealed by findings of ctap ( ct during arterial portography ) and ctha ( ct during hepatic angiography ) and correlated with histological analysis explain why high - grade dns and early - stage - well - differentiated hcc are hypodense relative to the surrounding liver . both lesions have decreased portal tracts ( including normal hepatic arteries ) , without increased abnormal arteries . and , when the increased abnormal arterial supply compensated for the decreased normal hepatic arterial supply , they are isodense . understanding this blood supply pattern is important for early detection , characterization , and treatment for early - stage hcc . most hccs have a fibrous capsule that is usually hypodense on hepatic arterial phase and enhance on delayed phase . hcc may present as a solitary mass ( figure 5 ) , a dominant mass with daughter lesions ( multicentric type ) ( figure 6 ) , or as a diffusely infiltrating neoplasm ( figure 7 ) . less frequently , it is multifocal with small foci usually less than 2 cm in both hepatic lobes , which may mimic liver metastasis . hcc is very locally invasive and may extend to the bile ducts , portal vein ( figure 8) , inferior vena cava ( ivc ) ( figure 9 ) , and hepatic veins . distant metastasis from hcc may be seen in the lungs , adrenals , adjacent lymph nodes , and bones . ct is accurate in staging hcc by detecting the number of lesions and involved segments , regional adenopathy , vascular tumor invasion , and metastases . the use of mdct with dynamic contrast - enhanced triple - phase technique and reformatted images is essential to detect small hcc lesions ; however , it remains the most challenging area in imaging cirrhotic liver . this technique demonstrates up to 30% more tumor nodules and in approximately 10% of cases of hcc will be the only phase to demonstrate the lesion ( figure 10 ) . despite optimal arterial phase imaging , a large number of small ( < 1.5 cm ) hccs remain isodense relative to the background and go undetected on ct . reported sensitivity for dynamic triple - phase contrast enhanced ct ranges from 50% to 96% and the specificity from 75% to 96% . it is well known that to obtain the best conspicuity of lesions , thinner slices ( collimation , 1.5 mm ; image reconstruction interval , 3 mm ) and late arterial phase images ( 3035 seconds after injection of contrast medium ) should be acquired . on the basis of explanted livers , it has been reported that the detection of hepatocellular carcinomas smaller than 2 cm , using three - phase helical dynamic ct , was 60% and the detection of those larger than 2 cm was 82% . the reported sensitivity using previous state - of - the - art mr imaging technique and correlation with explanted liver pathologic results is also disappointing in detecting small hccs and dysplastic nodules smaller than 2.0 cm . considering that in this country 60%90% of hccs occur in cirrhotic livers and early - stage detection is difficult , the american association for the study of liver diseases ( aasld ) includes a recommendation for periodic imaging surveillance in patients with liver cirrhosis and stated that the diagnosis of hcc can be made safe if a mass larger than 1.0 cm shows typical features of hcc ( arterial hypervascularity and venous or delayed phase washout ) at contrast material enhanced ct or mri , obviating the need for biopsy if these features are present . in our institution , four - phase mdct is usually the preferred technique in our practice and contrast - enhanced mri is used for problem solving cases or in those patients with any contraindication for contrast - enhanced ct . arterioportal shunting is a well - known phenomenon that occurs in patients with cirrhosis and hcc . the typical imaging presentation is a wedge - shaped area of high attenuation seen on the arterial phase that becomes isodense to the liver parenchyma during the portal venous phase . the classic imaging finding is the presence of contrast material in distal portal branches , with minimal or no contrast in the proximal portal vein or superior mesenteric vein during the arterial phase . spontaneous rupture of hcc is an unusual complication identified in approximately 8% of the cases ( figure 11 ) . extrahepatic abnormalities associated with cirrhosis include portal hypertension , ascites , splenomegaly , diffuse intra and retroperitoneal edema , small bowel , and gallbladder wall thickening . portosystemic collaterals develop spontaneously , as blood flow is shunted away from the liver to low pressure systemic vessels ( hepatofugal flow ) ( figure 12 ) . gastrointestinal variceal bleeding is the most common clinical presentation in patients with altered flow dynamics . on unenhanced ct , varices may mimic adenopathy , masses , or nonopacified bowel loops . the left gastric venous collaterals ( coronary varices ) are seen in approximately 80% of cirrhotic patients . these vessels are located in the lesser omentum , between the medial wall of the upper gastric body and the posterior margin of the left lobe . a 5 - 6 mm left gastric vein on ct is an indicator of portal hypertension . esophageal varices are located in the wall of the lower esophagus and appear as intraluminal protrusions with scalloped borders ( figure 13 ) . paraesophageal varices are found around the esophageal wall and arise from the posterior branch of the left gastric vein , whereas the esophageal varices arise from the anterior branch . they appear as circular or tubular enhancing structures in the falciform ligament and are supplied by the left portal vein . caput medusae refers to collateral vessels that radiate from the umbilicus and are situated in the subcutaneous fat . these varices are easily identified by their axial orientation and position in the perisplenic fat . the retrogastric varices are seen in the posteromedial aspect of the gastric fundus near the cardia and may be difficult to diagnose . retrogastric varices drain into the left renal vein through the gastrorenal shunt , whereas perisplenic varices drain directly into the left renal vein via the splenorenal shunt ( figure 15 ) . after administration of contrast , the portal vein shows a central hypodensity corresponding to the intraluminal thrombus . in this situation , the hepatic arterial flow to the liver is increased , developing scattered peripheral transient high attenuation areas known as transient hepatic attenuation differences . in subacute and chronic portal thrombosis , a cavernous transformation of the portal vein may manifest as multiple tubular collaterals in the porta hepatis ( figure 16 ) . when the portal vein is occupied by tumor thrombus , intraluminal enhancement may be seen . portal hypertension is considered the most common cause of splenomegaly in the united states ( figure 17 ) . foci of hemosiderin deposition in the spleen are seen in about 9%12% of patients with portal hypertension . these foci are called gamna - gandy bodies , and their ct imaging pattern varies from hypo- to hyperdense spots , depending on the presence of secondary calcium deposition ( figure 18 ) . mesenteric edema is defined as increased attenuation of the adipose tissue that surrounds the mesenteric vessels or their branches . inflammation , hemorrhage , neoplastic infiltration , and hypoproteinemia due to hepatic insufficiency are the most frequent conditions identified . the frequency of mesenteric edema in patients with cirrhosis is 86% , and it is usually associated with omental and retroperitoneal edema . most of the patients with mesenteric , omental , or retroperitoneal edema demonstrate a patchy , infiltrative pattern of fat stranding . focal edema may simulate a soft tissue mass . the severity of mesenteric edema parallels other manifestations of fluid overload in patients with cirrhosis such as subcutaneous edema , pleural effusion , and ascites . gastrointestinal wall thickening occurs in 64% of cirrhotic patients , usually as a result of submucosal edema . the jejunum and the ascending colon are the most common sites of involvement ( figure 19 ) . in almost all cases , the pattern of wall thickening is concentric with homogeneous enhancement after administration of intravenous contrast . the exact pathophysiologic mechanism leading to edema of the gallbladder wall is uncertain , but it is likely due to elevated portal venous pressure , decreased intravascular osmotic pressure , hypoproteinemia , or a combination of these factors . it is the most common complication of cirrhosis . within 10 years of the diagnosis of compensated cirrhosis , the development of ascites is the final consequence of anatomic and pathophysiologic abnormalities occurring in patients with cirrhosis . the formation of ascites is governed by the same principles as edema formation at other sites : net capillary permeability and the hydraulic / oncotic pressure gradients . patients with cirrhosis but without portal hypertension do not develop ascites . typical ascitic fluid in cirrhotic patients is a yellow - amber transudate with a total protein concentration of less than 2.5 g / dl and with relatively few cells ( figure 20 ) . hcc , its most lethal complication , is seen in 11% of cases after 5 years of hepatitis related cirrhosis . a triple phase evaluation of the liver with ct is essential to detect small hccs . the recognition of the extrahepatic abdominal complications is vital for adequate clinical assessment and treatment . | hepatic cirrhosis is the clinical and pathologic result of a multifactorial chronic liver injury .
it is well known that cirrhosis is the origin of multiple extrahepatic abdominal complications and a markedly increased risk of hepatocellular carcinoma ( hcc ) .
this tumor is the sixth most common malignancy worldwide and the third most common cause of cancer related death . with the rising incidence of hcc worldwide , awareness of the evolution of cirrhotic nodules into malignancy
is critical for an early detection and treatment .
adequate imaging protocol selection with dynamic multiphase multidetector computed tomography ( mdct ) and reformatted images is crucial to differentiate and categorize the hepatic nodular dysplasia .
knowledge of the typical and less common extrahepatic abdominal manifestations is essential for accurately assessing patients with known or suspected hepatic disease .
the objective of this paper is to illustrate the imaging spectrum of intra- and extrahepatic abdominal manifestations of hepatic cirrhosis seen on mdct . |
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according to world health organization ( who ) ( 2012 ) more than 347 million people worldwide suffer from diabetes mellitus ( dm ) , of which 90% have type 2 diabetes ( t2d ) . both the prevalence and morbidity , especially of t2d , continue to grow globally , especially in developing countries . this growth has led to strains on healthcare systems worldwide as t2d remains one of the leading causes of cardiovascular disorders , blindness , renal failure , amputations , and hospitalizations . adiponectin is a circulating protein produced by adipose tissue that is a versatile regulator of energy homeostasis , insulin sensitisation , inflammation / atherosclerotic processes , and anti - ischemic cardioprotection . it is well established that adiponectin plays an important role in t2d , hypertension , multiple sclerosis , and dyslipidaemias . particularly , there is a strong association between hypoadiponectinemia and t2d : both adipose adiponectin mrna expression and circulating adiponectin levels are significantly reduced in most rodent models of t2d , as well as t2d patients ; the degree of glycosylated adiponectin and high - molecular weight / total adiponectin ratio , which correlates with insulin sensitivity , were significantly decreased in t2d patients compared to healthy controls ; high adiponectin levels are associated with reduced risk of developing diabetes . the most significant role adiponectin may play is that of sensitizing the liver and muscles to the action of insulin in both humans and rodents . adiponectin has an ameliorating function on glucose metabolism apart from insulin signalling . in animal models of obesity and diabetes , adiponectin affected both skeletal muscle and liver , promoting fatty acid oxidation in muscle and inhibiting glucose production from the liver , thereby leading to decreases in circulating free fatty acids ( ffas ) , triglyceride , and glucose levels [ 7 , 8 ] . ffas as well as tumor necrosis factor alpha ( tnf ) are factors released from adipose tissue that contribute to insulin resistance . in addition , lipid accumulation in beta - cells might lead to reductions in insulin secretion . in individuals with visceral obesity , there is a decrease in the insulin - mediated suppression of lipolysis , leading to increases in circulating ffas concentrations that contribute to both peripheral and hepatic insulin resistance either by impairing insulin signalling pathways , by competitive inhibition of muscle glucose uptake , by stimulation of endogenous glucose production , thus contributing to hepatic insulin resistance , or by combination of mentioned mechanisms [ 6 , 7 , 9 , 10 ] . also it was shown that adiponectin regulates the expression of several pro- and anti - inflammatory cytokines . its main anti - inflammatory function might be related to its capacity to suppress the synthesis of tnf and interferon gamma ( ifng ) and to induce the production of anti - inflammatory cytokines such as interleukin-10 ( il-10 ) and il-1 receptor antagonist ( il-1ra ) . tnf is a key modulator of adipocyte metabolism , with a direct role in several insulin - mediated processes , including glucose homeostasis and lipid metabolism [ 6 , 7 , 12 ] . the net effect of tnf is to decrease lipogenesis ( ffa uptake and triglyceride synthesis ) and to increase lipolysis . finally , there is substantial evidence of adiponectin effects on beta - cell function and survival , which are well known as key factors in the development of t2d along with insulin resistance . taking into consideration the above mentioned it could be conclude that adiponectin - targeted pharmaceutical strategies increasing circulating adiponectin levels may be therapeutic against t2d . subetta is drug containing release - active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide ( no ) synthase . this novel class of drug was demonstrated to have a fundamentally new proantigen ( cotargeted with antigen ) targeted activity . for the first time in the study of the repeated dilution process , it was found that the resulting ultra - dilution generates a novel activity absent in the starting material , called a release - activity . a distinctive feature of the release - active antibodies is their ability not to suppress , and to modify the activity of the antigen against which they were raised . in two dm animal models , streptozotocin - induced dm and spontaneous t2d in goto kakizaki rats , subetta showed antidiabetic effects similar to that of rosiglitazone . additionally , subetta has shown promising results in its first clinical use in both type 1 diabetes ( t1d ) patients with poor glycemic control receiving intensive insulinotherapy and t2d patients receiving metformin / combination of basal insulin with metformin ( unpublished data ) . subetta treatment led to a significant decrease in fasting plasma glucose and glycated haemoglobin ( hba1c ) levels , exerted a normalizing effect on the daily glycemic levels ( self - control of blood glucose and daily glucose monitoring ) , significantly lowered insulin resistance and plasma insulin levels , as well as lowering total cholesterol and low - density lipoproteins . the aim of this work is to investigate the ability of subetta to affect adiponectin secretion by mature human adipocytes . human preadipocytes ( lot sl0047 ) were provided by zen - bio , inc . human preadipocytes were mixture of subcutaneous depots ( abdomen , thigh , hip , flank , and breast ) obtained from 5 healthy donors ( sex : female ; age : 47.0 5.4 ) undergoing elective surgery that have signed informed consent , under existing institutional review board ( irb ) . the cells were pooled and grown together , then harvested and cryopreserved as the superlot ( multidonor ) that was used in the current study . prior to use the cells undergo quality control in a number of functional assays ( differentiation ( measured by total triglyceride ) , lipolysis , insulin - induced glucose uptake ) and also the results of the quality control have shown that there were not any differences between the depots . preadipocyte medium ( catalog number pm-1 ) , adipocyte differentiation medium ( catalog number dm-2 ) , and adipocyte maintenance medium ( catalog number am-1 ) were provided by zen - bio , inc . enzyme - linked immunosorbent assay ( elisa ) kit ( catalog number adip-1 ) was the product of zen - bio , inc . subetta , release - active dilutions ( rad ) of antibodies ( abs ) to the beta subunit of insulin receptor and abs to endothelial no synthase , rad of abs to cannabinoid receptor type i ( r - cbi ) , rad of rabbit nonimmune serum ( rbs ) , rad of purified water ( placebo ) , and purified water were manufactured and supplied by ooo npf all of ooo npf materia medica holding compounds , except for purified water , were manufactured using the method described previously using routine methods described in the european pharmacopoeia ( 6th edition , 2007 ) . starting substances were mixed with a solvent ( ethanol - water solution ) and shaken for 1 min to produce c1 dilution . all subsequent dilutions consisted of one part of the previous dilution to 99 parts of solvent ( ethanol - water solution for intermediate dilutions and distilled water for preparation of the final dilution ) , with succession between each dilution . solutions were prepared in sterile conditions , avoiding direct intense light , and were stored at room temperature . rabbit polyclonal antibodies to the beta subunit of insulin receptor and to endothelial no synthase were used as starting substances for subetta , rabbit polyclonal antibodies to cannabinoid receptor type i for rad of abs to r - cbi , rabbit nonimmune serum for rad of rbs . in case of placebo , human preadipocytes were plated at 40625 cells / cm in 150 l of preadipocyte medium . the cells were allowed to attach overnight in a 37c humidified incubator at 5% co2 . the following day , the plating medium was removed and replaced with 150 l of adipocyte differentiation medium and the cells allowed to incubate in a 37c humidified incubator at 5% co2 for 1 week . after 1 week , 90 l of the differentiation medium was removed from each well and 120 l of adipocyte maintenance medium was added . the cells were then allowed to incubate in a 37c humidified incubator at 5% co2 for 7 more days . prior to the assay , the cells were incubated in the absence of serum and hormones for three days . after starvation , test compounds ( 75 l ) were added and the cells were incubated at 37c in a humidified incubator at 5% co2 for three days . conditioned media was collected after 72 hours and either frozen for later evaluation or measured immediately . for the elisa , 20 l of the conditioned medium was added to 80 l of pretreatment solution and heated to 100c for 5 minutes . once the samples were cooled to room temperature , 50 l of each sample was added to 200 l diluent buffer to generate a 5-fold dilution . samples were mixed and 100 l was transferred to the coated wells of the elisa plate . samples were incubated at room temperature for 1 hour after which the plate was washed and secondary antibody applied and incubated at room temperature for 1 hour . the plate was washed again and detection antibody was added and allowed to incubate for 1 hour at room temperature . detection reagents were added and absorbance at 450 nm ( spectramax 250 : molecular devices ) was determined . the data are presented as mean value per group ( m ) standard deviation ( sd ) . one - way analysis of variance ( anova ) followed by tukey hsd test was used for statistical analysis and p values less than 0.05 were regarded as significant ( statistica 6.1 software ) . incubation of mature human adipocytes with subetta for 72 hours resulted in a statistically significant increase in adiponectin concentration in the culture medium ( table 1 ) . nonspecific controls , rad of abs to r - cbi and rad of rbs , did not significantly affect adiponectin secretion , resulting in adiponectin levels comparable to background values of the negative controls , placebo , purified water , and 0.1% dimethyl sulfoxide ( dmso ) . the reference drug rosiglitazone stimulated adiponectin secretion but to a lower level compared to that of subetta and its effect was not significant in comparison with dmso value . previously , subetta demonstrated the ability to reduce hyperglycemia and improve glucose tolerance in various experimental models of diabetes mellitus and its effects were similar to that of rosiglitazone [ 14 , 15 ] . subetta was given by oral gavage , when it was studied in vivo but in the current study the direct action of subetta on mature human adipocytes was estimated in vitro . based on the activity demonstrated for the whole class of novel drugs and on data from previous studies , we may conclude that the complex drug mainly exerts a modulating effect on the beta subunit of the insulin receptor regulating the insulin receptor 's kinase activity and consequently activating receptor - associated signal pathways . the ability of direct activation of insulin receptor and activate receptor - associated signal pathways in the absence of insulin was shown for l7 ( merck ) [ 17 , 18 ] . according to shehzad et al . specifically , insulin enhances adiponectin regulation and secretion selectively in adipocytes . while the exact mechanism of action of insulin on adiponectin biosynthesis remains unclear , it has been suggested that insulin binding to its receptor activates a cell cascade inhibiting the activity of foxo1 , a suppressor of peroxisome proliferator - activated receptor gamma ( ppar gamma ) , which in turn results in the induction of adiponectin biosynthesis . it is important that adiponectin has an ameliorating function on glucose metabolism apart from insulin signalling . taking into consideration the above mentioned , it could be assumed that subetta via direct effect on beta subunit of the insulin receptor of mature human adipocytes activates insulin receptor in the absence of insulin in culture medium , which in its turn exerts activating receptor - associated signal pathways . as a result activity of foxo1 is inhibited and thus adiponectin biosynthesis is inducted . to confirm this suggestion , further studies ( both in vitro and in vivo ) should be conducted in order to provide a detailed investigation of the mechanism of subetta influence on the production of adiponectin and also to assess the involvement of this process and its role in the mechanism of drug action . in summary , the novel complex drug subetta , containing rad of antibodies to the beta subunit of insulin receptor and antibodies to endothelial no synthase , stimulates adiponectin production by mature human adipocytes . this effect on adiponectin production is specific ; neither rad of abs to r - cbi nor rad of rbs affected adiponectin secretion by mature human adipocytes . therefore , the ability of subetta to exert its modulating effect on the insulin receptor even in absence of insulin might be the basis for stimulating adiponectin secretion and might serve as one of the mechanisms of the antidiabetic effect of this drug . the results of the current in vitro study give first insight on possible mechanism of action of subetta and serve as a background for further studies . | purpose . to investigate the mechanism of action in peripheral tissues of novel complex drug containing release - active dilutions of antibodies to the beta subunit of the insulin receptor and antibodies to endothelial nitric oxide synthase ( subetta ) , which has shown efficacy in animal models of diabetes
. methods .
human mature adipocytes were incubated either with subetta , with one of negative controls ( placebo or vehicle ) , with one of nonspecific controls ( release - active dilutions of antibodies to cannabinoid receptor type i or release - active dilutions of rabbit nonimmune serum ) , or with dimethyl sulfoxide ( dmso ) at 37c in a humidified incubator at 5% co2 for three days .
rosiglitazone was used as reference drug .
secretion of adiponectin was measured by quantitative enzyme - linked immunosorbent assay ( elisa ) .
results .
only subetta significantly stimulates adiponectin production by mature human adipocytes .
nonspecific controls did not significantly affect adiponectin secretion , resulting in adiponectin levels comparable to background values of the negative controls and dmso .
conclusion .
increasing adiponectin production in absence of insulin by subetta probably via modulating effect on the beta subunit of the insulin receptor might serve as one of the mechanisms of the antidiabetic effect of this drug .
these in vitro results give first insight on possible mechanism of action of subetta and serve as a background for further studies . |
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fatigue is a transitional state between awake and sleep which manifests itself as lack of alertness and deteriorated mental or physical performance and often associated with drowsiness . road accidents due to fatigue are often much more severe than other crashes , since the driver reaction time increases ( 1 ) . driver fatigue is deemed to account for up to 40% of road accidents ( 2 ) . it is conjectured that 1030% of road deaths are related to driver fatigue ( 3 ) . mental fatigue causes reactions become prolonged , more fluctuable , and more error tending ( 4 , 5 ) . grandjean defined fatigue as a state with decreased efficiency and lack of general willingness to work ( 6 , 7 ) . these can be classified as subjective , psychological , performance and physiological methods . in subjective methods , standard questionnaires such as f - vas and moreover , the use of behavioral and psychological techniques in mental fatigue investigation has been adopted in several preceding studies ( 1417 ) . among these studies , a set of video recordings of facial expressions , mannerisms and personality traits questionnaires were common methodologies with high reliability and validity ( 17 ) . in addition , some fatigue studies on driving simulator exploited performance features such as steering wheel angle and lane departure ( 1820 ) . some other researchers have focused on driver s physiological changes , such as the measurement of eye activity , heart rate , skin electrical potential and specially eeg activity as a means to detect cognitive states ( 21 , 22 ) . although several physiological indices available for assessing the alertness level , eeg signal may be one of the safest and most predictive , ( 2325 ) , since it immediately reflects brain activity . driving involves several tasks such as motion , reasoning , visual and auditory processing , decision - making , perception and cognition . driving is also under the influence of emotion , anxiety and many other psychological factors ( 24 ) . the brain electrical activity rhythms are classified according to frequency bands including delta , theta , alpha and beta waves ( 26 ) . alpha rhythm has the frequency range from 813 hz , which occurs during wakefulness , especially in the occipital cortex area of the brain . it typically appears during eye closure and reduced when eyes open and attenuates severely during attention tasks ( 27 ) . in previous researches , it is known that increase in delta power during internal processing is associated with mental task performance ( 28 ) . other researchers have reported that an increase in eeg theta power depends on decreased performance in monotonous tasks ( 29 ) . although some definite trends were observed in the delta , theta and alpha power frequency bands during fatigue , the results of different studies may be influenced by inter - individual and intra - individual variations in eeg data ( 30 ) . changes in eeg power spectra are associated with fluctuations in the alertness state ( 31 ) . monitoring physiological signals while driving can provide the possibility to detect and warn fatigue ( 32 ) . most investigations revealed that changes in delta and theta activity are related to the transition to fatigue . therefore , eeg monitoring during driving may be a promising variable for using in fatigue countermeasure devices ( 24 ) . it has been suggested that during driving at night , delta band varies significantly with the degree of fatigue ( 33 ) . some researchers have presented an eeg - based cerebral workload index based on the increase of eeg power spectra in the theta band over prefrontal areas and the immediate decrease of eeg power spectra over parietal areas in alpha band during driving ( 34 ) . while physiological mental fatigue level increases , the relative power of theta , alpha and beta rhythms decrease , but the relative power in delta rhythm increases ( 35 ) . the relative power of alpha increased while the attention level of the driver decreased ( 36 ) . however , research on the physiological links specially eeg frequency bands to driver fatigue is still exploratory and is an important area that needs further investigation . therefore , this study attempted to detect the driver mental fatigue through changes in eeg alpha power activity . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . twelve healthy male volunteer car drivers ( ranging 20 to 30 years old ) participated in an overnight study from 2 to 6 a.m. in virtual reality laboratory of khaje nasir toosi university of technology in 2013 . all the subjects held valid driving license with at least 2 years driving experience and had no history of prior brain injuries . the epworth sleepiness scale was used to measure the participants for any trait sleepiness . after taking informed written consent , the subjects were requested to stay awake 18 h before the experiments and refrain from caffeinated drinks or any other stimulant as well as cigarette smoking for 12 h prior to the experiments . the drivers should have regular sleep pattern ( i.e. sleeping not later than 1 a.m. and awaking not later than 9 a.m. ) and not get used to daily napping which was studied through sleep diary for one week before the experiment . this research employed a fixed - based car driving virtual reality simulator ( ci004 semi ) in a calm controlled room with fixed temperature and illumination conditions . 1 shows a snapshot of the car - driving simulator developed in mechatronics department of k.n . snapshot of the car - driving simulator used for the implementation of the proposed protocol a set of 110 km road sceneries had been designed and simulated . the first 90 km of the road was monotonous and straight with minimal side components to induce more fatigue and the last 20 km was winding mountainous road ( see fig . 2 ) . a scenery of the road captured from the car simulator lcd the designed road was from the most dangerous parts of real road pattern of iran , so called haram to haram road , photographed through google maps using autodesk autocad civil 3d ( version 2013 ) and autodesk 3ds max ( version 2012 ) softwares . a portable g.usb amp bio - signal amplifier with 16 channels was used with 256 hz sampling rate and 24 bit quantification with active electrodes for signal acquisition . during driving on the simulator , eeg and the electrode positions on different areas of the scalp were based on the 1020 international electrodes placement guideline . the main eeg signal channels were o1 , o2 , p3 , p4 , p7 , p8 , oz , fp1 , fp2 , cz , fz , t7 and t8 . the eeg data analysis involved pre - processing , artifact removal , and features extraction . the eeg signal was first processed executing a band pass filter between 0.5 and 60 hz . eeglab ( version 10.2.5.6a ) was utilized to remove muscular and ocular artifacts by visual inspection . all the signals were sequenced to epochs each lasting two seconds ( 1 epoch consisting of 512 samples = 2 seconds ) . this research exploits power spectrum density and fast fourier transform ( fft ) analyzing technique to determine the absolute and relative powers of alpha frequency band . relative alpha power was computed as the ratio between absolute alpha power and the total spectral power of the signal . we compared the absolute and relative alpha power variations in the first and last 10 minutes of driving with the f - vas scores . for further certainty , this study employed the double check validity method by comparing the extracted features from eeg signals with the video rating scores . before resuming the experiments , the car driver s sleepiness propensity was measured by the epworth sleepiness scale . then , eeg and eog signals were recorded from the subjects in relaxed sitting posture for 3 minutes with closed eyes ( imagining driving on a highway ) and 3 minutes with open eyes ( looking at a road picture on the screen ) as a baseline for alertness . the subjects were asked to drive the monotonous road at a constant speed of 90 km / h . at the same time , a continuous eeg and eog records were taken during the driving on the car simulator . meanwhile , the drivers faces and behaviors were monitored via video recordings in lateral and front views . these videos were used to rate the fatigue level of drivers on a four - point scale from 1(alert ) to 4 ( very tired ) by two trained observers . moreover , the self - rating of fatigue was performed during the driving using fvas scored from 0 ( no fatigue or energetic ) to 10 ( worst possible fatigue ) in every 10 minute interval . the descriptive statistics such as central and scattered indices were computed in order to describe the variables . pearson and spearman correlation coefficients were employed to explore the associations between f - vas scores and video ratings , fvas and absolute and relative alpha powers . paired - sampled t - test was utilized to compare the means of f - vas scores , video ratings , absolute and relative alpha powers in the initial and final 10 minutes of driving . the drivers mean age was 23.8 years ( sd = 1.44 years ) with mean body mass index ( bmi ) of 21.57 ( sd = 2.01 ) . other demographic characteristics and work - related information of the drivers drivers demographic and background characteristics data as shown in table 2 the self - report scale measured by means of f - vas suggested that the participants had not been fatigued during the first 10 minutes of driving and moderately to extremely fatigue during the final section of driving . compared with the initial section , the f - vas scores increase significantly ( p = 0.001 ) during the final section of driving task , which demonstrates that continuous monotonous driving may predispose the subjects to cognitive fatigue . different fatigue measures in i10d and f10d i10d : the initial 10 min of driving ; f10d : the final 10 min of driving furthermore , table 2 presents the video scores rated by trained observers in the initial and final 10 minutes of driving . paired - sampled t test suggests significant increase during the final 10 minutes of driving ( p = 0.001 ) . the results showed a mild , but significant correlation between f - vas and video rating scores in the initial 10 minutes of driving ( r = 0.404 , p = 0.001 ) . meanwhile , spearman s correlation test showed a relatively strong and significant association between f - vas and video rating scores in the final 10 minutes of driving ( r = 0.62 , p = 0.001 ) . table 2 also indicates the absolute and relative alpha powers in the initial and final 10 minutes of driving . there was a significant increase in the absolute alpha power during the final 10 minutes of driving ( p=0.006 ) . moreover , our findings showed no differences in absolute alpha power in different areas of the scalp in the initial and final 10 minutes of driving , except in the parietal area of the brain ( p=0.005 ) . paired - sampled t test suggests statistically significant difference in p4 site between absolute alpha powers in the initial and final 10 minutes of driving ( p = 0.026 ) . this study attempted to present a simple and reliable method for early detection of driver mental fatigue based on eeg alpha power in healthy sleep deprived drivers . in our experiment , different fatigue outcome measures including subjective self - report of fatigue ( f - vas ) , video ratings as well as eeg alpha powers were employed to evaluate the change in fatigue for drivers during the initial and final 10 minutes of driving . the experimental results suggested a significant increase in the subjective self - report of fatigue ( fvas scores ) during the final 10 minutes of driving . the same results were obtained from zhang ( 2008 ) study which found that the level of both subjective sleepiness and fatigue increase significantly from pre - task to post - task ( 37 ) . they studied the impacts of visual display terminal ( vdt ) task on autonomic nervous system and central nervous system through subjective self - report of sleepiness and some physiological measures such as power spectral indices of hrv and wavelet packet parameters of eeg . it should be regarded that our findings from fvas scores showed lowered variability ( all participants reached the state of fatigue and extremely fatigue ) in the final section of driving . this may be because a subject who is fatigue will be less likely to evaluate his / her state sufficiently . in other words , mental fatigue may impact the drivers judgment of their existing state . the findings of this preliminary research indicated a significant increase in absolute alpha power in the final section of driving . this is consistent with the known aspects of eeg alpha rhythm ( 38 , 39 ) . the increase in alpha rhythm in the final 10 minutes of driving depicted the decrease in the level of alertness and attention and the commencement of fatigue and drowsiness . the findings of the present study are well be in line with the findings of researchers in which they found significant difference between the alpha frequency band in the first and fifth section of driving ( 40 ) . in another study , delta and theta activity increased in the final 3 hours of 7 truck drivers during night driving ( 41 ) . nevertheless , our results revealed no significant difference between the relative alpha powers in the initial and final 10 minutes of driving . the reason for this may be due to the increase in delta and theta rhythms , which causes less relative alpha variability in the final section of driving . in our study , most changes occurred in the right parietal region ( p4 ) which was in contrast with the schier ( 2000 ) study that showed the greatest changes in the right frontal region ( f4 ) of the scalp ( 36 ) . however , this is consistent with the reported role of the right hemisphere via blood flow measurement in attention demanding tasks ( 42 ) . since the subjective self - report of fatigue was measured in the initial and final 10 minutes of driving , sudden variations can not be detected using f - vas technique . another limitation to employing this scale is that drivers verbal expression of their level of fatigue may alert them , thereby decreasing their fatigue level . meanwhile , it is almost unfeasible to obtain fatigue feedback from a driver in real driving conditions unless significantly distracting the driver attention from the driving task . our study benefited from using video rating of facial expressions and driver behaviors by trained observers that might not be able to see what goes on behind the facial behavior and expression . placing the electrodes on the scalp to obtain signals is an intrusive technique that should be addressed as another limitation of researches using wired biological signals . therefore , future projects should make the use of less - intrusive systems such as wireless electrodes and benefit the strengths of the various fatigue monitoring methodologies into a hybrid system to develop an efficient fatigue detection device in real driving situations . the present study specifically deals with eeg alpha power in the initial and final section of driving in partially sleep - deprived drivers - a very prominent aspect of driver fatigue analysis . the results of this study may serve as a baseline for the development of anin - vehicle device preventing fatigue related crashes on monotonous roads and greatly improving the transport industry in terms of socio - economic benefits and safety . this study suggests that alpha brain wave rhythm can be a good indicator for early prediction of driver fatigue . meanwhile , image processing of facial expressions may be a complementary method for road accident prevention . these indicators can be incorporated to develop a fatigue countermeasure device to prevent road accidents and reduce fatigue related costs . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | background : driver fatigue is one of the major implications in transportation safety and accounted for up to 40% of road accidents .
this study aimed to analyze the eeg alpha power changes in partially sleep - deprived drivers while performing a simulated driving task.methods:twelve healthy male car drivers participated in an overnight study .
continuous eeg and eog records were taken during driving on a virtual reality simulator on a monotonous road .
simultaneously , video recordings from the driver face and behavior were performed in lateral and front views and rated by two trained observers . moreover , the subjective self - assessment of fatigue was implemented in every 10-min interval during the driving using fatigue visual analog scale ( f - vas ) .
power spectrum density and fast fourier transform ( fft ) were used to determine the absolute and relative alpha powers in the initial and final 10 minutes of driving.results:the findings showed a significant increase in the absolute alpha power ( p = 0.006 ) as well as f - vas scores during the final section of driving ( p = 0.001 ) .
meanwhile , video ratings were consistent with subjective self - assessment of fatigue.conclusion:the increase in alpha power in the final section of driving indicates the decrease in the level of alertness and attention and the onset of fatigue , which was consistent with f - vas and video ratings .
the study suggested that variations in alpha power could be a good indicator for driver mental fatigue , but for using as a countermeasure device needed further investigations . |
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most of the children only require observation ; however , if amls are large or symptomatic , they may be embolized or resected . there was no systemic treatment available until 2008 , at which point mtor inhibition was introduced as a viable treatment option . although the treatment has rarely been used in children , either sirolimus ( sir ) or everolimus ( evr ) is recommended if the amls are larger than 3.0 cm . there have been no documented cases detailing the use of sir or evr to treat numerous amls causing substantial renomegaly and chronic pain . here , we describe a case of a patient with renal amls whose kidneys returned to a normal size following treatment with sir for 3.5 years . an 8-year - old girl antenatally diagnosed with tsc presented with numerous bilateral renal amls . the length of her left kidney was 1.6 standard deviations above the norm ( z - score = + 1.6 [ figure 1 ] ) . she was asymptomatic until she developed bilateral severe chronic flank pain at the age of 15 . the pain coincided with a renal length of over 13 cm ; the z - score for the length of her left kidney was + 5.04 [ figure 1 ] . her pain was considered secondary to the capsular pain emanating from the significant growth of hundreds of renal amls , the largest measuring 3.5 cm in diameter . her baseline pain was 6/10 and 10/10 at its worst , causing frequent emesis and the inability to sleep . the pain neither subsided nor lessened with the use of strong analgesics such as opioids . sir = sirolimus she was conscious and oriented on examination and did not exhibit any acute distress . her blood pressure was 103/56 mmhg ( 50 percentile for her height 112/64 mmhg ) and her heart rate was 54/min . an abdominal ultrasound showed numerous amls and enlarged kidneys ( right kidney : 13.4 cm and left kidney : 12.4 cm [ figure 1 ] ) . her kidney function was stable with a serum creatinine of 72 mol / l and a cystatin c estimated glomerular filtration rate of 119 ml / min/1.73 m. testing did not reveal any hematuria or proteinuria . sir was started at a dose of 0.03 mg / kg ( 2 mg taken once daily ) with a target sir level of 6 9 ng / ml ( via immunoassay ) or 5 8 ng / ml ( via mass spectrometry ) , conforming with renal transplant guidelines . unfortunately , another physician prescribed a macrolide and the drug interaction caused severe stomatitis and a maculopapular rash on her legs , which are adverse effects associated with sir . the dose of sir was decreased to 1 mg once daily to minimize these effects . the dose was continually adjusted over the course of 3 years to minimize ongoing adverse effects , primarily stomatitis . the highest dose she received was 4 mg once daily ( 0.05 mg / kg ) . although her sir levels were consistently maintained between 2 and 5 ng / ml , the highest measured level was 7.8 ng / ml . her renal length decreased bilaterally to 10.6 cm , and the z - score for the length of her left kidney decreased to + 2.42 [ figure 1 ] . at her last follow - up appointment , the largest lesion in her right kidney measured 2.8 cm 3.1 cm 2.4 cm and the largest lesion in her left lower pole measured 2.2 cm 1.7 cm 2.2 cm . still , despite successful medical treatment , her pain - associated depression did not subside , and consultations at a pain clinic and with a psychiatrist were necessary to emphasize the importance of employing a multidisciplinary approach to treat her depression . treatment with evr was considered because of its potential to maintain a higher therapeutic exposure while reducing adverse effects ; at 10 times the price of sir , the medication 's cost prevented the switch . of note , neither sir nor evr have any published recommended target levels to treat renal amls , and previous studies examining treatment with evr or sir for tumors have titrated the dosing based on their effect or tolerability . this case demonstrates that sir is a viable treatment option for numerous renal amls causing renal enlargement and capsular pain , which would otherwise not be amenable to any therapy . overall , further research is necessary to develop precise dosing guidelines since the therapeutic goal published in transplant literature may be too high . this case also raises the question of whether treatment with sir should be considered early in the course of the disease before the patient develops any secondary morbidity such as chronic pain . | tuberous sclerosis complex frequently results in the formation of renal angiomyolipomas ( amls ) .
sirolimus ( sir ) or everolimus can be used to treat large amls , although this treatment has rarely been described in children , particularly for multiple renal amls . a 15-year - old girl presented with bilateral severe chronic flank pain coinciding with increased renal size and hundreds of renal amls .
we opted to treat her with sir over the course of 3.5 years .
following her treatment , her renal size had substantially decreased and the amls had shrunk .
the patient 's pain subsided as well .
our case , which has never been described in published literature , demonstrates that a child with multiple renal amls can be treated with sir , and suggests that this treatment may be a viable option for preventing the development of secondary morbidities such as chronic pain . |
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alzheimer 's disease ( ad ) is a progressive and degenerative disease that affects brain cells , and its early diagnosis has been essential for appropriate intervention by health professionals . noninvasive in vivo neuroimaging techniques such as magnetic resonance imaging ( mri ) and positron emission tomography ( pet ) are commonly used to diagnose and monitor the progression of the disease and the effect of treatment . in this regard , the problem of developing computer aided diagnosis ( cad ) tools to distinguish images with ad from those of normal brains has been extensively addressed in the past years [ 112 ] . a review of a recent work follows . magnin et al . used the relative weight of gray matter versus white matter and cerebrospinal fluid in 90 regions of interests ( roi ) as features classified with svm . based on the bootstrap method , the svm obtained 94.5% average classification accuracy in the classification of 16 ad and 22 control ( healthy ) subjects , with a mean specificity of 96.6% and a mean sensitivity of 91.5% . proposed a classification system for ad based on the partial least square ( pls ) regression model for feature extraction ( identification of discriminative voxels ) and the random forest ( rf ) classifier . the pls - rf system yielded accuracy , sensitivity , and specificity values of 96.9% , 100% , and 92.7% , respectively , after classifying 41 normal and 56 ad images using the leave - one - out cross - validation method . salas - gonzalez et al . used welch 's t - test to identify voxels that provide higher difference between normal and ad images . the identified voxels formed the main features to classify , and a svm with linear kernel reached 96.2% accuracy in distinguishing 41 normal and 38 ad images using leave - one - out cross - validation method ; the sensitivity and the specificity were about 96% . sampled the brain with seven relatively small volumes that were filtered to give intensity and textural mri - based features . each filtered region was analyzed with a random forest classifier to extract relevant features , which were subsequently processed with a support vector machine . the system performance was evaluated on the classification of 144 ad patients and 189 controls . using receiver operating curve ( roc ) analysis for 144 ad patients and 189 controls , and 20-fold cross - validation , the result was 0.97 area under curve ( auc ) for discriminating the ad images from the normal ones , with 89% sensitivity and 94% specificity . chen et al . used large - scale network ( lsn ) analysis to classify subjects with alzheimer 's disease and normal subjects . the pearson product moment correlation coefficients were used to assess the connectivity between roi in the brain . classification of the ad group ( 20 subjects ) and the non - ad group ( 20 subjects ) using leave - one - out cross - validation led to 87% auc , 85% sensitivity , and 80% specificity . graa et al . used fractional anisotropy of diffusion tensor imaging ( dti ) data to obtain information about the magnitude of the water diffusion process at each voxel . based on leave - one - out cross - validation method , the linear kernel svm achieved overall accuracy of 100% for the classification of 25 healthy controls and 20 ad images . used the hippocampal volume ( hv ) , cortical thickness ( cth ) , tensor - based morphometry ( tbm ) , and features extracted from manifold - based learning ( mbl ) framework to discriminate healthy controls ( 231 subjects ) from subjects with ad ( 198 subjects ) . five percent of the evaluation subjects were kept for testing and the remaining 95% were used for training . zhang et al . proposed a multimodal data fusion and classification method based on features extracted from structural mri , functional imaging ( fdg - pet ) , and cerebrospinal fluid ( csf ) . the three modalities were , respectively , used to measure brain atrophy and to quantify hypometabolism and specific proteins linked to ad . consequently , 93 volumetric features were extracted from 93 roi automatically labeled by an atlas warping algorithm . the linear svm was used to evaluate the classification accuracy using 10-fold cross - validation . the result of classifying 51 ad and 52 normal controls yielded classification accuracy of 93.2% , with a sensitivity of 93% and a specificity of 93.3% . daliri used the scale - invariant feature transforms ( sift ) to extract features from mr images that were clustered using the k - means algorithm . fisher 's discriminant ratio was used for ranking clustered features , and genetic algorithms performed feature subset selection . the validation data consisted of the mris from 98 normal subjects and from 100 subjects with ad . the svm achieved 86% correct classification rate using leave - one - out cross - validation method . gray et al . combined cross - sectional and longitudinal fdg - pet information for classification . particularly , the whole brain was segmented into 83 anatomically defined regions from which intensities and changes in signal intensity over the follow - up period were extracted . the svm achieved a classification accuracy of 88% for 200 patients with alzheimer 's disease and 200 healthy controls using fivefold cross - validation protocol . li et al . used longitudinal changes of cortical thickness to characterize ad pathology . in particular , three categories of features were extracted from each subject , including static cortical thickness , cortex thinning dynamics , and the correlation between the longitudinal thickness changes of different regions of interest ( roi ) in brain image . based on leave - one - out cross - validation method , the svm distinguished 37 ad patients from 40 nc with an accuracy of 96.1% . in a recent work , we proposed a fractal - based processing methodology to detect ad in brain mri . the system does not require image reduction or segmentation , and it relies on a simple three - step algorithm . first , the brain mri is transformed into a one - dimensional ( 1d ) signal by row concatenation . then , a three - component feature vector is extracted from the 1d signal to characterize its local and global fractal features as expressed by hurst 's exponent and the two results from the detrended fluctuation analysis ( dfa ) of the cumulated 1d signal : the scaling exponent and the total detrended fluctuation energy ( hurst 's exponent allows the evaluation of how a signal is self - affine , i.e. , can be made self - similar by an affine transformation for a given level of detail ( a self - similar image is one whose whole is similar to its parts ) ; dfa is a generalization that can also detect long - range power - law correlations in seemingly nonstationary signals . in particular , it can determine local trends in the signal and measure its level of persistence ) . in the last step , the obtained feature vector is classified by a svm with polynomial kernel . the validation with 10 normal brain mri and 13 corresponding to ad led to 100% classification accuracy by a svm with quadratic kernel . the obtained results suggested that the use of fractals to characterize the mri of normal patients and ones with ad held the promise of equal or better classification accuracy than the best alternative approach while being simpler to implement . in addition , the average processing time to extract the fractal features took over 400 seconds . this paper follows up the work in with a faster algorithm and a bigger database for validation . first , the features extraction processing time is reduced considerably by using multiscale analysis ( msa ) as alternative for hurst 's and dfa exponent estimation . indeed , the approach reported by lahmiri and boukadoum in determines the sought exponents by running polynomial regressions on multiple boxes or intervals of the 1d signal ; on the other hand , msa uses the generalized hurst 's exponent method , which determines the scaling properties of the signal by computing the qth - order moments of the distribution of the signal 's increments . this method combines the sensitivity to any type of signal dependence with a high computational efficiency due to its simple algorithm . as each scale q allows the estimation of a hurst 's exponent , varying this value allows obtaining the multifractals of the signal in a straightforward way . an additional contribution of this work is the use of a relatively larger database for validation in comparison to . finally , our approach does not require a predetermined region of interest as done in [ 1 , 5 , 8 , 10 ] and avoids extracting a large number of features to characterize the image in different modalities as done in [ 3 , 4 , 69 , 11 ] . instead , the multifractal analysis is performed on the whole brain mr image , and the extracted fractals are used to distinguish ad images from normal ones by the support vector machine . the svm is a pattern recognition technique based on the statistical learning theory that finds the optimal nonlinear hyperplane that minimizes the expected classification error . it was successfully applied in previous works [ 1 , 3 , 6 , 812 ] . section 2 presents the multiscale analysis used to obtain the generalized hurst 's exponents used to characterize brain mr images and the svm used for classification . in section 3 , we describe the obtained classification performance in terms of the accuracy rate , sensibility , and sensitivity . a collection of 93 axial , t2-weighted mr brain images of 256 256 size were downloaded from the harvard medical school webpage . the set included 51 images of normal ( healthy ) brains and 42 of abnormal ( unhealthy ) brains affected by ad . we notice that there is no indication in the database regarding the ad stage . figure 1 shows examples of healthy and ad images used in the experiments . consider a signal s(t ) t over a period t that is an integer multiple of v. the qth - order moments of the distribution that characterize the statistical evolution of s(t ) are defined as follows : ( 1)kq(d)=||s(t+d)s(t)||q|s(t)|q ,
where d [ v , dmax ] is a time interval and dmax is its predetermined upper limit . the generalized hurst 's exponent h(q ) is defined from the scaling behavior of kq(d ) according to the following empirical relation :
( 2)kq(d)(dv)qh(q ) . if kq(d ) and d satisfy a linear relationship for a given order q in log - log scale , hurst 's exponent h(q ) can be estimated by running a linear regression of log(kq(d ) ) versus log(d ) . the generalized hurst 's exponent h(q ) describes the long - memory dependence or persistence in the signal s(t ) . the multiscaling structure of signal s(t ) is related to different orders q of the exponent h(q ) . in general , when h(q ) > 0.5 , the signal fluctuations related to the order q are persistent . when h(q ) < 0.5 , the signal fluctuations related to order q are antipersistent . finally , the signal fluctuations are those of a random walk if h(q ) = 0.5 . notice that h(q = 2 ) corresponds to the classic hurst 's exponent . in this paper , higher moments could also have been considered as will be discussed in section 4 . the original mri is transformed into a one - dimensional ( 1d ) signal by row concatenation . hurst 's exponents h(q ) for q = 1 , , 6 are estimated by applying the msa algorithm . the resulting six - component feature vector forms the input of the svm classifier to perform the identification of ad images . introduced by vapnik , the support vector machine ( svm ) classifier is based on statistical learning theory . it implements the principle of structural risk minimization and has excellent generalization ability as a result , even when the data sample is small . the svm performs a classification tasks by constructing an optimal separating hyperplane that maximizes the margin between the two nearest data points belonging to two separate classes . given a training set { ( xi , yi ) , i = 1,2 , , m } , where the input xi r and class labels yi { + 1 , 1 } , the separation hyperplane for a linearly separable binary classification problem is given by
( 3)f(x)=wx+b ,
where w is a weight vector and b is a bias . the optimal separation hyperplane is found by solving the following optimization problem :
( 4)minimizew , b, 12ww+ci=1mi
subject to
( 5)yi(wxi+b)+i10 , i0 ,
where c is a penalty parameter that controls the tradeoff between the complexity of the decision function and the number of misclassified training examples and is a positive slack variable . the previous optimization model can be solved by introducing lagrange multipliers and using the karush - kuhn - tucker theorem of optimization to obtain the solution as
( 6)w=i=1miyixi . the xi values corresponding to positive lagrange multipliers i are called support vectors , and they define the decision boundary . once the optimal values of , * are found , the optimal hyperplane parameters w * and b * are determined . then , the discriminant function of the svm for a linearly separable binary classification problem is given by
( 7)g(x)=sign(i=1myiixix+b ) . in the nonlinearly separable case , the svm classifier nonlinearly maps the training points to a high dimensional feature space using a kernel function , where linear separation can be possible . the scalar product (xi ) (xj) is computed by mercer kernel function k as k(xi , xj ) = (xi ) (xj). then , the nonlinear svm classifier has the following form :
( 8)g(x)=sign(i=1myiikx , xi+b ) .
in this study , a polynomial kernel of degree 2 was used for the svm . as a global kernel , it allows data points that are far away from each other to also have an influence on the kernel values . the general polynomial kernel is given by
( 9)k(x , xi)=((xix)+1)d ,
where d is the order of the polynomial to be used . higher orders were ignored because of a higher computational burden with no substantial gain in classification accuracy from our experience . the validation experiments were conducted using the leave - one - out cross - validation method . then , the average and standard deviation of the correct classification rate ( ccr ) , sensitivity , and specificity were computed to evaluate the performance of the classifier . the three performance measures are defined as follows :
( 10)ccr = classified samplestotal number of samples , sensitivity = correctly classified positive samplestrue positive samples , specificity = correctly classified negative samplestrue negative samples ,
where positive samples and negative samples refer to ad and normal images , respectively . before processing , the grayscale images as shown in figure 1 are converted to double color format to perform msa ( see examples in figure 3 ) . figure 4 shows the obtained behavior of kq(d ) as a function of d ( see , ( 2 ) ) for the images of healthy patients and those with ad and for q varying from 1 to 6 , whereas d varies from 1 to 19 . figure 4 reveals that , for the images of normal brains , kq(d ) quickly reaches horizontal saturation for q = 1,2 , 3 ; this is not so for the ad images where kq(d ) increases monotonically for all d. in addition , the magnitude of kq(d ) for a healthy image is in general larger than that of an ad image . in summary , hurst 's exponents appear to be different at each scale for the two types of brain mris . as mentioned previously , the order of the polynomial kernel used in the svm was varied from 2 to 4 . the best classification performance was obtained with a fourth - order kernel , for which the correct classification rate , sensitivity , and specificity were 99.18% 0.0083 , 100% , and 98.20% 0.0182 , respectively . a visual analysis of the ad images revealed that seventeen of them looked markedly different from the example in figure 1 ( figure 6 shows three of them ) . removing them from the validation database led to 100% classification accuracy by the svm . finally , the msa running time was 5.64 seconds , and the overall image processing time was about 8 seconds on a 3.30 ghz core i5 - 2500 cpu using matlab 2012a codes , leading to an execution speed improvement of nearly two orders of magnitude in comparison to hurst 's exponent and dfa . this suggests that a multi - svm approach , where one or more classifier handles the misclassified images by the first svm , might lead to perfect classification accuracy in all situations . we intend to investigate this possibility . our approach for ad detection outperformed most of the studies found in the literature , where the reported classification accuracy was between 86% and 96.9% [ 15 , 713 ] . however , it is achieved with a database on only 45 mr images ( 25 healthy controls and 20 ad images ) , and the number of features used was higher than 1000 in comparison to the six used by our approach . we limited the number of features to six somewhat arbitrarily , and we have not investigated the effect of more or less features on the performance of the classifier . multiscale analysis - based hurst 's exponents were used for the classification of healthy brain images versus ad by a svm with fourth - order kernel . the obtained results show the potential of using multiscale fractal analysis to differentiate healthy brain images from ones affected by alzheimer 's disease . the msa algorithm took 5.64 seconds to analyze a brain mri while the detrended fluctuation analysis ( dfa ) took 400 seconds in our previous work . although we have obtained better result than the literature in general , it is difficult to draw definite conclusions since we used a different image database . in future work , we will explore a benchmark image depository such as the alzheimer 's disease neuroimaging initiative ( adni ) database . furthermore , we will investigate the effectiveness of msa to classify ad images versus mild cognitive impairment ( mci ) . indeed , the ability to correctly classify the ad and mci images based on msa hurst 's exponents might shed light on the ability to predict the conversion from mci to ad , which is of clinical interest . | we present a new automated system for the detection of brain magnetic resonance images ( mri ) affected by alzheimer 's disease ( ad ) .
the mri is analyzed by means of multiscale analysis ( msa ) to obtain its fractals at six different scales .
the extracted fractals are used as features to differentiate healthy brain mri from those of ad by a support vector machine ( svm ) classifier .
the result of classifying 93 brain mris consisting of 51 images of healthy brains and 42 of brains affected by ad , using leave - one - out cross - validation method , yielded 99.18% 0.01 classification accuracy , 100% sensitivity , and 98.20% 0.02 specificity .
these results and a processing time of 5.64 seconds indicate that the proposed approach may be an efficient diagnostic aid for radiologists in the screening for ad . |
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with recent improvements in optical components , the acquisition of polarized images has become easier and more cost effective . particularly , polarization imaging can reveal important optical properties of the imaged sample in addition to those revealed by a simple intensity imaging method . the fact that the polarization state of the light contains useful information has been shown in many literatures , for example , in [ 13 ] . rahmann and canterakis describe how the polarization state of light can be used for specular surface reconstruction to determine the shape of any three - dimensional ( 3d ) object . they use the fact that light reflected by dielectrics and metals becomes linearly polarized and that the direction of polarization depends on the orientation of the reflecting surface . demos and alfano demonstrate a technique based on polarization imaging that allows for optical imaging of a surface as well as structures beneath the surface . the interest of applying polarization imaging to study biological cells or tissues has been shared among many biomedical researchers from very early years to nowadays [ 415 ] . as early as in l949 , it was reported that the activity of nerve cells was associated with changes in their optical properties . when photons impinge on biological materials , their transmission depends on a combination of reflectance , scattering , and absorption effects . absorption occurs at specific wavelengths , determined by the molecular properties of the materials in the light path . the relatively good transparency of biological materials in the visible and near - infrared ( nir ) region of the spectrum permits sufficient photon transmission through organs in site for monitoring cellular events . it has been known for many years that some intrinsic changes in the optical properties of the tissue are dependent on electrical or metabolic activity [ 5 , 6 ] . changes in optical properties of brain cells have been reported in cell cultures , brain slices , as well as in intact cortical tissue . based on assessment of absorption and scattering , three types of activity - related signals have been recorded noninvasively : ( 1 ) changes in haemoglobin oxygenation , ( 2 ) changes in cytochrome - c - oxidase ( co ) , and ( 3 ) optical signals presumably related to changes in light scattering reflecting either membrane potential ( fast signals ) or cell swelling ( slow signal ) . villringer and chance claimed that the advantages of optical methods include biochemical specificity , a temporal resolution in the millisecond range , the potential of measuring intracellular and intravascular events simultaneously , and the portability of the devices enabling bedside examination . the light scattered by a tissue has interacted with the ultrastructure of the biological tissue , which imprinted some intrinsic properties of the tissue . tissue ultrastructure extends from membranes to membrane aggregates to collagen fibers to nuclei to cells . it has been demonstrated that light scattering can provide structural and functional information about the tissue [ 9 , 10 ] . one important biomedical application of optical imaging and spectroscopy is noninvasive or minimally invasive detection of precancerous and early cancerous changes in human epithelium , such as dysplasia or carcinoma in situ . recently , many researchers have proposed various optical sensing modalities that could potentially be used to aid in the diagnosis of superficial cancers and other dermatological conditions . in 2000 , jacques et al . demonstrated the use of polarized light for superficial tissue imaging . in their study , they showed that by simply collecting two polarization images through aligned and crossed polarizers and then computing the degree of linear polarization , image contrast can be significantly improved thus revealing superficial structures previously not apparent . in the methods proposed by other researchers , optical polarizers and retarders were varied to provide additional incident and analyzed polarization states , thus enabling the reconstruction of a two - dimensional ( 2d ) mueller matrix of biological samples [ 1215 ] . this paper presents a new approach to improved cell or tissue classification through the application of stokes imaging techniques and artificial neural networks [ 4 , 5 ] . measuring the polarization of backscattered light provides insight into the optical properties of the cell or tissue , which could lead to improved diagnosis of different tissue types . the polarization state of light can be represented by jones vectors or by stokes vectors . in particular , stokes vectors can represent fully polarized light as well as partially or unpolarized light , hence a natural choice for polarization image representation . in this paper , the intensity images , taken from the backscattered light of the samples , are first converted to stokes vector images . note that the stokes vector , one for each pixel location , fully describes the polarization state of the light at this particular spatial position . in addition , a broadband light source coupled with a tunable optical bandpass filter
allows for the illumination and collection of images at different optical wavelengths . artificial neural networks ( anns ) have been introduced to classify different cell / tissue types . in particular , multilayer perceptrons ( mlps ) are used to extract polarization signatures of the cells or tissues , through which a nonlinear decision boundary can be determined to classify the cells or tissues . to further improve the classification performance , we have also included a feature extraction step using wavelet transforms to derive a joint space and frequency representation of stokes images . in order to compare the classification performance of stokes imaging with conventional imaging technique , we have constructed three realistic phantoms using different sizes of polybeans to simulate the epidermal layer of skin . the classification performance , either using full stokes vector information or using intensity information only , is estimated by a cross - validation method ( i.e. , 3-fold
cross - validation ) to demonstrate an improved performance of the proposed polarization imaging system . , we will describe the design of polarization image device and its image analysis algorithms for classification . in section 3 , we will present a detailed report of preliminary experimental results using phantom studies and a plant cell study , especially on the results of using either single- or multispectral polarization information . finally , in section 4 we will conclude this paper with some future research directions . in this section , we will describe the principle , design , and algorithms of the proposed polarization system in detail . in the design , technically , a sequential acquisition procedure is proposed to acquire four intensity images with different polarization properties . four stokes images are then computed from the intensity images to give a full description of polarization states . in the data analysis subsystem , image analysis algorithms are developed for feature extraction and classification , in which wavelet transforms are used to extract polarization features and artificial neural networks are trained for binary classification . a detailed diagram of the polarization imaging device is shown in
figure 1 . as we can see from the figure , the device is composed of two aluminum cylindrical tubes for illuminator and detector , respectively . a 150-watt tungsten - halogen lamp is used as the light source providing a strong intensity over a broad spectrum ranging from ultraviolet ( uv ) ( 330 nm ) to near infrared ( nir ) ( 2 m ) . an optical fiber bundle guides the light to the illuminator consisting of a collimator , a 0-aligned polarizer and a filter . the tunable optical bandpass filter allows us to select a desired illumination wavelength for imaging , ranging from visible light to nir . the detector consists of an optical objective lens with an infinite focal length , followed by two optoceramic ( oc ) electro - optic phase retarders ( with their axes aligned at 45 and 22.5 , respectively ) , a 0-aligned linear polarizer , and a digital camera . two phase retarders are controlled independently by a computer through a two - channel driver , and a d / a module with usb interface . further , a three - dimensional ( 3d ) adjustable platform is used to hold phantoms and other testing samples . hence , the system is flexible enough to examine the sample with variable incident and collection angles . in order to acquire the polarization information , that is , the stokes images , four
intensity images ( i0i3 ) are taken sequentially , with two phase retarders ( i.e. , p1 and p2 ) controlled by a sequence of voltages as shown in figure 2 . specifically , i0 is taken first with 0 volt applied to both p1 and p2 ; i1 is taken second with a half - wave voltage v applied to p1 and 0 volt applied to p2 ; i2 is taken third with v is applied to both p1 and p2 ; finally , i3 is taken with a quarter - wave voltage v/2 applied to p1 and v applied to p2 . from the intensity images , that is , i0i3 , four stokes vector images s0s3 can be calculated by the following equations : figure 3 shows the diagram of the polarization image analysis subsystem . wavelet transforms ( wts ) are applied to stokes images to extract polarization features ; and artificial neural networks ( anns ) are then trained to classify the patterns based on the extracted features . below we will describe the principle of wts and anns briefly , their application to polarization image analysis , and their performance evaluation based on cross - validation . wavelet transforms are introduced to extract polarization features by providing the space and frequency information simultaneously , resulting in a space - frequency representation of the signal . the definition of a continuous wavelet transform for any 1-d signal f(x ) can be described as
( 2)w(a , b)=1af(x)*(xba)dx ,
where z denotes the complex conjugate of z , (x ) the analyzing wavelet , a the scale parameter , and b the position parameter . the wavelet function (x ) can be chosen as simple as the harr function or one of the popular daubechies functions . figure 4 shows the waveform of daubechies d4 function . by scaling and shifting the wavelet function (x ) , we can construct a family of analyzing functions a , b(x)=(xb / a ) to obtain a space - frequency representation of the original signal . in this project , we apply wavelet transforms to polarization images to extract detailed features or signatures for classification of different polarization properties . using the haar(or daubechies d4 ) function as a transform basis , an image can be decomposed into four separate bands ( denoted as ll1 , lh1 , hl1 , and hh1 ; see figure 5 ) . the ll1 band contains a scaled - down , low - resolution version of the original image and the remaining three bands ( lh1 , hl1 , and hh1 ) contain the detail information ( i.e. , horizontal , vertical , and diagonal orientation features ) about the original image . the process of the wavelet transform can be repeated by transforming the ll1 band into a
second - level representation : four subbands denoted as ll2,lh2 , hl2 , and hh2 . this repeating process , as illustrated in figure 5 , is also called pyramid decomposition . after the feature extraction step , we use multilayer perceptrons ( mlps ) , a type of nonlinear anns , to perform binary classification ( see figure 3 ) . mlps have been successfully applied to solve a variety of nonlinear classification problems . in our experiments , the so - called hidden nodes ( neurons ) in the middle layer can further extract diagnostic features from the input patterns for nonlinear classification . the connectivity weights are trained or learned in a supervised manner using the error back - propagation algorithm . one of the drawbacks of neural networks is that they do not perform well when the number of inputs is too large ( the so - called curse - of - dimensionality phenomenon ) . for this reason only the 16 lower band coefficients of the transformed blocks ( in a size of 3232 ) are selected as inputs to the mlp . since the neural network receives input values from four component images ( 16 input values from each component ) , the total number of inputs to the neural network is 64 . the neural network is trained with a standard steepest decent backpropagation algorithm , where its weights are initialized with small randomly selected values . the transfer function for both hidden layer and output layer is the sigmoid function f(x)=1/(1ex ) . the target values for two classes are 0 for the first class and 1 for the second class , respectively . to estimate the generalizable performance of our classification scheme ,
cross - validation is used to calculate classification error rates ( cers ) of the mlp . the input blocks are randomly divided into two sets , one set is used for training and the other is used for cross - validation . this random division of blocks into two sets was repeated 10 times and the neural networks are retrained and tested . the mean classification error rate ( cer ) and its standard deviation are then computed to evaluate the classification performance . in practice , we performed either l
leave - one - out ( loo ) test ( i.e. , holding out one block for testing ) or 3-fold
cross - validation ( i.e. , holding out 1/3 of the blocks for testing ) to estimate the classification error rates . to compare the performance of polarization imaging to that of unpolarization imaging , we computed the improvement of polarization imaging over unpolarization imaging using the following formula :
( 3)improvement = cerunpolarizedcerpolarizedcerunpolarized100% ,
where cerpolarized and cerunpolarized are the estimated cers of polarization imaging and unpolarization imaging , respectively . in this section , we will report our preliminary results of using polarization imaging and artificial neural networks for improved diagnostics . first , the polarization imaging device has been developed using two electrically tunable retarders for acquiring polarization images . second , different types of phantoms were built to simulate the epidermal layer of the skins for testing the performance of the proposed system . third , image analysis algorithms have been developed to extract polarization features and classify the phantoms and plant cells . the performance of classification accuracy was evaluated by cross - validation , and the improvement of performance was demonstrated by comparing the performance of the system using polarization information over that without using polarization information . figure 6 shows a photograph of the prototyped imaging device , showing an illuminator tube , a detector tube , and an adjustable platform for holding testing samples . the key component , electrically tunable retarder , which supports high - speed operation of the stokes polarization imaging system , is based on bati s newly breakthrough electro - optical ceramic material featuring high electro - optic effect , high operation speed , ruggedness , and ease of fabrication . as described in section 2 , a sequential image acquisition scheme has been implemented to acquire four intensity images with different polarization properties . from our experience , we learn that the accuracy of stokes polarization imaging is mainly determined by the accuracy of retardation on the phase retarders . therefore , a careful phase retarder characterization and calibration procedure is developed to minimize the errors between measured and desired phases . in our experiments , the error is less than 0.035 rad , which meets the requirement of the proposed system . three phantom samples , phantom-42 , phanom-74 , and phantom-99 , were prepared using polystyrene latex spheres with mean diameters of 42 m , 74 m , and 99 m , respectively . for all 42 m , 74 m , and 99 m spheres , distilled water was added to adjust the reduced scattering coefficient ( denoted as s ) to match the scattering property of the skin . india ink was also added to latex phantoms to make the absorption coefficient ( denoted as a ) to match that of the epidermis . the optical properties of the polystyrene phantoms were set at s = 2.0/mm and a = 2.46/mm . an intralipid solid phantom was then used to simulate the dermal layer of the skin . the intralipid solid phantom was made from agar ( a stiffening agent ) , distilled water , india ink , and 20% intralipid . the optical properties of the phantom were adjusted to the following numbers : s = 2.0/mm and a = 0.03/mm . the optical properties of the polystyrene phantom and intralipid solid phantom approximated the scattering and absorption of the epidermal and dermal layer of the skin in the range of 550 nm950 nm . for each sample , a diameter of 3 cm cup of intralipid solid phantom was placed below the incident light . to simulate a thin skin layer , a small volume of polystyrene phantom was placed onto the center of the solid phantom . this drop spread out in a uniform circle with a diameter that could be easily measured using a vernier caliper . with the original volume and area covered by the spheres , we can calculate the polystyrene thickness approximately . after waiting a few seconds for the drop area to become stable , , the thickness was controlled between 50 m and 170 m , which is comparable to the thickness of an epidermal layer of the skin ( varying from 70 m to 150 m for a thin skin ) . with phantom-42 , phantom-74 , and phantom-99 , different illumination wavelengths were chosen from a range of visible to near ir . in our experiments , we selected 550 nm , 650 nm , and 950 nm for this study . figure 7 shows examples of the four intensity images ( i0i3)of phantom-42 and phantom-99 collected by the imaging device . these two sets of images were acquired with an incident angle of 22.5 and a collection angle of 45.0. the wavelength of the incident light was 550 nm . figure 8 shows the stokes vector images of phantom-42 and phantom-99 , respectively , obtained from the intensity images by applying ( 1 ) . the s0 component image reflects the overall intensity ( polarized and unpolarized components combined ) . the component images s1 , s2 , and s3 contain intensity differences as defined in ( 1 ) and can contain positive as well as negative values . with the polarization images acquired using three phantoms ( phantom-42 , phanom-74 , and phantom-99 ) , we have conducted a series of experiments to study characteristics of the polarization system , for example , the incident / collection angle , image quality , and its impact on classification of phantoms . in this section , we will report the classification results on phantom-42 , phantom-74 , and phantom-99 , using either single wavelength or multiple wavelengths of polarization information . the stokes images were first processed by multiscale wavelet transforms to extract the discriminatory features for classification . the features were then fed to train a three - layer mlp to discriminate two phantoms . to estimate the generalizable classification performance , we used 3-fold cross - validation to compute the mean and standard deviation of the classification error rate . the performance of using polarization information ( i.e. , using s0s3 ) has been compared to that without using polarization information ( i.e. , using i0 only ) . from these results we have observed that a significant improvement can be gained by using the polarization information . table 1 summarizes the classification performance of various phantom studies with different phantoms ( phantom-42 , phantom-74 , and phantom-99 ) and different wavelengths ( 550 nm , 650 nm , and 950 nm ) . as can be seen , polarization imaging with stokes information has gained a significant improvement over unpolarization imaging . the improvements on reduction of classification error rate are ranging from 38.27% to 96.82% , resulting in an average improvement of 51.54% . note that for each case in table 1 , we use the best performances of unpolarized imaging and polarized imaging , respectively , for comparison . hence , the number of hidden neurons does vary in each case ; that is , the ann classifier is optimized for each case . however , since the performance is not very sensitive to the number of hidden neurons ( see tables 2 and 3 later ) , we believe that the comparison is reasonable and acceptable for this study . the results presented in this section show the classification performance on polarized phantom images taken at incident wavelengths of 650 nm , 850 nm , and 950 nm , respectively . the network was trained and tested only on images acquired with the same wavelength . in each wavelength category , all phantoms were illuminated at an incident angle of 22.5. the collection angles used were 22.5 , 45.0 , and 67.5. in order to use 3-fold cross - validation for estimating the network s classification performance , every image was subdivided into 64 by 64 pixel wide blocks . these blocks were randomly divided into a training set ( 2/3 of the total number of blocks ) and a test set ( 1/3 of the total number of blocks ) . from each of these blocks a total number of 81 overlapping windows , 32 by 32 pixels wide , were extracted as inputs to the classification system . the process of dividing the blocks into training and test sets was repeated 10 times to train and test the network . the resulting classification errors were used to calculate the mean classification error and its standard deviation . tables 2 - 3 show the results for the wavelengths of 650 nm and 950 nm , respectively . the classification error rates in the case of the images acquired at 650 nm show an improvement of the classification performance from 18% using intensity only to 11% using stokes vector images . the error rates in both cases are not dependent on the number of hidden neurons . while the error rate for the training set decreases when more hidden neurons are added ( table 2 ) , the testing error remains constant over a wide range . the standard deviation of the classification error rate is about 2.5% , which is relatively low . in the second case , the incident light wavelength of 950 nm , the classification performance of the stokes vector images is significantly better than that obtained by using intensity images only . as shown in table 3 , the classification error rate is about 25% when using the intensity information only . when the polarization information is used in addition to the intensity information , the classification error rate decreases to about 16% . table 3 also shows that the classification error rate is not dependent of the number of hidden neurons either . the following results show the classification performance on the two phantoms ( i.e. , phantom-42 and phantom-74 ) using multispectral polarization information . while the results in section 3.3.1 show the classification performance of the system when trained and tested at a single wavelength , the results in this section show the results when images taken at different wavelengths are combined to train the neural network for classification . specifically , the images used in this section were taken at the wavelengths of 550 nm , 650 nm , and 950 nm , respectively . as described in the previous section , 3-fold cross - validation was used to estimate the mlp s classification performance . these blocks were randomly divided into a training set and a test set . from each of these blocks a total number of 81 overlapping windows , 32 by 32 pixels wide , were extracted as inputs to the classification system . the process of dividing the blocks into training and validation set was repeated 10 times . the network was retrained and the classification performance was estimated using the test set . the mean classification error ( from cross - validation ) and its standard deviation are shown in table 4 where the number of hidden neurons varies from 1 to 10 . as we can see , the classification error rate decreases when additional hidden neurons are added to the neural network . this is the case for using the intensity image ( i0 ) only as well as for using four stokes images ( s0s3 ) . the network performs significantly better when trained on the stokes images ( 7.2% error rate with s0s3 ) than that using intensity image only ( 24.7% error rate with i0 only ) . it can also be clearly seen from table 4 that an increase of the number of hidden neurons improves the classification performance . from this limited preliminary study , it seems that the classification problem appears to be more complex when images taken at different wavelengths are combined , and the ann classifier with more hidden neurons might help extract polarization information for better classification . in this experiment , we collected two kinds of leaves to test our polarization imaging scheme . alcohol was used to clean the leave surfaces to get rid of the dusts and other containments . it is known that the plant cells can be viewed or sensed directly by collecting the transmission light . the transmission stokes imaging system shown in figure 9 was used for the experiment . the setup shown in figure 1 the working principle of the system is same , as that described in section 2.1 , except that the signal would be collected after the light passing through the sample . for those relatively thin and transparent samples , measurement with a transmission mode would be better than with reflective mode in achieving higher signal to noise ratio . as shown in figure 10 , the i0i3 images of leaf a and leaf b were acquired using the transmission mode as described above . figure 11 shows the stokes images ( s0s3 ) of the acquired polarization information , which clearly de - correlate the dependency between i0i3 . further processed by wavelet
transform and ann training , the discriminatory features from polarization imaging have been fully captured in hidden neurons for classification . the classification performance is detailed in table 5 , where the improvement over unpolarization imaging is also calculated . the classification error rate ( cer ) has been significantly reduced from 34.80% for unpolarization imaging to 11.3% for polarization imaging , resulting in an improvement of 64.04% in average . in this paper , we have presented a polarization imaging device to acquire a complete set of stokes vector images for improved diagnostics . an image analysis subsystem has also been developed to classify different types of phantoms based on the 2d discrete wavelet transforms ( 2d - dwts ) and multilayer perceptrons ( mlps ) . when trained and tested with the complete set of stokes images ( i.e. , using s0s3 ) , the classification performance is significantly improved compared to that with intensity image only ( i.e. , using i0 only ) . the results have confirmed that the polarization state contains important information that can be used to classify two different types of phantoms . while the results are encouraging and this study shows the potential of this imaging device , further study is needed . future work may include more realistic phantom studies and biological cell and tissue studies for validation . currently , polarization imaging of cell study has been under investigation in collaboration with ( georgetown university medical center dc , usa ) . some optimization procedure for anns ( such as the optimized mlp in ) will be explored to avoid the local minima problem existed in nonlinear classification problem . as also shown in our preliminary result in section 3 , the use of multispectral polarized images is another possible path to improve the classification performance . note that although we are encouraged by the initial results using multispectral polarization imaging , we have also realized that the problem seems to be complicated by many other factors , like the proper wavelength and illumination angle selection . to tackle this problem , we believe that some optimization approach needs to be developed in future to replace the current simple combining approach . | in recent years , there has been an increasing interest in studying the propagation of polarized light in biological cells and tissues .
this paper presents a novel approach to cell or tissue imaging using a full stokes imaging system with advanced polarization image analysis algorithms for improved diagnostics .
the key component of the stokes imaging system is the electrically tunable retarder , enabling high - speed operation of the system to acquire four intensity images sequentially . from the acquired intensity images ,
four stokes vector images can be computed to obtain complete polarization information .
polarization image analysis algorithms are then developed to analyze stokes polarization images for cell or tissue classification .
specifically , wavelet transforms are first applied to the stokes components for initial feature analysis and extraction .
artificial neural networks ( anns ) are then used to extract diagnostic features for improved classification and prediction . in this study ,
phantom experiments have been conducted using a prototyped stokes polarization imaging device . in particular
, several types of phantoms , consisting of polystyrene latex spheres in various diameters , were prepared to simulate different conditions of epidermal layer of skin .
the experimental results from phantom studies and a plant cell study show that the classification performance using stokes images is significantly improved over that using the intensity image only . |
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this is an observational study , of consecutive patients with biomicroscopic and fluorescein angiography appearance of maculopathy combined with optic pit . we obtained institutional review board approval and the study was conducted in accordance with the declaration of helsinki . all patients were examined with sd - oct ( spectralis , heidelberg engineering , germany or soct copernicus hr , optopol , zawiercie , poland ) . in copernicus hr , we performed 100 consecutive b - scans on a 7 7 mm area . the spectralis device makes 19 b - scans on an area of 4.5 6 mm . we always took two scans , one presenting the macula and the other presenting the optic disc . in sd - oct we recorded any changes in sd - oct appearance and/or visual acuity that occurred during the observation period . 19 patients ( 20 eyes ) with maculopathy associated with optic disc pit were included into the study . group 1 consisted of eyes with intraretinal fluid in outer retinal layers ( three eyes ) , group 2 ; eyes with intraretinal fluid in outer retina layers and subretinal fluid ( three eyes ) , group 3 ; eyes with intraretinal fluid in outer and inner retinal layers ( five eyes ) , group 4 ; eyes with an outer lamellar macular hole coexisting with fluid in inner and outer retinal layers ( three eyes ) , and group 5 ; eyes with elevation of all retinal layers in the macula ( six eyes ) [ fig . 1 ] . in five eyes the sd - oct appearance of one eye from group 1 ( fluid in outer retinal layers ) fluid migrated to the inner retinal layers and to the subretinal space [ fig . 2a - c ] and in four eyes from group 3 the sd - oct macular appearance changed to group 4 [ fig . ( a ) fluid in outer retinal layers , ( b ) fluid in outer retinal layers and subretinal space , ( c ) fluid in outer and inner retinal layers , ( d ) outer lamellar macular hole and fluid accumulated in inner and outer retina . on the left side infrared image of the fundus is presented , ( e ) subretinal fluid without intraretinal fluid accumulation . on the left side infrared image of the fundus ( a - c ) maculopathy associated with optic pit in one patient visualized with spectral oct . on scan a elevation of inner retinal layers , retino schisis and stria between inner and outer retinal layers the stria between inner and outer retinal layers tend to shorten and outer retinal layers are detached from retinal pigment epithelium and pulled towards already detached inner retinal layers . visual acuity improvement to 0.8 was noted at the time the scan was taken . on scan c , further movement of the outer retinal layers towards the inner retinal layers can be seen . ( d and e ) maculopathy associated with an optic pit in another patient . on scan d , scan e : four months later an outer lamellar macular hole developed 3-dimensional sd - oct ( 3d - sdoct ) imaging of the optic nerve head revealed the presence of a membrane in the bottom of the optic disc in 14out of 20 cases . in only one case was the membrane complete , with no defects in all 3-d scans of that eye . in the remaining 13 eyes , although the 3-d imaging mode showed the membrane to be complete in some scans , in others it did not [ fig . the arrow on b indicates a hypererreflective area , possibly a part of the membrane visible on a. on the left side above the sd - oct images infrared image of the fundus is presented in sd - octposterior hyaloid was visible in four cases with partial attachment to the optic nerve . a communication between the perineural and subretinal and/or intraretinal space at the margin of the optic disc existed in 17 out of 20 cases [ fig . a communication between the perineural and : a , subretinalspace in a patient with maculopathy combined with optic pit . b , intraretinal space in a patient with maculopathy combined with optic pit . on the bottom right side infrared image of the fundus is presented . arrows indicate the suspected flow of fluid sd - oct revealed several abnormalities of the optic disc , which have previously only been described histopathologically . first , the presence of hyporeflective spaces below the optic disc was observed in nine cases [ fig . whether these spaces represent an additional allocation of the perineural fluid , which did not pass to the intraretinal and/or subretinal space , or that they represent also fluid accumulated below the elschling membrane is open to discussion . secondly , in eight cases hyperreflective tissue was observed at the bottom of the optic nerve [ fig . the arrow indicates a hyporeflective area , possibly representing fluid accumulated below the optic nerve head optic disc appearance in a patient with optic pit associated maculopathy . this paper describes the morphology of the optic nerve and macula in optic pits visualized with 3d - sdoct . as maculopathy associated with optic pit is a rare event , many factors remain unknown . the origin of the subretinal and intraretinal fluid is a primaryinterest . avitreous origin was confirmed by histopathologic studies showing that alcian blue staining revealed mucopolysacharydes within the pit . further endorsement of the argument for a vitreous origin of the fluid is offered by the good results following pars plana vitrectomy ( ppv ) , which enables the relieving of tractions . at the 2007 american academy of ophthalmology ( aao ) optic nerve pit bubbles , a movie showing gas bubbles coming out from the optic nerve in optic pit maculopathy previously treated with ppv with gas . cerebrospinal origin of the fluid that enters the subretinal and intraretinal space was also confirmed . chang observed that contrast dye can pass from subarachnoid space to subretinal fluid in humans with morning glory syndrome , which is generally considered as another manifestation of optic pit . described a case of mri - documented intracranial silicone oil migration in an eye with optic pit . our sd - oct images confirm that intraretinal fluid may be both , cerebrospinal and vitreous in origin . we think that there may be a three - fold connection between the vitreous , perineural space and subretinal and intraretinal space . it may be that in individual cases , the paths of fluid differ in size and trajectory . vitrectomy , while relieving tractions may prevent the migration of vitreous fluid through the optic nerve into the subretinalor intraretinal space . laser burns create a scar between the outer retinal tissue and retinal pigment epithelium and this may prevent migration of fluid into the outer retinal space , but does not influence fluid migration into inner retinal layers . as multiple possible fluid pathways exist , vitrectomy and/or laser coagulation may be successful in some cases , and yet may fail in others . thus , an ideal procedure would combine the relieving of traction and prevention of fluid migration into the subretinal as well as into inner and outer retinal layers . we observed that fluid may be observed in the outer retinal layers , both in the outer retinal layers and subretinal , in outer and inner retinal layers , in a form of an outer lamellar macular hole or only subretinal [ fig . 1 ] . we additionally presented evolution of the fluid distribution with time [ fig . 3 ] . the second much discussed issue is whether maculopathy is a primary process , or is associated with posterior hyaloid detachment . some authors believe that macular detachment occurs only secondary to posterior hyaloid detachment , consisting either of detachment of the outer retinal layers from the retinal pigment epithelium without communication to the optic disc , or macular schisis with accumulation of fluid in the outer plexiform layer . other authors state that the posterior hyaloid remains attached in the majority of eyes , so macular detachment may be regarded as a primary process coexisting with the optic pit . we confirmed in this sd - oct study that partial vitreous detachment was observed only in 4/20 eyes . the role of sd - oct in the detection of posterior hyaloid detachment may be controversial . also , our observations during vitrectomy for optic pit associated maculopathy show that one of most difficult maneuvers was the induction of posterior hyaloid detachment ( unpublished data ) . a confirmation of the above thesis may be the fact , that we observed evolution of maculopathy in five cases without any signs of posterior hyaloid detachment . additionally , after vitrectomy , when the posterior hyaloid is already removed , the macula was reportedto reattach in several cases , which shows that vitreoretinal tractions are of minor importance in subretinal fluid accumulation . in view of these findings , we assume that macular detachment is primary to vitreous detachment . we observed a connection between the perineural and sub-/or intraretinal space in 17 out of 20 cases [ fig . earlier authors did observe such a connection , but not with such a high frequency , probably because of worse resolution of the oct devices used in earlier studies . in most cases in our group we observed a membrane in the bottom of the optic pit . in histopathologic studies a structure called the inner limiting membrane of elschnig ( a continuation of internal limiting membrane over the optic disc ) was observed on top of the optic disc in optic pit maculopathy . this membrane was described to consist of rudimentary retinal tissue including aberrant nerve fibers and pigmented tissue resembling retinal pigment epithelium . in an oct based study , doyle and coworkers identified a complete membrane traversing the optic disc cup in three of five eyes without maculopathy . the authors suggest that the membrane , consisting of neuroectodermal and astroglial tissue , may represent a barrier to the passage of fluid into or under the retina . as the membrane was absent or deficient in three eyes with maculopathy , they also suggested that the membrane may protect against the development of maculopathy . when we analyzed 3d - sdoct images , we noticed that even if the membrane seems intact in some b - scans , it is not complete in others several micrometers apart . this might not have been noticed by doyle and coworkers as they did not perform 3-dimensional reconstruction . in eight patients in our study we observed hyperreflective tissue in the excavation of the optic disc [ fig . this sd - oct finding may be either condensed vitreous or glial tissue as explained by histopathological studies . akiba observed a condensed vitreous strand ( cloquet 's canal ) that extended from the surface of the pit into the vitreous gel . cloquet 's canal pulsates with eye movements forming multiple microforamina in the membrane covering the optic pit . the liquefied vitreous is believed to enter the subretinal space through the optic pit . also , liquefied vitreous at the top of the optic pit some authors hypothetized that this glial tissue may contribute to fovea detachment . in our study , sd - oct revealed the presence of previously unreported hyporeflective areas at the bottom of the optic disc in nine cases [ fig . 5 ] . we suspect that these spaces represent an additional allocation of the perineural fluid , which did not pass to the intraretinal or subretinal space , although it is also possible that they represent fluid accumulated below the elschlnig 's membrane . in conclusion , 3d - sdoct scans revealed a three - fold connection , between subretinal and intraretinal space , perineural space , and the vitreous cavity . therefore , we suppose that intraretinal or / subretinal fluid in optic pit maculopathy may have both a vitreous and cerebrospinal origin . it may enter the subretinal space at the optic disc cup near to the vessels outcome . several other abnormalities of the optic disc are visible with sd - oct , such as a membrane overlying the bottom of the optic disc ( elschlnig 's membrane ) which does not seem to have a role in visual prognosis . additionally , we observed fluid accumulation below the margin of the optic disc and hyperreflective porous tissue in the optic disc excavation . avitreous origin was confirmed by histopathologic studies showing that alcian blue staining revealed mucopolysacharydes within the pit . further endorsement of the argument for a vitreous origin of the fluid is offered by the good results following pars plana vitrectomy ( ppv ) , which enables the relieving of tractions . at the 2007 american academy of ophthalmology ( aao ) optic nerve pit bubbles , a movie showing gas bubbles coming out from the optic nerve in optic pit maculopathy previously treated with ppv with gas . cerebrospinal origin of the fluid that enters the subretinal and intraretinal space was also confirmed . chang observed that contrast dye can pass from subarachnoid space to subretinal fluid in humans with morning glory syndrome , which is generally considered as another manifestation of optic pit . described a case of mri - documented intracranial silicone oil migration in an eye with optic pit . our sd - oct images confirm that intraretinal fluid may be both , cerebrospinal and vitreous in origin . we think that there may be a three - fold connection between the vitreous , perineural space and subretinal and intraretinal space . it may be that in individual cases , the paths of fluid differ in size and trajectory . vitrectomy , while relieving tractions may prevent the migration of vitreous fluid through the optic nerve into the subretinalor intraretinal space . laser burns create a scar between the outer retinal tissue and retinal pigment epithelium and this may prevent migration of fluid into the outer retinal space , but does not influence fluid migration into inner retinal layers . as multiple possible fluid pathways exist , vitrectomy and/or laser coagulation may be successful in some cases , and yet may fail in others . thus , an ideal procedure would combine the relieving of traction and prevention of fluid migration into the subretinal as well as into inner and outer retinal layers . we observed that fluid may be observed in the outer retinal layers , both in the outer retinal layers and subretinal , in outer and inner retinal layers , in a form of an outer lamellar macular hole or only subretinal [ fig . 1 ] . we additionally presented evolution of the fluid distribution with time [ fig . 3 ] . the second much discussed issue is whether maculopathy is a primary process , or is associated with posterior hyaloid detachment . some authors believe that macular detachment occurs only secondary to posterior hyaloid detachment , consisting either of detachment of the outer retinal layers from the retinal pigment epithelium without communication to the optic disc , or macular schisis with accumulation of fluid in the outer plexiform layer . other authors state that the posterior hyaloid remains attached in the majority of eyes , so macular detachment may be regarded as a primary process coexisting with the optic pit . we confirmed in this sd - oct study that partial vitreous detachment was observed only in 4/20 eyes . the role of sd - oct in the detection of posterior hyaloid detachment may be controversial . also , our observations during vitrectomy for optic pit associated maculopathy show that one of most difficult maneuvers was the induction of posterior hyaloid detachment ( unpublished data ) . a confirmation of the above thesis may be the fact , that we observed evolution of maculopathy in five cases without any signs of posterior hyaloid detachment . additionally , after vitrectomy , when the posterior hyaloid is already removed , the macula was reportedto reattach in several cases , which shows that vitreoretinal tractions are of minor importance in subretinal fluid accumulation . in view of these findings , we assume that macular detachment is primary to vitreous detachment . we observed a connection between the perineural and sub-/or intraretinal space in 17 out of 20 cases [ fig . earlier authors did observe such a connection , but not with such a high frequency , probably because of worse resolution of the oct devices used in earlier studies . in most cases in our group we observed a membrane in the bottom of the optic pit . in histopathologic studies a structure called the inner limiting membrane of elschnig ( a continuation of internal limiting membrane over the optic disc ) was observed on top of the optic disc in optic pit maculopathy . this membrane was described to consist of rudimentary retinal tissue including aberrant nerve fibers and pigmented tissue resembling retinal pigment epithelium . in an oct based study , doyle and coworkers identified a complete membrane traversing the optic disc cup in three of five eyes without maculopathy . the authors suggest that the membrane , consisting of neuroectodermal and astroglial tissue , may represent a barrier to the passage of fluid into or under the retina . as the membrane was absent or deficient in three eyes with maculopathy when we analyzed 3d - sdoct images , we noticed that even if the membrane seems intact in some b - scans , it is not complete in others several micrometers apart . this might not have been noticed by doyle and coworkers as they did not perform 3-dimensional reconstruction . in eight patients in our study we observed hyperreflective tissue in the excavation of the optic disc [ fig . 5 ] . this sd - oct finding may be either condensed vitreous or glial tissue as explained by histopathological studies . akiba observed a condensed vitreous strand ( cloquet 's canal ) that extended from the surface of the pit into the vitreous gel . cloquet 's canal pulsates with eye movements forming multiple microforamina in the membrane covering the optic pit . the liquefied vitreous is believed to enter the subretinal space through the optic pit . also , liquefied vitreous at the top of the optic pit some authors hypothetized that this glial tissue may contribute to fovea detachment . in our study , sd - oct revealed the presence of previously unreported hyporeflective areas at the bottom of the optic disc in nine cases [ fig . 5 ] . we suspect that these spaces represent an additional allocation of the perineural fluid , which did not pass to the intraretinal or subretinal space , although it is also possible that they represent fluid accumulated below the elschlnig 's membrane . in conclusion , 3d - sdoct scans revealed a three - fold connection , between subretinal and intraretinal space , perineural space , and the vitreous cavity . therefore , we suppose that intraretinal or / subretinal fluid in optic pit maculopathy may have both a vitreous and cerebrospinal origin . it may enter the subretinal space at the optic disc cup near to the vessels outcome . several other abnormalities of the optic disc are visible with sd - oct , such as a membrane overlying the bottom of the optic disc ( elschlnig 's membrane ) which does not seem to have a role in visual prognosis . additionally , we observed fluid accumulation below the margin of the optic disc and hyperreflective porous tissue in the optic disc excavation . | purpose : our purpose was to study the clinical manifestation and course of optic pit maculopathy using spectral domain optical coherence tomography ( sd- oct ) images.materials and methods : we used sd - oct to examine 20 eyes of 19 patients with a macular detachment in combination with an optic.results:we observed five different fovea appearances in regard to fluid localization . in five eyes , we recorded changes in the fluid distribution with sd - oct . in 17/20 eyes , we noted a communication between the perineural and subretinal and/or intraretinal space at the margin of the optic disc.conclusion:3-dimensional sd - oct ( 3d - sdoct ) scans revealed a three - fold connection , between subretinal and intraretinal space , perineural space , and the vitreous cavity .
therefore , we suppose that intraretinal or subretinal fluid in optic pit maculopathy may have both a vitreous and cerebrospinal origin . a membrane , covering the optic nerve was noted in 14 cases . even if it seems intact in some b - scans , it is not complete in others several micrometers apart . additionally , we observed fluid accumulation below the margin of the optic disc and hyperreflective porous tissue in the optic disc excavation .
those findings do not influence the course of maculopathy . |
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insulin therapy is recognized to possess the most striking and definite effect on glucose lowering in diabetic patients with the inevitable adverse effects such as hypoglycemia and weight gain . in this aspect , many previous studies have reported the efficacy and safety of combination therapy of oral hypoglycemic agents ( ohas ) and insulin , indicating advantages such as the reduction of the insulin dose , the prevention of weight gain or hypoglycemia , and the improvement of glycemic control . in most previous studies , however , insulin was added to ongoing ohas , and effects of add - on therapy of ohas to ongoing insulin therapy have seldom been discussed so far . sitagliptin is a selective dpp-4 inhibitor and it was approved for the treatment of type 2 diabetes mellitus ( t2 dm ) at the end of 2009 in japan . many reports have demonstrated the efficacy and safety of sitagliptin as monotherapy or in combination with other ohas , in which sitagliptin was generally well - tolerated and brought about a significant improvement of blood glucose control [ 2 , 3 ] . the efficacy and safety of sitagliptin when added to ongoing insulin therapy have recently been reported in countries other than japan [ 46 ] . since the combination therapy of sitagliptin with insulin was also approved in 2011 in japan , this add - on therapy has enthusiastically been discussed and recently some reports of japanese patients have been published . in most studies , a definite glucose - lowering effect without any severe adverse events ( saes ) has been demonstrated in either prospective or retrospective manner [ 79 ] . however , one report is limited to insulin monotherapy ( premixed or long acting insulin ) in spite of its prospective manner , and the other one is limited to combination therapy with only basal insulin . furthermore , effects of add - on therapy of sitagliptin on glucose fluctuation have not been discussed in a large number of patients even though it was evaluated in a small number of patients by calculating data based on self - monitoring of blood glucose ( smbg ) or continuous glucose monitoring ( cgm ) [ 7 , 10 ] . the aim of the present study is to evaluate the effect of add - on of sitagliptin on blood glucose control or glucose fluctuation as well as the tolerability of this combination therapy in more than 200 japanese type 2 diabetic patients treated with insulin and/or other oha in real clinical practice . two hundred forty - one type 2 diabetic outpatients who had been treated with insulin with or without other ohas and prescribed sitagliptin since december in 2009 were recruited in kurume university hospital and other satellite hospitals or clinics as candidates in the present study . the diagnosis of diabetes was confirmed according to the criteria of the japan diabetes society or by medical history of diabetes . patients with severe liver dysfunction ( ast > 100 u / l , alt > 100 u / l , or -gtp > 100 u / l , n = 8) , renal dysfunction ( serum creatinine concentration > 133 mol / l , n = 9 ) , known malignant disease ( n = 3 ) , or chronic inflammatory disease ( e.g. , rheumatoid arthritis , n = 1 ) were excluded from the study . the remaining 216 patients ( 126 men , 65 12 years old , bmi 24.9 4.5 , means s.d . ) finally participated in the present study . the study was performed in accordance with the declaration of helsinki and approved by the ethics committee of kurume university hospital . one hundred and eighteen patients had been treated with ohas in combination with insulin at the initiation of sitagliptin , including sulfonylurea ( su ) in 22 , biguanide ( bg ) in 93 , -glucosidase inhibitor ( -gi ) in 22 , and thiazolidinedione in 5 patients . a hundred and ninety patients were initiated by sitagliptin at the dosage of 50 mg , and remaining 26 patients were at 25 mg . types of insulin therapy were classified to three categories as follows:(1)twice or thrice daily injections of premixed insulin : n = 95;(2)multiple daily injections composed of long acting insulin once or twice and rapid acting insulin at each meal time ( mdi ) : n = 74;(3)once daily injection of basal insulin : n = 47 . twice or thrice daily injections of premixed insulin : n = 95 ; multiple daily injections composed of long acting insulin once or twice and rapid acting insulin at each meal time ( mdi ) : n = 74 ; once daily injection of basal insulin : n = 47 . a change of hba1c level from baseline value 1 and 3 months after the add - on of sitagliptin is a primary outcome variable for the assessment of efficacy and is evaluated in relation to predictive variables such as initial hba1c level , change in body weight , endogenous insulin reserve expressed by serum c - peptide level ( cpr ) , glucose fluctuation , and types of medications including ohas and insulin therapy . changes in body weight and insulin dose for 3 months are the other endpoints as well . at the initiation of s , coadministered ohas were ceased or reduced in 55 patients , and the dose of insulin was cut down in 9 patients according to the judgment of each physician . after the initiation of sitagliptin , the dose of coadministered ohas was fixed , and the dose of insulin was titrated based on the results of smbg to avoid hypoglycemia according to the judgment of each physician . parameters for the glucose fluctuation were calculated based on the results of smbg in 115 patients ( 69 men , 65 11 years old , bmi 25.0 4.8 ) . patients were selected based on the frequency of measurements ; that is , they had undertaken measurements of blood glucose at least 3 points from 7 points which include before and after each meal and before bedtime and at least 3 days per week . results of smbg for voluntary 3 days were collected before and 1 , 3 months after the add - on of sitagliptin . glucose fluctuations were expressed as coefficient of variations ( cv = standard deviation / mean ) of each measurement to avoid an influence of the size of absolute value . cv of blood glucoses in each day was calculated ; then an average of the cvs for 3 days was found as within - day variability of blood glucose . cv of blood glucose at each time point for 3 days was calculated ; then an average of the cvs at each time point was found as day - to - day variability as previously described ( modd : means of daily difference ) . the changes of hba1c , body weight , insulin dose , and within - day and day - to - day variation of blood glucose during 3 months were evaluated by repeated measures anova with bonferroni test . the difference of hba1c , body weight , and glucose fluctuation between before and 3 months after the initiation of sitagliptin was expressed as variable and calculated by the subtraction of the value before add - on of sitagliptin from the value after 3 months . a relationship between hba1c and each predictive variable was analyzed using student 's t - test or one - way anova for a nominal variable and pearson 's correlation coefficient for a continuous variable . for multivariate analysis on hba1c as an outcome variable , multiple stepwise regression analysis was performed . hba1c was significantly reduced as early as 1 month after the initiation of sitagliptin and further reduced after 3 months reaching the mean reduction of 0.68% in all patients . however , no improvement was observed in 79 patients whose hba1c levels did not decrease compared to those at baseline ( 8.33 1.32 to 8.49 1.244% , nonresponders ) . in spite of improvement of blood glucose level , after add - on of sitagliptin , total insulin dose per day was significantly cut down to avoid hypoglycemia according to the judgment of each physician . consequently , insulin dose could be decreased in 70 patients and insulin therapy could be ceased in 5 patients . regarding glucose fluctuation , day - to - day variability was significantly reduced 1 month after the add - on of sitagliptin and the reduction continued up to after 3 months , although within - day variability showed nonsignificant tendency of curtailment . the reduction of hba1c value ( hba1c ) was evaluated in relation to each predictive variable . as shown in table 3 , among nominal variables , the combination with -gi was negatively correlated and the use of premixed insulin was positively correlated to the reduction of hba1c . among continuous variables , the higher initial hba1c and fasting cpr level and the increase in body weight for 3 months the changes of parameters for glucose fluctuation were not significantly related to hba1c . in multiple stepwise regression analysis on hba1c as an outcome variable , initial hba1c level and fasting cpr level after the add - on of s , 15 patients including 9 treated with su complained of mild hypoglycemia without any assistance , while no severe hypoglycemic episode was reported . after an appropriate cutting down of coadministered insulin dose , all those patients could avoid a recurrence of hypoglycemia thereafter . no other aes were reported , and no patient was obliged to cease sitagliptin due to aes . the add - on of sitagliptin for 3 months showed a significant reduction of hba1c level in japanese patients with t2 dm whose glycemic control had not been adequate by insulin and/or ohas without a weight gain or an occurrence of severe hypoglycemia . sitagliptin add - on therapy could also reduce a fluctuation of blood glucose expressed as day - to - day variability . forty - eight percent of the variation in hba1c reduction after the add - on of sitagliptin was explained by the higher initial hba1c level and fasting cpr level . the add - on of sitagliptin to ongoing insulin therapy brought about the decrease in hba1c level of around 0.7% , which is comparable to those previously reported in t2 dm patients in japan [ 8 , 9 ] or in overseas countries [ 4 , 6 ] . three of them are randomized controlled studies [ 4 , 6 , 9 ] , and the remaining one is a retrospective observation study same as the present one . apart from the reports from overseas , one report is limited to insulin monotherapy ( premixed or long acting insulin ) in spite of its prospective manner and the other retrospective study consists of a relative small number in japanese t2 dm patients . even though this is a retrospective one , our current study revealed , for the first time , an efficacy of sitagliptin add - on therapy to insulin in real clinical practice among a large number of patients . weight gain or hypoglycemia is an inevitable adverse experience in t2 dm patients especially treated with su or insulin . during the add - on therapy of sitagliptin , body weight was stable in line with a previous report in which an increase of insulin dose significantly produced weight gain in spite of a stable weight observed in sitagliptin add - on therapy . same as most reports regarding sitagliptin add - on therapy [ 4 , 6 , 8 , 9 ] , a few reports of mild hypoglycemia were observed in the present study , indicating an excellent tolerability of this combination therapy . consequently , insulin doses were cut down in one - third of total patients and no recurrence of hypoglycemia was observed thereafter , suggesting the importance of an adjustment of insulin dose without delay referring to the results of smbg after the add - on of sitagliptin . to predict the efficacy of sitagliptin add - on therapy on the change of hba1c ( hba1c ) is a primary interest of the present study . initial hba1c level was the strongest predictor in line with most previous reports [ 8 , 13 ] . otherwise , increasing of body weight , the higher serum cpr , and the use of premixed insulin positively and the combination to -gi negatively contributed to the reduction of hba1c in univariate analysis . weight gain for 3 months might be a consequence of the blood glucose improvement . because the principal effect of dpp-4 inhibitor is an enhancement of endogenous insulin secretion as well as an inhibition of glucagon secretion and gastric emptying , it is quite conceivable that the higher endogenous insulin reserve positively contributes to glucose - lowering of this drug . although it is of interest which type of insulin therapy is more effective for glucose lowering when combined with sitagliptin , conflicting results have been reported concerning this issue so far . one observational study reported the more effectiveness with the combination of basal insulin therapy , whereas other two randomized control studies reported no difference among types of insulin therapy [ 4 , 9 ] . in the present study , premixed insulin therapy was the most effective on the reduction of hba1c , whereas the superiority diminished in multiple regression analysis together with weight gain and the combination to -gi . a fasting serum c - peptide level was selected as a significant explanatory variable together with an initial hba1c level after multivariate analysis , suggesting preserved endogenous insulin capacity is a clinically important factor for the prediction of the efficacy to sitagliptin add - on to ongoing insulin therapy . a recently published retrospective study which is similar to our study demonstrated that an increased total daily insulin dose and prolonged diabetes duration contribute to the attenuation of hba1c reduction independent of baseline hba1c level . in their study , however , endogenous insulin such as cpr was not measured and it is thus plausible that those two factors may have been a reflection of the lower insulin reserve in those patients . our secondary important outcome is the effect of sitagliptin add - on to insulin on glucose fluctuation which has been evaluated in a small number of patients by calculating data based on self - monitoring of blood glucose ( smbg ) or continuous glucose monitoring ( cgm ) [ 7 , 10 ] so far . although mean amplitude of glucose ( mage ) based on cgm data is a gold standard for the estimation of glucose fluctuation , it is inadequate for a large number of patients . in the present study , we calculated glucose fluctuation based on practically available smbg data in each patient instead of cgm data . sitagliptin add - on therapy significantly reduced day - to - day glucose variability , and this is the first report in a large number of japanese t2 dm patients . within - day variability tended to decrease but did not reach a significant difference , probably because points of measurements are limited ( 4.6 1.7 times per day ) and most patients preferred to measure their blood glucose levels before meal rather than after meal . the change of glucose fluctuation was not related to hba1c in the present study , suggesting an effect on glucose stability is independent of that on hba1c . this is a retrospective observation study and participating physician is limited to specialist in diabetes treatment , implying the possibility of selection bias for the patients . in other word , results in the present study could reflect the actual conditions of clinical practice for diabetes treated by specialists . because the changes of insulin dose were put into each physician 's hand to avoid hypoglycemia , the efficacy and safety of the add - on therapy of sitagliptin might be different from those in physicians who are not specialists in diabetes . as mentioned above , frequency and measure point of smbg were modestly different among patients and measurements were mainly performed before meal because they were put into each patient 's hands . smbg prior to meals , occasionally postprandially , is recommended as standards of medical care in diabetes in american diabetes association , and premeal measurements are easier and acceptable in many patients than postmeal ones . from the results obtained in the present study , add - on of sitagliptin to ongoing insulin therapy could be an effective and well - tolerated alternative to treat japanese patients with type 2 diabetes who had been inadequately controlled by insulin with or without other ohas . notably , this combination therapy could suppress glucose fluctuation as well as mean glucose levels . future studies of prospective manner with a large number of patients and with more frequent postmeal smbg data or with cgm data are warranted to confirm these findings . | aims . efficacy and safety of dpp-4 inhibitor , sitagliptin ,
add - on therapy to insulin were investigated in japanese patients with type 2 diabetes .
subjects and methods .
two hundred and sixteen patients ( 126 men , 65 12 years old , bmi 24.9 4.5 , means s.d . ) who had been treated by insulin alone or insulin combined with other oral hypoglycemic agents ( ohas ) were recruited , and sitagliptin was added for 3 months . results .
hba1c was significantly decreased after 3 months of add - on therapy as a whole ( 8.56 1.50% to 7.88 1.25% , p < 0.0001 ) .
body weight did not change and insulin dosage was significantly ( p < 0.0001 ) decreased for 3 months .
furthermore , day - to - day glucose variability was significantly reduced ( 18.3 9.1 to 16.1 8.1% , p < 0.05 ) . in stepwise multiple regression analysis on hba1c as an outcome variable , the higher baseline hba1c value and a preserved cpr were selected as significant predictive variables .
fifteen patients complained of mild hypoglycemia without any assistance during 3 months of sitagliptin add - on , while no severe hypoglycemic episode was reported .
conclusions .
add - on of sitagliptin to ongoing insulin therapy effectively reduced either hba1c level or glucose fluctuation and could be a practical and well - tolerated alternative to treat japanese patients with type 2 diabetes who had been inadequately controlled by insulin with or without other ohas . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
wheelchairs are important chairs that provide structural support for the trunk , pelvis , and
extremities in wheelchair users1 . proper
structural support allows them to perform activities of daily living under comfortable
conditions2 . of the various support
systems , an unsuitable backrest thickness
increases the load on some shoulder muscles and can lead to shoulder pain during wheelchair
propulsion4 . previous studies have
indicated that a proper backrest for a wheelchair can help reduce lumbar load and activation
of the upper extremity muscles4 , 5 . these results have important implications for maintain
active lifestyles providing function , comfort , and support for manual wheelchair users6 . therefore , the therapist must consider the
most suitable backrest when evaluating and recommending a wheelchair for patients . previous studies have examined the suitability of a backrest by measuring shoulder or trunk
muscles with surface electromyography ( semg ) . however , the use of semg has the limitations
of only providing data for a limited number of channels and only being able to scan the
movements of a few muscles groups . also , only movements made in a certain direction can be
analyzed when using emg7 . an accelerometer
is a subjective assessment tool that can detect acceleration and deceleration in response to
movements in one , two , or three directions ( uni- , bi- , or triaxial accelerometers ) as well
as energy expendixure8 . also , an
accelerometer is a valid and reliable tool for monitoring the level of various
activities9 . muscle movement changes
dependent on backrest thickness can be detected by using accelerometers attached to the
trunk and arm during arm propulsion10 , 11 . thus , measurement using accelerometers
may allow a selection of a suitable backrest thickness for wheelchair users . therefore , the purpose of this study was to investigate the effects of backrest thickness
on the trunk and shoulder muscles by assessing acceleration using accelerometers during
wheelchair propulsion . fourteen healthy people participated in this study . participants with upper extremity pain
or neuromuscular disorder were excluded . all participants were informed of the study s
purpose and procedures , and all signed informed consent forms voluntarily . the backrests used in this study had no lumbar
pad , a 3-cm - thick lumbar pad , or a 6-cm - thick lumbar , and the density of the lumbar pads was
27 kg / m . mma7260q the sensitivity of the accelerometers ranges from 6 to + 6 g. data for
measurement and storage in this study were set at 100 hz . we calibrated the single vector
magnitude ( svm ) by summing the acceleration of the three axes . the three backrest conditions
were tested in random order , and the lumbar pads were positioned such that they were aligned
at the mid lumbar level ( l3 ) . the two accelerometers were attached along the right upper arm
and lateral trunk using velcro straps . participants were asked to propel their own manual
wheelchairs 30 times under the three backrest conditions . using one - way repeated - measures analysis of variance ( anova ) , we compared the differences
in the levels of activities of the upper arm and trunk muscles according to lumbar pad
thickness . all data were
analyzed with a level of statistical significance of p<0.05 using the ibm spss
statistics , version 22.0 , software ( ibm corp . the results showed that the svm for the muscle activities with the 3-cm - thick lumbar pad
was significantly lower than that with no lumbar pad ( no lumbar pad , 35,350
3,652 cm / s ; 3-cm - thick lumbar pad , 30,600 3,855 cm / s )
( p<0.05 ) . also , the svm for the 6-cm - thick lumbar pad was significantly higher than that
for the 3-cm - thick lumbar pad ( 6-cm - thick lumbar pad , 37,640 3,769 cm / s ;
3-cm - thick lumbar pad , 30,600 3,855 cm / s ) ( p<0.05 ) . on the other hand ,
there was no significant differences in svm between no lumbar pad and the 6-cm - thick lumbar
pad ( p>0.05 ) . this study compared the differences in muscle activity of the trunk and upper arm according
to backrest thickness during wheelchair propulsion . for measuring muscle movements , we used
two accelerometers attached to the trunk and upper arm . the svm calculated from the
accelerometer measurements represented the actual intensity of movement of the trunk and
upper arm12 . the results of this study
indicated that among the three types of wheelchair backrest , the intensities of movement of
the upper arm and lateral trunk were decreased when participants used a wheelchair equipped
with a 3-cm - thick lumbar pad compared with no lumbar pad or a 6-cm - thick lumbar pad . it seems that the intensity of muscle
movement assessed by accelerometer is correlated with the emg amplitude of the proximal and
distal muscles7 . moreover , this
correlation also indicates that the timing variables that are changed are the push time and
recovery time , as increases in these times require more energy expenditure13 . , svm increases may be due to a less than optimal position
during wheelchair propulsion . consistent with this hypothesis , use of an appropriate
backrest enabled less expenditure of energy through effective arm and trunk movement14 . a previous study also showed that
suitable backward thoracic support can help an individual maintain a comfortable wheelchair
sitting posture , preventing or reducing the risks of back pain15 . also , shoulder muscle loads during manual propulsion by
wheelchair users can be decreased by using a suitable or adjustable backrest16 . these results indicate that an
appropriate backward support position can maintain neutral pelvic tit and lumbar
lordosis3 and provide a biomechanical
advantage to the shoulder4 . the use of
accelerometers in this study is very significant , especially considering that previous
studies have used accelerometers for monitoring physical activity or wheelchair
movement17 , 18 . accelerometers make it easy to measure variables such as movement
time and peak velocity , and it is also easy to obtain data such as emg records7 , 19 .
in the future , we must consider the appropriate backrest thickness for wheelchair users , and
assessment methods using accelerometers can provide feedback for appropriate wheelchair
measurement for wheelchair users . | [ purpose ] the purpose of this study was to investigate the effects of the thickness of a
wheelchair backrest provided for support and comfort on upper arm and trunk muscle load
during wheelchair propulsion by using accelerometers .
[ subjects and methods ] the fourteen
healthy participants were enrolled in this study .
the study compared effects of three
backrest conditions including no pad , a 3-cm - thick lumbar pad , and a 6-cm - thick lumbar
pad .
the instruments used for measurement were used two accelerometers .
the participants
were asked to propel their wheelchairs , which had been equipped with two accelerometers ,
30 times .
[ results ] the intensity of muscle movement with the 3-cm - thick lumbar pad was
significantly lower than the intensities with no lumbar pad and the 6-cm - thick lumbar pad .
the muscle intensity did not differ significantly between the no pad and 6-cm - thick lumbar
pad conditions .
[ conclusion ] an appropriately thick backrest has good effects on upper arm
and trunk muscles during wheelchair propulsion . in the future , we must consider the
appropriate backrest thickness for providing wheelchair users with a comfortable
wheelchair . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
halitosis is a term used to describe any undesirable odor in expired air , regardless of whether the odorous substances originate from oral or non - oral sources . bad breath is not only a personal problem , but also affects the public , as it occurs within a social and cultural context and it affects one 's body image and self - confidence . scientists studied the psychosomatic aspects of patients complaining of halitosis and they found that the patients had high scores for obsession and compulsion and interpersonal sensitivity , and the psychological conditions in terms of anger , inadequacy , sensitivity , decreased self - confidence , and insecurity in social and intimate relations were closely associated with these patients . the most fascinating problem regarding halitosis is the apparent inability of a person to know by him / herself whether he / she has halitosis and to what extent . they may not be aware of their breath bad odor due to a normally functioning olfactory system which has become desensitized to its own stimulants . telling people about their bad breath is very embarrassing and it is difficult to approach this issue as a person 's feeling may be hurt ; for this reason , dental professionals should be prepared and trained to practice in a sensitive and appropriate manner to deliver optimal oral health care and hygiene . a number of methods have been used to detect the presence of halitosis either directly or indirectly . direct tests include direct smelling of the exhaled air by judges ; this technique is called organoleptic or hedonic measurement and is considered the most logical measurement approach . it can also be detected by sniffing of bad breath and determination of the odoriferous sulfur - containing substances by volatile sulfur compounds ( vscs ) monitor or gas chromatography . it can be detected indirectly by identifying the odor - producing microorganisms or assessing their byproducts in vitro . halitosis is a multi - factorial problem that requires a well - defined approach to achieve definitive diagnosis and treatment . several studies analyzed the etiology of halitosis and found that 7590% of the cases had halitosis of intraoral sources , about 40% caused by tongue coating ( tc ) and 30% due to periodontal disease . few studies have documented the prevalence of oral malodor in a sample chosen to represent the entire population . moreover , there is no touchstone in defining a halituous patient , i.e. , there are no universally accepted standard criteria for data collection , evaluation of halitosis , instructions to patients before examination , and instruments used in diagnosis of halitosis , making comparisons between the results of these studies rather difficult if not impossible . the aim of this study was to investigate the awareness of halitosis in a sample of jordanians to assess its prevalence and to find the possible factors associated with this condition . before starting the study , an approval for the study and its protocol was obtained from the institutional review board ( irb ) at the jordan university for science and technology ( just ; approval number 2009/111 ) . the randomization was achieved using the random selection command of the computer program predictive analytics software ( pasw ) of statistical package for social sciences ( spss ) . each selected individual was handed a written invitation to participate in the study , including the consent and pre - examination instruction ; those who did not respond at the first time were re - contacted by phone . the participant should be 18 years of age or older , free from respiratory tract infections during examination , should not have been on antibiotics for at least 3 weeks prior to the assessment , refrain from eating odorous foods for 48 h before the assessment , refrain from ingesting any food or drink , omit their usual oral hygiene practices , mouth rinses , breath fresheners , and chewing gum for at least 2 h before their appointment , and refrain from smoking for at least 1 h before assessment . breakfast and tooth brushing without toothpaste were allowed in order to avoid confusion between breath malodor and morning breath . the examiner ( t. h. ) refrained from using any scented cosmetics for 2 months before the start of the examination period and throughout the examination period . the examiner also used flavorless toothpaste and refrained from drinking coffee and eating garlic , onions , and spicy foods during the examination days . candidates who agreed to take part were given an appointment at the post - graduate dental clinic of just . the clinical examination was performed by a calibrated examiner ( t. h. ) who was extensively trained and was calibrated by an experienced periodontist of university periodontal dentistry ( m. h. ) for 2 months before the start of the study to perform the halitosis and periodontal status examinations and diagnosis , until the supervisor was satisfied with the examiner 's ability for diagnosis . the questionnaire was modified from that used in the breath testing clinic in the university of british columbia . only the following questions were evaluated in the present study :
medical history regarding known blood , heart , respiratory , intestinal , liver , or renal diseases and allergies . also , a history of diabetes was recorded ( yes / no)dental and oral hygiene : how many times per day do you brush your teeth ? do you floss ? ( yes / sometimes / never)consumption of alcoholic beverages : never / rarely / daily / several times per daysmoking : exact number of cigarettes smoked per day for how many years?do you suffer from bad breath odor ? ( yes / sometimes / never)how did you know that you suffer from bad breath odor ? someone told me / people act funnily around me / i just know / my dentisthave you had any treatments for bad breath odor ? ( yes / no ) ; if you got it treated , what measurement did you use?do you have any social problems because of your bad breath odor?are you concerned about other people 's behavior toward yourself on account of your breath odor ? medical history regarding known blood , heart , respiratory , intestinal , liver , or renal diseases and allergies . also , a history of diabetes was recorded ( yes / no ) dental and oral hygiene : how many times per day do you brush your teeth ? do you floss ? ( yes / sometimes / never ) consumption of alcoholic beverages : never / rarely / daily / several times per day smoking : exact number of cigarettes smoked per day for how many years ? ( yes / sometimes / never ) how did you know that you suffer from bad breath odor ? ( yes / no ) ; if you got it treated , what measurement did you use ? are you concerned about other people 's behavior toward yourself on account of your breath odor ? ( yes / no ) . at the beginning , the oral hygiene of each participant was assessed using the plaque index ( pi ) of silness and le , 1964 and the gingival index ( gi ) of le and silness , 1963 . periodontal status of all teeth was assessed using probing pocket depth ( ppd ) and clinical attachment level ( cal ) . ppd and cal were measured at six sites per tooth for all teeth , excluding third molars . periodontitis was defined as the presence of four or more teeth with one site or more with ppd 4 mm and cal 3 mm . accordingly , it was assessed whether the subject was periodontally involved or not , regardless of the severity of the disease . then , the tc index of each recruit was graded using the modified grading scale . finally , the organoleptic assessment of halitosis was performed using the six - point organoleptic scale as described by rosenberg and coworkers . if halitosis did not exist , but the patient believed that he or she had oral malodor , the diagnosis was pseudo - halitosis . the organoleptic score measurement was carried out before all other measurements , i.e. tc and halimeter ( interscan corp . , chatsworth , ca , usa ) readings , to avoid any bias . to standardize the procedure and avoid adaptation of the examiner 's sense of smell to possibly detectable odors after the oral examination , halitosis was assessed with a portable industrial sulfide monitor , halimeter . according to the manufacturer , halitosis is diagnosed at a vsc value 140 ppb of the halimeter , and this value was used in the present study . after the clinical examination , in order to correlate organoleptic test ( olt ) scores with the halimeter readings , the olt scores were further categorized as : grade 0 = normal odor , grade 1 and 2 = mild odor , grade 3 = moderate odor , and grade 4 and 5 = severe odor . in order to make a diagnosis of the breath odor , we used the classification of halitosis proposed by yaegaki and coil . data were entered into a statistical computer program , spss , version 18 ( spss inc . , differences or associations with probabilities ( p - value ) of 0.05 level were considered statistically significant . before starting the study , an approval for the study and its protocol was obtained from the institutional review board ( irb ) at the jordan university for science and technology ( just ; approval number 2009/111 ) . the randomization was achieved using the random selection command of the computer program predictive analytics software ( pasw ) of statistical package for social sciences ( spss ) . each selected individual was handed a written invitation to participate in the study , including the consent and pre - examination instruction ; those who did not respond at the first time were re - contacted by phone . the participant should be 18 years of age or older , free from respiratory tract infections during examination , should not have been on antibiotics for at least 3 weeks prior to the assessment , refrain from eating odorous foods for 48 h before the assessment , refrain from ingesting any food or drink , omit their usual oral hygiene practices , mouth rinses , breath fresheners , and chewing gum for at least 2 h before their appointment , and refrain from smoking for at least 1 h before assessment . breakfast and tooth brushing without toothpaste were allowed in order to avoid confusion between breath malodor and morning breath . the examiner ( t. h. ) refrained from using any scented cosmetics for 2 months before the start of the examination period and throughout the examination period . the examiner also used flavorless toothpaste and refrained from drinking coffee and eating garlic , onions , and spicy foods during the examination days . candidates who agreed to take part were given an appointment at the post - graduate dental clinic of just . the clinical examination was performed by a calibrated examiner ( t. h. ) who was extensively trained and was calibrated by an experienced periodontist of university periodontal dentistry ( m. h. ) for 2 months before the start of the study to perform the halitosis and periodontal status examinations and diagnosis , until the supervisor was satisfied with the examiner 's ability for diagnosis . the participant should be 18 years of age or older , free from respiratory tract infections during examination , should not have been on antibiotics for at least 3 weeks prior to the assessment , refrain from eating odorous foods for 48 h before the assessment , refrain from ingesting any food or drink , omit their usual oral hygiene practices , mouth rinses , breath fresheners , and chewing gum for at least 2 h before their appointment , and refrain from smoking for at least 1 h before assessment . breakfast and tooth brushing without toothpaste were allowed in order to avoid confusion between breath malodor and morning breath . the examiner ( t. h. ) refrained from using any scented cosmetics for 2 months before the start of the examination period and throughout the examination period . the examiner also used flavorless toothpaste and refrained from drinking coffee and eating garlic , onions , and spicy foods during the examination days . candidates who agreed to take part were given an appointment at the post - graduate dental clinic of just . the clinical examination was performed by a calibrated examiner ( t. h. ) who was extensively trained and was calibrated by an experienced periodontist of university periodontal dentistry ( m. h. ) for 2 months before the start of the study to perform the halitosis and periodontal status examinations and diagnosis , until the supervisor was satisfied with the examiner 's ability for diagnosis . the questionnaire was modified from that used in the breath testing clinic in the university of british columbia . only the following questions were evaluated in the present study :
medical history regarding known blood , heart , respiratory , intestinal , liver , or renal diseases and allergies . also , a history of diabetes was recorded ( yes / no)dental and oral hygiene : how many times per day do you brush your teeth ? ( yes / sometimes / never)consumption of alcoholic beverages : never / rarely / daily / several times per daysmoking : exact number of cigarettes smoked per day for how many years?do you suffer from bad breath odor ? ( yes / sometimes / never)how did you know that you suffer from bad breath odor ? someone told me / people act funnily around me / i just know / my dentisthave you had any treatments for bad breath odor ? ( yes / no ) ; if you got it treated , what measurement did you use?do you have any social problems because of your bad breath odor?are you concerned about other people 's behavior toward yourself on account of your breath odor ? medical history regarding known blood , heart , respiratory , intestinal , liver , or renal diseases and allergies . also , a history of diabetes was recorded ( yes / no ) dental and oral hygiene : how many times per day do you brush your teeth ? do you floss ? ( yes / sometimes / never ) consumption of alcoholic beverages : never / rarely / daily / several times per day smoking : exact number of cigarettes smoked per day for how many years ? do you suffer from bad breath odor ? ( yes / sometimes / never ) how did you know that you suffer from bad breath odor ? someone told me / people act funnily around me / i just know / my dentist have you had any treatments for bad breath odor ? ( yes / no ) ; if you got it treated , what measurement did you use ? are you concerned about other people 's behavior toward yourself on account of your breath odor ? at the beginning , the oral hygiene of each participant was assessed using the plaque index ( pi ) of silness and le , 1964 and the gingival index ( gi ) of le and silness , 1963 . periodontal status of all teeth was assessed using probing pocket depth ( ppd ) and clinical attachment level ( cal ) . ppd and cal were measured at six sites per tooth for all teeth , excluding third molars . periodontitis was defined as the presence of four or more teeth with one site or more with ppd 4 mm and cal 3 mm . accordingly , it was assessed whether the subject was periodontally involved or not , regardless of the severity of the disease . then , the tc index of each recruit was graded using the modified grading scale . finally , the organoleptic assessment of halitosis was performed using the six - point organoleptic scale as described by rosenberg and coworkers . if halitosis did not exist , but the patient believed that he or she had oral malodor , the diagnosis was pseudo - halitosis . the organoleptic score measurement was carried out before all other measurements , i.e. tc and halimeter ( interscan corp . , chatsworth , ca , usa ) readings , to avoid any bias . to standardize the procedure and avoid adaptation of the examiner 's sense of smell to possibly detectable odors after the oral examination , halitosis was assessed with a portable industrial sulfide monitor , halimeter . according to the manufacturer , halitosis is diagnosed at a vsc value 140 ppb of the halimeter , and this value was used in the present study . after the clinical examination , in order to correlate organoleptic test ( olt ) scores with the halimeter readings , the olt scores were further categorized as : grade 0 = normal odor , grade 1 and 2 = mild odor , grade 3 = moderate odor , and grade 4 and 5 = severe odor . in order to make a diagnosis of the breath odor , we used the classification of halitosis proposed by yaegaki and coil . data were entered into a statistical computer program , spss , version 18 ( spss inc . , differences or associations with probabilities ( p - value ) of 0.05 level were considered statistically significant . out of the 300 approached employees , 221 responded ( 73.6% ) and of them , 208 were fit to participate according to the study criteria . they consisted of 105 males ( 50.5% ) and 103 females ( 49.5% ) and their ages ranged from 18 to 68 years with a mean ( sd ) age of 32.2 years ( 9.912 ) . a sample size of 208 from a population of 3000 achieves 100% power using a two - sided binomial test . most of the subjects reported brushing their teeth twice a day ( 43.9% ; 90 out of 205 ) , while only 6 participants mentioned that they never brush their teeth . tongue cleaning was reported by 79 subjects ( 49.4% ) , and 133 subjects ( 64.9% ) were using a mouth rinses . smoking was reported by 57 subjects ( 27.8% ; minimum one cigarette per day ) . none of the study participants said that they drank alcohol . regarding subjects self - estimation of halitosis , 157 subjects answered they never suffered from halitosis ( 75.8% ) and 48 persons ( 28.9% ) reported having experienced halitosis . of those persons suffering from halitosis ( 42 out of 48 ) , 37.5% ( n = 15/42 ) were aware of it by themselves either by smelling their breath or their saliva , while 7.5% ( n = 3/42 ) were made aware of it by their friends and relatives , but no one was made aware by his / her dentist . the most reported method used among aware halituous subjects to overcome halitosis problems was chewing gum ( 45.2% ) , followed by using mouth washes ( 33.3% ) . on the other hand , using tongue scrapper was the least used method ( 7.2% ) . regarding the impact of halitosis on the social and family relations of subjects who were aware of it , only 42.9% reported that they hesitate to talk to others . however , 85.7% of the subjects who were aware reported that their bad breath does not prevent them from meeting other people and neither interferes with their social and family life . about 83% of the sample subjects do care about others reaction toward their oral odor , while only 16.9% do not care about it . a high significant association was between diagnosis of halitosis by olt and halimeter readings ( p 0.001 ) . based on the olt categories , about 45 , 89 , 44 , and 30 subjects presented with normal , mild , moderate , and severe halitosis . respectively . based on the halimeter readings , about 48 , 89 , 42 , and 29 of the subjects presented with normal , mild , moderate , and severe halitosis , respectively . in order to correlate the halitosis status with clinical findings , the three - subject difference between the olt and the halimeter readings in the normal breath odor categories was excluded from further analysis . therefore , 160 subjects ( 78% ) out of the 205 included participants were diagnosed as having genuine halitosis and 45 ( 19.1% ) were healthy . among the healthy individuals , six subjects were diagnosed with pseudo - halitosis . among the 160 subjects who were diagnosed as halituous , only 42 ( 20.5% ) were aware of their problem and 118 ( 57.6% ) were not aware of it . olt categories were further recoded into either halituous or non - halituous cases in order to further investigate the association between halitosis and the various variables studied [ table 1 ] . prevalence and awareness of halitosis according to olt and vsc levels there were no significant differences between males and females regarding the prevalence ( 53.1% vs. 46.9% ) ( p = 0.12 ) and awareness of halitosis ( 52.4% vs. 47.6% ) ( p = 0.91 ) . tongue coating was present in 96.6% of the examined participants ( 198 of 205 study subjects ) . presence of tc and its severity were significantly associated with halitosis ( p < 0.001 ) . ninety - four percent and 71.1% of subjects with severe and moderate tc had halitosis , respectively , while only 27.8% of those with light tc had halitosis . the severity of tc was significantly associated with the level of vscs ( p < 0.001 ) . also , it increased among those who reported not practicing tongue brushing ( p < 0.001 ) [ table 2 ] . the association between tc , halitosis , vsc levels , and reported tongue brushing the mean pi and gi values recorded in the examined subjects were ~ 0.5 and ~ 0.4 , respectively . higher scores appeared among halituous compared to non - halituous subjects ; however , the difference was not significant ( p = 0.089 and p = 0.095 , respectively ) . only 20 subjects out of 205 were diagnosed as periodontally involved subjects ; however , a significant association between periodontal disease and the level of vscs was evident among these subjects ( p < 0.01 ) . the proportion of periodontally involved subjects increased from 2.2% in the normal oral odor group to 5.6 , 16.7 , and 24.1% in mild , moderate , and severe oral odor groups . pearson correlation coefficient that was calculated to evaluate the relationship between self - reported assessment and the clinical assessment ( vscs readings and olt ) of halitosis showed no correlation between them ( p > 0.01 ) [ table 3 ] . objectivity of self - estimation the majority of subjects who reported that they do brush their teeth uniformly had halitosis . however , all those who never brush their teeth were halituous ( p = 0.042 ) . there was no statistical association between the frequency of flossing ( p = 0.440 ) and use of mouth washes ( p = 0.144 ) with halitosis . there were no significant differences in the reported tooth brushing frequencies ( p = 0.822 ) and tongue brushing ( p = 0.925 ) dental flossing frequencies ( p = 0.36 ) and using mouth washes ( p = 0.816 ) among aware and non - aware subjects . out of the 300 approached employees , 221 responded ( 73.6% ) and of them , 208 were fit to participate according to the study criteria . they consisted of 105 males ( 50.5% ) and 103 females ( 49.5% ) and their ages ranged from 18 to 68 years with a mean ( sd ) age of 32.2 years ( 9.912 ) . a sample size of 208 from a population of 3000 achieves 100% power using a two - sided binomial test . most of the subjects reported brushing their teeth twice a day ( 43.9% ; 90 out of 205 ) , while only 6 participants mentioned that they never brush their teeth . tongue cleaning was reported by 79 subjects ( 49.4% ) , and 133 subjects ( 64.9% ) were using a mouth rinses . smoking was reported by 57 subjects ( 27.8% ; minimum one cigarette per day ) . none of the study participants said that they drank alcohol . regarding subjects self - estimation of halitosis , 157 subjects answered they never suffered from halitosis ( 75.8% ) and 48 persons ( 28.9% ) reported having experienced halitosis . of those persons suffering from halitosis ( 42 out of 48 ) , 37.5% ( n = 15/42 ) were aware of it by themselves either by smelling their breath or their saliva , while 7.5% ( n = 3/42 ) were made aware of it by their friends and relatives , but no one was made aware by his / her dentist . the most reported method used among aware halituous subjects to overcome halitosis problems was chewing gum ( 45.2% ) , followed by using mouth washes ( 33.3% ) . on the other hand , using tongue scrapper was the least used method ( 7.2% ) . regarding the impact of halitosis on the social and family relations of subjects who were aware of it , only 42.9% reported that they hesitate to talk to others . however , 85.7% of the subjects who were aware reported that their bad breath does not prevent them from meeting other people and neither interferes with their social and family life . about 83% of the sample subjects do care about others reaction toward their oral odor , while only 16.9% do not care about it . a high significant association was between diagnosis of halitosis by olt and halimeter readings ( p 0.001 ) . based on the olt categories , about 45 , 89 , 44 , and 30 subjects presented with normal , mild , moderate , and severe halitosis . respectively . based on the halimeter readings , about 48 , 89 , 42 , and 29 of the subjects presented with normal , mild , moderate , and severe halitosis , respectively . in order to correlate the halitosis status with clinical findings , the three - subject difference between the olt and the halimeter readings in the normal breath odor categories was excluded from further analysis . therefore , 160 subjects ( 78% ) out of the 205 included participants were diagnosed as having genuine halitosis and 45 ( 19.1% ) were healthy . among the healthy individuals , six subjects were diagnosed with pseudo - halitosis . among the 160 subjects who were diagnosed as halituous , only 42 ( 20.5% ) were aware of their problem and 118 ( 57.6% ) were not aware of it . olt categories were further recoded into either halituous or non - halituous cases in order to further investigate the association between halitosis and the various variables studied [ table 1 ] . prevalence and awareness of halitosis according to olt and vsc levels there were no significant differences between males and females regarding the prevalence ( 53.1% vs. 46.9% ) ( p = 0.12 ) and awareness of halitosis ( 52.4% vs. 47.6% ) ( p = 0.91 ) . tongue coating was present in 96.6% of the examined participants ( 198 of 205 study subjects ) . presence of tc and its severity were significantly associated with halitosis ( p < 0.001 ) . ninety - four percent and 71.1% of subjects with severe and moderate tc had halitosis , respectively , while only 27.8% of those with light tc had halitosis . the severity of tc was significantly associated with the level of vscs ( p < 0.001 ) . also , it increased among those who reported not practicing tongue brushing ( p < 0.001 ) [ table 2 ] . the association between tc , halitosis , vsc levels , and reported tongue brushing the mean pi and gi values recorded in the examined subjects were ~ 0.5 and ~ 0.4 , respectively . higher scores appeared among halituous compared to non - halituous subjects ; however , the difference was not significant ( p = 0.089 and p = 0.095 , respectively ) . only 20 subjects out of 205 were diagnosed as periodontally involved subjects ; however , a significant association between periodontal disease and the level of vscs was evident among these subjects ( p < 0.01 ) . the proportion of periodontally involved subjects increased from 2.2% in the normal oral odor group to 5.6 , 16.7 , and 24.1% in mild , moderate , and severe oral odor groups . pearson correlation coefficient that was calculated to evaluate the relationship between self - reported assessment and the clinical assessment ( vscs readings and olt ) of halitosis showed no correlation between them ( p > 0.01 ) [ table 3 ] . the majority of subjects who reported that they do brush their teeth uniformly had halitosis . however , all those who never brush their teeth were halituous ( p = 0.042 ) . there was no statistical association between the frequency of flossing ( p = 0.440 ) and use of mouth washes ( p = 0.144 ) with halitosis . there were no significant differences in the reported tooth brushing frequencies ( p = 0.822 ) and tongue brushing ( p = 0.925 ) dental flossing frequencies ( p = 0.36 ) and using mouth washes ( p = 0.816 ) among aware and non - aware subjects . the major aim of this cross - sectional study was to determine the awareness , distribution , and related determinants of halitosis in the target sample , in order to predict the future health care needs in controlling and solving this social and health problem , with no interest in generalizing our findings to the total jordanian population . since oral malodor is an olfactory stimulus , direct smelling of the exhaled air by judges is considered the most logical measurement approach . most researchers use either schmidt et al . scale , which is a three - point scale , or rosenberg et al . scale , which is a six - point scale . this scoring index has established itself as a gold standard and is used for treatment and research purposes throughout the world . nevertheless , reliability and reproducibility are problematic with this index , and efforts have been made to simplify it . intra- and inter - observer variations are also a frequent finding ; therefore , training and calibration of breath odor judges are needed for standardized examinations . to overcome this compromise , and also due to the fact that it is an uncomfortable procedure for both the examiner ( judge ) and the examined subject , it was decided clearly that olt should not be the sole method for defining patients with halitosis . this fact led several investigators to propose quantitative approaches based on measurement of vscs which are associated with halitosis . however , the arbitrary fixing of threshold measurements for halitosis using the halimeter may lead to wide variation between studies . used a threshold of 125 ppb for halitosis , roldan et al . used a level of 170 ppb , richter used a value of 150 ppb , and kazor et al . used a level of 200 ppb . in our study , we followed the most recent values set by the halimeter manufacturers . a complication of setting a halimeter threshold is that some patients with objectionable malodor organoleptically may have a halimeter reading below the manufacturer 's threshold , whereas others without organoleptically detectable halitosis may have a halimeter reading above the threshold . firstly , the halimeter is mainly sensitive to the vscs , hydrogen sulfide and methyl mercaptan . other compounds such as volatile fatty acids and the polyamines , putrescine and cadaverine , may be detected organoleptically , but not on using the halimeter . secondly , the halimeter is more sensitive to hydrogen sulfide than methyl mercaptan , but organoleptically methyl mercaptan is more objectionable . however , the results of the study show the high significant correlation between olt scores and halimeter values in the diagnosis of halitosis . few studies have documented the prevalence of oral malodor in a sample chosen to represent the entire population . lack of scientific data may be due to the differences in cultural and racial appreciation of odors for patients as well as for investigators . moreover , there are no universally accepted standard criteria for data collection , evaluation of halitosis , instructions for patients before examination , and instruments used in diagnosis of halitosis , making comparisons between the results of these studies rather difficult if not impossible . in addition , the surveyed samples rarely represented populations in general . the prevalence of halitosis in this jordanian population was 78% , which is higher than that reported by epidemiological studies in other communities that ranged between 2% and 40% , this may be due to the overall poorer oral hygiene and periodontal status of the subjects in our study . or the assumption by other authors that halitosis might be an underestimated oral health problem in the general population may be true . the most interesting observation of this study is that a majority ( 57.6% ) of the subjects with halitosis were not aware of it , and only 20.5% were aware of their problem . bad breath paradox where halitosis sufferers are completely unaware of their bad breath odor , because individuals become adapted to their own oral and body odors , but that does not prevent someone with halitosis from noticing it in others . people who are not aware of their oral bad breath may encounter social and professional rejection without knowing why . in light of lack of studies that measure the awareness of halitosis , it may be assumed that other communities have higher awareness , based on the presence of having halitosis centers in those communities and the lack of such centers in our community . in addition , the prevalence studies in other communities and the lack of such studies in ours may also support the assumption of higher awareness of halitosis in those communities . the percentage of pseudo - halituous subjects ( 2.9% ) in our study was much lower than that reported in other studies ; this may reflect other populations concern and awareness regarding the presence of such a social handicap and its consequences . age was not a risk factor for the increase in level of vscs in this study , and this is in agreement with many previous investigations . gender of the participants did not influence the awareness and presence of halitosis as detected both by halimeter and olt , and this is in agreement with other studies and in contrast with a recent cross - sectional survey from rio de janeiro which showed that the prevalence of persistent halitosis was nearly three times higher in men than in women , regardless of age . cigarette smoke itself contains significantly higher vsc levels which can be detected by the halimeter . furthermore , smoking has a negative effect on the periodontium , which may also promote halitosis . smokers in this study were significantly more aware of their halitosis problem than non - smokers probably because it is a common knowledge that smoking causes oral and breath odors . tongue coating was present in 96.6% of the study sample ( in 198 out of 205 subjects examined ) . the presence of tc and its severity were significantly associated with halitosis and vsc levels . these results are in agreement with other studies , that the dorsum of the tongue represents the primary source of vscs and its severity is significantly associated with halitosis . this significant association led winkel and tangerman to consider tc assessments as a useful method in detecting oral malodor . clinical studies comparing halitosis and periodontal diseases have produced conflicting results . in our study , we did not find a significant correlation between the gi or pi with odor judge scores or halimeter scores , which agrees with previous reports . however , there was a strong correlation between the presence of periodontal disease and the vsc levels . the proportion of periodontally involved subjects increased from 2.2% in the normal oral odor group to 5.6 , 16.7 , and 24.1% in the mild , moderate , and severe oral odor groups , respectively . found that pi was significantly correlated with halitosis and that periodontitis patients with halitosis had more severe disease than those without . delanghe et al . found that oral malodor is frequently ( 87% ) caused by an oral problem ( tongue coating > gingivitis > periodontitis ) . there was no association between self - reported tooth brushing frequency and halitosis ; this is in agreement with previous studies . the majority of those who reported regular tooth brushing were halituous ; on the other hand , all those who never brushed their teeth were halituous , and this may indicate that there might be some benefit of tooth brushing in solving this problem . the efficiency of brushing techniques used by the participants was not addressed in this study , so a confident conclusion can not be drawn about the role of tooth brushing in solving halitosis problem . the results also revealed that tongue brushing is more helpful and this was reported by other studies where tooth brushing accompanied with tongue cleaning resulted in a substantial reduction in vsc levels . removal of plaque from the tooth surfaces by brushing has been found to be less than half as effective in reducing oral malodor as tongue brushing alone . another aspect to be considered is how the halituous subjects who are aware of their condition notice their bad breath problem . it is interesting to point out that about 55% of those subjects noticed their halitosis problem by themselves or were told by others , including parents , spouses , other relatives , and friends , but no one was made aware of it by his / her dentist . besides , the majority of these subjects ( 83% ) stated that they are concerned about others reaction to their bad oral odor , and this may affect their body image and self - confidence . the american dental associasion ( ada 's ) survey showed that patients themselves initiated discussions of their halitosis with their oral health care providers . this is surprising in light of the potential embarrassment it causes . among those patients who were unaware of or unwilling to disclose their problem , a hygienist broached the subject 31% of the time , while a dentist brought it up 27% of the time . therefore , the rule of dentist in limiting this social and medical problem should be activated by training them to use sensory and instrumental examination tools , to treat the complaint of halitosis . the most reported method used by the halituous subjects , who were aware of their problem , to overcome the problem was chewing gum ( 67.9% ) , which is not a successful method . studies show that these halituous subjects make desperate attempts to mask the odor through the frequent use of mints and chewing gum , compulsive brushing , and repeatedly using flavored mouth rinses . others have chosen smoking as a way to mask the odor and become socially more acceptable . halitosis is widespread in the study population and is accompanied by a low level of awareness . tooth brushing alone is not effective in solving this problem , so tongue brushing should be part of daily oral hygiene and should be included in the oral hygiene instructions given to patients and the public . also , health care providers should pay more attention to health education regarding halitosis since halitosis from an extra - oral origin can be the sign of an underlying systemic disease . therefore , it is substantiated to organize halitosis consultations in a multidisciplinary setting , assembling periodontists , and ear nose throat specialists , specialists in internal medicine , and psychologists or even psychiatrists . | objectives : this study was conducted to estimate the prevalence and awareness of halitosis among the subjects of a population , and also to compare the results of halimeter readings to self - estimation of halitosis and to assess the relationship between halitosis and oral health.materials and methods : a sample of 205 employees from the jordan university for science and technology ( just ) were selected as study subjects .
a standardized questionnaire focusing on dental hygiene , self - reported halitosis , and smoking was filled by all participants . in the clinical examination
, the objective values for assessment of oral health and the presence of halitosis were gathered through an organoleptic test ( olt ) and the measurement of volatile sulfur compound ( vsc ) level by halimeter for each participant.results:the prevalence of halitosis was 78% , with low rate of awareness ( 20.5% ) .
the amount of tc played the most important role in increasing the concentration of vscs in mouth air ( p < 0.001).conclusion : a statistically significant correlation was found between olt and halimeter values ( p < 0.001 ) .
subjective patients opinion did not correlate with the objective evaluation of halitosis .
tc scores and smoking were the factors significantly associated to halitosis .
the halimeter showed promising characteristics regarding diagnosis of halitosis for clinical setting and field surveys . |
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both type 1 and type 2 diabetes show a direct relationship between the glycemic control and the risk of systemic complications . the american diabetes association ( ada ) promotes self - monitoring of blood glucose ( smbg ) to allow diabetic patients to achieve and maintain specific glycemic goals . ada recommends that all insulin - treated patients perform smbg to ( a ) achieve and maintain glycemic control , ( b ) prevent and detect hypoglycemia , ( c ) adjust changes in lifestyle . smbg is also used in establishing the need for insulin therapy in gestational diabetes mellitus . with the introduction of glucometers , there has been an ongoing , competition - driven development in both meter and strip technology , which has allowed for greater accuracy and reliability of results . however , despite the advances in technology , there is significant variation among these monitoring devices , which has necessitated the development of performance guidelines by organizations such as the ada and the international standardization organization ( iso ) . the iso guidelines recommend that the accuracy criteria for values < 100 mg / dl to 10 mg / dl and 20% for values 100 mg / dl . glucometers which are available in hospital settings are different from the portable glucometers used by patients at home and are not available in the open market due to marketing policies . the methods used for the estimation of blood glucose levels are similar in both the hospital based and out - patient glucometers . however the hospital based glucometers are modified taking into consideration various other parameters such as ph , oxygenation , temperature , humidity , hematocrit , hyperlipidemia , etc . some use glucose oxidase - peroxidase ( god - pod ) enzyme while some use the glucose dehydrogenase ( gdh ) enzyme for the estimation of blood glucose . we have chosen six commonly used glucometers ( three hospital based and three outpatient based ) and compared their reading with the laboratory values . the primary objective of the study was to compare the accuracy and efficacy of six glucometers commonly used in the hospital and out - patient settings . three hospital based glucometers - breeze 2 by bayer , accuchek performa by roche , surestep by johnson and johnson and three from the out - patient setting glucometers - contour ts by bayer , accuchek active by roche , one touch ultra 2 by johnson and johnson were compared with the laboratory readings . a total of 105 patients were randomly selected from out - patient and inpatients settings . efforts were made to cover all ranges of plasma glucose in the samples that were collected . glucose levels were estimated in fasting and post prandial state from diabetic and non - diabetic patients . upon phlebotomy , the whole blood venous sample was dropped in six different glucometers and the same venous sample was sent to the laboratory for analysis of glucose levels . the blood sample was analyzed within sixty minutes of collection by the reference laboratory god - pod method ( synchron lx systems ) . the calibration and the use of the glucometer strips were done according to the manufacturer instructions . the johnson and johnson one - touch surestep and one touch ultra 2 test strips employs a dry reagent technology based on the god method , which is specific for d - glucose . when a small drop of whole blood is applied to a surestep strip , god on the strip triggers the oxidation of glucose in the blood sample producing gluconic acid and hydrogen peroxide . pod on the test strip then causes the hydrogen peroxide to react with dyes to produce a blue color in the presence of oxygen . this blue color is visible through the confirmation dot on the back of the test strip . the color intensity of this dot , which is directly proportional to the level of glucose is measured and reported as a plasma - calibrated glucose result . the manufacturer 's packaging insert stated that this strip method system was compared with that obtained from the ysi 2700 glucose analyzer to obtain the calibration curve . the accuchek performa and active ( roche diagnostics ) is a new generation blood glucose strip that uses both ac and dc current to measure blood glucose levels in capillary blood . it utilizes gdh via a pyrroloquinoline mediator to measure glucose levels in the sample , which are in turn reported based on internal strip specific calibration curves . this calibration curve has been constructed using a plasma ( glucose hexokinase ) reference method . the breeze 2 and contour ts ( bayer health care ) is a third generation glucose monitoring system . the contour ts uses the gdh - flavin adenine dinucleotide chemistry with capillary filling test strips . the strip contained god and an electron transfer mediator , ferrocene , which replaced oxygen in the original god reaction ; the reduced mediator was reoxidized at the electrode to generate a current detected by an amperometric sensor . the laboratory uses a synchron system which determines glucose concentration by an oxygen rate method employing a bechman coulter oxygen electrode . a precise volume of the sample is injected in a reaction cup containing a god solution in a fixed proportion ( ratio : 1 part sample : 76 parts reagent ) . the peak rate of oxygen consumption is directly proportional to the concentration of glucose in the sample . the standard deviation for in - patient and out - patient glucometers is summarized in tables 1 and 2 respectively . standard deviation of inpatient glucometers standard deviation of outpatient glucometers the accuracy of glucometers was determined using correlation coefficient [ graphs 1 and 2 ] and bland altman scatter plots [ graphs 3 - 8 ] . the correlation coefficient data of in - patient and out - patient glucometers is shown in tables 3 and 4 respectively . correlation of performa , breeze 2 and surestep with laboratory correlation of contour , active and ultra 2 with laboratory scatter plot of performa , breeze 2 and surestep with laboratory for values < 100 scatter plot of with laboratory contour ts , active and ultra 2 for values < 100 scatter plot of breeze 2 , performa and surestep with laboratory for values 100 scatter plot of contour ts , active and ultra 2 with laboratory for values 100 scatter plot of breeze 2 , performa and surestep with laboratory with 5% variability scatter plot of contour ts , active and ultra 2 with laboratory with 5% variability correlation coefficient of inpatient glucometers correlation coefficient of outpatient glucometers the extent to which the glucometers deviated from the reference method is represented in the bland altman scatter diagram . the sub analysis was performed to laboratory values < 100 mg / dl [ graphs 3 and 4 ] and 100 mg / dl [ graphs 5 and 6 ] . we analyzed our data using this criterion and found that it was not practical as it was too stringent and most of the values were falling outside the valid range [ graphs 7 and 8 ] . this is an internationally recognized way of evaluating the compatibility of a blood glucose self - monitoring system with a laboratory reference . evaluation is based on comparison of clinical consequences using the test strip method and the reference . this analysis partitions the total blood glucose range into zones , based on the effects of glucose variations on diabetes treatment .
zone a : results to clinically correct treatment decisions either in hypoglycemia or hyperglycemia range.zone b : > 20% deviation from the reference ; represents values that would lead to benign or no treatment error.zone c : represents values would lead to treatment decisions opposite to that called for by the blood glucose levels.zone d : represent a failure to detect and treat errors.zone e : is a clinically more serious error zone , glucose meter generated values that failed to detect hypoglycemia or hyperglycemia . values are opposite the reference values resulting in corresponding treatment decisions opposite to those needed . zone a : results to clinically correct treatment decisions either in hypoglycemia or hyperglycemia range . zone b : > 20% deviation from the reference ; represents values that would lead to benign or no treatment error . zone c : represents values would lead to treatment decisions opposite to that called for by the blood glucose levels . zone e : is a clinically more serious error zone , glucose meter generated values that failed to detect hypoglycemia or hyperglycemia . values are opposite the reference values resulting in corresponding treatment decisions opposite to those needed . in our study , we found the following results in the error grid analysis [ tables 5 and 6 ] . error grid analysis of inpatient glucometers error grid analysis of outpatient glucometers all the glucometer readings were in zone a , b or c and none in zone d or e. however among the indoor glucometers , accucheck performa had maximum readings in zone a and none in zone c , d or e. similarly among the outdoor glucometers , johnsons one touch ultra had maximum readings in zone a [ graphs 9 and 10 ] . clarke error grid analysis of breeze 2 , performa and surestep clarke error grid analysis of contour ts , active and ultra 2 this is an internationally recognized way of evaluating the compatibility of a blood glucose self - monitoring system with a laboratory reference . evaluation is based on comparison of clinical consequences using the test strip method and the reference . this analysis partitions the total blood glucose range into zones , based on the effects of glucose variations on diabetes treatment .
zone a : results to clinically correct treatment decisions either in hypoglycemia or hyperglycemia range.zone b : > 20% deviation from the reference ; represents values that would lead to benign or no treatment error.zone c : represents values would lead to treatment decisions opposite to that called for by the blood glucose levels.zone d : represent a failure to detect and treat errors.zone e : is a clinically more serious error zone , glucose meter generated values that failed to detect hypoglycemia or hyperglycemia . values are opposite the reference values resulting in corresponding treatment decisions opposite to those needed . zone a : results to clinically correct treatment decisions either in hypoglycemia or hyperglycemia range . zone b : > 20% deviation from the reference ; represents values that would lead to benign or no treatment error . zone c : represents values would lead to treatment decisions opposite to that called for by the blood glucose levels . zone e : is a clinically more serious error zone , glucose meter generated values that failed to detect hypoglycemia or hyperglycemia . values are opposite the reference values resulting in corresponding treatment decisions opposite to those needed . in our study , we found the following results in the error grid analysis [ tables 5 and 6 ] . error grid analysis of inpatient glucometers error grid analysis of outpatient glucometers all the glucometer readings were in zone a , b or c and none in zone d or e. however among the indoor glucometers , accucheck performa had maximum readings in zone a and none in zone c , d or e. similarly among the outdoor glucometers , johnsons one touch ultra had maximum readings in zone a [ graphs 9 and 10 ] . clarke error grid analysis of breeze 2 , performa and surestep clarke error grid analysis of contour ts , active and ultra 2 self - monitoring of blood glucose using reagent impregnated strips is a simple and integral component of diabetes care and management . we chose to analyze these glucometers with the iso criteria , as among the various guidelines it seemed to be more practical . it can be adjudicated from our results that the glucometers available for out - patient care are far better in terms of accuracy and precision as compared to glucometers available for in - patient care at least in research settings . the limitations of this study are that other parameters that alter or affect the glucometer readings such as hematocrit , temperature , hypoxia etc . , validation of glucometers allows medical personnel and patients to choose the glucometers more objectively , thereby improving the quality - of - care . | background : glucometers allow self - monitoring of blood glucose in a convenient manner . with the availability of various glucometers
, there is a persistent attempt to improve the accuracy and the precision of these glucometer readings , so as to match the laboratory values of blood glucose.objective:we compared the glucometers used in hospital and out - patient settings with the laboratory reference method.materials and methods : we analyzed a total of 105 blood samples collected from in - patient and out - patient from our tertiary care hospital .
venous blood samples were collected and checked on six glucometers and the same blood sample was sent to the laboratory for glucose estimation .
the laboratory value was used as a reference for comparison .
the accuracy was evaluated by the iso criteria .
the results were evaluated by bland altman graphs , correlation coefficients , scatter plots and clarke 's error grid analysis.results:we observed good correlation between bed side glucometer and laboratory automated analyzer . among the in - patient glucometers breeze 2 ,
performa and surestep , the correlation coefficient was 0.97 , 0.96 and 0.88 respectively . among the outpatient glucometers
one touch ultra 2 , active and contour , the correlation coefficient was 0.97 , 0.97 and 0.95 respectively.conclusions:there is a good correlation between different glucometers and laboratory values especially in the out - patient settings . among all in - patient
glucometers , surestep by johnson and johnson had least correlation coefficient , whereas all three out - patient glucometers correlated well with the laboratory values . |
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the cbct data of a 11-year - old female was referred to evaluate radiolucency in the apical area of the maxillary left lateral incisor . the patient presented with no clinical symptoms and had a non - contributory medical history . the cbct scan was obtained with i - cat unit ( imaging science , hatfield , pa , usa ) . the digital imaging and communications in medicine ( dicom ) deidentified data was sent through a health insurance portability and accountability act - compliant secure email ( brightsquid , ross technology centre , calgary , alberta , canada ) and the data was evaluated using invivo5.4.3 ( anatomage , san jose , ca , usa ) . all data was evaluated by two board - certified oral and maxillofacial radiologists ( saz , sar ) . it was observed that the sagittal view was optimal for the visualization of this entity because of its extension in the anterioposterior direction across the clivus . radiographically cbm can be described as a well - defined , corticated and channel - like hypodense radiolucency extending from the pharyngeal aspect of the basiocciput to the intracranial aspect of the clivus . 1 ) . a 63-year - old female patient with a significant medical history of arthritis , thyroid disease , sinusitis , and gastrointestinal disease was referred for a cbct scan . , atlanta , ga , usa ) , and the patient was referred for the evaluation of a proposed implant site in the maxillary region . the data was analyzed using multiplanar reformatted slices by a board - certified oral and maxillofacial radiologist ( mm ) . the volumetric data set revealed an unusual finding on the basiocciput of the clivus . in the sagittal view the intracranial part of the clivus showed discontinuity , the radiographic presentation of this osseous defect presentation can be described as a well - corticated , hypodense channel extending in the superior - inferior direction along the intracranial aspect of the clivus ( fig . 2 ) . on initial inspection it appeared to be a fracture of the clivus . however , further close inspection revealed no other signs of osteolytic destruction on the posterior aspect of the clivus ( intracranial surface ) . based on the volumetric appearance of the defect , a radiographic impression of cbm was made . the cbct data of a 11-year - old female was referred to evaluate radiolucency in the apical area of the maxillary left lateral incisor . the patient presented with no clinical symptoms and had a non - contributory medical history . the cbct scan was obtained with i - cat unit ( imaging science , hatfield , pa , usa ) . the digital imaging and communications in medicine ( dicom ) deidentified data was sent through a health insurance portability and accountability act - compliant secure email ( brightsquid , ross technology centre , calgary , alberta , canada ) and the data was evaluated using invivo5.4.3 ( anatomage , san jose , ca , usa ) . all data was evaluated by two board - certified oral and maxillofacial radiologists ( saz , sar ) . it was observed that the sagittal view was optimal for the visualization of this entity because of its extension in the anterioposterior direction across the clivus . radiographically cbm can be described as a well - defined , corticated and channel - like hypodense radiolucency extending from the pharyngeal aspect of the basiocciput to the intracranial aspect of the clivus . a 63-year - old female patient with a significant medical history of arthritis , thyroid disease , sinusitis , and gastrointestinal disease was referred for a cbct scan . atlanta , ga , usa ) , and the patient was referred for the evaluation of a proposed implant site in the maxillary region . the data was analyzed using multiplanar reformatted slices by a board - certified oral and maxillofacial radiologist ( mm ) . the volumetric data set revealed an unusual finding on the basiocciput of the clivus . in the sagittal view the intracranial part of the clivus showed discontinuity , the radiographic presentation of this osseous defect presentation can be described as a well - corticated , hypodense channel extending in the superior - inferior direction along the intracranial aspect of the clivus ( fig . 2 ) . on initial inspection it appeared to be a fracture of the clivus . however , further close inspection revealed no other signs of osteolytic destruction on the posterior aspect of the clivus ( intracranial surface ) . based on the volumetric appearance of the defect , the term cbm is used to describe transclival defect of the basicocciput of the clivus . cbm presents itself in various configurations such as keyhole defect , transverse or longitudinally fissures in the clivus.12 radiological features of cbm can be described as a well - defined , corticated , osseous transclival defect located in the basiocciput of the clivus . in the axial view it is located in close approximation to the anterior rim of the foramen magnum.10 cbm 's reported prevalence in the literature is is 2%-3% for adults and 3 ) were described in the literature : three complete varieties ( superior , inferior , and bifurcated ) and three incomplete varieties ( a thin , long channel ; and either a superior recess ; or an inferior recess).910 the complete type of cbm is characterized by the presence of a channel or tract like hypodense region extending either anterio - posteriorly or superior - inferior direction occasionally , connecting the intracranial and pharyngeal portions of the clivus . incomplete type will traverse partially within the clivus and they fail to communicate with pharyngeal portion or intracranial of the portion of the clivus . cbm in general is considered an anatomical variant without any clinical significance.89 however , few studies indicate that they could serve as potential pathways for the progression of the disease such as meningitis.13 hemphill et al.11 and martinez et al.12 reported a transclival bony defect that was associated with a meningocele and caused recurrent meningitis . however , authors argued that a mere coexistence of cbm and meningitis may not have caused detrimental effects such as optic neuropathy and the episode of sterile meningitis as noted in their case.9 lohman et al.13 reported a cbm in association with a tornwaldt cyst for the first time in the literature . literature is providing conflicting reports about cbm 's association with other pathological entities such as meningitis , tornwaldt 's cyst and its detrimental effects as noted in the above description . first case described was incomplete type and second case was complete type of cbm , both cases were assessed using cbct . in both the cases , the occurrence of cbm was not associated with meningitis or other problems . our experience , as well as the literature as a whole , emphasizes the need for thorough analysis of skulls regardless of whether defects are detected or not when diagnostic tools such as cbct , ct , and mri are readily available.25 we recommend that all data should be interpreted by a board - certified oral and maxillofacial radiologist to prevent misdiagnoses and to avoid potential complications.7 | the aim of this report is to present two cases of canalis basilaris medianus as identified on cone - beam computed tomography ( cbct ) in the base of the skull .
the cbct data sets were sent for radiographic consultation . in both cases , multi - planar views revealed an osseous defect in the base of the skull in the clivus region , the sagittal view showed a unilateral , well - defined , non - corticated , track - like low - attenuation osseous defect in the clivus .
the appearance of the defect was highly reminiscent of a fracture of the clivus .
the borders of osseous defect were smooth , and no other radiographic signs suggestive of osteolytic destructive processes were noted .
based on the overall radiographic examination , a radiographic impression of canalis basilaris medianus was made .
canalis basilaris medianus is a rare anatomical variant and is generally observed on the clivus . due to its potential association with meningitis , it should be recognized and reported to avoid potential complications . |
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shivering , a common post - anaesthesia occurrence is defined as an involuntary , repetitive activity of skeletal muscles . the incidence of shivering has been found to be quite high , approximately 40 - 50% in different studies . shivering also increases intraocular and intracranial pressure , and may contribute to increased wound pain , delayed wound healing , and delayed discharge from post - anaesthetic care . apart from being an uncomfortable experience , its deleterious effects deserve primary prevention and rapid control on occurrence . shivering is a physiological response to core hypothermia in an attempt to raise the metabolic heat production . the main causes of intra / post - operative shivering are temperature loss , increased sympathetic tone , pain , and systemic release of pyrogens . spinal anaesthesia significantly impairs the thermoregulation system by inhibiting tonic vasoconstriction , which plays a significant role in temperature regulation . it also causes a redistribution of core heat from the trunk ( below the block level ) to the peripheral tissues . the non - pharmacological management is by external heating like the use of forced air warming , warming blankets , warmed fluids etc . , according to the results of a meta - analysis , the most frequently reported pharmacological interventions include clonidine , pethidine , tramadol , nefopam and ketamine . unfortunately , no gold standard treatment is known for shivering as the administration of all the available drugs is associated with various adverse effects . during the last decade , tramadol has become a favoured and commonly used drug for post - spinal anaesthesia shivering . however , it has many adverse effects like nausea , vomiting , dizziness etc . , which cause further discomfort to the patient . various studies , which have been conducted to compare them have concluded that clonidine has better efficacy and less adverse effects as compared to tramadol . it has been used as a sedative agent and is known to reduce the shivering threshold . few studies which have explored its anti - shivering potential have inferred that dexmedetomidine is an effective drug without any major adverse effect and provides good haemodynamic stability . hence , we planned to do a comparative study of the efficacy , haemodynamic , and adverse effects of tramadol and dexmedetomidine when used for the control of post - spinal anaesthesia shivering . this was a prospective , randomised , double - blind study , which was conducted at a tertiary care centre after taking approval from institutional ethics committee . 50 american society of anaesthesiologists ( asa ) grade i and ii consenting patients of either gender aged 18 - 65 years scheduled for elective as well as emergency lower abdominal , lower limb , orthopaedic and plastic surgeries under spinal anaesthesia were included in the study . patients with known hypersensitivity to dexmedetomidine or tramadol , cardio - pulmonary , renal or hepatic disease , hyperthyroidism , psychiatric disorder , urinary tract infection , severe diabetes or autonomic neuropathies , known history of substance or alcohol abuse , patients receiving any pre - medication were not included in the study . all patients who fulfilled the inclusion criteria and developed post - spinal anaesthesia shivering were enrolled and randomised using computer generated chart with allocation ratio of 1:1 into either of the two groups . group d ( n = 25 ) were administered dexmedetomidine 0.5 g / kg intravenous ( iv ) and group t ( n = 25 ) received tramadol 0.5 mg / kg iv as per randomisation by index anaesthesiologist ( not a part of the study ) who prepared either of the drugs , at the onset of shivering ( detailed below ) . the anaesthesiologist conducting the case as well as recording the data were unaware of the drug being administered . upon arrival in the operation theatre , an 18 g venous cannula was inserted and preloading done with ringer 's lactate solution 10 ml / kg before giving spinal anaesthesia and maintained at 6 ml / kg / h after spinal anaesthesia . before starting the procedure , standard monitors were attached and all the baseline parameters such as heart rate ( hr ) , non - invasive blood pressure ( nibp ) , oxygen saturation ( spo2 ) , electrocardiography ( ecg ) , and body temperature ( axillary ) were recorded . subarachnoid anaesthesia was administered with 0.5% heavy bupivacaine ( 15 mg ) at l3 - 4 or l4 - 5 interspace using 26 g quincke 's spinal needle under aseptic conditions . supplemental oxygen was administered to all the patients at the rate of 5 l / min with face mask and patients were covered with drapes but not actively warmed . vital parameters such as hr , nibp , and spo2 were recorded at intervals of every 5 min for first 30 min and every 15 min for the rest of the observation period . : grade 0 : no shivering , grade 1 : one or more of the following : piloerection , peripheral vasoconstriction , peripheral cyanosis , but without visible muscle activity , grade 2 : visible muscle activity confined to one muscle group , grade 3 : visible muscle activity in more than 1 muscle group and grade 4 : gross muscle activity involving the whole body . the drugs were diluted to a volume of 5 ml in a 5 ml syringe and presented as coded syringes as per randomisation list by an anaesthesiologist who was not aware of the group allocation . the attending anaesthesiologist recorded the time in minutes at which shivering started after spinal anaesthesia ( onset of shivering ) , severity of the shivering , time to the disappearance of shivering and response rate ( shivering ceasing within 15 min after treatment ) . duration of surgery was recorded and duration of spinal anaesthesia was noted by assessing spontaneous recovery of sensory block using the pin - prick method and observing spontaneous movements of limbs in the post - operative period . there was recurrence of shivering , patients were treated with an additional dose of dexmedetomidine ( 0.5 g / kg iv ) or tramadol ( 0.5 mg / kg iv ) in the respective groups . adverse effects such as nausea , vomiting , bradycardia ( < 50/min ) , hypotension ( > 20% of baseline ) , dizziness ; and sedation score were noted . the degree of sedation was graded on a four point scale as per filos et al . : grade 1 : awake and alert , grade 2 : drowsy , responsive to verbal stimuli , grade 3 : drowsy , arousable to physical stimuli , grade 4 : unarousable . nausea and vomiting were treated with injection metoclopramide 10 mg iv as and when required . power analysis was based on the detectable difference between the means of time taken for cessation of post - spinal shivering after treatment with dexmedetomidine or tramadol = 180 s. with a standard deviation ( sd ) of 180 s considering = 0.05 and power of 90% ( two - tailed test ) , sample size calculated to be 21.02 patients per group . to increase the power of study we enrolled 25 patients per group . numerical data were presented as mean sd and categorical data as proportions ( % ) . the comparison of the mean levels of all variables between two groups was made by the unpaired t - test . p value was calculated , and p < 0.05 was considered to be statistically significant . in the present study , a total of 50 patients out of 69 consecutive patients met the inclusion criteria and consented for study . these 50 patients were randomized into two groups of 25 each . out of the total patients , 25 were male , and 25 were female . as it was an intra - operative study , no patient was lost to follow - up [ figure 1 ] . patient flow ( according to consort chart ) both groups were comparable with respect to age , gender , asa grade , duration of surgery and the duration of spinal anaesthesia . duration of spinal anaesthesia ranged from 90 min to 150 min [ table 1 ] . there was no statistically significant difference in time for the onset of shivering between the two groups . however , the difference in the time interval between administration of drug after the onset of shivering and cessation of shivering was significantly shorter in the dexmedetomidine group when compared to tramadol group . there was recurrence of shivering in 1 patient in dexmedetomidine group and 2 patients in tramadol group . the patients were given rescue doses of dexmedetomidine or tramadol , respectively [ table 2 ] . parameters for post - spinal anaesthesia shivering nausea and vomiting was observed only in tramadol group , and there was no incidence in the dexmedetomidine group . almost similar number of patients were sedated in both groups and the sedation score in all the patients was 2 . there was no evidence of respiratory depression , hypotension or bradycardia in any of the patients . hr , mean blood pressure , body temperature , and spo2 remained within normal limits throughout the procedure in both groups [ table 3 ] . intra and post - operative shivering is a distressing experience for the patient . the exact mechanism of shivering during spinal anaesthesia has not been fully recognized . the possible mechanisms include impairment of central thermoregulation , internal redistribution of body heat , and heat loss to the environment . potential risk factors for hypothermia in spinal anaesthesia include ageing , level of sensory block , temperature of the operation theatre and iv solutions . in this study , all operation theatres ( ots ) maintained an ambient temperature of 23 - 25c , and all fluids and drugs were at room temperature during the surgery . demographic factors such as age , gender , duration of anaesthesia and surgery have also been matched to reduce any confounding bias . the neurotransmitter pathways involved in shivering are multiple and involve opioids , 2 adrenergic , serotenergic , and anticholinergic receptors . hence , drugs acting on these systems which include opioids ( pethidine , nalbuphine , or tramadol ) , ketanserin , propofol , doxapram , clonidine , ketamine and nefopam are utilized in the treatment of shivering . however , adverse effects such as hypotension , hypertension , sedation , respiratory depression , nausea and vomiting limit their use . tramadol is an opioid analgesic with opioid effect mainly mediated via mu receptor with minimal effect on kappa and delta receptors . the anti - shivering action of tramadol is probably mediated via its opioid or serotonergic and noradrenergic activity or both . it is a well - established agent in the treatment of post - anaesthetic shivering . dexmedetomidine is an 2 adrenoceptor agonist , with antihypertensive , sedative , analgesic , and anti - shivering properties . the anti - shivering effects of alpha adrenoceptor agonists are mediated by binding to 2 receptors that mediate vasoconstriction and the anti - shivering effect . dexmedetomidine comparably reduces the vasoconstriction and shivering thresholds , thus suggesting that it acts on the central thermoregulatory system rather than preventing shivering peripherally . it has been successfully used as an adjunct to local anaesthetics in spinal anaesthesia and peripheral nerve blockade , for the sedation of mechanically ventilated patients in the intensive care unit , as well as supplementation of post - operative analgesia . the role of dexmedetomidine in the treatment of shivering has been evaluated in a few studies . it may be a good choice because of its dual effects related anti - shivering and sedation . with same dose , that is , 0.5 mg / kg of tramadol , the response rate reported by shukla et al . was 92.5% , by reddy and chiruvella as 95.56% and by tsai and chu , 87% . maheshwari et al . reported similar recurrence rate with tramadol as in our study ( 8% ) but the dose used in their study was 1 mg / kg . the recurrence rate in the study by shukla et al . the incidence of nausea and vomiting with tramadol in our study was 28% and 20% , respectively . the results correspond with that of other studies by reddy and chiruvella , tsai and chu ; bansal and jain however , in the study by shukla et al . , the incidence of nausea was quite high ( 77.5% ) , whereas wason et al . maheshwari et al . have reported a very high incidence of sedation to the extent of 84% , which as mentioned earlier could be due to the higher dose as opposed to 28% sedation in our study . in a study by easley , all children who had post - anaesthesia shivering were treated with a single iv bolus dose of dexmedetomidine 0.5 g / kg over 3 - 5 min . all children had cessation of shivering behaviour within 5 min following the completion of dexmedetomidine administration . we found a response rate of 100% and apart from sedation , there was no other adverse effect observed . but there was recurrence in 1 patient who had to be given a rescue dose . all other studies have used higher dose of dexmedetomidine ( 1 g / kg ) , so comparison of results would not be appropriate . in our study , intra - operative dexmedetomidine infusion caused negligible sedation inspite of using a loading dose of 1 g / kg followed by a maintenance infusion of 0.5 g / kg / h . the other agonist clonidine is associated with a high incidence of hypotension and bradycardia . in this respect the incidence of adverse effects like nausea and vomiting was found to be higher in case of tramadol compared to dexmedetomidine . more studies need to be undertaken with varying dose ranges to extrapolate the results of this study . there was not much difference in the number of patients who were sedated in either group . the sedation seen with dexmedetomidine , in the absence of nausea and vomiting , is beneficial for the surgeon , anaesthetist as well as the patient . it provided more comfort to the patient , maintained more cardio - respiratory stability , improved surgical conditions and also provided amnesia during surgery . another limitation was that the present study included short duration surgeries as the mean duration of surgical period was calculated to be approximately 1 h in both groups . the anti - shivering effect of dexmedetomidine needs to be seen in surgeries of longer duration where chances of developing hypothermia are more . also , we did not assess different doses of dexmedetomidine ; further studies are needed to evaluate the anti - shivering and haemodynamic effects of dexmedetomidine with various doses . the search continues for drugs that sufficiently improve the tolerance of thermoregulation without simultaneously producing respiratory depression , or haemodynamic instability . dexmedetomidine might prove to be a valuable addition in the current armamentarium of the available drugs . both dexmedetomidine ( 0.5 g / kg ) and tramadol ( 0.5 mg / kg ) are effective in treating patients with post - spinal anaesthesia shivering , but time taken for complete cessation of shivering was shorter with dexmedetomidine as compared to tramadol , the difference being statistically significant . more studies of different dose ranges of dexmedetomidine need to be conducted in order to cement its position as an efficient anti - shivering agent . | background and aims : dexmedetomidine ( 2 adrenergic agonist ) has been used for prevention of post anaesthesia shivering .
its use for the treatment of post - spinal anaesthesia shivering has not been evaluated .
the aim of this study was to evaluate and compare the efficacy , haemodynamic and adverse effects of dexmedetomidine with those of tramadol , when used for control of post - spinal anaesthesia shivering.methods:a prospective , randomised , and double - blind study was conducted in 50 american society of anaesthesiologists grade i and ii patients of either gender , aged between 18 and 65 years , scheduled for various surgical procedures under spinal anaesthesia .
the patients were randomised in two groups of 25 patients each to receive either dexmedetomidine
0.5 g / kg or tramadol 0.5 mg / kg as a slow intravenous bolus .
grade of shivering , onset of shivering , time for cessation of shivering , recurrence , response rate , and adverse effects were observed at scheduled intervals .
unpaired t - test was used for analysing the data.results:time taken for cessation of shivering was significantly less with dexmedetomidine when compared to tramadol .
nausea and vomiting was observed only in tramadol group ( 28% and ; 20% respectively ) .
there was not much difference in the sedation profile of both the drugs.conclusion:we conclude that although both drugs are effective , the time taken for cessation of shivering is less with dexmedetomidine when compared to tramadol
. moreover , dexmedetomidine has negligible adverse effects , whereas tramadol is associated with significant nausea and vomiting . |
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before 2000 , no cases of p. falciparum had occurred in chinese immigrants living in northern and central italy , despite a large immigrant population . an initial cluster of 22 cases was described during summer 2000 in the lombardy region ( 3 ) . a cluster of six cases was detected in tuscany during the same period ( 4 ) . in both outbreaks , all patients were exposed to malaria during a prolonged journey to europe ( 39 months ) through a number of asian and african countries . from 2000 to 2002 , a total of 10 sporadic cases were reported to the italian ministry of health in 2001 ( l. vellucci , directorate for prevention , ministry of health , italy , pers . the 2003 cluster prompted us to examine hospital records from august 2002 , where we identified an additional , previously undetected , cluster of 12 malaria cases in four of our study hospitals ( data not included in the table ) . the ministry of health had 26 confirmed p. falciparum cases during 2002 ( l. vellucci , pers . comm . ) , suggesting an ongoing ( and possibly increasing ) influx of chinese laborers . some differences exist between the later cluster and the 2000 cluster . in the 2003 cluster , the proportion of severe cases was lower than in the previous reports , with a patient with a fatal case first admitted to a general hospital where diagnosis of malaria was not considered ; in the others , awareness of the possibility of malaria had been raised by the earlier cluster ( 3,4 ) and led to prompt diagnosis and treatment , with favorable outcome . a single african country , cte divoire , was the transit country for most of the patients . visa processing for entry to europe was arranged by the courier organization in cte divoire . although italy was the final destination , at least some immigrants entered through france , which also has had reports of p. falciparum cases in chinese immigrants ( f. legros , centre national de rfrence de lepidmiologie du paludisme , france , pers . use of clandestine travel by air to emigrate from china , where sudden acute respiratory syndrome ( sars ) is present , poses a threat for the african countries , where the introduction of sars virus could have devastating consequences on their health systems with a potential overlap with the hiv epidemic . other diseases could be spread or acquired by the immigrants in the countries of transit . while curtailing the huge , illegal immigrant system to europe is difficult , we can not overemphasize the need for a sound surveillance on imported infectious diseases in this continent . both clusters of malaria were detected early through salute internazionale regione lombardia ( sirl ) , a network on imported diseases of the lombardy region , in conjunction with the european network on imported infectious disease surveillance ( tropneteurop ) . any physician in europe who sees a chinese patient with a history of recent travel and a high fever should exclude malaria , besides considering the possible diagnosis of sars . respiratory symptoms are also frequent in uncomplicated malaria ( 5,6 ) , and acute respiratory distress syndrome has long been recognized as one of the main features of severe malaria ( 7,8 ) . | between november 2002 and march 2003 , 17 cases of malaria ( 1 fatal ) were observed in illegal chinese immigrants who traveled to italy through africa . a further cluster of 12 was reported in august , 2002 .
several immigrants traveled by air , making the risk of introducing sudden acute respiratory syndrome a possibility should such illegal immigrations continue . |
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in the past years , bcl-2 , the mammalian homologue of ced-9 , the antiapoptotic gene initially described in caenorhabditis elegans , has emerged as a major checkpoint controlling the escape of cells from their death fate ( kroemer 1997 ) . in mammals , bcl-2 is a member of a broad family of related gene products , with various degrees of sequence identity and different functions . while bcl-2 and some members of the family ( e.g. , bcl - x ) protect the cells from apoptosis , others ( e.g. , bax ) have the opposite effect . however , while there is a general consensus on the role of bcl-2 , its mechanism of action remains elusive . on one hand , the various members of the family have been shown to dimerize and to interact with cofactors of caspases , key executors of the apoptotic machinery ( li and yuan 1999 ) ; on the other , the specific localization of the protein on the er and outer mitochondrial membrane ( lithgow et al . 1994 ) suggests that these proteins may interfere with some specific functions localized in these organelles . in particular , bcl-2 has been shown to prevent the release of cytochrome c and aif from mitochondria ( kluck et al . 1997 ) , events that are considered essential in triggering apoptosis , whereas expression of bax has opposite effects ( jurgensmeier et al . thus , the intriguing possibility has emerged that members of the bcl-2 family affect the apoptotic process by modulating ion fluxes in these organelles ( minn et al . in particular , recent work in cell clones stably expressing the oncogene suggests that bcl-2 causes an overload of ca within the er ( lam et al . . however , this effect appears in contrast with the observation that bcl-2 insertion in artificial lipid bilayers results in the formation of ionic channels of low selectivity ( minn et al . such property should in fact cause a reduction in the er ca content ( due to an increased leak ) rather than excess accumulation . in this report , we have undertaken a detailed investigation of ca homeostasis at the subcellular level using the recombinant aequorin ( aeq ) approach developed in our laboratory ( rizzuto et al . we conclude that bcl-2 overexpression affects ca handling by reducing the state of filling of the intracellular ca stores . the implications of these results for the protective action of bcl-2 on apoptosis are discussed . hela cells were grown in dme supplemented with 10% fcs in 75-cm falcon flasks . before transfection , cells were seeded onto 13-mm glass coverslips and allowed to grow to 50% confluence . at this stage , transfection with 4 g of dna ( 3 g bcl-2 + 1 g aeq ) was carried out as previously described ( rizzuto et al . for fura-2 measurements , the cells were seeded onto 24-mm coverslips and transfection was carried out with 8 g plasmid dna ( 6 g bcl-2 + 2 g mtgfp ) . for cytosolic aeq ( cytaeq ) and mitochondrial aeq ( mtaeq ) , the coverslip with the cells was incubated with 5 m coelenterazine for 12 h in dme supplemented with 1% fcs , and then transferred to the perfusion chamber . for reconstituting with high efficiency , the aeq chimeras targeted to the golgi apparatus and the er ( goaeq and eraeq , respectively ) this was obtained by incubating the cells for 1 h at 4c in krb ( krebs - ringer modified buffer : 125 mm nacl , 5 mm kcl , 1 mm na3po4 , 1 mm mgso4 , 5.5 mm glucose , and 20 mm hepes , ph 7.4 , at 37c ) supplemented with coelenterazine 5 m , the ca ionophore ionomycin , and 600 m egta . after this incubation , the cells were extensively washed with krb supplemented with 2% bsa and 1 mm egta , and then aeq reconstitution was carried out as described . agonists and other drugs were added to the same medium . in experiments with permeabilized cells , a buffer mimicking the cytosolic ionic composition ( intracellular buffer , ib : 140 mm kcl , 10 mm nacl , 1 mm k3po4 , 5.5 mm glucose , 2 mm mgso4 , 1 mm atp , 2 mm sodium succinate , 20 mm hepes , ph 7.05 , at 37c ) was employed . this solution was supplemented with either 100 m egta or a fixed [ ca ] ( buffered with 2 mm egta ) . the experiments were terminated by lysing the cells with 100 m digitonin in a hypotonic ca - rich solution ( 10 mm cacl2 in h2o ) , thus discharging the remaining aeq pool . the light signal was collected and calibrated into [ ca ] values as previously described ( brini et al . , a 13-mm round coverslip with the transfected cells was placed in a perfused , thermostated chamber located in the close proximity of a low - noise photomultiplier , with built - in amplifier discriminator . the output of the discriminator was captured by a thorn - emi photon counting board and stored in an ibm - compatible computer for further analyses . the aeq luminescence data were calibrated off - line into [ ca ] values , using a computer algorithm based on the ca response curve of wild - type and mutant aeqs , as previously described ( brini et al . 1997 ) . in some experiments using eraeq , after reaching the plateau level , an artifactual slow decrease in [ ca ] was observed . such an artifact is due to a small fraction of missorted aeq in a low [ ca ] compartment and a simple practical cure for it has been described by maechler et al . 4 , cells transfected with eraeq were used to determine the steady [ ca]er obtained by incubating the cells in low ca medium . two protocols were adopted that gave identical results . in the first , cells incubated in normal medium were depleted according to the standard protocol , but the ca concentration during the refilling was reduced to 100 m . in the second protocol , the cells were preincubated in 100 m cacl2 for 16 h , depleted , and finally refilled with an extracellular ca concentration of 100 m . the coverslip with the cells was incubated with 5 m fura-2 ( added to dme + 1% fcs ) at 37c for 30 min , and after a brief washout was then transferred to the thermostated stage of a zeiss axiovert inverted microscope equipped with a sutter filterwheel and 340/380 excitation filters . the fluorescence data were collected with a princeton instruments back - illuminated camera and expressed as emission ratios , using metafluor software ( universal imaging ) . within the microscope field , transfected and untransfected cells were identified before carrying out the fura-2 monitoring by revealing mitochondrial - targeted green fluorescent protein ( mtgfp ) fluorescence ( excitation 480 nm , emission 510 nm ) . hela cells were grown in dme supplemented with 10% fcs in 75-cm falcon flasks . before transfection , cells were seeded onto 13-mm glass coverslips and allowed to grow to 50% confluence . at this stage , transfection with 4 g of dna ( 3 g bcl-2 + 1 g aeq ) was carried out as previously described ( rizzuto et al . for fura-2 measurements , the cells were seeded onto 24-mm coverslips and transfection was carried out with 8 g plasmid dna ( 6 g bcl-2 + 2 g mtgfp ) . for cytosolic aeq ( cytaeq ) and mitochondrial aeq ( mtaeq ) , the coverslip with the cells was incubated with 5 m coelenterazine for 12 h in dme supplemented with 1% fcs , and then transferred to the perfusion chamber . for reconstituting with high efficiency , the aeq chimeras targeted to the golgi apparatus and the er ( goaeq and eraeq , respectively ) , the luminal [ ca ] of these compartments first must be reduced . this was obtained by incubating the cells for 1 h at 4c in krb ( krebs - ringer modified buffer : 125 mm nacl , 5 mm kcl , 1 mm na3po4 , 1 mm mgso4 , 5.5 mm glucose , and 20 mm hepes , ph 7.4 , at 37c ) supplemented with coelenterazine 5 m , the ca ionophore ionomycin , and 600 m egta . after this incubation , the cells were extensively washed with krb supplemented with 2% bsa and 1 mm egta , and then aeq reconstitution was carried out as described . agonists and other drugs were added to the same medium . in experiments with permeabilized cells , a buffer mimicking the cytosolic ionic composition ( intracellular buffer , ib : 140 mm kcl , 10 mm nacl , 1 mm k3po4 , 5.5 mm glucose , 2 mm mgso4 , 1 mm atp , 2 mm sodium succinate , 20 mm hepes , ph 7.05 , at 37c ) was employed . this solution was supplemented with either 100 m egta or a fixed [ ca ] ( buffered with 2 mm egta ) . the experiments were terminated by lysing the cells with 100 m digitonin in a hypotonic ca - rich solution ( 10 mm cacl2 in h2o ) , thus discharging the remaining aeq pool . the light signal was collected and calibrated into [ ca ] values as previously described ( brini et al . , a 13-mm round coverslip with the transfected cells was placed in a perfused , thermostated chamber located in the close proximity of a low - noise photomultiplier , with built - in amplifier discriminator . the output of the discriminator was captured by a thorn - emi photon counting board and stored in an ibm - compatible computer for further analyses . the aeq luminescence data were calibrated off - line into [ ca ] values , using a computer algorithm based on the ca response curve of wild - type and mutant aeqs , as previously described ( brini et al . 1997 ) . in some experiments using eraeq , after reaching the plateau level , an artifactual slow decrease in [ ca ] was observed . such an artifact is due to a small fraction of missorted aeq in a low [ ca ] compartment and a simple practical cure for it has been described by maechler et al . 4 , cells transfected with eraeq were used to determine the steady [ ca]er obtained by incubating the cells in low ca medium . two protocols were adopted that gave identical results . in the first , cells incubated in normal medium were depleted according to the standard protocol , but the ca concentration during the refilling was reduced to 100 m . in the second protocol , the cells were preincubated in 100 m cacl2 for 16 h , depleted , and finally refilled with an extracellular ca concentration of 100 m . the coverslip with the cells was incubated with 5 m fura-2 ( added to dme + 1% fcs ) at 37c for 30 min , and after a brief washout was then transferred to the thermostated stage of a zeiss axiovert inverted microscope equipped with a sutter filterwheel and 340/380 excitation filters . the fluorescence data were collected with a princeton instruments back - illuminated camera and expressed as emission ratios , using metafluor software ( universal imaging ) . within the microscope field , transfected and untransfected cells were identified before carrying out the fura-2 monitoring by revealing mitochondrial - targeted green fluorescent protein ( mtgfp ) fluorescence ( excitation 480 nm , emission 510 nm ) . to directly investigate the role of bcl-2 in ca homeostasis , we have selectively measured the [ ca ] in different cell compartments , i.e. , the cytoplasm and the organelles acting as sources ( er and golgi apparatus ) or targets ( mitochondria ) of the ca signal . 1992 ; brini et al . 1995 ; montero et al . 1995 ; pinton et al . the first is the high subcellular specificity of the probes , which allows to clearly identify the effects of bcl-2 expression on the signaling properties of the various compartments ; the second is that the ca probe can be cotransfected with bcl-2 . we and others have previously shown that in transient cotransfection , the two recombinant proteins are expressed by the same subset of cells , thus avoiding the risks associated with the use of stable clones or single cells ( brini et al . 1995 ) . the effect of bcl-2 expression on the cytosolic ca signal elicited by an agonist , atp , acting on receptors coupled , through gq proteins , to the production of inositol 1,4,5 trisphosphate ( ip3 ) was first investigated . in the experiment shown in fig . 1 , hela cells , either coexpressing bcl-2 and cytaeq ( bcl-2transfected ) or expressing only cytaeq ( controls ) were challenged with atp , which acts on a gq - coupled p2y receptor . both in control and bcl-2transfected cells , atp stimulation causes a rapid rise in cytoplasmic ca concentration ( [ ca]c ) , followed by a gradually declining sustained plateau . in bcl-2transfected cells , the [ ca ] increases evoked by stimulation with atp are significantly smaller than in controls ( peak amplitude 1.53 0.22 vs. 2.06 0.13 m , n = 10 ; fig . 1 a ) . since bcl-2 is localized in the mitochondria also , and thus could , at least in principle , enhance ca uptake in this organelle , a simple explanation for this result would be that cytosolic ca is more rapidly cleared by mitochondria , acting as an immobile buffer , placed in closed proximity to ip3-gated channels . indeed , bcl-2 overexpression does not increase , but rather reduces , the mitochondrial [ ca ] rise induced by atp ( peak amplitude 6.56 0.72 in controls vs. 4.42 0.46 m in bcl-2expressing cells ; n = 10 ) . interestingly , the reduction caused by bcl-2 overexpression appears larger in the mitochondria than in the cytosol , both in absolute terms and as percent ( 26 vs. 33% reduction , respectively ) . while not responsible for the alteration of the cytosolic ca signal , the effect of bcl-2 on mitochondrial ca homeostasis appears of major functional relevance , given that key events occurring in the mitochondrial matrix , such as the stimulation of atp production ( jouaville et al . 1999 ) and possibly the opening of the permeability transition pore , ptp ( bernardi 1999 ) , are regulated by [ ca]m . a second possible explanation for the reduction in the agonist - dependent [ ca]c ( and [ ca]m ) increases was next considered , i.e. , a decrease in the amount of ca released by the agonist - sensitive ca stores . to investigate this possibility , the [ ca ] in the lumen of the er was measured in cells cotransfected with bcl-2 and an eraeq chimera ( montero et al . in addition , since we recently have demonstrated that the golgi apparatus shares many of the ca homeostatic properties of the er ( pinton et al . 1998 ) , this latter organelle was also investigated . 2 shows the calibrated [ ca ] values in the two compartments . to obtain reliable quantitative estimates of the [ ca ] in the lumen of these two organelles , their [ ca ] needs to be decreased during both the reconstitution of aeq with coelenterazine and the subsequent initial phase of perfusion with krb / egta in the luminometer ( see materials and methods ) . under those conditions , the [ ca ] was < 10 m in both organelles . when the [ ca ] in the perfusion medium was switched to 1 mm , the [ ca ] in the lumen of the two compartments gradually increased , reaching in control cells plateau values of 452 46 m ( n = 7 ) in the er and 262 47 m ( n = 7 ) in the golgi apparatus , respectively . in bcl-2 transfected cells , lower steady state values were attained in both compartments ( 304 38 m , n = 7 for the er , and 186 34 m , n = 7 for the golgi apparatus , respectively ) . when the cells were treated with atp , rapid decreases in the [ ca ] of the two compartments the decrease of [ ca ] was larger and faster in controls compared with bcl-2expressing cells , reflecting the higher filling state and thus the more rapid flow through the ip3-gated channels . the data reported above differ substantially from those reported in wehi7.2 clones , where bcl-2 overexpression results in an augmentation of the er ca content ( lam et al . this discrepancy may be due to the use of transient vs. stable transfections , in which the results might reflect unique properties of the selected clone and/or the long - term expression of a foreign gene may result in adaptation phenomena that are only indirect consequences of the expressed protein . this interpretation might also explain the results obtained by kuo and coworkers in clones of mcf10a cells overexpressing bcl-2 , in which an increase in serca2 ( sarcoendoplasmic reticulum ca atpases 2 ) protein leads to accelerated ca uptake and enhanced ca loading ( kuo et al . 1998 ) . on the other hand , it is possible that , although the duration of these experiments is compatible with the time course of the apoptotic process , the events observed in transient experiments could include only some of the relevant effects of bcl-2 on the mechanisms controlling programed cell death . given that the steady - state [ ca ] in both organelles depends on the equilibrium between active accumulation and passive leak , the effect of bcl-2 could be on either process . on one hand , bcl-2 could reduce ca uptake by the serca , either by a direct effect on the pump or by reducing the resting cytosolic [ ca ] . on the other , it could increase the passive diffusion of ca from the er . to clarify this critical issue , we investigated these possibilities independently . at first , the possibility that bcl-2 affected primarily cytosolic [ ca ] ( and hence [ ca]er only indirectly ) was verified . given that aeq is not accurate enough to reveal small differences of [ ca ] 10 m , the resting [ ca]c was measured with the fluorescent indicator fura-2 ( grynkiewicz et al . to identify bcl-2overexpressing cells in single - cell imaging experiments , the cells were cotransfected with mitochondrially targeted gfp , mtgfp(s65 t ) ( rizzuto et al . gfp - positive cells were distinguished from controls by the typical fluorescence emitted upon illumination with blue light . no difference in the resting cytoplasmic [ ca ] between bcl-2transfected and control cells was observed ( data not shown ) . then , we investigated whether bcl-2 overexpression had any direct effect on the activity of the serca pump . for this purpose , the kinetics of ca accumulation were studied in permeabilized cells ( fig . 2 , bcl-2transfected and control cells were transferred to the luminometer chamber and perfused with a buffer ( ib , see materials and methods ) mimicking intracellular ionic conditions , supplemented with 100 m egta ( ib / egta ) . after permeabilization with 100 m digitonin added to the same medium and wash in ib / egta , the medium was switched to ib , containing 200 nm [ ca ] ( ib / ca ) . under those conditions , [ ca]er gradually increased , reaching , in control cells , a plateau value of 728 60 , n = 5 . in bcl-2transfected cells , the maximal rate of ca accumulation was the same as in control cells ( 13 2 vs. 12 3 m / s , n = 5 ) , arguing that the ca pumping activity is not modified . however , the steady - state value eventually attained was distinctly lower ( 504 52 m , n = 5 ) . therefore , these data exclude that the alteration of er ca homeostasis is due to a reduction in the activity of the sercas or to soluble cytosolic factors ( that are released into the medium after permeabilization with digitonin ) . rather , by demonstrating a difference only when er refilling approaches the high [ ca]er values of intact resting cells , i.e. , in conditions in which passive leak becomes significant , they suggest that bcl-2 acts on the latter process . we sought direct evidence for this interpretation by measuring the ca leak rate from the er of bcl-2expressing and control cells . after the depletion protocol , the er was first refilled by exposing the cells to extracellular [ ca ] ranging from 0.23 mm , thus resulting in different levels of steady - state [ ca]er . the serca blocker , 2,5-di-(tert - butyl)-1,4-benzohydroquinone ( tbubhq ) , was then added that initiates the release of stored ca . given that , by definition , the rates of ca uptake and leak in steady state are equal , the rate of [ ca]er decrease upon blockade of the serca must reflect the rate of ca cycling across the er membrane and , thus , of the leak rate at any given steady - state [ ca]er . b shows the relationship between [ ca]er and leak rate , which can be fitted by a power equation . in bcl-2overexpressing cells , the data are more scattered , but for each [ ca]er value , the rate of ca efflux is faster than in control cells . this result is consistent with the evidence that bcl-2 can induce cation channels of low selectivity in artificial lipid bilayers ( minn et al . previously , we have demonstrated that a reduction of 30% in steady - state [ ca]er results in a substantial activation ( > 50% ) of the so - called capacitative ca influx ( hofer et al . the reduction in steady - state [ ca]er caused by bcl-2 overexpression is thus expected to cause an activation of this pathway . however , indirect evidence ( namely , the observation that the resting cytosolic ca level is indistinguishable from controls ) suggests that this is not the case . to further investigate the problem , we measured the capacitative ca influx in bcl-2transfected and control hela cells expressing cytaeq ( fig . the cells were treated with the serca blocker ( 10 m tbubhq ) while perfused with ca - free medium ( krb / egta ) . this procedure first evoked a small transient increase in [ ca]c due to release of ca from the stores . when the release of stored ca was complete ( 3 min ) , ca was readded to the medium . this maneuver evoked a second , larger [ ca]c increase via the capacitative pathway , which was markedly lower in bcl-2transfected than in controls cells ( 1.19 0.11 in controls vs. 0.68 0.13 m in bcl-2expressing cells , n = 6 ) . the question then arises as to the mechanism(s ) through which bcl-2 downregulates capacitative ca influx . two possibilities were considered : one was that the reduction of this ca influx pathway is an adaptive mechanism to the long lasting reduction in steady - state [ ca]er ; the second was that the reduction is a direct consequence of bcl-2 overexpression . to distinguish between these two possibilities , we verified whether a long - term reduction of [ ca]er , obtained independently of bcl-2 , could cause a comparable downregulation of the capacitative ca influx . capacitative ca influx was thus compared in controls and in cells whose [ ca]er was reduced to values similar to those of bcl-2overexpressing cells by prolonged incubation with a low ( 100 m ) extracellular [ ca ] . in cells that had been maintained at low external [ ca ] , and thus experienced a long - term reduction in the resting [ ca]er levels , the [ ca]c peak evoked by activation of the capacitative ca influx was markedly lower than in controls ( 0.59 0.11 m , n = 5 ) . of interest , the level of steady - state [ ca]er in cells incubated in low extracellular ca was very similar ( 269 46 m , n = 4 ) to that measured in bcl-2overexpressing cells incubated in normal ( 1 mm ) extracellular ca concentration . the simplest explanation of these results is that the decrease in capacitative ca influx is an adaptive consequence to the prolonged reduction in steady - state [ ca]er , though , at the moment , it can not be excluded that other factors ( e.g. , a reduction in plasma membrane potential ) contribute to this effect . this reduction in capacitative ca influx , on one hand , appears critical to maintain a resting [ ca]c indistinguishable from that of control cells , despite a major reduction of [ ca ] in the er ; on the other hand , it could account , at least in part , for a higher resistance of cells expressing bcl-2 to apoptotic cell death , as the reduction of this ubiquitous ca influx pathway may prevent the overload of ca , considered one of the key events in apoptosis . in agreement with this view , ma and coworkers recently demonstrated that serca overexpression , by causing er ca overload , increases spontaneous apoptosis ( ma et al . finally , our data are not in contrast with previous results showing that the serca inhibitor , thapsigargin , activates programed cell death ( bian et al . 1997 ) . in this latter case , in fact , the ca depletion is acute ( 12 min ) and complete , whereas in bcl-2transfected cells , the drop in [ ca]er is modest and develops slowly . thus , thapsigargin not only causes a massive activation of the capacitative ca influx pathway , but also such a drastic reduction in the level of [ ca]er to interfere with the basic activity of er chaperonins . we have demonstrated that overexpression of bcl-2 causes a reduction of the steady - state [ ca ] levels , both in the er and in the golgi apparatus , the two main agonist - sensitive ca stores . we also showed that this alteration affects the ca signal to which the effector systems are exposed , in particular those located in local environments highly dependent on the filling state of the intracellular stores ( such as the mitochondria ) . the chronic reduction in [ ca ] of the er results in a downregulation of the capacitative ca influx . the alterations in ca handling provoked by bcl-2 overexpression could be part of the mechanisms through which bcl-2 exerts its role in the control of cell growth . targets of bcl-2 effects on ca homeostasis could be : the er itself , where sorting and trafficking of proteins , e.g. , enzymes or membrane receptors , are modulated by luminal [ ca ] ( booth and koch 1989 ) ; the mitochondria , where ca accumulation regulates the production of atp ( robbgaspers et al . 1998 ; the concentration of which is a key determinant of the apoptosis vs. necrosis choice , leist et al . 1997 ) , and , possibly , the release of the apoptotic cofactor cytochrome c ( krajewski et al . 1999 ) ; or the cytoplasm , where bulk or local [ ca ] increases activate enzymatic effectors , such as the ca - dependent protease , calpain ( carafoli and molinari 1998 ; squier et al . 1999 ) , or the phosphatase , calcineurin ( wang et al . 1999 ) . whether , and how , these routes interact , and how they are affected by the alteration in ca homeostasis caused by bcl-2 , awaits further investigation . | the mechanism of action of the oncogene bcl-2 , a key regulator of the apoptotic process , is still debated .
we have employed organelle - targeted chimeras of the ca2 + -sensitive photoprotein , aequorin , to investigate in detail the effect of bcl-2 overexpression on intracellular ca2 + homeostasis . in the er and the golgi apparatus , bcl-2 overexpression increases the ca2 + leak ( while leaving ca2 + accumulation unaffected ) , hence reducing the steady - state [ ca2 + ] levels . as a direct consequence ,
the [ ca2 + ] increases caused by inositol 1,4,5 trisphosphate ( ip3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria .
bcl-2 overexpression also reduced the rate of ca2 + influx activated by ca2 + store depletion , possibly by an adaptive downregulation of this pathway . by interfering with ca2 + -dependent events at multiple intracellular sites
, these effects of bcl-2 on intracellular ca2 + homeostasis may contribute to the protective role of this oncogene against programed cell death . |
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any rising prostate - specific antigen ( psa ) level above 0.2 ng / ml in patients who have undergone prior radical prostatectomy ( rp ) should be considered as a recurrence [ 1 , 2 ] . because the site of relapse , either local or distant , is sometimes equivocal [ 3 , 4 ] , absolute psa value and psa kinetic parameters such as psa velocity at recurrence , interval to psa failure , postoperative psa doubling time [ 79 ] , and preoperative psa velocity are often used to assess clinical outcome , although not always suitable to predict the type of relapse . in case of local relapse , salvage radiation therapy is able to cure selected patients [ 11 , 12 ] . nevertheless , given the risk of morbidity associated with the use of conventional three - dimensional conformal radiation therapy ( 3d - crt ) in the postoperative setting [ 1315 ] , most clinical trials have investigated the delivery of low - level radiation doses ranging from 60 to 66 gy [ 1618 ] . in contrast , more recent studies have evaluated the use of intensity - modulated radiation therapy ( imrt ) in conjunction or not with image - guided rt ( igrt ) at radiation doses up to 76 gy and demonstrated acute and late toxicity profiles similar to those obtained with conventional 3d - crt using standard doses . in our institution , we decided to exploit the dosimetric advantages of imrt , primarily not to deliver escalated doses , but to better spare the organs at risk ( oar ) and to reduce the incidence of acute and late toxicities . in this paper , we present the safety results and preliminary data regarding the short - term biochemical control of patients treated with salvage imrt to the prostatic fossa . from january , 2007 , to september 2007 , the first ten patients were treated with postoperative imrt to the prostate bed in our center . the very favorable toxicity profile observed in this series enabled us to use their dosimetric plans to set our constraints afterwards . a total of 57 consecutive patients were treated from january 2007 to february 2010 with imrt to the prostate bed for biochemical relapse after rp . patients were seen every week during the course of treatment , and acute genitourinary ( gu ) and gastrointestinal ( gi ) toxicities were scored according to the common toxicity criteria for adverse events scale ( ctcae 3.0 ) . after completion of the treatment , patients were followed every 3 to 6 months by routine examination and psa test . acute toxicity was defined as any side effect that occurred during treatment or within 3 months of the initiation of radiotherapy , and the late toxicity included new or persisting symptoms occurring 3 months or later after the start of the treatment . biochemical relapse after salvage radiotherapy was defined as a psa value of more than nadir + 0.2 ng / ml or a continued rise in the serum psa despite imrt . the use of concomitant androgen deprivation was left to the physician 's discretion , and the decision based on clinical history , pathological findings , initial staging , and patient agreement . adt consisted of lhrh agonists , sometimes associated for the first month with anti - androgen to prevent the flare - up effect . in total , 77% of patients underwent androgen deprivation , but this treatment was short course ( < 9 months ) in 84% of them ( 37/44 patients ) . patients were instructed to empty their bladder before the scan according to our in - house protocol . they underwent computed tomography ( ct)-based virtual simulation in the supine position , with knee and feet supports , but no custom immobilization device was used . online in the middle of the prostatic fossa by the treating physician immediately after the scan , while the patient waited in the treatment position on the scan table . structures were manually contoured on the ct scan according to the recommendations stemming from major randomized trials , described in more detail by the eortc radiation oncology group , the australian and new zealand radiation oncology genito - urinary group , and the rtog group [ 2022 ] . the clinical target volume ( ctv ) included the prostate bed encompassing the vesicoureteral anastomosis , and target volume delineation was based on surgical clips , preoperative imaging , operative findings , and additional information from surgical pathology . the final length of the ctv was at least 3 cm and at most the length of the prostate gland on the pathology report or preoperative imaging . the ctv was extended to the seminal vesicles in case they were still visible on the planning ct scan and included the seminal vesicle bed in case of invasion on the rp specimen . two planning target volumes ( ptvs ) were defined to account for daily set - up errors and internal motion and to provide a smooth differential dose to the ctv : ptv1 consisted of the ctv + a 3d margin of 1 cm and ptv2 consisted of the ctv + a 3d margin of 0.5 cm . the bladder was contoured in its entirety , and the rectum as a whole organ but starting 2 cm above and below the ctv . femoral heads were drawn from the top of the acetabulum to the small trochanter inferiorly . prescription doses were 68 gy in 34 fractions for 54 patients , including the patients of the dosimetric study , and 70 gy in 35 fractions for 3 patients . treatment plans were generated using eclipse software ( varian , palo alto , ca , usa ) without heterogeneity correction . patients were treated using either conventional imrt or volumetric intensity - modulated arc therapy ( rapidarc ) . for conventional imrt , the 3d - imrt beam geometry consisted of five coplanar fields with gantry angles of 60 , 95 , 180 , 265 , and 300. an 18-mv linear accelerator was used ( 21 ex , varian , palo alto , ca , usa ) and imrt was delivered using the sliding - window mode of the multileaf collimator ( mlc millenium 120 , varian , palo alto , ca , usa ) . the rapidarc technique was used in the last patients ( n = 12 ) with an optimization version 8.5 . the treatment was delivered in a 360-degree arc with a simultaneous variation of the gantry speed , dose rate , and leaf position allowing imrt dose distribution with reduced delivery time as compared to the fixed - gantry imrt solution . energy of 18 mv with a maximum dose rate of 400 monitor units per minute was used , and a collimator rotation of 45 was fixed for all patients . the minimum doses to ptv1 and ptv2 were 64 gy and 68 gy , respectively . igrt was associated with imrt using on - board digital imaging devices such as cone beam ct and kilovoltage x - ray , or megavoltage x - ray imaging . the dvh generated for the first 10 patients ( figure 1 ) demonstrated adequate coverage of the target volumes with 95% of volume ptv1 and ptv2 receiving at least 95% of the prescribed doses ( 60.4 gy and 64.6 gy , resp . ) . detailed dosimetric data are reported in table 2 for this series . in order to obtain a cohort of homogeneously treated patients , we then applied the same constraints and reached the same dose levels for all 57 patients . as summarized in table 3 , no patient developed acute gu or gi toxicity greater than grade 2 , and no patient required treatment breaks because of acute radiation - induced toxicity . grade 2 acute toxicity was reported in five patients ( 8.8% ) ( 3 gu , 1 gi , 1 both gu and gi ) . all reactions could be managed with symptomatic treatments in ambulatory setting . with a median follow - up of 21 months , there was only one case of late grade 3 toxicity ( table 3 ) . this patient developed diarrhea up to ten stools per day four months after completion of the treatment , requiring 5 days of hospitalization for rehydration and symptomatic treatment . after one month , he had recovered with regular frequency , but persisting loose stools . a single late grade 2 urinary adverse effect was observed , which was frequent urinary leakage . the short - term oncologic control was very good with only 2 cases of relapse ( 4% ) at 21 months . one patient had both biochemical and clinical relapse , with progressive nodal disease responding to androgen deprivation treatment . failure rates for patients treated with or without concomitant androgen deprivation were 2.3% ( 1/44 ) and 7.7% ( 1/13 ) , respectively . postprostatectomy irradiation improves outcome either as salvage or adjuvant therapy , but at the cost of increased gi or gu toxicity compared to no irradiation . it is not clear however to what extent this treatment - related toxicity impacts on quality of life as compared to observation since the diagnosis of cancer recurrence itself , even if only biochemical , is associated with impaired physical function [ 23 , 24 ] . it is also questionable whether adjuvant radiotherapy , systematically given to high - risk patients with no known recurrence , is a better cost - effective option than salvage treatment given to relapsing patients . from a clinical point of view , the superiority of one approach over the other will necessarily imply minimizing side effects . the recently updated results of the eortc 22911 trial presented at estro 29 ( barcelona ) and at astro 2010 did not show any overall or metastasis - free survival benefit of adjuvant radiotherapy , possibly due to the use of early salvage therapy in the control arm . that is in contradiction with the results of the swog study , which had showed a reduced risk of metastasis and an increased survival in the adjuvant arm . to date , it can not be stated whether selective salvage radiotherapy is less effective than adjuvant radiotherapy , despite a clear benefit on biochemical control in favor of adjuvant radiotherapy [ 17 , 2628 ] . some clinical trials addressing that question are underway ( radicals , getug 17 ) [ 2931 ] , and hopefully , the results should help us to better identify the patients who may avoid adjuvant treatment and to distinguish those who would benefit more from one treatment strategy than the other . there is clear evidence that the delivery of adjuvant radiotherapy to all patients with high - risk features will cause more radiation - related toxicity than salvage treatment only applied to those who relapse . consequently , having regard to the fact that prostatectomy for advanced stage including t3 disease is becoming common [ 3335 ] , and until the results from ongoing clinical trials are available , it can be hypothesized that salvage radiotherapy will be increasingly used , emphasizing the need for improving clinical outcome after treatment , both in terms of disease control and side - effects . unlike the large number of studies conducted for the use of dose - escalated radiation therapy as exclusive treatment of prostate cancer some randomized controlled trials have assessed adjuvant radiotherapy using conventional technique with prescription doses ranging from 60 to 64 gy [ 16 , 17 ] . in the salvage setting , doses up to 66 gy are usually delivered with 3d - crt , with acceptable but improvable toxicity rates . nonetheless , it remains to be proven whether further dose escalation has beneficial effect on disease - free outcome . however , imrt allows high doses to be delivered to the target volumes while limiting the radiation dose to the oar [ 3741 ] . the role of imrt has been widely studied for the treatment of localized prostate cancer , and this treatment modality has now become a standard of care for this condition [ 4246 ] . in contrast , there was little research in the postoperative setting because of the concerns regarding cumulative toxicity . imrt treatment could nevertheless provide the best therapeutic ratio , since it has been shown to decrease radiation doses in normal tissues , including the rectum and bladder [ 47 , 48 ] . on that basis , we introduced imrt in our daily practice , at a dose of 68 gy , actually not for dose escalation , but instead to minimize toxicity . previous studies addressing the question of postprostatectomy imrt had focused on different aspects , namely , the impact of dose escalation on clinical outcome [ 19 , 49 , 50 ] , the effect on erectile function , the interest in specific radiation modalities such as the whole pelvis irradiation , or the comparison between adjuvant and salvage treatment [ 5355 ] . to our knowledge , our study is the first one addressing the dosimetric feasibility and toxicity of imrt without dose escalation for salvage treatment of prostate cancer . furthermore , more than half of our patients had positive margins , which is higher than other reported studies in the salvage setting . our results compare favorably with previously published studies , with better results than those using conventional 3d - crt and similar findings to those using imrt . results are promising in terms of toxicity , but careful follow - up is required to make sure that this treatment modality would not translate into worse oncologic outcome . it is too early , and follow - up is too short to draw definite conclusions from these results with respect to disease control . first , the number of patient is rather low , but in return , the cohort is homogeneous in characteristics and treatment . the follow - up is adequate for acute toxicity and sufficient for late gastro - intestinal toxicity as it usually manifests within 2 years from the treatment . however , our follow - up is too early for a full assessment of urinary toxicity and much too short for disease control evaluation . moreover , toxicity reporting using the ctcae 3.0 scale might have underscored urgency symptoms . regarding the concomitant use of androgen deprivation with postoperative radiotherapy the rtog 85 - 31 trial evaluated the effect of immediate androgen suppression in conjunction with standard rt versus rt alone in a group of men eligible for adjuvant treatment . this study did not allow definitive conclusions , as the results were positive for freedom from biochemical relapse , but no differences were observed for overall survival , distant failure , and local control . with regard to salvage treatment , one study from the french group getug ( getug 16 ) is addressing this question , and results are pending . initial results from the rtog 96 - 01 trial presented at astro meeting 2010 evaluating the benefit of adding bicalutamide to rt for salvage treatment tend to indicate an advantage for freedom from progression . on our side , we believe that patients undergoing salvage treatment should be regarded as part of a very - high - risk group , taking into consideration failure of the first treatment , potentially driving disease progression towards more aggressive forms . until final results from ongoing trials are published , short - course concomitant androgen deprivation ( < 9 months ) should be considered for those patients , and that is the reason why the majority of our patients currently receive this combination in this setting . salvage imrt for rising psa level after rp is feasible and results in very low acute and late toxicity . a longer follow - up | background . to assess the feasibility of salvage intensity - modulated radiation therapy ( imrt ) and to examine clinical outcome .
patients and methods .
57 patients were treated with salvage imrt to the prostate bed in our center from january , 2007 , to february , 2010 .
the mean prescription dose was 68 gy in 34 fractions .
forty - four patients received concomitant androgen deprivation . results .
doses to organs at risk were low without altering target volume coverage .
salvage imrt was feasible without any grade 3 or 4 acute gastrointestinal or urinary toxicity . with a median follow - up of 21 months ,
one grade 2 urinary and 1 grade 2 rectal late toxicities were reported .
biological relapse - free survival was 96.5% ( 2.3% ( 1/44 ) relapsed with androgen suppression and 7.7% ( 1/13 ) without ) .
conclusion .
salvage imrt is feasible and results in low acute and chronic side - effects .
longer follow - up is warranted to draw conclusions in terms of oncologic control . |
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although significant progress has been made on many of the millennium development goals , many sub - saharan countries have not been able to achieve millennium development goal 5 : to reduce maternal mortality by 75% ( un 2014).1 ethiopia s efforts to reduce their 10-year static maternal mortality rate appear to have finally paid off ; the latest estimate of 420 maternal deaths per 100,000 live births exists within a relatively peaceful environment with substantial investment in maternal health.2 however , in addition to maternal mortality , there is considerable long - term suffering from maternal morbidity . obstetric fistula , a condition that classically arises when the obstructed fetal head causes prolonged compression of the bladder and vagina on to the symphysis pubis , has been a relatively common phenomenon in rural sub - saharan african countries . in a study3 of the prevalence of vaginal fistula symptoms in 19 sub - saharan countries through a meta - analysis of demographic health surveys and multiple incidence cluster surveys , the rate ranged from 0.4 fistula per 1,000 women of childbearing age in burkina faso to 19.2 fistula per 1,000 women of childbearing age in uganda . conflict and infectious disease prevented the collection of data from countries such as the republic of sierra leone and the republic of mali . although the authors quoted a fistula rate of 7.1 per 1,000 women of reproductive age in ethiopia , without clinical evaluation , these estimates are likely to be higher as other causes of incontinence are not excluded . indeed , data obtained from a large ethiopian community - based study undertaken in 2005 suggested that the prevalence was 2.2 per 1,000 reproductive age women.4 accurate figures of the prevalence of obstetric fistula are also difficult to obtain as women are often reluctant to disclose their condition and tend to isolate themselves.5 given the difficult geopolitical situation in sub - saharan africa , the relative prevalence of obstetric fistula remains difficult to accurately assess and the incidence of new cases will fluctuate as a result of conflict or outbreaks of infectious disease , making travel even more difficult . since 2005 , however , the government of ethiopia has prioritized maternal health , and in the final two phases of the health sector development program6 took steps to dramatically increase the number of health centers and maternal health professionals and to provide a fleet of ambulances for transferring women in labor to higher level care facilities . recently published data suggest that these improvements have been effective , with the 2015 prevalence of fistula in ethiopia dropping to 0.6 per 1,000 reproductive age women.7 simultaneously , the government also increased the number of medical school places and introduced an accelerated training program in emergency surgery and obstetrics for health officers.6 a key aim of the health officer s surgical training program was to produce a new cadre of health professionals , based in rural health facilities and capable of performing cesarean sections . however , the majority of referrals to hospital from the health centers are likely to be women who have been in obstructed labor for many hours , with the uterine cervix being fully dilated and with a fetal malpresentation . in such situations , surgery is likely to be technically challenging , with an increased chance of bladder and ureteric trauma leading to iatrogenic urinary fistula . this paper looks specifically at the proportion of patients with fistulae that apparently follow cesarean section or emergency hysterectomy who subsequently present to hamlin fistula ethiopia for treatment . obstetric urinary trauma can be broadly divided into low ( figure 1 ) or high ( figure 2 ) urinary fistulae . low fistulae are traditionally caused by ischemic necrosis as a result of prolonged compression of the lower vagina , urethra , and bladder base between the fetal head and the symphysis pubis . very prolonged compression can lead to a circumferential urethral fistula in which the urethra becomes disconnected from the bladder base with variable but often significant tissue loss . high juxtacervical , intracervical , or ureteric fistula usually , though not always , follows operative intervention such as cesarean section . low fistulae can also follow a successful cesarean section undertaken to relieve the obstruction , but after tissue necrosis in the lower vagina has occurred . indeed , in a study of 146 women , matthieu et al8 reported that two - thirds of the women with a low obstetric fistula following prolonged labor had a cesarean section . there is little evidence about the magnitude of iatrogenic fistula following cesarean section , although raassen et al9 in a study of 5,959 women with fistulae , from eleven countries over 18 years from 1994 , reported that 13.6% of these women were judged to have an iatrogenic fistula , the majority following cesarean section . similarly , in a retrospective analysis of 576 women presenting with an obstetric fistula between 2005 and 2007 in the democratic republic of the congo , 229 ( 40% ) were following cesarean section and 24% of these were considered to be iatrogenic.10 we present a study of secular trends in the changing incidence of low and high obstetric fistula presenting to hamlin fistula ethiopia in addis ababa over a 4-year period from january 2011 to december 2014 . this is a retrospective review of the surgical logbook in three of the hamlin fistula hospitals : addis ababa ( capital city ) , metu ( west ethiopia ) , and yirgalem ( south ethiopia ) . hamlin fistula ethiopia is a charitable organization with one main hospital in addis ababa , the capital city of ethiopia , and five outreach hospitals in different areas across the country . the treatment is completely free of charge , with transport costs and any rehabilitation needs also being catered for . it is the largest fistula organization and provides around 80% of the fistula treatments in ethiopia.8 all women attending one of the study hospitals for surgical treatment of a new ( previously untreated ) obstetric urinary fistula over the 4-year study period were included in the study . data relating to the type of fistula , treatment provided , age , and parity were retrieved from the hospital operating logbook where they were routinely recorded . patients were placed into the following groups based on age : under 20 , 2125 , 2630 , 3135 , 3640 , and greater than 41 years . fistulae were classified as either low noncircumferential , low circumferential , or high fistula on the basis of the operative record by the principle researcher . high fistulae were further subdivided into whether there was a ureteric injury or not ( table 1 ) . we did not use either of the two main classification systems for obstetric fistula ( the goh11 classification and the waadijk12 system ) as both classification systems rely on a great degree of subjectivity , and the fistulae had not been accurately scored at the time of surgery . moreover , the focus of the classification systems is on the likelihood of postclosure continence rather than etiology , and our study was looking specifically at the changing etiology of vesicovaginal fistula . the data were entered into spss version 20 ( ibm corporation , armonk , ny , usa ) for analysis . pearson s chi - squared test was used to determine relationships between parity , age , and fistula type . haenszel chi - square test was used to test for linear association between the change in fistula type over the 4 years . a p - value of < 0.05 was regarded as significant . as this was an audit of surgery carried out at a hamlin fistula ethiopia hospital by an employee of that hospital and patient anonymity was preserved , ethical clearance was not considered necessary by the ethical committee of hamlin fistula ethiopia . ethiopia did not require patient consent to be obtained as this was a retrospective study and patient anonymity was preserved . hamlin fistula ethiopia is a charitable organization with one main hospital in addis ababa , the capital city of ethiopia , and five outreach hospitals in different areas across the country . the treatment is completely free of charge , with transport costs and any rehabilitation needs also being catered for . it is the largest fistula organization and provides around 80% of the fistula treatments in ethiopia.8 all women attending one of the study hospitals for surgical treatment of a new ( previously untreated ) obstetric urinary fistula over the 4-year study period were included in the study . data relating to the type of fistula , treatment provided , age , and parity were retrieved from the hospital operating logbook where they were routinely recorded . patients were placed into the following groups based on age : under 20 , 2125 , 2630 , 3135 , 3640 , and greater than 41 years . fistulae were classified as either low noncircumferential , low circumferential , or high fistula on the basis of the operative record by the principle researcher . high fistulae were further subdivided into whether there was a ureteric injury or not ( table 1 ) . we did not use either of the two main classification systems for obstetric fistula ( the goh11 classification and the waadijk12 system ) as both classification systems rely on a great degree of subjectivity , and the fistulae had not been accurately scored at the time of surgery . moreover , the focus of the classification systems is on the likelihood of postclosure continence rather than etiology , and our study was looking specifically at the changing etiology of vesicovaginal fistula . the data were entered into spss version 20 ( ibm corporation , armonk , ny , usa ) for analysis . pearson s chi - squared test was used to determine relationships between parity , age , and fistula type . the mantel haenszel chi - square test was used to test for linear association between the change in fistula type over the 4 years . a p - value of < 0.05 was regarded as significant . as this was an audit of surgery carried out at a hamlin fistula ethiopia hospital by an employee of that hospital and patient anonymity was preserved , ethical clearance was not considered necessary by the ethical committee of hamlin fistula ethiopia . the ethical committee of hamlin fistula ethiopia did not require patient consent to be obtained as this was a retrospective study and patient anonymity was preserved . a total of 2,593 new cases of vesicovaginal fistulae were identified in the three study centers over the 4-year period . there was a significant ( p<0.001 ) decline in the annual number of fistula cases throughout the 4-year study period , dropping by 20% between 2011 and 2012 , by a further 13% from 2012 to 2013 , and a further 20% from 2013 to 2014 ( figure 3 ) . as might be expected , there was a strong relationship between low parity and young age : =722.372 , p<0.001 . the majority of women presented with a low fistula , with 1,041 ( 41% ) women having a low noncircumferential fistula and 804 ( 31% ) having a low circumferential fistula . the proportion of low noncircumferential fistula remained fairly constant over the 4 years , whereas the proportion of low circumferential fistulae declined over the study years , with 36.5% of the cases being low circumferential in 20112012 , reducing to 26.8% in 20142015 ( p<0.001 ) . a greater proportion of primiparous women ( 68.6% ) had a low circumferential fistula compared with multiparous women ( 31.4% ) ( p<0.001 ) . parity , however , was not related to the presence of a low noncircumferential fistula , with similar proportions of primiparous women ( 46.7% ) and multiparous women ( 43.3% ) presenting with low noncircumferential fistulae ( table 2 ) . a total of 638 ( 24.6% ) women had a high bladder fistula , and 110 ( 4.2% ) women had a high ureteric fistula . the proportion of high fistula increased over the years , with 26.9% of the cases being high fistula in 20112012 and 36.2% in 20142015 ( p<0.001 ) . there was a significant linear association between the increasing annual rate of high fistula and decreasing annual rate of low fistula over the years ( p=0.014 ) . a greater proportion of multiparous women had a high bladder fistula ( 70.3% ) compared with primigravid women ( 29.7% ) ( p<0.001 ) . a total of 2,593 new cases of vesicovaginal fistulae were identified in the three study centers over the 4-year period . there was a significant ( p<0.001 ) decline in the annual number of fistula cases throughout the 4-year study period , dropping by 20% between 2011 and 2012 , by a further 13% from 2012 to 2013 , and a further 20% from 2013 to 2014 ( figure 3 ) . as might be expected , there was a strong relationship between low parity and young age : =722.372 , p<0.001 . the majority of women presented with a low fistula , with 1,041 ( 41% ) women having a low noncircumferential fistula and 804 ( 31% ) having a low circumferential fistula . the proportion of low noncircumferential fistula remained fairly constant over the 4 years , whereas the proportion of low circumferential fistulae declined over the study years , with 36.5% of the cases being low circumferential in 20112012 , reducing to 26.8% in 20142015 ( p<0.001 ) . a greater proportion of primiparous women ( 68.6% ) had a low circumferential fistula compared with multiparous women ( 31.4% ) ( p<0.001 ) . parity , however , was not related to the presence of a low noncircumferential fistula , with similar proportions of primiparous women ( 46.7% ) and multiparous women ( 43.3% ) presenting with low noncircumferential fistulae ( table 2 ) . a total of 638 ( 24.6% ) women had a high bladder fistula , and 110 ( 4.2% ) women had a high ureteric fistula . the proportion of high fistula increased over the years , with 26.9% of the cases being high fistula in 20112012 and 36.2% in 20142015 ( p<0.001 ) . there was a significant linear association between the increasing annual rate of high fistula and decreasing annual rate of low fistula over the years ( p=0.014 ) . a greater proportion of multiparous women had a high bladder fistula ( 70.3% ) compared with primigravid women ( 29.7% ) ( p<0.001 ) . although this study reports a decline in the overall number of new cases of urinary fistula presenting for treatment , the type of fistula is changing , with an increased proportion of high , iatrogenic fistulae and a decreased proportion of low fistula . of particular note is the decline in the proportion of low circumferential fistulae , which are largely experienced by young primiparous women and are associated with exceptionally poor treatment outcome.14 iatrogenic fistulae , on the other hand , are generally associated with good treatment outcome , with the vast majority being cured by their surgery.9 the implication of this study , however , is that women are increasingly able to reach a place of treatment before an ischemic low fistula can occur , but some are now suffering an iatrogenic high fistula as the necessary skills or facilities to perform a cesarean section for obstructed labor when the presenting part is impacted and causing necrosis as a result of soft tissue compression . this is the first large study reporting changes in the types of new fistula being presented for treatment over a period of 4 years . although our data reflect a large proportion of the fistula treatments being provided in ethiopia , the findings are limited to the population seeking treatment at three hamlin hospitals rather than the population seeking fistula treatment across the whole of ethiopia . there are , however , only two main organizations providing the vast majority of fistula treatment within ethiopia , and the hamlin fistula ethiopia provides around 80% of the care.12 moreover , although we only have data from three of the six hamlin hospitals , these hospitals represent about two - third of the fistula workload within hamlin , and the patterns are likely to be similar in the other centers . we are , of course , unable to report on the types of fistula experienced by women who do not present for treatment . since hamlin fistula ethiopia is the biggest provider of fistula care across ethiopia , our finding of an annual reduction in number of women attending for treatment of a new fistula points toward an overall national decline in fistula prevalence . this , however , may not be the case in other sub - saharan countries where access to medical care is even poorer , although the study by matthieu et al8 suggests that in areas where there is some , if limited access to cesarean section , there is still a significant proportion of women still developing a low obstetric fistula despite surgery suggesting that referral may be delayed . indeed , a recent large community - based study in ethiopia in which the fistula was confirmed by clinical examination reports a fistula prevalence of 0.6 per 1,000 reproductive age women,7 which is a sizeable reduction from the 2.2 per 1,000 reproductive age women reported in 2005.4 the overall decline in the number of new cases by around 50% is gratifying , and the decreasing percentage of low fistulae suggests that women are gaining access to help earlier when labor becomes obstructed . the increased provision of maternal health services , improved road infrastructure , and availability of ambulances in rural areas will have undoubtedly contributed greatly to these improvements . as and when similar conditions can be established in more troubled areas of sub - saharan africa , it is hoped that similar declines in incidence will be observed . it is however important to recognize that while the rate of low circumferential fistulae is on the decline , our data show that this type of fistula is predominantly experienced by young women under the age of 25 years , with one - third of the women being under the age of 20 years . the chances of ever achieving complete continence following a circumferential fistula are poor,14 and therefore , helping women and health care professionals to recognize the signs of obstructed labor and improving the referral process to appropriate health facilities is paramount . in addition to the appalling maternal trauma , stillbirth is almost always associated3 with loss of life and increasing misery . a recent study of fistula patients15 has suggested that a simple and easily performed assessment of the pelvic outlet size could identify the group of women at particular risk of disproportion . identifying women at particular risk of fistula during antenatal assessment and encouragement to deliver their baby in a health facility might further reduce the number of women presenting with circumferential fistula . the increasing number of high fistula is more difficult to explain , and previous surgery , either cesarean section or hysterectomy , was not routinely recorded , although given our finding that high fistula was more common in multiparous women , it is likely that the fistulae occurred following operative deliveries for malpresentation at full cervical dilatation for many hours or following uterine rupture rather than absolute bony disproportion . in either of these situations , the delivery will be technically very challenging and probably beyond the capacity of newly qualified doctors with limited surgical experience or surgically trained health officers . although there appears to be limited evidence about the incidence of iatrogenic obstetric fistula , a recent systematic review and meta - analysis of six sub - saharan african studies investigating maternal and perinatal outcomes following cesarean section by doctors and surgical health officers found that while there was an increased risk of wound infections and dehiscence among women whose cesarean section was performed by surgically trained health officers , there was no increased maternal or perinatal mortality.16 none of the studies reported data on iatrogenic fistula . moreover , only one study reported on the years of experience that the doctors and health officers had , and it may be possible that outcomes equalize only after several years of practice . although this study reports a decline in the overall number of new cases of urinary fistula presenting for treatment , the type of fistula is changing , with an increased proportion of high , iatrogenic fistulae and a decreased proportion of low fistula . of particular note is the decline in the proportion of low circumferential fistulae , which are largely experienced by young primiparous women and are associated with exceptionally poor treatment outcome.14 iatrogenic fistulae , on the other hand , are generally associated with good treatment outcome , with the vast majority being cured by their surgery.9 the implication of this study , however , is that women are increasingly able to reach a place of treatment before an ischemic low fistula can occur , but some are now suffering an iatrogenic high fistula as the necessary skills or facilities to perform a cesarean section for obstructed labor when the presenting part is impacted and causing necrosis as a result of soft tissue compression . this is the first large study reporting changes in the types of new fistula being presented for treatment over a period of 4 years . although our data reflect a large proportion of the fistula treatments being provided in ethiopia , the findings are limited to the population seeking treatment at three hamlin hospitals rather than the population seeking fistula treatment across the whole of ethiopia . there are , however , only two main organizations providing the vast majority of fistula treatment within ethiopia , and the hamlin fistula ethiopia provides around 80% of the care.12 moreover , although we only have data from three of the six hamlin hospitals , these hospitals represent about two - third of the fistula workload within hamlin , and the patterns are likely to be similar in the other centers . we are , of course , unable to report on the types of fistula experienced by women who do not present for treatment . since hamlin fistula ethiopia is the biggest provider of fistula care across ethiopia , our finding of an annual reduction in number of women attending for treatment of a new fistula points toward an overall national decline in fistula prevalence . this , however , may not be the case in other sub - saharan countries where access to medical care is even poorer , although the study by matthieu et al8 suggests that in areas where there is some , if limited access to cesarean section , there is still a significant proportion of women still developing a low obstetric fistula despite surgery suggesting that referral may be delayed . indeed , a recent large community - based study in ethiopia in which the fistula was confirmed by clinical examination reports a fistula prevalence of 0.6 per 1,000 reproductive age women,7 which is a sizeable reduction from the 2.2 per 1,000 reproductive age women reported in 2005.4 the overall decline in the number of new cases by around 50% is gratifying , and the decreasing percentage of low fistulae suggests that women are gaining access to help earlier when labor becomes obstructed . the increased provision of maternal health services , improved road infrastructure , and availability of ambulances in rural areas will have undoubtedly contributed greatly to these improvements . as and when similar conditions can be established in more troubled areas of sub - saharan africa , it is hoped that similar declines in incidence will be observed . it is however important to recognize that while the rate of low circumferential fistulae is on the decline , our data show that this type of fistula is predominantly experienced by young women under the age of 25 years , with one - third of the women being under the age of 20 years . the chances of ever achieving complete continence following a circumferential fistula are poor,14 and therefore , helping women and health care professionals to recognize the signs of obstructed labor and improving the referral process to appropriate health facilities is paramount . in addition to the appalling maternal trauma , stillbirth is almost always associated3 with loss of life and increasing misery . a recent study of fistula patients15 has suggested that a simple and easily performed assessment of the pelvic outlet size could identify the group of women at particular risk of disproportion . identifying women at particular risk of fistula during antenatal assessment and encouragement to deliver their baby in a health facility the increasing number of high fistula is more difficult to explain , and previous surgery , either cesarean section or hysterectomy , was not routinely recorded , although given our finding that high fistula was more common in multiparous women , it is likely that the fistulae occurred following operative deliveries for malpresentation at full cervical dilatation for many hours or following uterine rupture rather than absolute bony disproportion . in either of these situations , the delivery will be technically very challenging and probably beyond the capacity of newly qualified doctors with limited surgical experience or surgically trained health officers . although there appears to be limited evidence about the incidence of iatrogenic obstetric fistula , a recent systematic review and meta - analysis of six sub - saharan african studies investigating maternal and perinatal outcomes following cesarean section by doctors and surgical health officers found that while there was an increased risk of wound infections and dehiscence among women whose cesarean section was performed by surgically trained health officers , there was no increased maternal or perinatal mortality.16 none of the studies reported data on iatrogenic fistula . moreover , only one study reported on the years of experience that the doctors and health officers had , and it may be possible that outcomes equalize only after several years of practice . ensuring that there are appropriately trained and skilled obstetricians in rural areas is a particular challenge , but addressing both this and the detection of pelvic contraction would have the potential to make the reduction in obstetric fistula the rarity it should be . these figures also point to the need for an audit of indications for and outcome of cesarean sections performed in these health facilities , similar to the maternal death surveillance programs now being carried out . repair of high fistulae , though technically challenging , has good prognostic functional outcomes , as the bladder base and urethra are not irretrievably damaged . low and circumferential fistulae occur in young women and despite treatment have much poorer outcomes in terms of persistent problems with incontinence . ethiopia has made significant steps to reduce obstetric fistula.13 other sub - saharan countries should be able to follow suit . early recognition of obstruction or malpresentation and prompt competent surgery would solve the problem of obstetric fistula . while the overall incidence of obstetric fistula appears to be declining , the number of women having iatrogenic fistulae has increased over the past few years . the reduction in fistula has likely arisen from increased uptake of maternal health services for delivery . however , the increase in iatrogenic fistula suggests that women may be presenting late at health facilities and requiring complex cesarean sections . although surgical training for health officers offers a possible solution , they may not yet have the experience to undertake complex cesarean deliveries in what are often difficult surgical circumstances . | objectiveto examine the incidence and type of obstetric fistula presenting to hamlin fistula ethiopia over a 4-year period.study designthis is a 4-year retrospective survey of obstetric fistula treated at three hamlin fistula hospitals in ethiopia , where approximately half of all women in the country are treated .
the operation logbook was reviewed to identify all new cases of obstetric fistula presenting from 2011 to 2015 .
new cases of urinary fistula were classified by fistula type ( high or low ) , age , and parity of the woman.resultsin total , 2,593 new cases of urinary fistulae were identified in the study period .
the number of new cases fell by 20% per year over the 4 years ( p<0.001 ) .
a total of 1,845 cases ( 71.1% ) were low ( ischemic ) fistulae , and 804 cases ( 43.6% ) of these had an extreme form of low circumferential fistula .
a total of 638 ( 24.6% ) women had a high bladder fistula , which predominantly occurs following surgery , specifically cesarean section or emergency hysterectomy , and 110 ( 4.2% ) women had a ureteric fistula .
the incidence of high fistulae increased over the study period from 26.9% to 36.2% ( p<0.001 ) .
a greater proportion of multiparous women had a high bladder fistula ( 70.3% ) compared with primigravid women ( 29.7% ) ( p<0.001 ) .
conversely , a greater proportion of primiparous women experienced a low circumferential fistulae ( 68.6% ) compared with multiparous women ( 31.4% ) ( p<0.001).conclusionthere appears to be a decline in the number of ethiopian women being treated for new obstetric urinary fistulae .
however , the type of fistula being presented for treatment is changing , with a rise in high fistulae that very likely occurred following cesarean section and a decline in the classic low fistulae that arise following obstructed childbirth . |
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during recent years , infection of acinetobacter baumanii showed a rapid growth in hospitals and community . baumanii infection mainly occurs in the lung and blood , the notable feature being that multi - drug resistant strains cause outbreaks in hospitals . large outbreaks of a. baumanii infection have been reported in hospitals from usa , france , spain , belgium , britain and south korea , which brought great economic loss to the patients and hospitals ( 15 ) . recently , a. baumanii infection increases the mortality rate by 7.823% through cohort and case - control studies ( 6 ) . in the past few years , reports about breaks of a. baumanii infection have also increased in china . since the outbreak among over 20 wards in beijing union medical college hospital in 2004 , similar cases have been reported in a number of cities , including chongqing and hangzhou ( 710 ) . when pathogens invade the body , the natural immune system recognizes foreign pathogens through pattern recognition receptors . so far , toll - like receptors ( tlrs ) , a member of the interleukin-1 receptor ( il-1r ) superfamily , are regarded as the most important pattern recognition receptors . tlr-1 , tlr-6 and tlr-10 do not recognize the ligand directly , and play their roles by forming heterodimers with other tlrs . although tlr-7 and tlr-8 were reported to interact with imidazoquinoline , the synthetic and anti - viral small molecule , their natural ligands have not been identified . currently , researches about tlr-11 are few , and lipopolysaccharide ( lps ) , pgn or poly ( i : c ) can not stimulate nf-b activation in tlr-11-expressing cells . therefore , tlr-2 , tlr-4 , tlr-5 and tlr-9 are the most important tlr members related to the immunal protection against bacteria in the body . more and more studies indicate that single nucleotide polymorphisms ( snps ) in tlr-2 , tlr-4 , tlr-5 and tlr-9 are closely related to the susceptibility to diseases in humans . tlr-2 has a molecular weight of 89.8 kda , forms heterodimers with tlr-1 or tlr-6 , and interacts with bacterial ligands such as lipopeptides , peptidoglycan and membrane fatty acid of gram - positive bacteria ( 11 ) . one variant of tlr-2 ( g2477a / arg753gln ) , with an incidence of 2.79.4% in the population , is associated with the susceptibility to asthma , atopic dermatitis and the infection of mycobacterium tuberculosis , staphylococcus aureus and candida ( 12 , 13 ) . another variant of tlr-2 ( c2029t / arg677trp ) is related to the pathogenesis of lepromatous leprosy in the asian population . furthermore , the levels of interleukin-12 ( il-12 ) are decreased in peoples carrying this polymorphism ( 14 ) . tlr-4 , a protein with 839 amino acids and a molecular weight of 95.6kda , can form homodimers . tlr-4 recognizes heat shock proteins and cilia of gram - positive bacteria , envelope glycoproteins of retroviruses , heat - labile compositions of mycobacteria , as well as muramic acids of spirochetes ( 15 ) . furthermore , tlr-4 plays a definite and critial role during recognizing lps of escherichia coli , neisseria meningitidis , salmonella and legionella pneumophila , as well as subsequent signal transduction ( 14 ) . two common snps in tlr-4 , a1287g / asp299gly and c13174t / thr399ile , change its extracellular structure , slow its response to lps and increase the incidence of septic shock ( 16 ) . besides , these snps are also closely related to the pathogenesis of severe respiratory syncytial virus ( rsv ) bronchiolitis ( 17 ) . another two common snps in tlr-4 ( a896 g and c1196 t ) have protective effects against the infection of legionella pneumophila ( 18 ) . tlr-5 contains 858 amino acids with a molecular weight of 97.7 kda , and can form homodimers or heterodimers with tlr-4 . tlr-5 recognizes flagellin , and induces production of high levels of interleukin-8 ( il-8 ) . one tlr-5 variant ( 1174c / t ) is related to the susceptibility to infection of l. pneumophila(19 ) . it was reported that a polymorphism in the tlr9 promoter ( 1237t / c ) is related to the susceptibility to asthma , eczema , and crohn s disease ( 14 ) . despite the prevalence of a. baumanii infection in many hospitals and communities , the reason underlying a. baumanii outbreaks remains unclear . previous studies mainly focus on the mechanism of drug resistance of bacteria , ignoring the role of the interaction between the susceptibility to infection and the baterial genotype . in this study , we investigated the mechanism of a. baumanii infection from the perspective of host genetics . we screened snps in tlr-2 , tlr-4 , tlr-5 and tlr-9 , and studied whether polymorphisms in tlrs were associated with the susceptibility to a. baumanii infection . the study was approved by the review board / ethics committees of the chinese pla general hospital . cases of a. baumanii infection were collected in major cities of china using the national hospital infection surveillance network . three hundred and eighty five uninfected patients from the same ward and with the same gender were selected as the controls . besides , the ages of infected and uninfected patients from the same ward were less than 2 years . to reduce the interference of population stratification , all the patients in this study were han chinese . genomic dna was extracted using qiagen blood mini kit ( qiagen , ca , usa ) , and stored at 80 c . thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed ( ta . snps were selected according to their frequencies and positions , as well as their associations with infectious diseases reported previously . snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique ( genesky biotechnologies inc . , the genotyping success rates of rs3804099 , rs7656411 , rs76112010 , rs1927914 , rs10759932 , rs11536889 , rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 , rs17163737 and rs187084 were 99.5% , 99.3% , 99.5% , 99.4% , 99.5% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% and 99.5% , respectively . the association of a. baumanii infection with gender and age were examined using the pearson chi - square test and the t test , respectively . the chi - square tests were used to determine whether each polymorphism was in hardy weinberg equilibrium . a multiple logistic regression model was used to investigate the individual effect of tlr-2 , tlr-4 , tlr-5 and tlr-9 snps on a. baumanii infection . the analysis was based on additive , recessive and dominant models , and adjusted for age . the odd ratios ( ors ) with 95% confidence intervals ( cis ) were presented . the statistical analysis was performed using snpalyze v4.0 ( dynacom , kanagawa , japan ) . the study was approved by the review board / ethics committees of the chinese pla general hospital . cases of a. baumanii infection were collected in major cities of china using the national hospital infection surveillance network . three hundred and eighty five uninfected patients from the same ward and with the same gender were selected as the controls . besides , the ages of infected and uninfected patients from the same ward were less than 2 years . to reduce the interference of population stratification , all the patients in this study were han chinese . genomic dna was extracted using qiagen blood mini kit ( qiagen , ca , usa ) , and stored at 80 c . thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed ( ta . snps were selected according to their frequencies and positions , as well as their associations with infectious diseases reported previously . snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique ( genesky biotechnologies inc . , the genotyping success rates of rs3804099 , rs7656411 , rs76112010 , rs1927914 , rs10759932 , rs11536889 , rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 , rs17163737 and rs187084 were 99.5% , 99.3% , 99.5% , 99.4% , 99.5% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% , 99.8% and 99.5% , respectively . the association of a. baumanii infection with gender and age were examined using the pearson chi - square test and the t test , respectively . the chi - square tests were used to determine whether each polymorphism was in hardy weinberg equilibrium . a multiple logistic regression model was used to investigate the individual effect of tlr-2 , tlr-4 , tlr-5 and tlr-9 snps on a. baumanii infection . the analysis was based on additive , recessive and dominant models , and adjusted for age . the odd ratios ( ors ) with 95% confidence intervals ( cis ) were presented . the statistical analysis was performed using snpalyze v4.0 ( dynacom , kanagawa , japan ) . we selected 13 snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes ( table 1 ) . the pearson chi - square test showed that the susceptibility to a. baumanii infection was not associated with the gender significantly ( p = 0.98 ) . however , a. baumanii - infection increased with ages , as indicated by the t test ( p = 8.6 10 ) . characteristics of the studied snps at the tlr-2 , tlr-4 , tlr-5 and tlr-9 genes maf , minor allele frequency ; hwe , hardy weinberg equilibrium ; statistically significant values are shown in bold . the positions and minor allele frequencies ( mafs ) of the snps all snps are in hardy - weinberg equilibrium ( hwe ) , except for rs1341987 ( p = 0.046 in control patients ) and rs1640827 ( p = 0.021 in control patients ) of tlr-5 and rs187084 of tlr-9 ( p = 0.021 in control patients ) . the associations of tlr2 , tlr4 , tlr5 and tlr9 gene polymorphisms with a. baumanii infection after logistic regression analysis are shown in table 2 . the snp of tlr-9 , rs187084 , was related to a. baumanii - infection under the additive model ( 2a / a + g / a , to g / g , p = 0.05 , or = 0.82 , 95% ci = 0.671.00 ) and recessive model ( g / g , to a / a + g / a , p = 0.0064 , or = 0.59 , 95% ci = 0.400.86 ) after adjustment with age . however , as mentioned above , rs187084 did not obey the hwe rule in control patients . association of tlr-2 , tlr-4 , tlr-5 and tlr-9 gene polymorphisms with ab infection after logistic regression analysis or : odd ratio ; ci : confidence interval . we found a significant association of the haplotype gcg of tlr-4 with a. baumanii - infection ( p = 0.027 ) . no significant differences were detected in the distribution of tlr-2 and tlr-5 haplotypes between a. baumanii - infected patients and controls . compared with the common haplotype atg of tlr-4 , the haplotye gcg contained two mutations both in the promoter region ( table 1 ) . haplotype distribution of tlr-2 , tlr-4 and tlr-5 polymorphisms in ab infected patients and controls haplotype : tlr-2 [ rs76112010 , rs3804099 , rs7656411 ] ; tlr-4 [ rs1927914 , rs10759932 , rs11536889 ] ; tlr-5 [ rs1861172 , rs5744174 , rs2241097 , rs17163737 ] . frequency : the haplotypefrequency in the whole population including ab - infected patients and controls . or : odd ratio ; ci : confidence interval . the major risk factors of a. baumanii infection include the severity of the underlying disease , surgical operations , a large area of trauma ( especially burns ) , premature birth , ever living in icu , prolonged hospitalization , contact with contaminated ward or equipment , invasive procedures ( such as mechanical ventilation , in - dwelling tubes , etc . ) , and ever using antibiotics . although these factors are statistically associated with a. baumanii infection , they can not accurately predict the risk of exposure , it is difficult to explain why some patients exposed to these factors are infected , and some are not . tlrs are the most important pattern recognition receptors , through which the natural immune system recognizes foreign pathogens . many studies indicate that gene polymorphisms in tlrs are closely related to the susceptibility to diseases in humans . although tlr9 detect bacterial dna and recognize dsdna from viruses and the genomes of protozoa ( 20 ) , and the innate immune system appears to use tlr9 for detecting unmethylated cpg dinucleotides , which are relatively common in bacterial and viral genomes but are highly methylated and rare in vertebrate genomes ( 21 , 22 ) , but in this study , the snp of tlr-9 , rs187084 , was significantly related to a. baumanii infection especially under the recessive model . however , this snp was not in hwe in control patients . therefore , more random and more extensive sampling may be required to confirm further this association . interestingly , the haplotye gcg contained two mutations both in the promoter region , compared with the common haplotype atg . we speculate that these two snps in the tlr-4 promoter , rs1927914 and rs10759932 , influence its transcriptional regulation . one snp of tlr-9 ( rs187084 ) and the haplotype gcg of tlr-4 are significantly associated with a. baumanii infection . our studies provide insights to the mechanism of a. baumanii infection from the perspective of the host . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | background : during recent years , infection of acinetobacter baumanii showed a rapid growth in hospitals and community .
toll - like receptors ( tlrs ) are the most important pattern recognition receptors , which play a critical role during recognizing invading pathogens by the natural immune system .
our objective was to determine the associations of tlrs polymorphisms with the susceptibility to a. baumanii infection in a chinese population.methods:we carried out a case - control study , genotyping 13 polymorphisms of tlr-2 , tlr-4 , tlr-5 and tlr-9 genes on 423 a. baumanii - infected patients and 385 exposed controls .
thirteen snps at the tlr-2 ( rs3804099 , rs7656411 and rs76112010 ) , tlr-4 ( rs1927914 , rs10759932 and rs11536889 ) , tlr-5 ( rs1341987 , rs1640827 , rs1861172 , rs2241097 , rs5744174 and rs17163737 ) and tlr9 ( rs187084 ) genes were analyzed .
snp genotyping was performed using an improved multiplex ligation detection reaction ( imldr ) technique.results:the snp of tlr-9 , rs187084 , was related to a. baumanii - infection significantly under recessive model ( g / g , to a / a + g / a , p = 0.0064 , or = 0.59 , 95% ci = 0.400.86 ) after adjustment with age . besides , the haplotype gcg of tlr-4 was significantly associated with a. baumanii infection ( p = 0.027).conclusion : tlr-4 and tlr-9 may be related to the susceptibility to a. baumanii infection in a chinese population . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the beef tapeworm ( taenia saginata ) is a common infection of both humans and cattle around the world , especially in areas where beef is eaten . areas where the infection has high prevalence include sub - saharan africa , southeast asia and the middle east . it does occur in the united states , but it is not common due to high sanitation standards . a 47-year - old woman was admitted to our outpatient clinic with a 2-month history of intermittent epigastric pain . her physical examination and the results of routine blood tests were unremarkable . her abdominal x - ray and abdominal ultrasonography was normal . on upper gastroduodenal endoscopy , a live tapeworm was found ( fig.1 ) , extending from the proximal corpus to the third portion of the duodenum , during the endoscopy worm curls around the endoscope as it supposes the scope is the same of its kind . but after the scope hits and pursues the worm , it escapes . in the upper gastroduodenal endoscopy ; there was pale antral mucosa with a shiny surface and mild nodularity , which gastric biopsy revealed antral atrophic gastritis with h. pylori infection . the patient was treated with single dose ( 600 mg ) of praziquantel for t. saginata infestation and we decided to follow up the patient for her chronic atrophic gastritis . after 1 month , she was symptom free and specific exams showed no trace of the parasite . the patient was given a single dose of praziquantel : 15 mg / kg body weight . parasitological controls ( two series of three fecal samples each ) , perfor - med two months later , were negative for proglottides in stool specimens . taenia saginata , or the beef tapeworm , is a parasite of both cattle and humans , which can only reproduce in humans . adult worms residing in the small intestine of a human will lay eggs in the proglottids , which are then released in feces . once proglottids eaten by cattle , the eggs released in the stomach , the digestive enzymes will break down the eggs , allowing the development of oncos - pheres , or zygotes . the zygotes penetrate through the mucous lining of the digestive tract and circulate through the blood stream , where they develop into small larvae . larva will develop into cysts with the muscle tissue and have been found in the liver and the lungs of the intermediate host . in humans , once the stomach acids break down the cysticercus ( cyst containing larva ) , the worm will move into the intestine to evolution . the worm after about two months , reaches an average size of about sixteen feet . the beef tapeworm can reach an average body length between thirteen and thirty - two feet , although some individuals can grow to be thirty - nine feet in length . it can live to be twenty - five years of age within a host ( 1 , 2 ) most taeniasis infections are asymptomatic . when symptoms occur , hosts may experience abdominal pain , headaches , dizziness , weight loss , diarrhea , nausea , constipation , and chronic indigestion . in humans , the tapeworm may cause a blockage in the intestine or cause an allergic reaction when it releases antigens . taeniasis due to t. saginata is relatively common in africa , some parts of eastern europe , the philippines , russia , and latin america . it is rare in the united states , except in places where cattle and people are concentrated and sanitation is poor . in iran , taeniasis is common particularly on western and central regions of the caspian sea ( 3 ) . other reported compli - cations include intestinal obstruction ( 2 ) , obstruction of bile ducts or pancreatic duct ( 4 ) , abnormal vaginal bleeding ( 5 ) and rarely anastomotic leak ( 6 ) or granulomatous gastritis ( 7 ) . taeniasis can be treated using medicines including praziquantel ( 10mg / kg ) ( 9 ) which paralyzes the worm and allows the host to flush it out of the intestines . in order to prevent an infection in humans , it is important to exactly cook beef products . salting or freezing the beef products will also kills the infective larvae within the meat , but other measures , like sanitary disposal of human waste , should be taken . in this patient , t. saginata upward migrated from small intestine to the stomach . because of the high gastric acidity , worm skedaddled but in this case because of ppi use and coexistence h. pylori infection , acidity of stomach ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | abstracta case of a 40-year - old female , in whom a 6-meter long worm ( taenia saginata ) was found in stomach , is reported here . in this patient ,
t. saginata upward migration of the worm to the stomach , its rare phenomenon , worm mostly seen in the small intestine .
this is mainly because of the high gastric acidity . in this patient
, we believe proton pump inhibitor ( ppi ) use caused hypochlorhydria and coexistence h. pylori infection caused chronic atrophic gastritis , which resulted in the retrograde migration of the tapeworm to the stomach in our patient . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
giant uterine fibroid is a rare tumor of the uterus , usually symptomatic requiring surgical intervention . in this study , a case of 31 year old nullipara was reported who presented to the gynaecology unit of enugu state university teaching hospital ( esuth ) , nigeria with a history of progressive abdominal swelling of 8 years duration , six years duration of infertility and weight loss and dyspnoea on exertion . physical examination and the surgical option of management should consider the fertility preservation and aspiration of couples especially in developing countries . giant uterine leiomyomata are those tumors weighing 11.4 kg or more . giant uterine fibroids are rare and usually symptomatic . uterine leiomyomata are the commonest benign tumors of the female reproductive tract arising from smooth muscle cells of the uterus it is estimated that by their fifth decade , as many as 50% of black women will have leimyomata ( 1 , 2 ) . although uterine leiomyomata are asymptomatic in 50% of cases , they may cause menorrhagia , dysmenorrhoea , abdominal swelling and pressure symptoms ( 3 ) . it is a common gynaecological tumor in nigeria ( 4 , 5 ) . in enugu , nigeria , although there have been reports of giant uterine fibroid from different parts of the world , our literature search did not yield any previous report of giant uterine fibroid from the south eastern region of nigeria . in this study , a case of a nullipara was reported who had a giant uterine fibroid which was managed by a myomectomy and the woman subsequently achieved a live birth . a 31 year old nulliparous housewife presented initially to the gynaecology unit of enugu state university teaching hospital ( esuth ) , nigeria in february 2012 with a history of progressive abdominal swelling of 8 years duration , six years duration of infertility and weight loss and dyspnoea on exertion . she had been diagnosed to have uterine fibroid 6 years prior to presentation and had refused to have surgery due to her belief that the fibroid and infertility were spiritually inflicted on her by her enemies . she took local medications with the belief that it would dissolve the fibroid . physical examination revealed a cachectic woman , who was pale and had a massively protuberant abdomen . there was a firm nodular abdominal mass measuring 52 cm from the pubic symphysis to the epigastrium . the patient 's abdomen at presentation the patient had hemoglobin level of 7.3 g / dl ( pcv = 22% ) . the marker of ca-125 , ct scan and mri could not be done due to their cost and unavailability . she was counseled for surgery . she consented to myomectomy with an option of hysterectomy as a life saving procedure , as she desired to have children . intra - operative findings included very thin subcutaneous tissue , 300 ml of ascitic fluid , collateral blood vessels involving the omentum and rectus muscle . large conglomerate fibroid masses measuring 2432 cm with a broad stalk connecting it to the fundus of the uterus ( figure 2 ) was seen . the aggregate weight of all the fibroid extracted was 16.8 kg . estimated blood loss was 1000 ml . intra - operative picture of the uterus delivered through the incision site before the removal of the fibroid masses a size 16 urethral catheter was tied round the uterus at the isthmic region i.e. level of the internal os to control bleeding at surgery . the pedun - culated one was then excised at the base . three subserous fibroids measuring 4.56.2 cm , 6.06.2 cm , 5.48.7 cm respectively were found and two intramural fibroid seedlings were enucleated . abdominal skin stitches were removed on the 7 th post - operative day ( figure 3 ) . the abdominal wall after removal of skin stitches on the 7th post operative day she was followed up at the gynaecology outpatient clinic . seven months later , she presented to the clinic with history of nausea and amenorrhea . she had an elective caesarean section at 38 weeks of gestational age and delivered a live female baby that weighed 2.8 kg . giant uterine fibroid has been reported to be exceedingly rare ( 2 , 6 ) . however , cases have been reported by different authors from different parts of the world ( 1 , 7 ) . report shows that the largest uterine tumor ever removed from a patient that survived the procedure weighed 45.4 kg ( 8) . prior to this feat , a uterine fibroid mass that weighed 63.3 kg had earlier been removed in a women postmortem ( 8) . the uterine fibroid extracted from our patient had an aggregate weight of 16.8 kg . it is a common indication for hysterectomy in nigeria ( 4 , 5 ) . about 36.8% of women who had symptomatic uterine fibroid had total abdominal hysterectomy with or without bilateral salpingo- oophorectomy in enugu , nigeria ( 5 ) . due to the distensibility of the anterior abdominal wall and the large volume of the abdominal cavity , uterine fibroid can grow into extremely large sizes . recently , cases of a giant leiomyoma mimicking an ovarian malignancy were reported ( 9 , 10 ) . imaging studies such as ultrasound , computer tomography , magnetic resonance imaging and tumor markers have been reported to be helpful in determining the extent of the mass and likelihood of malignancy ( 8) . in nigeria , like in many low income countries , except for ultrasound , these facilities are not readily available . there is no single best approach to uterine fibroid treatment . however , giant uterine fibroids are best treated surgically and require adequate pre - operative preparations and an experienced and skillful surgeon . many have reported total abdominal hysterectomy with or without salpingo- oophorectomy for giant uterine fibroids ( 1 , 2 , 6 ) . however , in nigeria , like in many developing countries , there is a strong desire among women to have children . in most of these countries , reproductive failure is attributed to women who are childless ( 11 ) , making hysterectomy a less desirable option for some patients . the surgical option for management of giant uterine fibroids should be individualized and where possible , conservation of the uterus for those women who earnestly desire future child bearing should be considered . our patient had successful myomectomy and achieved spontaneous conception about seven months after the surgery . post - operative complications are major challenges that could be prevented through adequate pre - operative preparation of the patient , especially through optimisation of haematological status , good surgical skills ( with a careful attention to collateral vessel for proper ligation ) and close post - operative management which would enable early detection and management of post - operative complications ( 12 ) . although giant uterine fibroid is relatively rare , deciding on the best surgical option of management that considers the fertility wish and aspiration of couples in developing countries could be challenging . the surgical option for the management of giant uterine fibroid in developing countries should be individualized to the best interest of the patient . | backgroundgiant uterine fibroid is a rare tumor of the uterus , usually symptomatic requiring surgical intervention .
uterine fibroid is generally common among black women.case presentationin this study , a case of 31 year old nullipara was reported who presented to the gynaecology unit of enugu state university teaching hospital ( esuth ) , nigeria with a history of progressive abdominal swelling of 8 years duration , six years duration of infertility and weight loss and dyspnoea on exertion .
physical examination and transabdominal ultrasound revealed a huge abdominopelvic mass .
she had myomectomy .
she had a histologically confirmed giant uterine fibroid that weighed 16.8 kg .
she subsequently achieved a live birth.conclusiongiant uterine fibroids are relatively rare .
it poses great challenges in its management .
the surgical option of management should consider the fertility preservation and aspiration of couples especially in developing countries . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
our aim was to create a tool that would allow the construction of gene lists with homology to any specified protein domain , coupled with the capability to analyze the expression levels of these genes as provided by gene microarray experiments . our motivation to develop such a resource originated from the general lack of such interactivity between existing highly used online resources . we specifically intended to develop a resource that could rapidly provide exhaustive gene - type classification information , concomitantly with expression information of the entire set of members within a specific species type . such a working profile suits numerous areas of investigation , as it is often the case that a priori knowledge of gene family involvement in a given biological process is available , but the individual gene member is unknown . however , if exhaustive gene family lists could be generated with ease , the coupling of such lists to an extensive gene expression base , should help in narrowing down family members playing tissue - specific roles . if such a tool was available , we reasoned it would benefit investigators in a diverse range of disciplines . the core knowledgebase of genespeed is the comprehensive homology categorization of all pfam ( 1 ) protein domain homology within the unigene transcriptomes ( 2 ) of human , mouse , worm and fly , without which its development would have been impossible . this dataset facilitates the generation of gene lists with homology to any specified protein domain by first allowing the user to identify a single protein domain that may characterize a certain protein family of interest and then searching the transcriptome of one of these species for all translations containing homology to this domain . the current version of genespeed only allows queries of single domains ; however , we plan to implement the ability for multiple domain searches in a future version . the resultant list will contain all genes in the transcriptome with homology to the protein domain of interest ( displayed with decreasing similarity ) . in order to assist the user in identifying an initial protein domain of interest the first takes advantage of a custom / manually curated transcription factor characterization of all domains within pfam ( currently 7973 domains ) so that an investigator may browse by transcription factor type . second , we incorporated the gene ontology ( go ) characterization of domains ( 3 ) , which allows browsing by the go organizational tree and the ability to generate a gene list from any specified go node . the third technique allows a more traditional query by gene / domain name , symbol or accession identifier . finally , the capability to search the genespeed database by fold expression level or by tissue specificity . this is made possible through the incorporation of the novartis symatlas2 dataset ( 4 ) , containing expression data from 280 microarrays of 61 mouse and 79 human tissues . these integrated expression data also serve an exploratory role , in that the genes within gene lists generated by the other query techniques mentioned above may be analyzed for fold - change from median , tissue specificity as well as absolute expression level . all resulting datasets and tools were organized into a lamp ( linux / apache / mysql / php ) based dynamic website named genespeed and is available at . the process for populating the genespeed database is illustrated in figure 1 , and is nearly fully automated . ( i ) the first step involves obtaining the full set of pfam domains from the pfam database ( currently representing 7973 domain families ) . ( ii ) a single domain family is selected out of the total of 7973 ( in this example the c2h2-zinc finger domain will be used ) . full pfam alignment file for the c2h2-zinc finger domain ( containing 32 874 distinct c2h2-zinc finger protein sequences from a multitude of different species ) is generated . this process involves using the tblastn algorithm ( 5 ) to search for homology for each of the 38 874 c2h2-zinc finger domains within the unigene est databases for mus musculus , homo sapiens , caenorhabditis elegans and drosophila melanogaster . in order to preserve protein id / aa - aa / e - score value custom perl and python scripts were utilized to perform all tblastn homology searches using the blast pbh cluster maintained at massively parallel technologies inc . ( v ) the tblastn output for this pfam domain is subsequently parsed for expectancy score ( e - score ) , unigene i d , genbank i d , pfam i d , domain range and domain size . these results are sorted first by e - score and then by unigene i d and subsequently all redundancy was eliminated by retaining only the most significant e - score containing record for each unique homologous unigene hit . ( vii ) this process is then repeated for each of the 7973 pfam domains . ( viii ) in addition to the results of the tblastn population process , several other sources of data were incorporated into the genespeed database . information from external databases including unigene ( 2 ) , interpro ( 3 ) , go ( 6 ) and the novartis symatlas2 ( 4 ) were included . from the most recent build of the unigene database we incorporated the unigene i d , gene symbol , gene name , entrez gene , chromosome and cytoband location . in addition , through retired unigene information tables , we implemented a tool that automatically checks for unigene ids that have been retired from the database . when uploading a custom gene list into genespeed , this script scans the list for all retired or changed unigene identifiers . once a retired unigene i d is discovered , the most recent identifier(s ) is displayed for the user . from interpro , we obtained all interpro ids and gene ontology ( go ) information relating domain families to go categories . this information allowed the development of a go browsing tool to search for genes containing domains that have an associated go classification ( 3,6 ) . as a result , these incorporated go classifications may allow for gene - to - gene inference based on an original go - member domain . however , care should be exercised , as protein homology does not always equate functional similarity , especially at the lower go - node levels . as given in the figure : ( 1 ) all domain information and alignments in the pfam database are downloaded from pfam ; ( 2 ) a single domain is selected ( c2h2-zinc finger in this example ) ; ( 3 ) the full alignment ( in this case the c2h2-zinc finger domain full alignment contains 32 874 distinct protein sequences ) is used in a ( 4 ) batch ncbi - based tblastn using the massively parallel pbh blast server ; ( 5 ) tblastn output is parsed and redundancy eliminated ; ( 6 ) non - redundant data are banked into a custom mysql database ; ( 7 ) the process is repeated for all domain families in pfam ( currently 7973 ) ; ( 8) other datasets are integrated with the database . as mentioned above , array data for the 79 human and 61 mouse tissues ( in duplicate ) of the novartis symatlas2 dataset ( 4 ) were included into the genespeed database . these array data were downloaded from novartis ( ) in its mas5 ( affymetrix ) normalized form . in addition to incorporating the absolute expression normalized values for each probeset into the database , we also pre - computed the fold - change differential from median as well as performing an anova statistical test to determine a p - value for tissue specificity . this anova computation compared the mean expression value of a gene for one tissue against the mean expression value of the same gene in all other tissues . such a calculation was repeated for all genes within each tissue type for both human and mouse tissues . all computations were performed within the r statistical environment ( ) running on a linux mandrake 10.2 operating system ( ) . given personal interests in the regulatory dna - binding factors , we also incorporated a custom transcription factor classification of all 7973 pfam protein domains . modeling the outline of a previous classification scheme ( 7 ) , domains within the pfam database were manually evaluated for their relatedness with transcription factors and classified into three hierarchies of superclass , class and subclass . relatedness in this case refers to any domains characteristic of or associated with known transcription factors ( i.e. associated with text queries of relatedness , however , does not imply an absolute transcription factor classification for any particular domain . to clarify this point with an example , the user may browse the genespeed m.musculus division of the database for the transcription factor superclass of helix turn helix ( hth ) and class of fork head / winged helix. this yields a list of domains including the fork head domain as well as 13 other domains and a total of 480 genes . the fork head domain is the signature domain that definitively characterizes the hth and fork head / winged helix transcription factor ; however , 13 other domains have also been found to be associated with these transcription factor proteins . thus , if an interpro - based sub - search of this list is performed for all genes in genespeed that only contain the fork head domain ( pf00250 , ipr001766 ) , the gene list shrinks from 480 to 38 . in translation , 480 genes were identified that contain domains ( albeit some with very low similarity ) that have been associated with fork head / winged helix transcription factors and 38 genes contain a definitive fork head domain . an update consists of running the complete database generation process from the most recent versions of all information / database sources . a general navigation overview for the genespeed database is illustrated in figure 2 . in order to permit users to save custom gene lists while searching the genespeed database , all users of the database , they may browse genespeed , perform queries , create custom gene lists and save these lists into their account . guest users have full access to all database content ; however , to save gene lists , a free account must be established . there is also an upload tool implemented allowing users to upload their own gene lists into the database . this tool receives ( as upload from user ) a unigene gene list , scans the list for all retired or changed unigene identifiers and updates all retired unigene identifiers to the most recent build of the unigene database . any discrepancies , such as retired or divided clusters , will be displayed to the user . once the user 's external list has been uploaded into the database , all the tools of genespeed may be used on the new uploaded list . the primary rationale for including the import tool is to provide rapid domain assignment and other annotation within the genespeed environment for gene lists obtained from external experiments . users may log back into the database at any time to access their previously saved custom gene lists . each user 's gene list information is protected by login name and password and may not be observed by any other user . if using genespeed as a guest user , only a single gene list may be created and manipulated at a given time . ( a. search ) a search is started by first selecting the organism and then the search type . ( b. results ) the results page will display the number of unigene clusters ( genes ) found and the user then has the option to select additional specific information they would like displayed concerning these genes . ( c. sub - searches / tool ) other sub - searches / tools are available to refine the original search including domain sub - search ( finds all domains in a specific gene ) , interpro sub - search ( finds all genes with a specific domain ) , external links ( www links to outside databases ) and novartis symatlas2 ( investigate the expression level of any number of genes with the data contained within the norvartis symatlas2 dataset including 79 human and 61 mouse tissues ) . ( d. user account ) each user is provided with a private account , in which they may store any number of user - specified gene lists to keep for analysis at any later time point . upload tools are also provided , allowing users to analyze any gene list not generated within the genespeed database . a new search ( figure 2 , a. search ) starts with selecting the organism and then the type of search , which includes ( i ) keyword , search the gene name and ( ii ) i d or accession , search by unigene , entrez gene , chromosome or interpro . ( iv ) transcription factor classification , to browse genespeed 's custom transcription factor classification hierarchy . ( v ) microarray expression data , allowing the user to query the database by fold - change from median as well as tissue specificity ( anova p - value ) . once a search has produced a new list ( or a previously saved list is selected from the account page ) the user is prompted to select what information to display for the genes in the resulting list ( figure 2 , b. results ) . output choices include various identifiers ( unigene , pfam , interpro , ensembl , entrez , go ) , gene descriptors ( gene symbol , gene name ) , domain descriptors ( name , size , number ) , mapping information ( chromosome , cytoband ) , transcription factor classification ( superclass , class , subclass ) and blast homology information ( e - score , genbank i d and amino acid range of the original blast query sequence ) . the domain descriptors refer to the name and size of each domain as well as the number of different domains containing homology in each gene sequence . the genbank i d and amino acid range of all original blast queries are also provided so that homology with any domain may be traced back to its original source . the e - score field provides a drop down menu of different e - score values allowing the user to set the e - score cutoff for the resultant output . elaborating on the e - score further , when designing the genespeed database we sought to allow a flexible and open environment allowing the user to have precise control over various selection criteria . because genespeed is a similarity - driven database , the most important criterion for the evaluation of domain sequences in the transcriptome is the expectation - score ( e - score ) . the e - score describes the number of hits that one can expect to encounter by chance when searching by homology in a database . consequently , the score is influenced by the size of the database , and by the size of the query sequence . for any particular hit , as the e - score approaches 0 ( becomes smaller ) , the hit exhibits a more significant degree of similarity with the query . the purpose of an e - score cutoff is to exclude hits generated from the similarity search that do not have sufficient resemblance to the original domain of interest and thus do not represent a homologous domain . unfortunately , due to the dramatic size differences between domain families , and the variations in conserved residues even between similarly sized domains , there is not a clearcut e - score cutoff that may be used for all domains and their corresponding hits in the database . rather , we leave such critical decisions up to the user because both size and content characteristics of each domain must be taken into consideration when evaluating an appropriate e - score cutoff . despite these complications , in most cases , an inverse correlation exists between the domain size and e - score ; therefore , a suitable cutoff may be selected using these criteria . in other less common situations , domain size may not be a suitable tool for selecting the e - score cutoff value . explanations and recommendations for choosing an appropriate e - score cutoff may be found on the genespeed website in the background section . after the results have been displayed , the user has a choice of using other links and tools to further investigate the genes in their list ( figure 2 , c. sub - searches / tools ) : ( i ) domain sub - search identifies all the domains with homology in a specified gene . ( ii ) interpro sub - search identifies all other genes in the genespeed database within the currently selected species displaying homology to the specified domain . ( iv ) novartis symatlas2 , when working in the human or mouse section of the database , the average absolute expression , fold change above median and anova tissue specificity statistical information pre - computed from the symatlas2 dataset may be accessed and visualized for genes contained within a gene list . we have provided several additional resources on the genespeed website to help users navigate and use the database with success . these include a section of real biological scenarios , where we present possible scientific questions and full descriptions on how genespeed may answer these questions . similarly , we have included an faq page as well as a glossary of helpful definitions and descriptions . in addition , we have also included a tutorial section that provides several short tutorials on how to effectively use the tools within the genespeed database . we here describe the construction and use of genespeed , a database providing an extensive homology compilation of all protein domains within the transcriptomes of human , mouse , worm and fly . at present , we have restricted this domain compilation to these four organisms . although unigene databases exist for many species ( at present 76 ) , the transcriptome sequencing effort for most is not approaching the saturated level of the four chosen organisms , and incorporating these additional species , although not limited in a practical sense , was deemed premature . these can later be added , as the sequencing effort and their respective genomics platforms become expanded and further utilized . the homology assemblage employed in the creation of genespeed is based on a computationally demanding batch tblastn population process , which to our knowledge has not been used previously for this purpose . when developing this population process it was necessary to choose an efficient homology detection algorithm . others have shown that a hidden markov model ( hmm ) based approach achieves better performance than gapped blast techniques for homology identification ( 8,9 ) . we , on the other hand , have observed that by using a very large number of domains ( each used as an individual query ) derived from all types of species in the similarity search , a greater number of true hits is possible with the blast algorithm than hmm - based techniques ( results to be published elsewhere ) . by using a large batch of query sequences from a diversity of organisms all representative of a particular protein domain family , it may be possible to more accurately reflect the natural diversity and evolution of amino acid combinations within that family ( details of these experiments and their results are available on the genespeed website ) . included with the domain analysis of these transcriptomes , genespeed provides a toolset for the exploration of these domains , their protein families and the genes in which they reside . relatedness with known transcription factors , go classification , full name , average size , blast domain data and various protein domain identifiers ( pfam , interpro and genbank ) . moreover , with the incorporated blast e - score cutoff tool , the user has control governing the amount of homology displayed when analyzing query results in order to accommodate any domain type or the specific biological question at hand . this is both a unique and indispensable feature of the database , as an appropriate cutoff may significantly fluctuate depending on the size of a domain , type of domain or even the taxonomic limitation / category of a particular protein domain . we have found it highly useful to be able to follow low levels of homology as this often carries certain functional relevance , which may link very distantly related members and often allows bridging between protein superfamilies . such information is normally not available at other domain - based sites as only near - perfect matches are curated as true members ( additional discussion on this topic is available at the genespeed website ) . in essence , genespeed provides users with a very domain - centric online suite for analyzing the transcriptome . it is this domain focus that affords the genespeed database its many unique benefits , such as providing the user the ability to infer protein type classification of a gene based on the composition and homology of its protein domain content . this is particularly noticeable in almost any genespeed query result , where many of the resultant hits are genes that have not been classified in any of the common databases ( genbank , entrez gene , unigene , etc . ) . as an example , a genespeed query for the homeobox domain in human yields 217 genes with significant e - scores . of these , 24 have not been characterized as homeoboxes and 16 have no previous classification associated with them . in such instances the domain content of these genes may be quickly and easily analyzed in the genespeed environment , thereby revealing distinct clues about the possible classification of these unknown genes . to our knowledge , few online biological knowledgebases are dedicated to the task of helping investigators define all genes in a particular proteomic category , which is both species specific and non - redundant , as well as linking this information to searchable gene expression information at this level of detail . only recently have large databases such as ncbi and embl started to interlink genetic , functional and expression datasets . even so , there are still no tools allowing the analysis of large user defined gene lists or the ability to save this information in a private account within the database of interest . furthermore , there is no current resource where an investigator may study protein domain families in a non - redundant manner and within a select organism of choice . we have developed genespeed in an attempt to fulfill these needs within a single database and provide access from a globally accessible website . we have used the genespeed database with significant success in our own research not only to identify particular protein families based on domain content , but also to analyze these gene lists for expression level in our tissue of interest in order to define candidates for follow up in our laboratory . the success by which genespeed has facilitated this process for our research should exemplify its potential value for other investigators . | the genespeed database ( ) is an online database and resource tool facilitating the detailed study of protein domain homology in the transcriptomes of homo sapiens , mus musculus , drosophila melanogaster and caenorhabditis elegans .
the population schema for the genespeed database takes advantage of howard parallel cluster technology ( ) and performs exhaustive tblastn searches covering all pre - assigned pfam domain classes in all species ( currently 7973 domain families ) against the respective unigene est databases of the selected four transcriptomes .
the resulting database provides a complete annotation of presumed protein domain presence for each unigene cluster . to complement this domain annotation
we have also performed a custom transcription factor - family curation of all pfam domains , incorporated the gene ontology classifications for these domains as well as integrated the novartis symatlas2 dataset for both human and mouse which provides rapid and easy access to tissue - based expression analysis .
consequently , the genespeed database provides the user with the capability to browse or search the database by any of these specialized criteria as well as more traditional means ( gene identifier , gene symbol , etc . ) , thereby enabling a supervised analysis of gene families through a top - down hierarchical basis defined by domain content , all directly linked to an optimized gene expression dataset . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the percutaneous endoscopic gastrostomy ( peg ) has been in clinical use for more than thirty years now . a recent innovation , the loop - gastrostomy , makes the tube more secure and easier to change . thus it is more suitable for developing countries . in the typical peg , one end of the hollow tube is fitted with a dilator ( for pull - through ) while the other end has a mushroom- or dome - shaped retention disc . the loop - gastrostomy tube , however , has both ends fitted with dilators and the exit opening is at the midpoint [ figure 1a ] . insertion of the loop - gastrostomy tube is similar to the typical peg except that two gastric punctures are needed and both ends are pulled - through simultaneously , after which the ends are locked together [ figure 1e ] . loop - gastrostomy with gastropexy ( a ) formation of the secondary loop ( b ) simultaneous pull - through of both ends of the loop - gastrostomy tube ( c ) apposition of the stomach wall to abdominal wall using the secondary loop and lock ( d ) matured track with fibrous adhesions between stomach and abdominal walls ( e ) after release of the secondary loop , only the standard loop - gastrostomy tube remains gastropexy , the apposition of the anterior stomach wall to the anterior abdominal wall , enhances the safety of a tube gastrostomy . it is typically achieved with suturing , t - fasteners , and a bumper - bolster combination in surgical , radiologic and endoscopic gastrostomies , respectively.[35 ] fibrous adhesions between the stomach and abdominal wall will develop after a while and maintain the gastropexy . ideally the gastropexy for the loop - gastrostomy should be simple , effective and low - cost . mentally retarded , he was cared for by his mother . he had been fed orally until aspiration pneumonia precipitated his admission into the hospital . as his swallowing reflex had deteriorated , to prevent recurrence of aspiration pneumonia , his doctor recommended tube - feeding . unfortunately , naso - gastric intubation proved to be impossible ; the tube kept coiling in the throat . the family gave written informed consent for the loop - gastrostomy instead of a standard peg . pre - procedure fasting , initial gastroscopy , skin preparation , selection of puncture sites , etc . were performed in the usual manner . only one modification was made : prior to pull - through insertion of the tube ( looppeg 3 g ) a secondary loop was fashioned at the mid - portion with vicryl ( polyglactin 910 ) 3/o ligatures , as illustrated in figure 1a . pulling the tube ends apposed the stomach wall to the abdominal wall [ figure 1b ] . when doing so , care was taken to avoid strangulating the tissues . then the lock was applied to the two ends as per the instructions of the manufacturer [ figure 1c ] . the endoscopic view of the secondary loop in the stomach is shown in figure 2 . endoscopic view of the secondary loop in the stomach of the patient the patient tolerated the procedure well . this was to be expected because only sedation and local anaesthesia were used , as is the case for most peg insertions . this case proves that a gastropexy can be added to the loop - gastrostomy via the tube . fashioning the secondary loop was a simple matter because only two reef knots per ligature were needed . in the acidic environment of the stomach , upon degradation of the ligatures by hydrolysis , the secondary loop will be released , and the tube may be changed percutaneously without risk . release of the secondary loop is easily detected because the lock will be lifted off the skin [ figure 1e ] . if non - absorbable ligatures are used , the loop - gastrostomy tube must be changed endoscopically . it is plain to see that the secondary loop and lock are the counterparts of the bumper and bolster of the standard peg . clinical experience with the peg allowed us to predict that the secondary loop and lock would be safe and effective . | the percutaneous endoscopic gastrostomy has been in clinical use for more than three decades .
a recent innovation , the loop - gastrostomy , is more suitable for developing countries because the tube can not be dislodged and is easy to change . gastropexy and gastrostomy are separate but related moieties .
we describe a novel technique to add a gastropexy to the loop - gastrostomy , using it successfully in a man with permanent dysphagia .
it involved creating a secondary loop at the mid - portion of the looppeg 3 g tube with absorbable ligatures . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
patients with diffuse dme in fundus fluorescein angiography ( ffa ) , best corrected visual acuity 20/200 , glycated hemoglobin 7.5 mg / dl were included . eyes that had the following features were excluded : i ) only focal macular edema attributable to focal leaks from micro aneurysm , ii ) presence of any other macular pathology like armd or any vascular occlusive diseases affecting macula , iii ) optic disc pathology due to chronic glaucoma , vi ) previously treated with pan retinal photocoagulation ( prp ) and grid laser within last six months , v ) those with evidence of vitreomacular traction vi ) angiographic evidence of widening or irregularity of the foveal avascular zone suggestive of ischemic maculopathy . patients with uncontrolled diabetes , hypertension , chronic renal failure and history of stroke were excluded from the study . the number of anterior chamber cells observed in cases of ocular inflammation was determined by slit - lamp examination . zero cells indicated that no cells were visible in any optical section when the slit - lamp beam ( 1x1 mm ) was swept across the anterior chamber , trace cells indicated that one to three cells were observed , 1
cells three to 10 cells , 2
cells 10 to 25 cells , 3
cells 25 to 50 cells and 4
cells > 50 cells and or hypopyon present . each patient underwent best corrected distance va measurement with early treatment diabetic retinopathy study ( etdrs ) chart and ophthalmic assessment including slit - lamp biomicroscopy . all the patients underwent anterior segment examination , biomicroscopic evaluation with fundus non contact + 90d lens and ffa . central macular thickness was measured with optical coherence tomography ( oct iii , stratus oct , carl zeiss , germany).three vertical and horizontal manually assisted oct scans were obtained to locate the fovea and foveal thickness . the study parameters were evaluated three months and six months after the second intravitreal injection . all the injections were performed in a strict aseptic fashion and prophylactic topical antibiotics were given for five days post injection . study parameters included i ) etdrs visual acuity , ii ) central macular thickness as measured by oct and iii ) incidence of side - effects which included rise in intraocular pressure ( iop ) , inflammation and endophthalmitis . systemically all the patients were monitored for blood pressure rise , chest pain and thromboembolic events ( weakness in limbs ) . statistical analysis for descriptive statistics repeated measures analysis of variance test ( 2 ways anova ) was applied for the analysis . all the 20 eyes had received some form of laser photocoagulation previously as shown in
table 1 . the baseline characteristics included a mean va of 20/494 ( logmar=1.3380.455 ) , mean central macular thickness of 492.8167.2 m and mean iop of 15.332.7 mmhg . statistically significant changes in va and central macular thickness were observed at month 3 after second intravitreal injection . va at month 3 improved to a mean of 20/295 ( logmar= 1.0940.254 ) , a difference from baseline that was highly significant (
p = 0.008 )
[ fig . . mean central macular thickness at month 3 was 377.3113.57 , a difference from baseline that was also highly significant (
p = 0.001 )
[ fig . 2 ] . none of the injected eyes had 2
or more cells . at month 6 after the second injection the mean va was 20/304 ( logmar=1.1240.219 )
[ fig . 1 ] . though the mean va worsened marginally the difference was still statistically significant compared to baseline ( p = 0.029).the mean central macular thickness was 379104 at the end of six months follow - up ( p = 0.001 )
[ fig . 2 ] . the mean iop was 16.6 mmhg at the end of six months . during the entire follow - up of six months , there were no cases of clinically evident inflammation , endophthalmitis , increased iop , retinal detachment . figs . 3 and
4 show baseline and final oct images of two separate eyes . all the 20 eyes had received some form of laser photocoagulation previously as shown in
table 1 . the baseline characteristics included a mean va of 20/494 ( logmar=1.3380.455 ) , mean central macular thickness of 492.8167.2 m and mean iop of 15.332.7 mmhg . statistically significant changes in va and central macular thickness were observed at month 3 after second intravitreal injection . va at month 3 improved to a mean of 20/295 ( logmar= 1.0940.254 ) , a difference from baseline that was highly significant (
p = 0.008 )
[ fig . mean central macular thickness at month 3 was 377.3113.57 , a difference from baseline that was also highly significant (
p = 0.001 )
[ fig . 2 ] . at month 6 after the second injection the mean va was 20/304 ( logmar=1.1240.219 )
[ fig . 1 ] . though the mean va worsened marginally the difference was still statistically significant compared to baseline ( p = 0.029).the mean central macular thickness was 379104 at the end of six months follow - up ( p = 0.001 )
[ fig . 2 ] . the mean iop was 16.6 mmhg at the end of six months . during the entire follow - up of six months , there were no cases of clinically evident inflammation , endophthalmitis , increased iop , retinal detachment . figs . 3 and
4 show baseline and final oct images of two separate eyes . we report the results of 20 consecutive eyes with diffuse dme treated with intravitreal bevacizumab which resulted in both anatomic and functional improvement . our results also show that bevacizumab was well tolerated and no systemic adverse events were noticed during the study . intravitreal steroids reduce macular edema for which several theories were proposed , including local reduction of inflammatory mediators , lower levels of vegf , increased diffusion by an effect on calcium channels and improved blood retinal barrier function ;
19 it however remains plagued by a considerably high percentage of side - effects , namely cataract progression in a number of eyes and rise in iop ( 10 to 50% ) . 3 recently , the results of a phase ii study of pegaptanib have been reported with encouraging results for eyes with dme . 6 a strict comparison between safety , efficacy and end points of bevacizumab and pegaptanib is not possible because of the limited number of patients , study size and study design . however , in a limited comparison of adverse events , intravitreal bevacizumab appears to have a profile comparable to that of pegaptanib injection . another finding that had surfaced during this study , which has already been shown by other studies , is the prompt regression of neovascularization in the five eyes with active pdr . these eyes underwent a milder form of prp four to six weeks post intravitreal bevacizumab injection . in this small study which was carried out in an indian population , the mean central macular thickness reduced to 369 from 492 and the visual acuity also showed a modest improvement from a baseline of 20/494 to 20/295 at the end of six months . 20 the capillary permeability seen in dme is secondary to release of vegf , primarily vegf - a whose release is inhibited by the pan anti - vegf monoclonal antibody , avastin .
4 similar to the study published by haritoglou
et al . ,
20 our study also demonstrated significant improvement in va and decrease of central macular thickness after two intravitreal bevacizumab injections . but in contrast to their study ( six weeks follow - up ) our study population was more homogenous and we followed up all the patients for six months . we also monitored for any post injection ocular inflammation in the form of anterior chamber cells and any systemic adverse events caused by bevacizumab . additionally , our study show the beneficial effect of intravitreal bevacizumab in patients with dme associated with active pdr . the prp laser requirements were reduced in this subset of patients following the intravitreal injection . our preliminary study provides evidence that inhibition of vegf associated with both pathological ocular neovascularization and increased retinal vascular permeability in diabetic retinopathy may produce a clinically meaningful and statistically significant benefit in the treatment of dme . though the positive results of this prospective , nonrandomized study preclude any estimation of the long - term efficacy or safety of intravitreal bevacizumab , they are quite promising and suggest the need for further longer prospective randomized studies . | background : to report the anatomic and visual acuity response after intravitreal bevacizumab ( avastin ) in patients with diffuse diabetic macular edema.design : prospective , interventional case series study.materials and methods : this study included 20 eyes of metabolically stable diabetes mellitus with diffuse diabetic macular edema with a mean age of 59 years who were treated with two intravitreal injections of bevacizumab 1.25 mg in 0.05 ml six weeks apart .
main outcome measures were 1 ) early treatment diabetic retinopathy study visual acuity , 2 ) central macular thickness by optical coherence tomography imaging . each was evaluated at baseline and follow - up visits.results : all the eyes had received some form of laser photocoagulation before ( not less than six months ago ) , but all of these patients had persistent diffuse macular edema with no improvement in visual acuity .
all the patients received two injections of bevacizumab at an interval of six weeks per eye .
no adverse events were observed , including endophthalmitis , inflammation and increased intraocular pressure or thromboembolic events in any patient .
the mean baseline acuity was 20/494 ( log mar=1.3380.455 ) and the mean acuity at three months following the second intravitreal injection was 20/295 ( log mar=1.0940.254 ) , a difference that was highly significant ( p = 0.008 ) .
the mean central macular thickness at baseline was 492 m which decreased to 369 m ( p = 0.001 ) at the end of six months.conclusions : initial treatment results of patients with diffuse diabetic macular edema not responding to previous photocoagulation did not reveal any short - term safety concerns .
intravitreal bevacizumab resulted in a significant decrease in macular thickness and improvement in visual acuity at three months but the effect was somewhat blunted , though still statistically significant at the end of six months . |
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parkinsonism is a common symptom , but early diagnosis of parkinson s disease ( pd ) and dementia with lewy bodies ( dlb ) is very difficult even for neurologists because clinical history or physical examination is nonspecific . metaiodobenzylguanidine ( mibg ) is a physiological analog of noradrenaline ( norepinephrine ) and has been used to evaluate postganglionic cardiac sympathetic innervation . many studies have reported that cardiac mibg uptake is reduced in the case of lewy body disease ( lbd ) . the studies have proven that the heart to mediastinum ( h / m ) ratio of i - mibg cardiac scintigraphy is a useful diagnostic tool for indicating the significant reduction in myocardial mibg uptake in lbd as compared with a control group . mibg scintigraphy is performed twice in the early phase ( from 10 to 30 min after injection of the radioisotope ) and in the delayed phase ( from 3 to 4 h after injection of the radioisotope ) . all previous studies on lbd examined the patients twice , and the h / m average count ratio was obtained for both early and delayed images mainly using planar imaging . however , only the delayed h / m ratio was used to evaluate the diagnostic performance in most of the previous studies 2 . to our knowledge , there are no reports comparing the diagnostic performance of h / m ratio statistically between early and delayed phases 3,4 . therefore , the potential role of early h / m ratio has not been established . the aim of our study was to compare the diagnostic performance of the h / m ratio in early - phase i - mibg scintigraphy with that in delayed - phase imaging . if the diagnostic performance of the h / m ratio in the early phase is found comparable to that of the delayed phase , the total time taken by the examination to evaluate lbds would be much shorter . our institutional review board approved the study and the need for informed consent was waived . between april 2012 and december 2013 , 217 consecutive patients with suspected degenerative parkinsonism underwent i - mibg cardiac scintigraphy . the exclusion criteria for this study were a history of tricyclic antidepressive medication or heart disease . the latter included coronary heart disease , infarction , and heart failure . the final diagnosis of lbd that is , pd or dlb was made by neurologists who took into account the patient s history , the clinical presentation of key features , and a diagnostic brain mri to exclude symptomatic parkinsonism such as multiple arteriosclerotic changes or hydrocephalus . the uk pd society brain bank criteria 5 were used for the clinical diagnosis of pd , and the third report of the dlb consortium 3 was used to make the clinical diagnosis of dlb . patient characteristics all patients underwent i - mibg myocardial scintigraphy after an intravenous injection of 111 mbq of i - mibg ( fujifilm ri pharma co. ltd , tokyo , japan ) . anterior images of the chest were obtained after 15 min and again after 4 h using a dual - headed gamma camera ( toshiba e.cam duet ; toshiba medical systems , kawasaki , japan ) equipped with low - to medium - energy general - purpose collimators . the cardiac uptake of i - mibg was determined by manually drawing a region of interest ( roi ) that encircled the left ventricle . the mediastinal uptake was determined by setting a rectangular roi drawn by a skilled medical radiation technologist in the upper mediastinum . the h / m ratio was calculated as the ratio of the average counts per pixel in the heart roi divided by that in the mediastinum roi . we used software for transport and analysis of myocardial planar images ( stamp ver1.3 ; fujifilm ri pharma co. ltd ) to calculate the h / m ratio . normality of the h / m ratios of the early and delayed images was examined by means of the shapiro the difference in h / m ratios between the early and delayed images was examined for statistical significance using the mann we conducted conventional receiver - operating characteristic ( roc ) analysis for assessing the diagnostic performance of the h / m ratio in the early and delayed phases . the area under the curve ( auc ) was calculated for each phase and the difference in auc was analyzed . the optimal cutoff value of the h / m ratio for both early and delayed phases was chosen to maximize the youden index ( sensitivity+specificity1 ) . our institutional review board approved the study and the need for informed consent was waived . between april 2012 and december 2013 , 217 consecutive patients with suspected degenerative parkinsonism underwent i - mibg cardiac scintigraphy . the exclusion criteria for this study were a history of tricyclic antidepressive medication or heart disease . the latter included coronary heart disease , infarction , and heart failure . the final diagnosis of lbd that is , pd or dlb was made by neurologists who took into account the patient s history , the clinical presentation of key features , and a diagnostic brain mri to exclude symptomatic parkinsonism such as multiple arteriosclerotic changes or hydrocephalus . the uk pd society brain bank criteria 5 were used for the clinical diagnosis of pd , and the third report of the dlb consortium 3 was used to make the clinical diagnosis of dlb . all patients underwent i - mibg myocardial scintigraphy after an intravenous injection of 111 mbq of i - mibg ( fujifilm ri pharma co. ltd , tokyo , japan ) . anterior images of the chest were obtained after 15 min and again after 4 h using a dual - headed gamma camera ( toshiba e.cam duet ; toshiba medical systems , kawasaki , japan ) equipped with low - to medium - energy general - purpose collimators . the cardiac uptake of i - mibg was determined by manually drawing a region of interest ( roi ) that encircled the left ventricle . the mediastinal uptake was determined by setting a rectangular roi drawn by a skilled medical radiation technologist in the upper mediastinum . the h / m ratio was calculated as the ratio of the average counts per pixel in the heart roi divided by that in the mediastinum roi . we used software for transport and analysis of myocardial planar images ( stamp ver1.3 ; fujifilm ri pharma co. ltd ) to calculate the h / m ratio . normality of the h / m ratios of the early and delayed images was examined by means of the shapiro the difference in h / m ratios between the early and delayed images was examined for statistical significance using the mann we conducted conventional receiver - operating characteristic ( roc ) analysis for assessing the diagnostic performance of the h / m ratio in the early and delayed phases . the area under the curve ( auc ) was calculated for each phase and the difference in auc was analyzed . the optimal cutoff value of the h / m ratio for both early and delayed phases was chosen to maximize the youden index ( sensitivity+specificity1 ) . the h / m ratios of the early and delayed images did not show a normal distribution . the h / m ratio of the lbd group ranged from 1.1 to 3.88 ( median 1.8 ) in the early phase and from 0.97 to 4.7 ( median 1.45 ) in the delayed phase . the h / m ratio of the nondiseased group ranged from 1.17 to 4.18 ( median 2.93 ) in the early phase and from 0.85 to 4.77 ( median 3.18 ) in the delayed phase . both early and delayed h / m ratios of the lbd group there was no normality in the h / m ratios in the early and delayed images . ( a ) the comparison of early h / m ratio for lbd and nondiseased patients . early h / m ratios of lbd were significantly lower than those of the nondiseased group ( p<0.0001 ) . ( b ) the comparison of delayed h / m ratio for lbd and nondiseased patients . delayed h / m ratios of lbd were significantly lower than those of the nondiseased group ( p<0.0001 ) . the boxes are bound on the top by the third quartile , and on the bottom by the first quartile . the upper whisker extends from the third quartile to the third quartile+1.5iqr ( interquartile range ) , and the other extends downward from the first quartile to the first quartile1.5iqr . h / m , heart to mediastinum ratio ; iqr , interquartile range ; lbd , lewy body disease . the roc curves for both phases are shown in fig . the auc for the early phase was 0.871 and that for the delayed phase was 0.893 . the optimal cutoff value of the h / m ratio for the early and delayed phases was 2.28 and 1.91 , respectively . these are higher because our collimator and the way of drawing rois were different from the past report . the sensitivity and specificity of the early phase were 80.2% ( 65/81 ) and 91% ( 101/111 ) , respectively . the sensitivity and specificity for the delayed phase were 81.4% ( 66/81 ) and 96.7% ( 106/111 ) , respectively . in contrast , five nondiseased participants were classified correctly as nondiseased only with the early phase ( their h / m ratio was greater than the cutoff value ) . receiver operating characteristic curves of the h / m ratio in the early and delayed phases . the auc for the early phase was 0.871 and that for the delayed phase was 0.893 . auc , area under the curve ; h / m , heart to mediastinum ratio . to our knowledge , this is the first research to statistically compare the diagnostic performance of early and delayed phases . although previous studies 810 have reported the sensitivity and specificity of early and delayed phases , they did not compare the difference statistically . our results reveal that the diagnostic performance of i - mibg scintigraphy was not significantly different between early and delayed phases . the results for i - mibg cardiac scintigraphy in the evaluation of lbd have been obtained both in the early ( 1530 min ) phase and in the delayed ( 35 h ) phase . however , the delayed phase was weighted heavily in the meta - analysis part of a previous study 2 . early myocardial uptake of mibg reflects the integrity and distribution of the presynaptic sympathetic system 11 . however , the neuronal accumulation of mibg uptake reaches its peak 34 h after injection and the delayed uptake may reflect the functional status such as the relative level of neuronal uptake or the degree of washout of norepinephrine from sympathetic nerve terminals 8 . 12 reported that the h / m ratio in the lbd group was significantly lower in the delayed phase than in the early one , and the h / m ratio in the control group was higher in the delayed phase than in the early one . this means that the difference in h / m ratio between the lbd and control groups was larger for the delayed phase than for the early one . regardless of these explanations , the previous studies actually revealed that the h / m ratio in the early phase was also significantly lower in the lbd group than in the control group 1,9,11 . 8 conducted an roc analysis for each of the two phases and showed similar aucs ( 0.86 for the early phase and 0.85 for the delayed phase ) , although they were not statistically compared . therefore , once the optimal cutoff value of the h / m ratio for the early phase is determined , early - phase imaging can provide comparable diagnostic performance to delayed - phase imaging . the comparable diagnostic performance of early - phase imaging could change the protocol for patients suspected of having lbd . at present , it takes about 4 h to complete the procedure because patients have to wait to undergo delayed - phase imaging . early - phase imaging would shorten the time from injection to completion of imaging to less than 1 h and would make testing available to patients who can not tolerate long hours because of their parkinsonism or dementia . nowadays , i - ioflupan dopamine transporter visualization using single photon emission computed tomography ( spect ) is being increasingly used to assist in the evaluation of parkinsonism . low dopamine transporter uptake in the basal ganglia is one of the suggestive features of parkinsonism according to the third report of the dlb consortium , whereas abnormally low uptake in mibg scintigraphy is only a supportive feature 3 . this means that i - ioflupan spect is a more specific examination for lbd compared with i - mibg cardiac scintigraphy . the sensitivity and specificity of i - ioflupan spect for pd were 86.5 and 93.6% , respectively , and that for dlb were 98 and 67% 13,14 . therefore , the diagnostic performance of i - ioflupan spect for lbd relative to that of i - mibg cardiac scintigraphy may be somewhat superior but not overwhelmingly so . second , setting rois on i - ioflupan spect images has not been standardized and the optimal cutoff value for diagnosis has not been established . third , it is reported that mibg can differentiate lbd from multiple systemic atrophy or progressive supranuclear palsy , but i - ioflupan can not 13,14 . in addition , i - ioflupan spect takes longer than early - phase i - mibg scintigraphy ; that is , the data of i - ioflupan are usually collected from 3 to 6 h after injection of the radioisotope . therefore , we believe that cardiac i - mibg scintigraphy will not be completely replaced by i - ioflupan spect . it is retrospective in nature and the results of i - mibg scintigraphy could have affected the clinician s diagnosis . currently , an roi is placed automatically over the heart by the new software smart mibg ( fujifilm ri pharma co. ltd ) . although an experienced technician placed the roi in this study , third , the patients were referred to the university hospital , and hence pretest probability may be high . early h / m ratio has almost the same diagnostic performance as delayed h / m ratio . early - phase i - mibg scintigraphy may be optional for patients who can not tolerate a long interval from injection to completion of imaging . | objectivesthe aim of the study was to compare the diagnostic performance of early - phase 123i - metaiodobenzylguanidine ( mibg ) scintigraphy with that of delayed - phase imaging in lewy body disease ( lbd).methodsa retrospective cohort study of 123i - mibg scintigraphy was carried out in 192 patients who were suspected of having lbd .
clinical diagnosis was obtained using the uk parkinson s disease brain bank criteria in some cases or the third report of the dementia with lewy bodies consortium in others .
the participants consisted of 81 patients with lbd and 111 nondiseased patients .
an injection of 111 mbq of 123i - mibg was used .
planar images were obtained in an early phase and again in a delayed phase and the heart to mediastinum count ratio was calculated for both phases .
diagnostic performance was compared using a receiver - operator characteristic analysis .
the cutoff value was chosen to maximize the youden index .
the sensitivity and specificity of each phase were calculated from the optimal cutoff value.resultsthe heart to mediastinum ratio of the lbd group ( median 1.8 and 1.45 for early and delayed phases , respectively ) was significantly lower than that of the nondiseased group ( median 2.93 and 3.18 for early and delayed phases , respectively ) .
the area under the receiver - operating characteristic curve was not significantly different between the early and delayed phases ( 0.871 vs. 0.893 ; p=0.0914 ) .
sensitivity and specificity were 80.2 and 91% for early - phase imaging ( cutoff value at 2.28 ) and 81.5 and 95.5% ( cutoff value at 1.91 ) for delayed - phase imaging , respectively.conclusionthe diagnostic performance of 123i - mibg scintigraphy was not significantly different between early - phase and delayed - phase imaging . |
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the world health organization estimated in 2010 8.8 million new tuberculosis ( tb ) cases globally . more than 2 billion people ( about one - third of the world s population ) are estimated to be infected with mycobacterium tuberculosis ( latent tuberculosis infection ) . of this infected population , 10% will develop active tb disease throughout their lives.1,2 there are several risk factors that promote the development of active tb disease , such as immunologic response , substance abuse ( tobacco , alcohol , and drugs ) , nutritional status ( underweight , vitamin d deficiency ) , systemic diseases such as diabetes , high blood pressure , and renal impairment.2 further , migration and endemics of the disease are important identifiable factors in tb infection.3 increased travel from endemic tb countries into the usa , represents a challenge to tb control . mexico represents one of the top countries with the majority of foreign - born active cases in the usa.3,4,6 the centers for disease control and prevention ( cdc ) , along with the us department of state , established that any foreign applicant for an immigrant visa for the usa must be screened outside the country for infectious diseases such as tb as part of the legal immigration process . panel physicians.5,8 evaluation and diagnosis procedures for tb are based on clinical or radiological grounds.4 once clinical or radiological suspicion is established , microbiological assessment is used to confirm the diagnosis of active tb disease . the diagnosis of tb is confirmed by isolation of the organism from secretions or tissue.7 while culturing remains the international gold standard for the diagnosis of tb disease , in many low - income countries sputum smears , which are known to be less sensitive and specific , remain the only and most accessible tool with which to diagnose active tb disease . a retrospective study was performed on the medical records of 122 active tb cases diagnosed at a clinic in ciudad juarez , chihuahua , mexico , from january 2009 to december 2012 . all of the active tb cases were mexican immigrants bound for the usa who needed to be screened as part of the legal immigration process established by the us department of state . the medical files were reviewed extensively to determine whether each patient had a positive smear besides a positive culture for m. tuberculosis complex , which was identified by dna probe . neelsen technique , and culturing on solid ( lwenstein jensen agar ) and liquid ( mycobacteria growth indicator tube ) media were utilized with every sample collected , as per the cdc technical instructions for panel physicians . after the data were gathered and grouped by smear results , each case was analyzed to determine parameters such as age , sex , city of origin , first smear result , and first culture result ( which was defined for the purposes of this research as the first positive test obtained for at least one of three initial samples on consecutive days ) . a database containing all this information was created and analyzed using epi info ( v 7.0 , cdc , atlanta , ga , usa ) . the study had no ethical conflicts and access to information was granted by the clinic and the patients themselves , who provided informed consent . a total of 122 medical files were analyzed , of which 58% were male and 42% were female , giving a 1:1 ratio . active tb disease was most prevalent in the mexican state of jalisco , followed by in chihuahua , guerrero , and baja , california , all of which have a high immigration index rate . on the smear results , 80% ( 97 ) of the active tb cases had negative sputum smears , while only 20% ( 25 cases ) had positive smears . the fact that 80% of the tb cases were smear negative and 20% smear positive shows that there is a clear gap between the actual state of active tb disease within patients under screening conditions , meaning that eight out of ten actual cases are being missed when sputum smear is the only diagnostic tool in asymptomatic patients with abnormal chest x - rays . in screening mexican immigrants bound for the usa , culturing as a diagnostic tool was extremely useful to identify active tb cases before they crossed the border into the usa . currently in mexico , culturing is not standardized via the national health regulations as a routine process for the diagnosis of tb and smears are indicated for the asymptomatic population . based on the results of this study , it is highly recommended that mexico and other low - income countries that have not standardized culturing as the gold standard for the diagnosis of active tb do so by solid media ( lwenstein jensen agar ) and mycobacteria growth indicator tube so that tb cases which may endanger global public health are not missed . it is also recommended that further studies be undertaken to determine the clinical background of the patients diagnosed by smear and culture to identify a direct relationship between clinical signs and symptoms and the smear result . | backgroundthe world health organization estimated in 2010 that 8.8 million new tuberculosis ( tb ) cases .
about one - third of the world s population is infected and 10% will develop active tb disease . while cultures remain the international gold standard for diagnosing tb disease , in many other low - income countries , sputum smears remain the only and most accessible tool with which to diagnose active tb disease . as a consequence , in patients with tb who have negative smears ,
their tb remains undetected.aimthe objective of the study reported here was to demonstrate the proportion of smear - positive / culture - positive cases compared with smear - negative / culture - positive tb cases in mexican immigrants bound for the usa.methodsa retrospective study was undertaken of the medical records of 122 active tb cases diagnosed at a clinic in ciudad juarez , mexico , from 2009 to 2012 .
all cases were confirmed by culture , regardless of the sputum smear results.resultsof the cases , 80% ( 97 active tb cases ) had negative sputum smears , while only 25 cases ( 20% ) had at least one positive smear .
all of the cultures were confirmed as positive for mycobacterium tuberculosis complex.conclusionthe fact that 80% of the tb cases were smear negative and 20% smear positive shows that there is a clear gap between the actual state of active tb disease within patients under screening conditions , meaning that eight out of ten actual cases are being missed when sputum smear is the only diagnostic tool in asymptomatic patients with abnormal chest x - rays . based on these results ,
it is highly recommended that countries that have not standardized culturing as the gold standard for the diagnosis of active tb do so , so that tb cases which may endanger global public health are not missed .
it is also recommended that further studies be undertaken to determine the clinical background of the patients diagnosed by smear and culture to identify a direct relationship between clinical signs and symptoms and the smear result . |
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an elderly male patient in his 60 s with a 150 pack year history of smoking , chronic obstructive pulmonary disease , hiatal hernia , gastroesophageal reflux disease , and coronary artery disease presented to the emergency room complaining of a three - week history of incessant forceful cough and right - sided chest pain in the absence of trauma . he required emergent uneventful endotracheal intubation and mechanical ventilation for hypoxic respiratory failure upon arrival to the emergency room . physical examination revealed an obese male with a heart rate of 110 beats per minute and blood pressure 135/80 mmhg . decreased breath sounds over the right lung with overlying severe right anterior chest wall ecchymosis and subcutaneous edema were noted . laboratory testing included an arterial blood gas with a ph 7.28 , pao2 107 mmhg ( 100% fio2 ) , pco2 43 mmhg , hco3 20 meq / l , hemoglobin 11.5 g / dl , and white blood cell count 23,100 cm . 1 ) revealed minimal opacity at the right lung base and blunting of the left costophrenic angle . computed tomography ( ct ) of the chest ( fig . 2 ) revealed a dramatic lateral herniation of the right lung at the seventh intercostal space without evidence of any overlying rib fractures . current radiologic findings were compared to images from a prior ct scan performed five months earlier which demonstrated symmetrically developed thoracic cage muscles without evidence for fracture or herniation . initial management in the medical intensive care unit ( icu ) included intravenous sedation and low tidal volume ventilation with attention to peak airway pressures and permissive hypercapnea given the high risk for incarceration and potential advancement of the lung herniation . an initial conservative strategy was opted for given the patient s overall co - morbidities contributing to high surgical risk and having no evidence for impending lung incarceration . the previously identified lung herniation was no longer visualized but an extensive extra pleural collection consistent with a large hematoma remained . patient remained on intravenous antibiotics during this time but blood and sputum cultures showed no growth of organisms . 3 ) . radiological studies supported evidence for contralateral acute lung injury , making mechanical ventilation and oxygenation very challenging . other icu complications included persistently high fevers despite broad - spectrum antibiotics , intra - cerebral hemorrhage due to cerebral amyloid angiopathy , and st segment elevation myocardial infarction . his hospital course was further complicated by anoxic encephalopathy due to inability to maintain adequate gaseous exchange . lung herniation is an uncommon medical problem with only about 300 cases cited to date.13 it has been defined in the literature as a displacement of lung tissue outside the thoracic cavity through a defect in the pleura of the chest wall , diaphragm , or mediastinum.4 the morel - lavallee system developed in 1845 is used to classify lung hernias on the basis of location and etiology.5 location is further broken down into cervical , diaphragmatic , and thoracic . if acquired , it may be due to traumatic , spontaneous , pathological , or post - surgical causes.5 spontaneous lung herniation due to coughing is related to a combination of two processes : weakness of the boundaries of the thoracic cavity and increased intra - thoracic pressure . weakness in the intercostal muscles may be a consequence of corticosteroid use , congenital abnormalities , or trauma ; however , chronic cough , valsalva maneuver , or playing wind instruments can contribute to increased intra - thoracic pressures.6 interestingly , there is a report in the literature of a spontaneous lung herniation after a single cough.7 most patients present clinically with crepitus and a bulging thoracic mass that increases in size with valsalva maneuver , coughing , or straining . patients may or may not have pain.8 diagnosis is usually clinical although ct is helpful in delineating the exact size and location of the hernia.4 treatment is still debated in asymptomatic patients , especially given the low incidence of this clinical entity . surgical intervention is considered to be the treatment of choice if the hernia causes extreme pain , incarcerates the pulmonary parenchyma , or results in infection.911 a conservative , nonsurgical approach is generally recommended for asymptomatic patients ; however this dogma may be changing . surgery may be the treatment option of choice even for asymptomatic patients to prevent complications or extension of the defect into the abdominal wall.1214 this case represents a rare fulminant and atraumatic complication of violent and pervasive coughing . initially , there was evidence of only unilateral lung herniation which we believe may have contributed to a systemic inflammatory response with contralateral lung injury followed by several icu complications . we believe that lung injury due to extra - thoracic herniation was complicated by ventilator - induced lung injury . the mechanism of this process relates to cyclical stretching of alveoli which leads to an ensuing cascade of inflammatory processes that are associated with cytokine upregulation.15 the subsequent leukocyte attraction and activation of polymorphonuclear neutrophils ( pmns ) from elevated levels of proinflammatory cytokines including tumor necrosis factor alpha ( tnf)- , interleukin ( il)-6 , il-8 , and il-1 which are produced by bronchial , bronchiolar , and alveolar epithelial cells may have led to a systemic inflammatory response and multiple organ dysfunction syndrome ( mods).15,16 furthermore , due to the direct mechanical trauma to the lung parenchyma from herniation , a state of alveolar leakage of proteins may have led to some degree of surfactant dysfunction exaggerating the lung injury.15 ventilation and oxygenation were very challenging due to development of bilateral lung injury . while surgery is advised in cases of symptomatic lung herniation , given the patient s multiple co morbidities he was deemed to be a poor surgical candidate . to the best of our knowledge , this is the first report of unilateral spontaneous lung herniation associated with contralateral acute lung injury . we propose that patients with lung herniation who require mechanical ventilation may benefit from early surgical intervention to reduce the inflammatory response that can predispose to contralateral lung injury , thus minimizing morbidity and mortality . | lung herniation is a relatively rare clinical entity that is most commonly either congenital or acquired traumatically .
we describe a case of spontaneous lung herniation secondary to acute cough in an obese male smoker complicated by contralateral acute lung injury and systemic inflammatory response syndrome ( sirs )
. mechanisms of lung herniation , classification , diagnosis , and management will be discussed . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
total energy expenditure ( tee ) in humans is determined by resting energy expenditure ( ree ) , dietary - induced thermogenesis , and energy cost of physical activity [ 1 , 2 ] . ree is the major fraction of tee in sedentary people , accounting for approximately 70% of tee . several studies showed that ree is low in underweight patients with an compared with normal - weight controls [ 316 ] and long - term recovered an patients . the low ree seems mainly due to the loss of lean body mass , a major determinant of ree , and to a lesser extent , to the effect of several neuroendocrine changes ( e.g. , thyroid hormones , adrenal hormones , and leptin ) observed in underweight an patients . the accurate measurement of the ree in underweight an patients is clinically useful , because it may help us ( i ) predict the energy level necessary to promote weight restoration and ( ii ) optimize nutritional rehabilitation preventing severe medical complications such as the refeeding syndrome . indirect calorimetry , performed with the subject in supine position after an overnight fast , is the most valid method used to measure ree . unfortunately , this technology is not available in the majority of the hospitals , because it requires skilled technicians and sophisticated methodologies that are costly and difficult to apply in standard clinical settings . predictive formulas of ree may be used as an alternative to indirect calorimetry that may be utilized by clinicians . the most cited and used predictive formula is the harris - benedict equation which includes age , stature , and body weight to estimate ree . unfortunately , data available on an patients indicate that harris - benedict equation overestimates ree [ 15 , 16 , 23 , 24 ] . to overcome this problem , a correction of this equation was derived specifically for individuals with an and validated in 37 hospitalized female an patients . however , subsequent studies found that the schebendach formula is useful for adolescents but seems to underestimate the ree in young adults patients with an . a method that might overcome the limits of predictive formulas requires the use of fat - free mass ( ffm , kg ) , and fat mass ( fm , kg ) to estimate ree . by using this procedure , mller et al . developed different formulas for different range of body mass index ( bmi ) , including one for a bmi < 18.5 kg / m . however , this procedure has never been implemented in an patients by ffm and fm values assessed by means of gold standard body composition methods , such as dual energy x - ray absorptiometry ( dxa ) . recently , advancements in technology have led to the development of relatively inexpensive devices , such as bodygem and the fitmate , designed for estimating ree by measuring only oxygen consumption that might make the use of the indirect calorimetry more popular in clinical settings . although they have been shown to be accurate when compared with the douglas bag system [ 27 , 28 ] , the data obtained in severe underweight an patients are somewhat controversial . for instance , systems for indirect calorimetry that assumed fixed respiratory quotient showed a significant bias with increasing rmr . aim of this study was to compare the results obtained by douglas bag system in assessing ree with the fitmate method , the harris - benedict equation , and the mller et al . 15 patients ( 14 females and 1 male ) with an participated in the study . all patients were voluntarily and consecutively admitted to the eating disorder inpatient unit of villa garda hospital during 2010 . the patients were referred from all over italy by general practitioners or by outpatients ' eating disorder specialists . indications for admission were the failure of less intensive treatments ( e.g. , outpatient treatment ) or the presence of an eating disorder of clinical severity not manageable in an outpatient setting . patients with active substance abuse , schizophrenia , and other psychotic disorders were not included . the indications and contraindications for the inpatient treatment were evaluated during an eligibility interview completed by a senior specialist in the field ( rdg ) . the eating disorder examination interview ( ede ) 12.0d was used to generate operational definitions of the dsm - iv diagnoses of an . before participation , written informed consent was obtained from all subjects ( or by the legal guardian for those less than 18 years old , in accordance with our institution 's requirements ) . the protocol was approved by the institutional review board of villa garda hospital , verona . data collection included weight and height measurement , dxa body composition measurement , indirect calorimetry with the douglas bag and the fitmate methods and harris - benedict and the mller et al . equations . body weight was measured on a medical balance and height with a stadiometer by a medical doctor involved in the study . the bmi was determined according to the usual formula of body weight divided by the squared of height in meters . indirect calorimetry was performed the second day of admission using the douglas bag and the fitmate methods in a single session early in the morning before breakfast . the day before the test , all participants consumed with the assistance of a dietitian a diet of 1,750 kcal ( protein 21% , carbohydrate 46% , and fat 33% ) . participants were informed to fast overnight , to avoid drinking caffeinated beverages for at least 12 hours , and to abstain from physical activity prior the tests . upon their arrival in the laboratory , participants rested on a medical bed with the upper part of the body partially raised ( + 3/4 ) and assumed a comfortable position while the instruments were prepared and calibrated and environmental data were recorded . then , after 10 minutes at rest , the measurements were performed for 11 minutes during which time the participants were instructed to lay quietly , to remain awake and to avoid fidgeting and hyperventilating . the douglas bag method involves collection of the expired air in a large impermeable rubber bag and subsequent volume and analysis of the expired gases [ 33 , 34 ] . it has been served as the gold standard method for many studies in the last decades [ 28 , 3537 ] . expired gases were collected by using a mask connected to a two - way , low - resistance respiratory valve whose expiratory outlet was fed to a 100 liter douglas bag . after gas collection , expired gas composition and volume were assessed using a paramagnetic o2 analyser ( oxynos 100 , rosemount analytical , usa ) , an infrared co2 meter ( binos 1 , leybold - heraeus , d ) , and a dry gas meter ( mcs , s.i.m . gas analysers were calibrated before each experimental trial using gas mixtures of known and certified composition . vo2 and vco2 in stpd conditions were calculated by applying standard equations implying the haldane correction for inspired ventilatory volume . respiratory quotient ( rq ) was calculated as vco2/vo2 and on the basis of the measured values of rq , the corresponding value of the energy equivalent of vo2 in kj was calculated . then , ree was obtained on the basis that 1 kcal equals 4,186 kj . the fitmate is a small device designed for measurement of oxygen consumption and energy expenditure during rest and exercise ( cosmed , rome , italy ) . it uses a turbine flow meter for measuring ventilation and a galvanic fuel cell o2 sensor for analysing only the fraction of oxygen in expired gases . it is considered to retain the performance of a metabolic cart with a standard mixing chamber or canopy . the fitmate uses standard metabolic formulae to calculate oxygen uptake , and energy expenditure is calculated using a fixed respiratory rq of 0.85 . body composition was assessed by using dxa ( idxa luner general electric ) in the third day of the admission . no special preparation was required , with the exception that participants wore lightweight clothing for these measures and did not have any metal accessories . the harris - benedict equations for women [ 655 + ( 9.6 weight in kg ) + ( 1.8 height in cm ) ( 4.7 age in years ) ] and men [ ( 66.47 + ( 13.75 weight in kg ) + ( 5.0033 height in cm ) ( 6.755 age in years ) ] , and the mller et al . equation for individuals with bmi < 18.5 kg / m [ 0.08961 ffm ( kg ) + 0.05662 fm ( kg ) + 0.667 ] 238.84 were used to estimate ree . the bland - altman method was used to study the concordance between the douglas bag method with the fitmate method , and the harris - benedict , and mller et al . the z - test was used to evaluate whether the mean of the differences between the values obtained by the three methods , with respect to douglas bag , was or was not significantly different from zero . the same test was also utilised to evaluate whether the respiratory quotient obtained with douglas bag was significantly different from the fixed value of 0.85 utilised in the calculations by the fitmate method . furthermore , the wilcoxon signed - ranks test was performed to compare the mean ree values of the douglas bag method with those of the fitmate method , then with those of the harris - benedict equation , and finally with those of the mller et al . table 1 shows the 15 participants ' data , with data summarized for age , height , weight , bmi , and ree values . age ranged from 15 to 45 years , and bmi ranged from 11.96 kg / m to 16.9 kg / m , with 60% of participants having a bmi < 15 kg / m . figure 1 shows the bland - altman plots reporting the differences between ree values measured with douglas bag and those obtained by using the other methods ( fitmate method and harris - benedict and mller et al . the mean of the differences between the ree values estimated with harris - benedict equation and those measured with douglas bag ( bias = 284 kcal / day ) was significantly different from zero ( 6.990 ; p < 0.005 ) . precision amounted to 158 kcal / day and the 95% limits of agreement ranged from 600 to 30 kcal / day . for the other two comparisons , the means of differences were not significantly different from zero . ( fitmate method douglas bag method = 1.85 ; mller et al . bias for the fitmate versus douglas bags was 87 kcal / day , and precision ( sd ) turned out to be 181 kcal / day . in turn , the 95% limits of agreement ranged from 448 to 275 kcal / day . as far as the mller and douglas bag comparison is concerned , the values were 5 kcal / day , 155 kcal / day , and 305/310 kcal / day , for bias , precision , and limits of agreement , respectively . in table 2 , the data of ree measured with the two systems utilised for ic of rq obtained with the douglas bag are reported for each participant in the study . rq measured with douglas bag was on the average equal to 0.88 0.07 and turned out to be not significantly different ( p > 0.3 ) from 0.85 , namely , the fixed value of rq assumed by fitmate for calculations . the wilcoxon signed - ranks test showed no significant differences between the mean ree values estimated with the douglas bag method and the mean values estimated with the fitmate method ( z = 1.70 , p = 0.088 , effect size = 0.65 ) and the mller et al . equation ( z = 0.23 , p = 0.820 , effect size = 0.04 ) , but significant differences were found comparing mean ree values estimated with the douglas bag method and with the harris - benedict equation ( mean ree values : 920.5 124.0 versus 1205.0 99.2 , resp . , z = 3.35 , the principal finding of this study on severe underweight an patients is that the fitmate method and the mller et al . equation gave an acceptable ree estimation , while the harris - benedict equation overestimated the ree , when compared with the douglas bag method . the principal strengths of the study are the use of the douglas bag method , the dxa , and the ede interview , three instruments considered the gold standard to assess ree , body composition , and eating disorder diagnosis , respectively . limitations of the study include the small number of participants , a common problem when studying rare disorder as an , and the absence of a control group and of longitudinal evaluation . in addition , since patients were tested in a single session , reliability of the single methods for estimating ree was neither evaluated nor quantified . participants in the study had severe underweight ( mean bmi 14.5 kg / m ) , marked reduction of fm ( mean fm% 9.70 ) , and low ree ( mean ree 920.00 kcal / day estimated with douglas bag method ) . these data confirm that patients with an admitted in specialist inpatient units have a condition of severe underweight and , as previously reported , a marked loss of fm . they also show that the underweight and the alteration of body composition are associated with an hypometabolic status as consequence of the adaptation to undereating and underweight . our data confirm that harris - benedict equation overestimates the ree in underweight patients with an [ 15 , 16 , 23 ] and that it should not be used with this population . using the harris - benedict equation to assess the energy need for refeeding underweight an patients may led to prescribe an excessive energy intake that increase the risk to produce severe negative consequences , such as the refeeding syndrome . this syndrome may include minor complications ( e.g. , transient pedal edema ) or serious complications requiring immediate care ( e.g. , a prolonged qt interval or hypophosphatemia with associated weakness , confusion , progressive neuromuscular dysfunction , and cardiovascular collapse ) . the good news of our data , if confirmed by other studies , is that ree expenditure in underweight an patients may be estimate with discrete accuracy , after just one day of refeeding , by the fitmate method , and by the mller et al the fitmate method , in comparison with the douglas bag method , is inexpensive , does not requires skilled technicians , and can be used by a wide variety of health professionals to determine the energy need of underweight an patients . the results reported in the present study confirm and extend those obtained on 60 not underweight healthy adults ( n = 30 males , n = 30 females ) , where no significant differences between douglas bag method and fitmate method for oxygen consumption and ree were found . however , the moderate effect size ( 0.65 ) of our comparison indicates the need of a bigger sample size to confirm this conclusion . fitmate utilizes in the calculations a fixed value of rq set equal to 0.85 . similar devices have shown a poor agreement between ree calculated by assuming a constant and fixed value of rq ( 0.85 ) and that calculated by means of deltatrac , which was considered wellestablished as a valid and reliable criterion reference system . specifically , if we do not consider vco2 , we are prone to underestimate of ree when rq is between 0.85 and 1.00 and to overestimate it if rq is between 0.70 and 0.85 . this might have brought about systematic underestimation of ree in our population , because an patients seem to be characterized by elevated rq values larger than 0.85 . however , while previous studies tested participants in severe caloric restriction , our participants have been tested after 24 hours of refeeding . this may explain why the mean rq of our participants was not significantly larger than 0.85 even though eleven patients out of fifteen had an rq larger than 0.85 . our data are in agreement with those presented in an patients after one week of refeeding and characterized by bmi overlapping those of our population . in addition , although fitmate in the present case seemed to overestimate ( and not to underestimate ) ree , ree values measured with the two approaches were not significantly different . also , in this case , however , the small sample size , coupled with the inherent inaccuracy of the experimental data , prevents us to draw any clear conclusion and confirm the prediction of the underestimation of ree when assuming a fixed rq of 0.85 with the fitmate . equation is another method that may be used in eating disorder units to estimate ree . this because the dxa , measuring the fm and the ffm required by the mller et al . equation , is a widely used in eating disorder units to assess the bone mineral density of underweight patients . in conclusion , equation , but not the harris - benedict equation , to estimate ree in severe an patients after short - term refeeding . | introduction . aim of this study was to compare the resting energy expenditure ( ree ) measured by the douglas bag method with the ree estimated with the fitmate method , the harris - benedict equation , and the mller et al .
equation for individuals with bmi < 18.5 kg / m2 in a severe group of underweight patients with anorexia nervosa ( an ) .
methods .
15 subjects with an participated in the study . the douglas bag method and the fitmate method were used to measure ree and the dual energy x - ray absorptiometry to assess body composition after one day of refeeding .
results .
fitmate method and the mller et al .
equation gave an accurate ree estimation , while the harris - benedict equation overestimated the ree when compared with the douglas bag method .
conclusion .
the data support the use of the fitmate method and the mller et al .
equation , but not the harris - benedict equation , to estimate ree in an patients after short - term refeeding . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
carcinosarcoma of the prostate gland is a rare malignancy characterized by an admixture of malignant epithelial and mesenchymal components . because it was presumed that the origin of these tumors was a carcinoma with varying degrees of differentiation including a sarcomatoid pattern , these tumors were first called sarcomatoid carcinoma . the contemporary classification , carcinosarcoma , is a more accurate description of this tumor because it comprises components of both adenocarcinoma and sarcoma . in contrast , a sarcomatoid carcinoma would apply to pure spindle cell tumors with epithelial differentiation . the incidence of carcinosarcoma is estimated to be less than 0.1% , with only 50 documented cases in the english literature . of interest , 48% to 55% of patients diagnosed with prostatic carcinosarcoma have a history of adenocarcinoma of the prostate . the objectives of this paper are to report a case of carcinosarcoma of the prostate resected by robotic - assisted radical cystoprostatectomy as well as discuss this new minimally invasive procedure as the first reported use for prostatic adenocarcinoma . a 73-year - old man who had undergone transurethral needle ablation of the prostate in the past underwent transurethral resection of the prostate ( turp ) for obstructive voiding symptoms refractory to alpha blockade . the pathology was notable for 2 foci of gleason 3 + 3=6 adeno - carcinoma and several foci of high - grade prostatic intraepithelial neoplasia . a cystoscopy at that time demonstrated extensive prostatic regrowth , and the patient underwent a second turp . the pathology from the second turp revealed adenocarcinoma with gleason 5 + 4=9 involving approximately half the prostatic chips . at this point , androgen ablation therapy was initiated and a psa nadir of 0.3ng / ml was achieved . one year later , a third turp was required for recurrent urinary retention . at this time a metastatic evaluation including whole body pet ct scan and mri of the head were negative and psa was < 0.1 ng / ml . at this point , after cystoscopic examination that demonstrated extension of the tumor into the bladder neck , a robotic - assisted laparoscopic cystoprostatectomy , bilateral pelvic lymph node dissection , and ileal conduit urinary diversion were performed . operative time was 6 hours . estimated blood loss was 250 ml , and 2 units of packed red blood cells were given intraoperatively for a hemoglobin of 9.6mg / dl and a hematocrit of 26.8% compared with preoperative values of 13.3 mg / dl and hematocrit of 38.1% . the patient had an unremarkable postoperative course and was discharged from the hospital on postoperative day 7 . he was fully recovered 3 weeks following the procedure and traveled to mexico on a family vacation . the gross pathology demonstrated tumor protruding into , but not invading , the bladder ( figure 1 ) . microscopic examination revealed leiomyosarcoma and a separate focus ( 1% of specimen ) of adenocarcinoma , gleason grade 4 ( figure 2 ) . one postulation is that both carcinoma and sarcoma develop simultaneously from different areas of the prostate . postradiation sarcoma ( prs ) of the prostate was described by canfield et al in 2001 . carcinosarcoma was defined as prostatic cancer containing poorly differentiated adenocarcinoma mixed with sarcomatous elements , sometimes associated with a history of radiation or hormone ablation , less than 10 years before diagnosis . pure prs of the prostate was described as sarcoma alone , in a previously irradiated patient , 25 years after the original neoplasm ( one testicular cancer and one prostate cancer , histology not reported ) . the study concluded that with only 2 cases of prs of the prostate reported , the entity was extremely rare . considering this extremely small number and the fact that only a fraction ( 14% to 21% ) of patients with carcinosarcoma have a history of radiation therapy , radiation likely plays a minor , if any , role in development of carcinosarcoma . similarly , as the number of patients treated by androgen deprivation therapy greatly exceeds the number of cases of carcinoma arising in patients with previous androgen deprivation therapy , hormone therapy is unlikely to have a role in development of carcinosarcoma . in a series of 21 patients with carcinosarcoma by dundore et al in 1995 , only 5 of 21 patients ( 24% ) had a history of androgen deprivation therapy . this may be explained by the undifferentiated nature of the sarcoma cells , hence the inability to produce psa . histologically , the epithelial portion is adenosarcoma with a variable sarcoma component . a review of 41 cases2 demonstrated osteosarcoma to be the most common ( 50% ) , followed by chondrosarcoma ( 33% ) , leiomyosarcoma ( 17% ) , rhabdosarcoma ( 12% ) , malignant fibrous histiocytoma ( 10% ) , fibrosarcoma ( 7% ) , spindle cell sarcoma ( 7% ) , myosarcoma ( 5% ) , undifferentiated sarcoma ( 2% ) and angiosarcoma ( 2% ) . up to 41% of reported cases had more than one type of sarcoma . a review of 41 cases of carcinosarcoma found the lungs to be the most common site ( 43% ) followed by bone or spine ( 26% ) , lymph nodes ( 19% ) , liver ( 17% ) , brain ( 10% ) , and 2% each of peritoneum , pancreas , spleen , penis , kidney , retroperitoneal space , pleura , and adrenal glands . radical surgery is recommended for carcinosarcoma due to its malignant nature and poor prognosis ; however , radiation , chemotherapy , and hormonal therapy are also utilized . overall prognosis with any treatment is poor , with a survival period of approximately 7 months , ranging from 34 days to 5 years . no factor , including age , history of radiation , or androgen deprivation therapy , histologic subtype , percentage of necrosis , percentage of sarcoma , sarcoma grade , or gleason grade of adenocarcinoma , has been found to be predictive of outcome . robotic - assisted laparoscopic radical prostatectomy has become widely adopted and robotic - assisted laparoscopic cystoprostatectomy is now being developed in several centers . gill et al reported the initial two cases of laparoscopic radical cystoprostatectomy with ileal conduit performed completely intracorporeally in 2000 . in 2003 , menon et al reported a series of 17 robotic - assisted radical cystoprostatectomies and urinary diversions for bladder cancer focusing on nerve preservation . they used a 3-step technique of robotic pelvic lymphadenectomy and cystoprostatectomy followed by externalization of bowel and extracorporeal neobladder reconstruction , and internalization of the neobladder with robotic urethravesical anastomosis . balaji et al reported one case of robotic - assisted radical cystoprostatectomy and ileal conduit urinary diversion performed totally intracorporeally . blood loss was 500ml , operative time 828 minutes , and hospital stay 10 days . concurrently , yohannes et al reported 2 cases of robotic - assisted radical cystoprostatectomy with ileal conduit urinary diversion with intracorpo - real ileal conduit construction . estimated blood loss was 435ml and 1800ml , operative time 10 hours and 12 hours , and hospital stay 6 days for both patients . to date , all reported robotic - assisted cystoprostatectomies have been performed for bladder cancer . our acceptable operative time and short convalescence suggest that the robotic interface may facilitate minimally invasive cystoprostatectomy . larger studies are certainly needed to determine whether robotic - assistance provides any advantages over standard open surgery . | carcinosarcoma of the prostate is a rare neoplasm with malignant epithelial and mesenchymal components . herein , we report the case of a patient who underwent multiple transurethral resections of the prostate showing adenocarcinoma initially then carcinosarcoma .
he underwent a robotic - assisted laparoscopic cystoprostatectomy , bilateral pelvic lymph node dissection , and ileal conduit urinary diversion and was discharged on postoperative day 7 .
carcinosarcoma is discussed as an extremely rare malignancy of the prostate , with less than 50 cases reported in the literature .
robotic - assisted radical cystoprostatectomy is also discussed as a new procedure in minimally invasive surgery and as the first reported use for prostatic carcinosarcoma . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
nowadays , everyone in their daily lives will experience some form of stress and inevitably tries to utilize a unique way to response . stress represents a normal , necessary and unavoidable life phenomenon that can generate temporary discomfort , as well as long - term consequences . scientific information confirms the idea that personality traits are an important factor in identifying , responding and approaching stress events . personality traits are as prepreparation for thinking or acting in a similar style in response to a variety of different stimuli or situations . maladaptive personality traits ( e.g. , neuroticism ) is related with increased exposure to stressful life events and likely to make individuals susceptible in experiencing negative emotion and frustration , while , adaptive personality traits ( e.g. , high extraversion and conscientiousness ) were less affected by daily stresses . also , personality traits could predict coping styles and influence the coping style we choose . coping is a regulatory process that can reduce the negative feelings resulting from stressful events . coping is like the changing of thoughts and actions to manage the external and/or internal demands for a stressful event . indeed , coping is a dynamic process that fluctuates over time in response to changing demands and appraisals of the situation . three main coping styles are problem - focused coping , emotion - focused coping , and avoidant coping . problem - focused coping ( e.g. , problem engagement and positive re - interpretation and growth ) involves altering or managing the problem that causes the stress and is highly action - focused . emotion - focused coping styles are quite varied , but they all diminish the negative emotions associated with stressor , thus those coping are action - orientated . adaptive forms of emotion - focused coping are seeking support and accepting responsibility . avoidant coping can be described as cognitive , and behavioral efforts directed toward minimizing , denying or ignoring dealing with a stressful situation . although some researchers are placed avoidant coping and emotion - focused coping in a group , the styles are conceptually distinct . some studies have shown that adaptive personality traits are significantly positively associated with active coping styles , while maladaptive personality traits ( neuroticism ) are positively associated with avoidance coping . the association between personality and coping styles suggest that individuals with maladaptive personalities are at a greater risk for experiencing psychological distress as they probably use a maladaptive coping style such as avoidant coping . however , not all the findings regarding the relationship between personality and coping have been consistent . some researcher have failed to find a significant relationship between some personality traits ( agreeableness , conscientiousness , and openness ) and coping . for example , the significant relationship has not found between extraversion and either problem - focused coping or generally adaptive forms of emotion - focused coping such as seeking support and accepting responsibility . accordingly , the main goal of this study is more comprehensive examining the association of personality traits and coping styles according to the stress level in a large sample . the current study was conducted within part of the study on the epidemiology of psychological - alimentary health and nutrition ( sepahan ) project . this project was a community - based program designed to study the epidemiology of functional gastrointestinal disorders ( fgids ) in iran in 2011 . furthermore , the role of different lifestyle , nutritional , and psychological factors in fgids symptoms and their severity was investigated . the current study is a part of the sepahan ( ref ) . in this cross - sectional study , the studied sample was selected using multistage cluster sampling and convenience sampling in last stage among 4 million people in 20 cities across isfahan province . in sepahan study , data were collected in two separate phases to increase the accuracy , as well as the response rate . in the first phase , all participants were asked to complete a self - administered questionnaire about demographic and lifestyle factors including nutritional habits and dietary intakes . in the second phase , further information on gastrointestinal functions and different aspects of psychological variables were collected using another bunch of self - administered questionnaires ( response rate : 86.16% ) . in the current analysis , we used data from 4,763 adults who had completed data on demographic data , personality traits , life event , coping with stress , social support , and psychological outcome such as depression and anxiety . the protocol of the study was approved by the ethics committee of iums and was clarified for all the participants , and a written informed consent was obtained from all participants . the protocol of study was approved by the medical research ethics committee of iums ( # 189069 , # 189082 , and # 189086 ) , and it was clarified for all the participants and a written informed consent was obtained from all them . after assuring to individuals about the confidentiality of the information , data on demographic characteristics , personality traits and coping styles were collected by standardized self - administered questionnaires . demographic factors applied in this study were age , sex as male and female , marital status as unmarried ( single , widow and divorce ) and married , educational level as 0 - 12 years ( undergraduate ) , and > 12 years ( graduate ) . each item is rated on a four - point scale ( less than usual , no more than usual , fairly more than usual and much more than usual ) . the system used to score the ghq-12 questionnaires was the 0 - 0 - 1 - 1 method . using this method , a participant could have been scored between 0 and 12 points ; a score of 4 or more was used to identify a participant with high - stress level . validity of the ghq-12 is good and it has the satisfactory internal consistency ( a = 0.87 ) . it consisted of 60 items grouped into five subscales : extraversion , neuroticism , agreeableness , openness to experience and conscientiousness . respondents rate each item on a one ( strongly disagree ) to five ( strongly agree ) scale . the reliability for the entire scale ( a = .70 ) and subscales ( as > 68 ) were adequate . a multicomponent self - administered coping strategies questionnaire was used to assess the cope with stressful life event . it consisted of the 23 items grouped into five subscales : positive reinterpretation and growth , problem engagement , acceptance , seeking support and avoidance . the reliability of the questionnaire was determined using cronbach 's alpha coefficient ( a = 0.84 ) . each item was scored on a 3-point scale ( never = 0 , sometimes = 1 , and often = 2 ) . for each scales , descriptive analysis of the study population was performed ( i.e. , mean standard deviation for continuous variables and percentages for discrete variables ) , and differences between groups were analyzed with t - test and chi - square test . pearson correlation coefficient was used to evaluate the correlation of personality traits with coping styles . moreover , for evaluating of the normality of data , kolmograph - smirnov test was used . a binary logistic regression analysis was performed to separately find the association between personality traits and coping styles with stress level . the dependent variable was stress level ( low / high ) and the independent variables were personality traits and coping styles . ors were reported with the corresponding 95% confidence intervals . the statistical package for the social sciences version 15.0 ( spss inc . , the current study is a part of the sepahan ( ref ) . in this cross - sectional study , the studied sample was selected using multistage cluster sampling and convenience sampling in last stage among 4 million people in 20 cities across isfahan province . in sepahan study , data were collected in two separate phases to increase the accuracy , as well as the response rate . in the first phase , all participants were asked to complete a self - administered questionnaire about demographic and lifestyle factors including nutritional habits and dietary intakes . in the second phase , further information on gastrointestinal functions and different aspects of psychological variables were collected using another bunch of self - administered questionnaires ( response rate : 86.16% ) . in the current analysis , we used data from 4,763 adults who had completed data on demographic data , personality traits , life event , coping with stress , social support , and psychological outcome such as depression and anxiety . the protocol of the study was approved by the ethics committee of iums and was clarified for all the participants , and a written informed consent was obtained from all participants . the protocol of study was approved by the medical research ethics committee of iums ( # 189069 , # 189082 , and # 189086 ) , and it was clarified for all the participants and a written informed consent was obtained from all them . after assuring to individuals about the confidentiality of the information , data on demographic characteristics , personality traits and coping styles were collected by standardized self - administered questionnaires . demographic factors applied in this study were age , sex as male and female , marital status as unmarried ( single , widow and divorce ) and married , educational level as 0 - 12 years ( undergraduate ) , and > 12 years ( graduate ) . each item is rated on a four - point scale ( less than usual , no more than usual , fairly more than usual and much more than usual ) . the system used to score the ghq-12 questionnaires was the 0 - 0 - 1 - 1 method . using this method , a participant could have been scored between 0 and 12 points ; a score of 4 or more was used to identify a participant with high - stress level . validity of the ghq-12 is good and it has the satisfactory internal consistency ( a = 0.87 ) . it consisted of 60 items grouped into five subscales : extraversion , neuroticism , agreeableness , openness to experience and conscientiousness . respondents rate each item on a one ( strongly disagree ) to five ( strongly agree ) scale . the reliability for the entire scale ( a = .70 ) and subscales ( as > 68 ) were adequate . demographic factors applied in this study were age , sex as male and female , marital status as unmarried ( single , widow and divorce ) and married , educational level as 0 - 12 years ( undergraduate ) , and > 12 years ( graduate ) . each item is rated on a four - point scale ( less than usual , no more than usual , fairly more than usual and much more than usual ) . the system used to score the ghq-12 questionnaires was the 0 - 0 - 1 - 1 method . using this method , a participant could have been scored between 0 and 12 points ; a score of 4 or more was used to identify a participant with high - stress level . validity of the ghq-12 is good and it has the satisfactory internal consistency ( a = 0.87 ) . it consisted of 60 items grouped into five subscales : extraversion , neuroticism , agreeableness , openness to experience and conscientiousness . respondents rate each item on a one ( strongly disagree ) to five ( strongly agree ) scale . the reliability for the entire scale ( a = .70 ) and subscales ( as > 68 ) were adequate . in iranian sample , a multicomponent self - administered coping strategies questionnaire was used to assess the cope with stressful life event . it consisted of the 23 items grouped into five subscales : positive reinterpretation and growth , problem engagement , acceptance , seeking support and avoidance . the reliability of the questionnaire was determined using cronbach 's alpha coefficient ( a = 0.84 ) . each item was scored on a 3-point scale ( never = 0 , sometimes = 1 , and often = 2 ) . for each scales , descriptive analysis of the study population was performed ( i.e. , mean standard deviation for continuous variables and percentages for discrete variables ) , and differences between groups were analyzed with t - test and chi - square test . pearson correlation coefficient was used to evaluate the correlation of personality traits with coping styles . moreover , for evaluating of the normality of data , kolmograph - smirnov test was used . a binary logistic regression analysis was performed to separately find the association between personality traits and coping styles with stress level . the dependent variable was stress level ( low / high ) and the independent variables were personality traits and coping styles . ors were reported with the corresponding 95% confidence intervals . the statistical package for the social sciences version 15.0 ( spss inc . , in this study , 4628 individuals with mean age 36.3 7.91 ; 2604 ( 56.26% ) female ; 2585 ( 55.8% ) graduate ; 3658 ( 79.1% ) married were examined . the scores on stress level were recorded into two categories , namely , low stress and high stress . individuals with high stress ( 1097 , 23.1% ) significantly were younger , female , undergraduate and married . descriptive statistics , means and sd of demographic characteristics , personality traits and coping style according to stress level ( n = 4628 ) correlations between personality traits and coping styles were computed . as shown in table 2 , extraversion , openness , agreeableness and conscientiousness were positively correlated with problem engagement , seeking support , positive reinterpretation and growth and acceptance , and negatively with avoidance ; while neuroticism was negatively correlated with problem engagement , seeking support , positive reinterpretation and growth and acceptance , and positively with avoidance . pearson correlations between personality traits and coping styles to examine the association of personality traits and coping styles according to stress level , a binary logistic regression was conducted with stress level serving as the dependent variable . the results are shown in table 3 . in crude analysis , neuroticism was a risk factor for stress level with or , 95% confidence intervals : 1.24 ( 1.22 , 1.26 ) ; but other personality traits were protective factors . the most protective factor was extraversion with 0.83 ( 0.82 , 0.85 ) . also , active coping styles were protective factors for stress level , and positive reinterpretation and growth was the most effective of coping style with 0.64 ( 0.60,0.69 ) . in model 1 , with adjusting covariates of demographics characteristics ( age , sex , marital status and educational level ) similarly , in model 2 , with adjusting covariates of demographic characteristics , and the rest of personality traits did nt show sensible changing in or stress . also , in model 3 , with adjusting covariates of demographic characteristics , and the rest of coping styles did nt show sensible changing in or stress . in this study , the association of five personality traits and coping styles was examined . as excepted , results showed adaptive personality traits were positively associated with active coping styles , and negatively with avoidance coping ; and maladaptive personality trait ( neuroticism ) was negatively associated with active coping styles , and positively with avoidance coping . openness and conscientiousness had the most significantly positive correlation with problem engagement , and extraversion and agreeableness had the most significantly positive correlation with positive reinterpretation and growth . studies have shown individuals with neuroticism use passive coping strategies but extravert individuals utilize active copings . costa et al . , reported that neuroticism is negatively related to the use of some effective coping styles such as problem - focused and active coping , and positively associated with avoidance coping . furthermore , most research shows that extraversion is positively related to active coping styles like problem - focused coping styles and looking for social support , and it predicts avoidance negatively . conscientious is significantly positively associated with problem - focused coping and its various components like planning , restraint coping and acceptance of responsibility . agreeableness is positively associated with social support seeking , active coping , planning and positive reappraisal , and negatively associated with self - blame , avoidance and wishful thinking . also , research findings show positive relationships between openness and active coping and positive reinterpretation , and negative correlations with avoidance coping . considering the research , it can be elucidated that neuroticism has been associated with more subjective reports of stress symptoms and the occurrence of stressful life events . individuals with high neuroticism are susceptible to psychological helplessness and irrational thoughts and have less ability to control their impulses . they have a tendency to experiencing negative emotions and , therefore , may be to direct their coping efforts toward managing those painful emotions . so , it is more possible that these individuals get involved in passive and maladaptive coping styles . past efforts have indicated that certain heritable personality attributes make individuals naturally more resistant or susceptible to eustress or distress and its benefits or disadvantages . specifically , elements of neuroticism and more protective traits like conscientiousness have been linked to differential interpretations of stimuli , eustressful or distressful , challenging or threatening . it is believed that conscientiousness results in challenge appraisal or eustressful condition because of sharing in rational solution formation while neuroticism leads to threat appraisal or distressful experience because it is associated with negative reactions . some authors assume that coping styles can directly be derived from personality traits , indeed , coping is personality in action . so , it is supposed that personality traits may influence the effectiveness of coping styles . it means the styles that are useful for some individuals may be less effective or even harmful for individuals that have different personality traits . thus , there is a possibility that high - neuroticism individuals are emotionally more reactive because they choose maladaptive coping styles , or that they choose similar styles to those chosen by low - neuroticism individuals ( problem - focused coping ) that they are ineffective at alleviating their distress . however , it is believed that deeper understanding of the role of personality in the coping process requires an assessment of personality traits and specific coping strategies , and use of laboratory and daily report studies . the strengths of this study are the large sample of respondents and the application of validated instruments . limitations are the cross - sectional design , self - report questionnaires and non - controlling other factors that may affect stress level . the current research provided a more complete picture of the relationship of personality traits with coping ways in stressful situations . it showed that adaptive traits with active copings and maladaptive traits with passive copings were associated , and traits associated with passive copings cause high - stress level . so , it could be concluded that improve and strengthen effective coping strategies in individual with maladaptive traits should be considered as a crucial component of prevention and control programs of stress . also , the findings could be used for determining specific training programs for managing psychological distresses . but , it seems that the active and effective copings require a systematic work considering the role of personality traits in them , especially in at risk traits . | background : some personality traits and coping styles could be as risk factors in stressful situations .
this study aimed to investigate the association of personality traits and coping styles according to the stress level.meterials and methods : this cross - sectional study was performed in 2011 .
a total of 4628 individuals over 20 years were selected by random sampling from nonacademic employees that working in 50 different centers across isfahan province .
data were collected using 12-item general health questionnaire ( ghq-12 ) , big five personality inventory short form and coping strategies scale , and individuals were divided into high and low - stress groups in term of ghq-12 . to analyze the data , a binary logistic regression analysis was conducted.results:mean age of participants was 36.3 7.91 years and 56.26% ( 2604 ) of them were female .
neuroticism with adjusting covariates of demographic characteristics and the rest of personality traits was a risk factor for stress level with odds ratios ( or ) or:1.24 ; but other personality traits were protective . also ,
active coping styles were protective factors for or of stress level with adjusting covariates of demographic characteristics and the rest of coping styles , and positive reinterpretation and growth was the most effective of coping style with or:0.84.conclusion : some personality traits are associated with passive copings and cause high - stress level .
so , it could be concluded that improve and strengthen effective coping strategies in individual with maladaptive traits should be considered as a crucial component of prevention and control programs of stress . |
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celiac disease ( cd ) is an immune - mediated inflammation of the small intestine caused by sensitivity to dietary gluten in genetically sensitive individuals . the disorder is common , occurring in 0.5 to 1 percent of the general population in most countries ( 1 ) . overall , the global distribution of the disease seems to parallel the distribution of hla genotypes that predispose to cd , provided that the population is also exposed to gluten ( 2 ) . the naspghan recommends serologic screening of cd be performed in the following groups of children with a gluten - containing diet ( 1 ) . patients with failure to thrive , persistent diarrhea , chronic constipation , recurrent abdominal pain , or vomiting , dental enamel hypoplasia of permanent teeth , idiopathic short stature , significant pubertal delay , iron deficiency anemia not responsive to supplementation , dermatitis herpeticformis - like rash , recurrent aphthous stomatitis , fracture not explained by the level of trauma , abnormal liver biochemical tests , chronic fatigue , and all members of the following high - risk groups : first - degree relatives of patients with cd , autoimmune thyroiditis , type 1 diabetes , down syndrome , turner syndrome , williams syndrome , selective iga deficiency , and autoimmune hepatitis ( 3 ) . diagnosis of cd for most patients is recommended with measuring iga antibodies to human recombinant tissue transglutaminase ( ttg ) . this test is highly specific and sensitive . with using second - generation elisa technology , the sensitivity and specificity of anti - ttg antibodies for biopsy - proven cd are generally above 96 percent ( 1 ) . measurement of iga antibodies to endomysium are equally accurate , but this test is more expensive and somewhat dependent on operator interpretation ( 4 ) . therefore , total iga should be measured in children with negative results of iga - ttg but a high clinical suspicion of cd ( 5 ) . all individuals with positive ttg antibodies or anti endomysial antibodies should have an intestinal biopsy to establish the diagnosis of cd . multiple biopsies should be taken from the distal duodenum and duodenal bulb and interpreted by an expert pathologist ; the disease may have a patchy distribution ( 6 ) . the assessment of intestinal biopsies requires a certain level of expertise and skill , and the diagnostic accuracy can be affected by variability in sample quality and subjective interpretation ( 7 ) . as the difficulties in histological diagnosis of cd in clinical practice have been well documented ( 8) , the appropriate use of simpler and more accurate tools would add reliability to the diagnosis of cd . a new diagnostic standard based on serology alone , which could accurately detect patients in any clinical setting , has been previously suggested ( 9 ) . thus , based on the very high positive predict values ( ppvs ) of reliable serological tests , some authors have proposed that intestinal biopsy could no longer be mandatory for the diagnosis of cd in some patients . our aim in this prospective study was to explore the performance of serology - based algorithms that can potentially obviate the need for intestinal biopsies for diagnosis . the subjects were selected among individuals with suspected cd ( with symptoms such as chronic diarrhea ( 3 cases ) , chronic abdominal pain ( 3 cases ) , failure to gain weight ( 52 cases ) , and short stature ( 32 cases ) , referring from other clinics , and high - risk individuals , namely cases with type 1 diabetes ( 24 cases ) and first - degree relatives of patients with cd ( 1 case ) . the subjects were evaluated after physical examinations . in total , 121 subjects ( 69 female and 52 male subjects ) within the age range of 2 to 18 years were included in the study . patients under the age of 2 years were eliminated from the study , given the low accuracy of serologic screening with ttg - iga at this age . in patients , ttg - iga and the new generation of aeskulisa kit ( germany ) , along with elisa method , was applied for all patients . upper endoscopy , along with duodenal biopsy ( biopsy from bulb and discrete sites of duodenum ) , was performed on 5 samples . the samples were interpreted by an expert pathologist and findings were classified based on marsh - oberhuber categorization . according to pathologic findings we used the area under the receiver operating characteristic ( roc ) curve to investigate the hypothesis and spss ( ver.11 ) for the statistical analyses . the number of male and female subjects was not significantly different ( p = 0.804 ) . among 121 cases , 40 subjects ( 33.1% ) had positive pathologic results for cd and 81 ( 66.9% ) had negative pathologic results . the lowest ttg - iga titer was 19 ml / iu and the highest was 980 ml / iu ( table 1 ) . 25 cases were in the high - risk group , which included patients with diabetes mellitus ( 24 cases ) and first - degree relatives of patients with cd ( 1 case ) . in the high - risk group , 9 patients had positive pathologic results , with mean serological level of 217i u / ml and standard deviation 129.6 , while 16 subjects had negative pathologic results with mean serological level of 164.6i u / ml and sd 211 . despite the lowert tg - iga titer in subjects with negative pathologic results , compared to those with positive pathology , the difference was not statistically significant ( p = 0.121 ) . the serological titer was higher in the high - risk group ( mean = 183 and sd 185.1 ) , compared to other subjects ( mean = 109 and sd 133.2 ) ; also , the difference between the high - risk group and other patients was statistically significant ( p = 0.007 ) . in the high - risk group , by using receiver operating characteristic ( roc ) curve , the cut - off point for serological titer was 126 iu / ml ( sensitivity 7% and specificity 75% ) . according to espghan guidelines ( 5 ) , a serological titer of 3 upper limit normal ( uln ) is considered for avoiding biopsy in the high - risk group . so with serology titer of 54 ( 3 uln , normal : 18 ) , sensitivity 88.9% and specificity 9% was concluded . a statistically significant relation was observed between serological titer and positive pathologic results in patients with suspected cd . in other words , patients with positive pathologic results had higher serological titers ( mean = 190 iu / ml and sd 180.5 ) and p < 0.001 . maximum sensitivity and specificity were estimated at 65% and 65.4% with a serological titer of 81.9 iu / ml , respectively ( serologic titers above and lower this titer ( 81.9 iu / ml ) were not suitable for maximum sensitivity and specificity ) . among patients with positive pathologic results ( 40 individuals ) , 26 ( 65% ) cases had a serological titer of 81.95 iu / ml ; if the titer was higher or equal to 81.95 iu / ml , pathologic results were considered positive . intestinal biopsy could be avoided in 21.48% of cases witha positive predictive value ( ppv ) of 48.15% and a negative predictive value ( npv ) of 79.10% . according to espghan guidelines ( 5 ) , a serological titer equal to or higher than 10 uln therefore , based on a titer of 180 , intestinal biopsy could be avoided in 14.4% of cases with ppv = 70.8 and npv = 76.29 ; titer sensitivity was estimated at 42% and specificity was found to be 14% . on the other hand , a significant correlation was observed between failure to gain weight and serological titer in patients . in fact , subjects with failure to gain weight had lower serological titer ( mean serological titer of 93.4 with sd 95 ) , compared to subjects without problems in gaining weight ( mean serological titer of 147.9 with sd 174.9 ) ( p = 0.045 ) . serological titer was not significantly correlated with chronic diarrhea or chronic abdominal pain in patients . moreover , no significant relationship was observed between short stature of patients and serological titer . however , serological titer in patients with diabetes mellitus ( mean = 182.83 with sd 189 ) was higher than that reported in other subjects ( mean = 110 with sd 132.8 ) ; this difference was statistically significant ( p = 0.012 ) . evaluation of serological titer in first - degree relatives of celiac patients did not indicate any significance , given the low number of these subjects . no meaningful association was found between serological titer and type of pathology according to kruskal - wallis test ; however , higher serological titer was correlated with higher pathologic grading . the mean serological titer in patients with marsh grade 2 ( 1 case ) was 60.5 iu / ml and 174 - 182 iu / ml with sd 147 - 109.8 in patients with marsh grade 3 ( 39 cases ) ( p = 0.722 ) . moreover , serological titer and pathology grading were not significantly associated in patients with short stature , type 1 diabetes , or failure to gainweight . additionally , no statistically significant relation was observed between positive pathological results and short stature , chronic diarrhea , failure to gain weight , chronic abdominal pain , or type 1diabetes and first - degree relatives of patients with cd . final diagnosis of cd is established via small bowel biopsy . in fact , this procedure is still considered the gold standard , despite the fact that morphological features are non - specific and other conditions can produce similar findings ( 10 ) . however , according to conducted studies , diagnosis of cd can be made using serological titer to avoid intestinal biopsy ; via this method , results can be obtained more rapidly and invasive operations can be avoided . in previous studies , for the diagnosis of this condition , serological results were compared in cases with positive pathology in order to avoid biopsy in patients . in these studies , evaluation was performed using only one serological test ( or several tests ) , combined with genetic studies . intestinal biopsies could be avoided in 65% to 92% of cases in these studies ( 11 ) . in our study , one serological test ( ttg - iga ) , along with positive pathologic results , was used ; therefore , in comparison with previous studies , fewer serological tests were performed ( 12 ) . by selecting one serological test ( ttg - iga ) , with higher ( > 95% ) sensitivity and specificity were obtained and other tests such as endomysial antibodies ( ema ) could be avoided . in fact , in comparison with ema , the obtained results were equally accurate , though ema test is more expensive and somewhat dependent on the operator s interpretation ( 13 ) . in our study , a statistically significant relation was observed between higher serological titers and positive pathologic results ; however , in our study , higher serological titers had lower sensitivity and specificity , compared to other studies . with a serological titer of 81 iu / ml , specificity and specificity were estimated at 65% and 65.4% , respectively ; this finding suggests that further studies are required . in our study , application of a single serological test ( ttg - iga ) for the diagnosis of cd was controversial , contrary to other studies ( 11 , 14 ) . in fact , patients with positive pathologic results had higher serological titer ( mean 190 iu /ml and sd 180.5 ) , compared to patients with negative pathologic results ( mean 91 iu / ml and sd 116.4 ) ( 11 ) . the high - risk group ( patients with diabetes mellitus and first - degree relatives of patients with cd ) had higher serological titers , compared to others ; moreover , a significant relation was found . this finding ( higher serological titers ) was in match with espghan guidelines , which indicate that in high - risk individuals with lower serological titers ( 3 uln ) in comparison with other patients suspected for cd ( 10 uln ) , biopsy can be avoided ( 5 ) . patients with failure to gain weight ( mean 93 iu / ml ) had lower serological titers , compared to other subjects ( mean 147 iu / ml ) ; this finding suggests that more interventions are required for the diagnosis of cd in these individuals . a similar finding was reported in patients with chronic diarrhea ; however , considering the low number of these patients , confirmation of the findings needs further analysis . also , considering the low number of patients with chronic abdominal pain and chronic diarrhea , and first - degree relatives of patients with cd , further analysis is required moreover , in the high - risk group , serological titer in subjects with positive pathologic findings was higher than in patients with negative pathology ; however , the difference was not statistically significant . in our study , considering the lower sensitivity and specificity of serological titers and lower success in avoiding intestinal biopsy , in contrast with other studies , the utility of one serological test for the accurate diagnosis of cd is not sufficient and further analysis is required . to obtain the best serological algorithm , future studies need to evaluate as many tests and test combinations as possible furthermore , a diversity of patient populations needs to be evaluated by taking different relevant aspects ( e.g. , disease prevalence and the magnitude of intestinal mucosal damage ) into consideration . in our study , considering the lower sensitivity and specificity of serological titers and lower success in avoiding intestinal biopsy , in contrast with other studies , the utility of one serological test for the accurate diagnosis of cd is not sufficient and further analysis is required . to obtain the best serological algorithm , future studies need to evaluate as many tests and test combinations as possible . furthermore , a diversity of patient populations needs to be evaluated by taking different relevant aspects ( e.g. , disease prevalence and the magnitude of intestinal mucosal damage ) into consideration . | background : celiac disease is an immune - mediated inflammation of the small intestine caused by sensitivity to dietary gluten in genetically sensitive individuals.objectives:in this study , we aimed to evaluate the predictive value of tissue transglutaminase ( ttg ) antibodies for the diagnosis of celiac disease in a pediatric population in order to determine if duodenal biopsy can be avoided.patients and methods : the subjects were selected among individuals with probable celiac disease , referring to a gastrointestinal clinic . after physical examinations and performing tissue transglutaminase - immunoglobulin a ( ttg - iga ) tests , upper endoscopy was performed if serological titer was higher than 18 iu / ml . therapy started according to pathologic results.results:the sample size was calculated to be 121 subjects ( 69 female and 52 male subjects ) ; the average age of subjects was 8.4 years .
a significant association was found between serological titer and pathologic results ; in other words , subjects with high serological titer had more positive pathologic results for celiac disease , compared to others ( p < 0.001 ) . maximum sensitivity ( 65% ) and specificity ( 65.4% ) were achieved at a serological titer of 81.95 iu / ml ; the calculated accuracy was lower in comparison with other studies .
as the results indicated , lower antibody titer was observed in patients with failure to gain weight and higher antibody titer was reported in diabetic patients.conclusions:as the results indicated , a single serological test ( ttg - iga test ) was not sufficient for avoiding intestinal biopsy . |
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protein - losing gastroenteropathy ( plge ) is a disease that induces excessive plasma protein loss from digestive organs into the gastrointestinal ( gi ) tract . causes of plge include primary diseases , such as mntrier disease or intestinal lymphangiectasia and secondary diseases that develop along with other diseases . although several reports have been published recently on plge associated with collagen diseases [ 2 , 3 , 4 , 5 ] , it is relatively rare for plge to develop together with sjgren syndrome ( ss ) . few patients with ss require steroid pulse or immunosuppressive therapy because of resistance to the moderate or high amounts of steroid , as in this case . here the patient was a 59-year - old female with a medical history of rheumatoid arthritis developed at the age of 40 and hashimoto thyroiditis developed at the age of 51 . since a few years , she has been free from rheumatoid arthritis symptoms such as morning stiffness , fatigue , and joint pain , and on examination active arthritis was absent . she noticed facial edema around march 2010 and was referred to our hospital in april 2010 for further examination and treatment . at admission her height was 153.2 cm , weight was 74.4 kg , body temperature was 35.8c , blood pressure was 121/90 mm hg , and she had a regular pulse rate of 72/min . furthermore , no cardiac murmurs or pulmonary rales were heard , and edema on the upper eyelids and lower legs with pitting was observed . laboratory findings at admission including levels of total protein and albumin were 5.9 g / dl and 2.8 g / dl , respectively , suggesting hypoproteinemia . however , the quantitative urinary protein was 34.2 mg / dl , and no abnormal protein leakage into the urine was observed . thyroid hormones were controlled within the normal range ( tsh 2.812 iu / ml , ft3 1.71 pg / ml , and ft4 1.23 ng / dl ) with 100 g / day levothyroxine sodium . the level of the anti - thyroglobulin antibody was 1,630 u / ml and that of the anti - microzome antibody was positive at a dilution of 1:1,280 . the levels of complement c3 and c4 were 35.6 mg / dl ( normal 75 - 130 mg / dl ) and 20 mg / dl ( normal 16 - 31 mg / dl ) , respectively . serum igg level was 658 mg / dl ( normal 932 - 1,976 mg / dl ) , and stool fat was detected by sudan iii staining . chest x - ray on admission revealed a right - sided pleural effusion , and an echocardiogram revealed a pericardial effusion , although no abnormality was found in cardiac function . after admission , the patient was concomitantly treated with intravenous albumin infusion and diuretic injection ( fig . 1 ) , and further examinations were performed to detect the cause of her symptoms . upper and lower gi endoscopic examinations were performed to find the cause of protein leakage from the digestive organs . upper gi endoscopy revealed mild swelling of the entire gastric mucosa without any tumor or erosive ulcerative lesions or giant folds such as those manifested in mntrier disease and other conditions . although the mucosal surface appeared normal , lower gi endoscopy revealed mild edema and congestion of the entire colon ; however , no tumor formation or erosive ulcerative lesion was observed . tc - human serum albumin ( tc - hsa ) scintigraphy revealed early radioisotope accumulation in the stomach ( fig . 2 ) and a1-antitrypsin ( a1-at ) clearance was high at 205 ml / day ( normal < 13 ml / day ) , and thus a diagnosis of plge was made . furthermore , complication of her condition by ss was suspected because she had eye and oral dryness for more than a month before admission . using the gum test , 23 ml of saliva was collected and schirmer 's test results were 6 and 8 mm for the right and left eyes , respectively . according to the diagnostic criteria for ss in japan , diagnosis of ss was confirmed by a positive result for anti - ro / ssa antibodies ( anti - ro / ssa antibody level was 355 u / ml and anti - la / ssb antibody level was < 7 u / ml ) and salivary gland biopsy . the diagnosis of plge complicated by ss was made as described above and 50 mg / day prednisolone ( psl ) was administered ( fig . hypoproteinemia was aggravated with total serum protein at 2.8 g / dl and albumin at 1.0 g / dl , and worsening of edema and development of diarrhea were observed . therefore , steroid pulse therapy ( 1.0 g / day methylprednisolone for 3 days ) was initiated as an alternative treatment . the levels of total serum protein and albumin increased and facial edema and digestive tract symptoms disappeared . the dose of psl was tapered off after reducing it to 60 mg / day . however , a flare - up was observed . we obtained informed consent from the patient and performed steroid pulse therapy for the second time together with cyclophosphamide ( cpa ) pulse therapy and oral administration of 150 mg / day mizoribine ( mzr ) . no relapse occurred , and she was discharged when her total serum protein and albumin had been maintained at nearly normal levels . after treatment , radioisotope accumulation previously observed in tc - hsa scintigraphy disappeared and a1-at clearance improved to 17.3 ml / day with reduced protein loss from the digestive organs into the gi tract . plge is a disease that induces excessive loss of plasma proteins into the gi tract . the causes of plge include various diseases such as digestive tract diseases , cardiac diseases , or collagen diseases . for autoimmune plge , the number of reports regarding plge cases associated with collagen disease has been increasing in japan [ 3 , 4 , 5 ] , especially after the study of pachas et al . in 1971 of systemic lupus erythematosus ( sle ) complicated by plge . primary collagen diseases include sle which is observed most commonly - , mixed connective tissue diseases , ss and scleroderma . to the best of our knowledge , from 1986 until 2010 , only 22 cases of plge associated with ss were reported in japan ( including minutes in a conference and our case ) [ 3 , 5 ] . among these , 86.4% of patients , including our case , were females with a mean age of 45.3 years . in most cases ( 90.9% ) , edema was the main symptom and digestive tract symptoms , such as diarrhea , were rare ( 22.7% ) . the most common site of protein loss , if detectable , is the small intestine . protein loss from the stomach , as presented in this case , is rare ; only 4 cases have been reported until date . the mean a1-at clearance was 219.1 ml / day ; the level was likely to be higher than the 100 ml / day in plge cases that developed repeatedly along with inflammatory bowel disease with mucosal disorders . upper gi endoscopic examination performed before treatment revealed neither giant folds at the greater curvature of the body of the stomach nor viscous gastric fluid secretion as that manifested in mntrier disease , indicating that abnormality in the mucosal surface was absent . furthermore , lymphangiectasia was not detected in biopsies of the stomach and duodenum , from where protein loss was suspected . the mechanisms of protein loss from the gi wall involve abnormality of the intestinal lymphatic system , increased papillary permeability , or abnormality of gastric mucosal epithelium . however , the exact mechanism responsible for plge in association with collagen disease remains unclear . a majority of the gi endoscopic findings in previously reported cases were edematous changes of lesions with a very mild redness , and the biopsy findings were often mild inflammatory cell infiltration and mild lymphangiectasia . with regard to pathological observations , deposition of immunoglobulins and complement components , such as c3 , were observed with immunostaining on the capillary walls in the lamina propria . it has been reported that complement activation may induce increased capillary permeability [ 3 , 4 , 10 ] resulting in protein loss . in this case , deposition of igg , c3 , c1q , and fibrinogen was detected on submucosal capillaries during immunostaining of biopsy samples having no lymphangiectasia , suggesting that the condition was induced by increased capillary permeability . with regard to treatment methods , all 22 patients with plge associated with ss have been reported to be treated with steroid therapy [ 3 , 5 ] . among the available data , in 13 cases , the patients were treated with moderate to high levels of steroid and the treatment was effective in 10 patients ( 76.9% ) . thus , the outcome was relatively satisfactory . however , in the present case , the patient was resistant to high levels of steroid ( 50 mg / day psl ) and required steroid pulse therapy ( fig . 1 , point a ) . although steroid pulse therapy was partially effective , it did not adequately improve symptoms in our patient ( fig . 1 , point b ) . finally , her symptoms improved with the use of immunosuppressive agents ( cpa pulse therapy and oral administration of mzr ) that followed the two - fold steroid pulse therapy ; the steroid dose was eventually reduced . therefore , more aggressive immunosuppression may increase the overall response of patients with plge associated with collagen disease resistant to a moderate to high dose of psl . in some cases , it has been reported that cpa pulse therapy or administration of oral azathioprine ( aza ) was effective in autoimmune plge cases associated with mixed connective tissue disease or sle [ 11 , 12 ] . although it has been used in various non - neoplastic autoimmune diseases , some reports have shown an association of immunosuppressive drugs with an increased risk of hematological and lymphoreticular malignancies . we employed intravenous cpa pulse therapy to treat patients because some reports have suggested the efficacy of this therapy for plge associated with sle . in addition , other reports suggest that treatment of plge with a combination of psl and aza is effective and well tolerated by the patients . we used mzr ( an imidazole nucleoside ) , which has frequently been used instead of aza as a component of immunosuppressive drug regimens ( renal transplantation patients , severe lupus nephritis , steroid - resistant nephritic syndrome , and rheumatoid arthritis patients ) . furthermore , the efficacy of mzr for ameliorating glandular symptoms has been reported through improvements in immune abnormalities in patients with ss . we present the first report of a patient with plge associated with ss that shows a favorable therapeutic effect of mzr . this patient showed improvement with the use of cpa pulse therapy , and consequently , mzr followed the steroid pulse therapy . therefore she has been free from facial edema as well as hypoproteinemia and has maintained complete remission after the combination therapy of low - dose psl and mzr . we suggest that a combination of immunosuppressive agents may be effective in patients with plge - associated autoimmune diseases . however , these treatments are still controversial and to support efficacy require more evidence with regards to the types of immunosuppressive agents , dose and duration of the treatment . | we report the case of a 59-year - old female who developed facial edema together with hypoproteinemia . on the basis of 99mtc - human serum albumin scintigraphy and a1-antitrypsin clearance ,
she was diagnosed with protein - losing gastroenteropathy .
furthermore , she was diagnosed with sjgren syndrome on the basis of eye and oral dryness , positive result with anti - ssa antibody , and salivary gland biopsy .
her symptoms improved with the use of immunosuppressive agents following steroid pulse therapy . therefore , steroid pulse therapy and immunosuppressive agents should be considered as possible effective treatment strategies for protein - losing gastroenteropathy associated with autoimmune diseases . |
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to describe the 1-year success of a trabecular stent implant in a pseudophakic glaucoma patient . we report the case of a patient who was implanted and followed - up for 1 year at the dipartimento di fisiopatologia clinica . the patient s intraocular pressure was controlled with topical beta - blockers for 6 months and without therapy for 6 months . identification of the implantation site is challenging and reduction of efficacy over time may occur . glaucoma in pseudophakic eyes is not uncommon ; both trabeculectomy and deep sclerectomy failure have been reported in the literature with higher rates in pseudophakic eyes than in unoperated eyes ( fontana et al 2006 ) . the istent ( glaukos corp , laguna hills , ca , usa ) is a heparin - coated titanium device that can be inserted ab interno in the trabecular meshwork under gonioscopic control , driving the aqueous into the schlemm s canal ; it can lower intraocular pressure ( iop ) in cultured human anterior segments ( bahler et al 2004 ) . the device inserted ab interno has shown to be clinically effective when used concomitantly with phacoemulsification ( feijoo et al 2006 ) . we report a case of a pseudophakic glaucoma patient who achieved target pressure after the implant of the trabecular bypass stent . a 68-year - old patient with primary open - angle glaucoma ( poag ) underwent uncomplicated phacoemulsification ( od ) and presented with elevated iop in the operated eye 1 month postoperatively . her prior medical history is as follows : renal colic with a diagnosis of nephrolitiasis , which was medically controlled and a previously reported intolerance to prostaglandin analogues . after the initial phaco surgery , her postop regimen consisted of steroids for 1 week , tapered over 2 weeks . at day 10 follow - up , iop in the operated eye was 19 mm hg ( 14 mm hg in the contralateral eye ) . also at day 10 , the right visual field had a fascicular scotoma impending on the macular region , and a superior notch of the optic nerve head . by 1 month postoperation , her iop had increased to 30 mm hg od ( and to 15 mm hg in the nonoperated eye ) . at that time , she was on dorzolamide tid in both eyes , with the addition of brimonidine in the right eye only twice daily . at this follow - up , there were no signs of inflammation and steroids were not felt responsible for the iop rise because they had already been stopped and iop was normal while she was on steroid treatment ( ie , previous control at 10 days ) . she was followed every 2 weeks for an additional 2 months ( 3 months after initial phaco ) . target pressure ( iop < 18 mm hg ) was achieved ( iop ranged from 12 to 17 mm hg ) with added medical therapy ( acetazolamide 250 mg tablet tid ) . following a significant eyelid and conjunctival allergic reaction , she was switched to preservative - free carteolol 2% , brimonidine , and topical steroids . inflammation returned after steroid use was discontinued . to attempt better management of her glaucoma , her therapy was changed to preservative - free carteolol 2% bid and acetazolamide 250 mg tid . she was able to achieve target iop of < 18 mm hg on maximum therapy , but did not want additional invasive surgery after an unsuccessful argon laser trabeculectomy was performed to avoid the use of systemic drugs . following a renal cholic , the patient agreed to the istent implantation , as she did not view the procedure as invasive . the device itself is a small l - shaped hollow tube designed to be implanted into schlemm s canal by having the surgeon make a slit through the trabecular meshwork . the istent is designed for nasal placement and is guided into schlemm s canal using ab interno gonioscopy . in this patient , a nasal clear corneal 2 mm incision was made . the anterior chamber was then filled with a viscoelastic agent to reform the anterior chamber and provide more clearance in the angle . the surgeon ( amf ) ensured the orientation of the stent on the applicator , placed the gonioscope on the cornea , and positioned the microscope to enable visualization of the trabecular meshwork . the leading edge of the istent was eased through the meshwork and into the canal , with the tip directed inferiorly . on the first postoperative day , her vision od was maintained ( 25/20 ) , few red cells were present in the anterior chamber , iop was well controlled ( 12 mm hg ) , and the stent was correctly placed in the angle ( figure 1 ) . when pressure was applied to the goniolens , small blood reflux could be seen sideways from the stent and some blood was reabsorbed by the stent snorkel ( video ) . pressure was monitored monthly for 6 months and was always lower than 17 mm hg ( range : 1216 mm hg ) on carteolol ; iop remained < 17 mm hg during the day . at 6 months , the stent remained in place and unobstructed ( figure 2 , left ) ; the ubm ( figure 2 , right ) demonstrated that the snorkel of the stent was adequately placed in the angle . topical therapy was then discontinued od and the patient followed for an additional 6 months ( 1 year after stent implantation ) . the iop in the right eye remained < 18 mm hg ( mean 14.9 1.7 ; range : 1317 mm hg ) . a diurnal curve without therapy demonstrated iop < 17 mm hg ( range : 1216 mm hg ) . we are reporting on our own successful experience using a trabecular stent in a pseudophakic eye using an ab interno approach . our experience mimics that of other surgeons using the stent in patients who were not pseudophakic ( spiegel et al 2007 ) , suggesting the stent s efficacy in controlling glaucoma may not be limited to just virgin eyes . there is currently under way a larger clinical study of 47 patients with cataract and open - angle glaucoma to directly compare results in iop lowering between those implanted with stents to those undergoing cataract surgery only and using medical therapy to manage their glaucoma . of note , in combined surgery , the ability for the surgeon to use the clear corneal incision created during cataract surgery can be advantageous , as there is no need for cautery and the conjunctiva and sclera are not disturbed . in this technique , a traditional glaucoma surgical procedures such as trabeculectomy would not be negatively affected ( samuelson 2007 ) . in this case study , the patient presented with a high iop 1 month following uneventful cataract surgery and without any sign of inflammation . it is unlikely that steroids might have caused this iop rise because they had already been tapered . the patient had a trabecular bypass stent inserted through a 2 mm temporal corneal incision . a key to a successful surgery is the need of correct identification of schlemm s canal and therefore proper placement of the stent . although in enucleated eyes , the mean postoperative iop averaged 12.4 mm hg . in our patient , the mean iop was slightly higher ( 14.9 mm hg ) , possibly due to limited circumferential flow in schlemm s canal . additional stent placement might result in further iop lowering as suggested by in vitro models ( bahler et al 2004 ) . longer - term iop reduction has to be assessed with regard to continuous lumen clearance or possible future trabecular fibrosis . | purposeto describe the 1-year success of a trabecular stent implant in a pseudophakic glaucoma patient .
the stent is implanted through a small corneal incision under gonioscopic control .
intraocular pressure control was reported in cultured human anterior segment.designobservational case report.methodswe report the case of a patient who was implanted and followed - up for 1 year at the dipartimento di fisiopatologia clinica .
the main outcome measures were intraocular pressure and visual field.resultsthe patient s intraocular pressure was controlled with topical beta - blockers for 6 months and without therapy for 6 months .
two diurnal curves demonstrated achievement of target pressure during the day .
the 1-year visual field was unchanged.conclusionswe are not aware of previous reports of trabecular stent implantation in pseudophakic patients .
the trabecular stent had minimal peri - operative morbidity .
identification of the implantation site is challenging and reduction of efficacy over time may occur . |
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over the past several years , schools of nursing have been called upon to restructure education programs to better prepare graduates for increasingly complex and rapidly changing health care environments [ 13 ] . according to benner and associates , nursing education programs must be redesigned to prepare nurses for new responsibilities and challenges in these health care environments . to accomplish this , the practice - education gap must be addressed by major shifts in both curricula and teaching methods . the call to revise nursing education programs is paralleled by similar calls from other organizations including the robert wood johnson foundation ( rwjf ) and the institute of medicine ( iom ) . the institute of medicine 's 2001 report , crossing the quality chasm : a new health system for the 21st century , recommended that leaders in health professions should develop strategies for restructuring clinical education to be consistent with the principles of the 21st - century health system throughout the continuum of undergraduate , graduate , and continuing education for medical , nursing , and other professional training programs the recent consensus report of the rwjf in collaboration with the iom , initiative on the future of nursing : leading change , advancing health , emphasizes that nurses should achieve higher levels of education and training through an improved education system that promotes seamless academic progression . recently , partnerships between schools of nursing and service settings in hospitals and other health care agencies led to the development of new approaches to improve clinical learning . the goal of these partnerships is to facilitate students ' understanding of both the responsibilities of the nurse and the environments in which nurses work as they acquire clinical knowledge and proficiency necessary for competent practice . one common approach in hospital settings is the dedicated education unit ( deu ) . pioneered in australia , deus are health care units either enhanced or developed through partnered commitments by clinical faculty and nursing staff to enrich student nursing clinical education . to help students complete learning objectives , an environment is created to provide optimal learning opportunities using a variety of teaching and learning strategies [ 713 ] . in the deu model , education responsibilities may not be shared equally between the clinical faculty and clinical nursing staff . while clinical faculty are present to assist with student learning and model good teaching practices , the clinical staff assumes more responsibility for direct instruction of students assigned to them . thus , not all students experience this educational concept during their program of study in nursing . those not participating receive the more traditional model where the clinical faculty assumes nearly all responsibility for clinical instruction with less intensive involvement of staff nurses . in the traditional approach , clinical faculty and guests on the units that host students as neither the faculty or students are fully integrated into unit functioning . outcomes of the deu concept of clinical education for undergraduate nursing students are not extensive , and the concept continues to be explored . extant findings [ 9 , 10 , 12 , 13 ] document positive outcomes to this approach in clinical education . collectively , outcomes show that students report feeling welcomed and supported by nursing staff , and included as members of the health team . they express confidence in patient assessment and communication skills and report taking greater responsibility for their own learning . clinical nursing staff comment that students are more accountable for patient care than traditional model student . clinical staff report they are comfortable that the quality of care is upheld in the new approach and are pleased to see that students are advancing clinically . in addition , clinical staff members report that they became more aware of the curriculum and clinical education requirements . in 2007 , the university of michigan school of nursing ( umsn ) and nursing services at the university of michigan health system ( umhs ) formed a partnership called the initiative for excellence in clinical education , scholarship and practice . the partnership was designed to leverage the resources and expertise of both organizations to advance all three mission areas . to begin the work , the partnership engaged in an extensive change process using danemiller tyson associates ' whole - scale change . whole - scale change is large organizational change that occurs through the power of microcosms , groups comprised of members who represent and bring a perspective of every level and area of their organization , and nonmembers who represent those who rely on their organization to meet their needs . the assumption is that these stakeholders collectively have knowledge of the work of the whole system and their wisdom can bring about a new identity for how the organization will function in the future . for our two partnering organizations , stakeholders ( n = 120 ) included members from all levels of staff nurses and nurse administrators , all levels of faculty including administrators and graduate student nursing instructors , nursing and medical students , physicians , patients , financial and human resource representation , and the vice - chair of the nurses ' collective bargaining unit at umhs . at a two - day retreat called an event , stakeholders were placed in small groups and asked to define the current state of our working relationship with respect to clinical education , practice , and scholarship , to envision a different future state for our relationship in these mission areas and to propose actions to move toward the future state . a facilitator skilled in conducting from the considerable data generated throughout discussions , six major themes emerged : teams , innovative strategies , shared vision , physical facilities , human resource development , and outcomes . a six - member task force with representation from both organizations was charged to elaborate each theme with actions culled from the larger dataset generated at the event . these themes and actions subsequently would serve as the foundation for further work on each of the three mission areas clinical education , practice , and scholarship . developing a clinical education model became the first priority for our work . by embedding students in the clinical setting with faculty who have clinical and research expertise education this link is critical both to advancing practice and solving clinical problems to improve patient outcomes . the purpose of this article is to describe the first phase of our collaboration , the development of the clinical education model , with attention given to the recommendations put forward by the iom , rwjf , and others . a three - member project team consisting of two faculty from the umsn and one health system nursing director was charged to use the themes from the event to develop a new clinical education model that was vetted and ready for pilot implementation in one year . in developing the new model , it was explicitly stated that the proposed model must align with values and goals of each organization and with current economic resources , that existing contractual and other legal and regulatory requirements must be honored , and that the model must align with the current undergraduate course curriculum . in addition , the new model was to reflect recommendations of organizations calling for change in how nurses were educated in order to better prepare them for current health care environments . an advisory committee to the project team was formed comprised of members from both organizations including the dean , the cno of the health system , and students . the role of the advisory committee was to provide advice , consultation , feedback , and recommendations throughout development of the model and plan for pilot implementation . that this is a true partnership between our organizations was demonstrated as the project team consulted with various faculty , students , and clinical partners in the health system including clinical nurse specialists , educational nurse coordinators , supervisors , and staff nurses for suggestions and endorsement throughout development of the model . feedback from these stakeholders was instrumental in revising ongoing work . circling back allowed stakeholders to see how their suggestions and concerns were handled . this approach to the work also permitted participants in the process to develop relationships over time and increase knowledge of health system units , patient populations , nursing practice , student instruction , and the curriculum . over the year of model development , 18 presentations and interactive sessions were held with various stakeholder groups . final vetting occurred with both organizations including the nurses ' collective bargaining unit in the health system . the new model consists of five components : clinical unit clusters , clinical teams , student skill development , clinical immersion , and student portfolio . maximizing umsn student placement capacity at umhs was highly desired as the environment provides strong evidence - based practice and quality of care is paramount . at the time of model development , many umsn students received clinical education in multiple hospitals and agencies . in the new model at umhs , all 36 inpatient clinical units , comprised of adult acute care , pediatrics , icus , and specialty services , were used to construct three multiunit clusters ( see figure 1 ) comprising key components of students ' clinical requirements . each cluster ( blue , yellow , green ) includes students of all academic levels . students organized in these clusters progress through undergraduate clinical courses over their four years of study . continuums of care areas available to all clusters are clinical areas in addition to inpatient units that provide care and services to patients as needed . maximizing the use of these additional clinical areas affords students opportunities to participate in and follow patient care throughout the health system as they learn about nurses ' roles in a variety of settings and reduces the need for placements at external agencies . in the current model , a few sections of obstetrical nursing and psychiatric nursing continue to be external due to current limited capacity of these services at umhs . community health nursing continues to be community based . upon admission to the school of nursing in the freshman year , students are assigned to one cluster and will have diverse clinical experience designed to meet all clinical course objectives across the four years of the curriculum . students who transfer from community colleges at the junior level are entered into clusters when admitted . the assignment to clusters familiarizes students with a defined set of units , their operations , and the work of nurses as the context for clinical learning . in addition , the clinical cluster approach creates cluster identity among faculty , staff , and students thereby enhancing relationships among these individuals in such a way that both education and patient care can benefit . cluster unit familiarity has high potential to increase identification of clinical problems for further study both within and across units in the cluster . health system clinical unit profiles , descriptions of each clinical unit that include common patient populations , diagnoses , medications , and treatments , are given to students for each unit in their cluster . profiles provide background information which orients students to the cluster of units to which they are assigned . in the model , a clinical team ( see table 1 ) is the relational structure between faculty , clinical mentor , clinical resources , and students in which clinical education of the students occurs . five roles are described for each clinical team : ( 1 ) faculty of record , ( 2 ) clinical faculty , ( 3 ) clinical resources , comprised of current unit - based clinical leaders known as clinical nurse manager , clinical nurse supervisor , clinical nurse specialist , and educational nurse coordinator who serve as sources of information about patient care issues on an on - need basis , ( 4 ) clinical mentor , and ( 5 ) student whose responsibility is to work closely with the clinical mentor and clinical faculty in providing patient care while progressing in a learning environment . students are involved in the mentor 's entire patient assignment over the course of the shift . all members of a clinical team are expected to develop strong relationships and function as an integrated group rather than through a hierarchical structure of accountability . clinical faculty are matched as much as possible where their clinical skills and knowledge are compatible with unit patient populations as they are responsible not only for clinical education but for participation in scholarship and practice development on these units as well . they may provide direct instruction of the student but are more likely to facilitate the student 's learning through work with the clinical mentors . importantly , they model appropriate teaching strategies , clinical expert practice , and an understanding of evidence - based practice and its application . the clinical mentor assumes a direct role in student education while using clinical faculty for guidance in conducting the student 's experience .
figure 2(a ) figure 2(b ) reflects a shift in teaching responsibility as clinical mentors work closely with students while they engage in clinical care . figure 2(c ) shows the direct role that clinical faculty bring to the partnership including oversight of student assignment and some direct instruction of the student , modeling teaching strategies to the mentor , ensuring that course objectives are being met , and participating with the mentor in assessing student performance . these figures demonstrate , at the unit level , the partnership that was envisioned at the whole - scale change event . in addition to preparation for the role of clinical mentor , a document developed by nursing staff and clinical unit leaders , with added input from the project team , specifies essential characteristics guiding the transition into the mentor 's new responsibilities . this document charts the growing responsibilities of mentors as they mature in the role . members of the clinical team together provide a rich environment in which to nurture and support student growth in clinical competence . in reframing the student 's role as a team member , faculty were aware from prior nursing staff feedback that increased skill preparation for students was necessary for successful student integration into the clinical team . following an intensive workshop for faculty and clinical partners on simulation use in student education , new simulations were designed and embedded in clinical courses to improve clinical reasoning in the delivery of patient care . successful completion increases the students ' ability to participate in patient care as an integral member of the clinical team . increasing the number of hours devoted to clinical practice is important for gaining competence and feeling confident in providing care . over time in addition , student clinical assignments often have been limited to the care of one or two patients not only early in clinical experience but throughout the entire curriculum . current clinical environments demand that the nurse be able to manage a larger assignment while providing exemplary evidence - based care . the clinical model incorporates intensive clinical experiences in which students are fully engaged in a practice environment providing care to patients and families . a paper portfolio consisting of course descriptions , clinical unit profiles , student skills checklists for both completed skills and those to be achieved , and midterm and final student clinical evaluation forms is given to students . a clinical mentor feedback form to serve as a communication tool between the mentor and student about the student 's performance and clinical course materials for subsequent courses are added as the student progresses in the curriculum . the portfolio is designed to be utilized by all clinical team members to facilitate student learning and performance . transition to an electronic portfolio is to be determined by the patterns of use , value of current materials , and compatibility with umhs systems . the clinical education model was pilot tested in the junior year of the generic undergraduate program from september , 2008 through april , 2009 when the academic term ended . since it was not reasonable at the outset to test the model for all four years of the curriculum , the decision was that the pilot would include only students in the junior year when they were enrolled in specialty clinical courses . half the students ( n = 31 ) enrolled concurrently in medical - surgical and pediatric nursing courses participated during the fall term pilot . they continued in the pilot during the winter term when enrolled in psychiatric and obstetrical nursing courses . an additional 30 students who completed the psychiatric and obstetrical nursing courses in the fall joined the pilot for the first time in the winter term for a one - term pilot experience . by the end of the winter term , half the junior class ( n = 61 ) had experience in the new clinical education model . the selection of clinical units to participate in the pilot was decided following conversation with unit leaders and staff . the decision was that highly enthusiastic units would be asked to participate as the likelihood for demonstrating success of the clinical model would occur with units that were more positive about change and willing to work to enhance the success of our partnership . in the fall term pilot , three medical - surgical units and one large pediatric unit participated . during the winter term , the large women 's birthing unit and one adult and one child psychiatric unit were added . orientation and training to the model and the operation of its components was developed for all pilot participants including clinical leaders , staff who would become clinical mentors , clinical faculty , and pilot students . introductory time was allotted to meet each other and hear the vision of the new partnership from the dean and the cno . planned exercises were designed to prepare participants to successfully inhabit new roles and responsibilities , and to increase understanding of communication strategies in the patient care settings . to create an identity for the initiative , a logo and color scheme was designed and used for all materials posted in common areas , distributed materials , presentations , and portfolio covers . all clinical faculty attending the orientation and training had been teaching in the traditional clinical program and would be continuing in the new education model . a series of upfront skills sessions , relevant for the clinical courses in which students were enrolled , was designed and took place during the first month of each semester before clinical experience began ( see table 2 ) . these skills and scenarios were in addition to those covered in the lecture portion of the clinical courses . faculty and clinicians from umhs presented material and demonstrated skills including use of high - fidelity mannequins . skills sessions were accompanied by scenarios of clinical events that could occur in patient care areas that required intervention . practice opportunities for students followed and included practice of the skill as well as clinical reasoning exercises to support decisions made during skill demonstration . materials were made available for student use during and following demonstration . during this month , students attended class lectures and completed course assignments . their clinical experience on the units began in the fifth week of the term . they were paired with a unit clinical mentor and began their role as a member of the clinical team . the clinical immersion experience ( table 2 ) consisted of two consecutive days per week alternating clinical specialties each week ( i.e. , medical - surgical nursing the first week and pediatric nursing the second ) for five weeks followed by three weeks of four consecutive clinical days . the remaining week of the term reverted back to a two - day experience to accommodate the academic calendar . in winter term , the same plan was followed for ongoing pilot students now enrolled in obstetrical and psychiatric nursing . new students to the pilot in winter term followed the same clinical schedule as their peers in fall term . the immersion was intended to actively engage students in clinical work , to increase competence , and to enhance their understanding of the nurse 's role in managing holistic change in patients over time . students were expected to be fully involved in the mentor 's entire patient assignment over the course of the shift . pilot students used the clinical portfolio to guide clinical work and collaborate with mentors on assessment of performance . pilot and nonpilot students were together for the didactic portion of each of the clinical courses . aside from shared class they began their clinical experiences at the start of the term and did not participate in the upfront skills sessions . they followed the typical student plan for clinical experiences of two days per week alternating clinical specialties each day ( i.e. , medical - surgical nursing the first day and pediatric nursing the second ) across the entire term . the clinical faculty member on the unit was largely responsible for selecting patient assignments , monitoring student performance , and assessing performance . nonpilot students were not part of a clinical team , and they did not use a student portfolio . clinical faculty solicited input from staff about students ' performance and provided this feedback to students and used it as needed in written evaluations . throughout the pilot of the clinical model , project team members visited clinical units to monitor the learning environment . discussion occurred with clinical faculty , mentors , and students to solve problems , answer questions about team functions , or work one on one with team members to support their respective roles in the clinical team . occasionally , clinical unit meetings were held to further explain the model and respond to questions . clinical faculty , clinical mentors , clinical resources , and pilot students participated in clinical model evaluation . nonpilot ( traditional students ) were not asked about their experiences in the traditional model or their thoughts about how not having the pilot opportunity influenced their perception of their education . feedback regarding the clinical cluster arrangement was gathered during meetings of unit representatives from each cluster group clinical resources , mentors , and clinical faculty . clinical cluster meetings were typically held twice a year , once per term , for discussion and confirmation of upcoming clinical placements . data from two discussion sessions with clinical faculty , clinical leaders , and clinical mentors ( n = 23 , fall and 25 participants winter ) constituted the method for data collection during and following the pilot . on average , representation was approximately 4 representatives from each pilot unit including the clinical faculty member . the number of attendees per unit attending the discussions is acceptable given that time away from the unit needed to be negotiated in advance to cover staffing and students . prior to the discussion sessions , attendees talked with others on their respective units to gather information in order to bring as many perspectives as possible to the discussions . components of the clinical education model clinical teams , student skills preparation , clinical immersion , and feedback was obtained at term 's end ( n = 25 ) for the fall term pilot students ( medical - surgical / pediatric courses ) and at the end of the winter term from this same group of students ( n = 25 ) now enrolled in obstetrical / psychiatric nursing courses . feedback from the one - term winter pilot students ( n = 24 ) in medical - surgical / pediatric nursing courses was also obtained at the end of the term . the one - hour focus groups were led by a skilled facilitator who was unknown to the students thus reducing worry about being candid . this number exceeds that typically recommended for sessions but did not restrict students from expressing , concurring , or differing with views expressed by peers . the facilitator summarized information after each discussion of a clinical model component to clarify that the information was an accurate representation of views . each session was taped , session notes were taken by a recorder , and a summary report was generated by the facilitator . these members supported the concept of clinical clusters and agreed to continue defining working relationships both within and across clusters . one immediate and very positive outcome of work across clusters was a more efficient clinical placement process of umsn students . during discussion sessions , faculty described needing to adjust their role from full responsibility for student education to shared responsibility with mentors . this change required modeling best education practices as well as facilitating mentors in their new role in the clinical team . initially , some mentors were tentative about their new role and engaged students less fully in the team ( see table 3 ) until , with faculty and unit leadership support , their comfort level increased . the experience level of mentor seemed not to affect ability to function in the role . clinical faculty and clinical mentors both indicated that students benefitted from being a member of a clinical team and they soon adjusted to their new role in the team . mentors , in conversations with faculty reported using more clinical reasoning strategies with students , and those who had worked with umsn students previously indicated it was their impression that students ' clinical performance was accelerated beyond previous cohorts of students . focus group discussions with students ( see table 4 ) revealed that they enjoyed working in the clinical team and most became fully integrated over the term . being fully integrated , according to students , meant that they were involved in all aspects of their mentor 's assignment . students agreed that through experience as a team member they became more skillful in organizing work , communicating with others , practicing with greater independence , and identifying and requesting learning opportunities . students also reported that they benefitted from frequent feedback from mentors during the day about how the day 's work was progressing . overall , the upfront presentation of clinical skills did not fare as well as expected . both clinical faculty and mentors agreed that skill preparation should be increased throughout the curriculum with unit - based skills emphasized during clinical conference time on the unit . mentors added that being able to receive and give report to others at shift changes is an essential skill to be included . students maintained during focus group discussions that time were not used well , sessions were long and with limited practice , and unit - based skills they felt were necessary were not included . during winter term , the addition of unit - specific skills to clinical unit orientation and clinical conferences was seen to facilitate learning and skill performance by students , especially when accompanied by clinical scenarios . as shown in table 3 , clinical faculty and mentors agreed that two consecutive clinical days and later four consecutive clinical days increased student competence over the term . toward the end of the term , during the four consecutive clinical day experience , faculty reported in that students ' time - management skills increased noticeably from the beginning of the term . students ( see table 4 ) reported having a better understanding of patients ' experiences and care with increased time and continuity of patient assignments . in addition , they reported their clinical capabilities had increased from the start of the term and patient relationships were strengthened over the course of the immersion experience . the utility of the portfolio as a whole was not commented on by clinical faculty , clinical mentors , or students . however , one document , the clinical mentor feedback form used jointly by the student and mentor to assess and summarize the student 's clinical day , was singled out and by all groups ( tables 3 and 4 ) . mentors found the 14 rating of student performance hard to use and tended to rate students highly as they were reluctant to use the lower range that might discourage students . they indicated that discussing expectations with faculty about student performance would help with using the scale wisely . both clinical faculty and mentors agreed that written comments were more informative than the rating scale . students preferred written comments and recommended that the form provide more space for these as they are more informative than numerical ratings . the vision for a new clinical education model has become a reality and the pilot has shown its potential as well as pointed out areas for improvement . the partnership envisioned by participants in whole scale was demonstrated at every level of model development , in planning for pilot implementation , and in carrying out the pilot . the enthusiasm of units willing to participate in the pilot ensured that every effort would be placed in making the model work . findings from the pilot revealed the components of the model that were working well and those that required additional effort . clustering students in a designated set of units for the entire curriculum projected calculations showed that the new centralized student placement process can accommodate student placement in other years of the curriculum aside from the junior year including freshman ( shadowing experience ) , sophomore , and senior students who need or select placements in our health system . the full potential of clusters supporting education of students housed within them , and in promoting clinical research , continues to be developed . the traditional faculty role of primary supervision of students changed to that of facilitating and supporting the mentor who , in turn , took greater responsibility for student learning . some faculty , early on , were not convinced that this shift in responsibility was appropriate . however , as they participated in the clinical team , they adapted to the new role that included an increase in role - modeling instructional strategies and support for mentor activities . clinical mentors varied in how quickly they accommodated to their revised role in student education , most assumed the new role fairly quickly and easily . annoyance was expressed by some mentors who indicated that education of students took too much time and tended to be a burden . with support from unit leadership , the project team , and faculty , most became experienced team members . in our model students , as part of a designated clinical team , participate in the mentor 's entire assignment . students in the pilot stated that clinical experiences enhanced their learning and that working with the mentor and other unit leaders gave them a strong sense of contributing to the work of the team while learning . additional support that the team approach to clinical education worked is that our findings were similar to those found using the deu . for example , students in our model , like those in the deu approach , felt they were included in the clinical team , supported by mentors , more confident in providing patient care and able to assume more responsibility for their own learning [ 9 , 10 , 12 , 13 ] . they benefitted from continuity of patient care , became more confident of their capabilities , and began to experience more fully the work of the staff nurse . student skill preparation has been changed , based on student and faculty input , to include greater emphasis on clinical unit relevant skills in addition to general skills taught in conjunction with courses . although the clinical education model was pilot tested with half of the junior students , the pilot was not conducted as an experimental study to compare the views and outcomes between pilot and nonpilot students . as a result , nonpilot students were not asked to provide their views about being a student in the traditional program while peers were receiving a different approach to clinical education . information from nonpilot students may have been useful to understanding their thoughts about the merits of the different approaches , or how being treated differently from their peers influenced their views about the quality of education they were receiving . some were angry that they were not asked how things were going for them clinically . some expressed relief from not having to attend the intense skill preparation sessions or complete the four - day clinical immersion which they stated seemed exhausting for their classmates . some also mentioned that having fewer clinical obligations at the end of the term left them more time to study . a number commented that they were happy not to be part of an experiment and felt comfort in knowing that the traditional curriculum was known to faculty and nurses in umhs with whom they worked . although nonpilot student comments may have been informative , they would not have changed the decision to move forward with the new clinical education model . the clinical education model is part of a larger vision of the partnership between the umsn and umhs about how we will work together in advancing the missions of excellence in education , research , and practice , with education as the anchor for advancing the other two missions . the new education model is consistent with calls from the iom and rwjf to restructure education programs to be consistent with 21st century health systems [ 4 , 5 ] , and to prepare nurses for new responsibilities in rapidly changing health care environments . partnerships and teams are emphasized by these organizations as being critical to improving health care . full implementation of the model for all junior students and to all 36 clinical units in umhs in 2009 followed the pilot . some changes in the model have occurred . because it became difficult to structure faculty workload to accommodate both the two - day and four - day clinical immersion over the term , a different approach to clinical immersion was taken . the undergraduate clinical curriculum was revised and approved to add and progressively increase clinical hours from freshman through senior year culminating in a three - day per week capstone clinical practicum over the term supported by seminar . to ensure skill readiness for beginning clinical work , students must be checked off each term on skills they learned in clinical courses the previous term before they can begin the new term 's clinical experience . last , and very important , two new clinical track faculty roles are now in place . clinical cluster leads are faculty educators who provide full - time leadership and education to faculty , staff , and students in a cluster . responsibilities include supporting coordination of clinical placements in the cluster , facilitating faculty in clinical education and practice , and engaging in and leading others in the conduct of clinical research . clinical educators are clinical faculty who , in addition to providing supervision and education of students in the clinical setting , will build strong partnerships with clinical mentors and through these partnerships conduct scholarly work designed to improve patient outcomes and advance nursing practice . these faculty and students comprise the structure of the clinical bridge that anchors and connects the mission areas all three mission areas . the vision for a new clinical education model has become a reality and the partnership between umsn and the umhs became stronger as the model was developed and implemented in the health system . it continues to be a work in progress with the goals of preparing nurses to provide state - of - the - art care to patients and families , adapt to new approaches in rapidly changing health care environments , use evidence to ground practice , and participate in exploring nurse sensitive solutions to clinical problems . | the university of michigan school of nursing and the health system partnered to develop an undergraduate clinical education model as part of a larger project to advance clinical education , practice , and scholarship with education serving as the clinical bridge that anchors all three areas .
the clinical model includes clusters of clinical units as the clinical home for four years of a student 's education , clinical instruction through team mentorship , clinical immersion , special skills preparation , and student portfolio .
the model was examined during a one - year pilot with junior students .
stakeholders were largely positive .
findings showed that clinical faculty engaged in more role modeling of teaching strategies as mentors assumed more direct teaching used more clinical reasoning strategies .
students reported increased confidence and competence in clinical care by being integrated into the team and the mentor 's assignment . two new full time faculty roles in the health system support education , practice , and research . |
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in this case - control study , all the 9 - 10 year - old boy students studying at third and fourth grades in boy schools in the 2nd educational district of shiraz , south of iran , were enrolled . among the 800 enrolled boys , 40 students were selected as the case group for they were diagnosed as having adhd . moreover , the laboratory tests evaluation of the children with adhd did not show any significant finding . we also selected 40 boys for the control group among the remaining children without adhd . all children lived with their parents . at first , all the students were evaluated using the child symptom inventory - teacher s form ( csi-4 ) developed by gadow and spafkin ( 21 , 22 ) based on the dsm - iv adhd criterion ( 21 ) ; its farsi version has enough validity and reliability ( 23 ) . the mothers of those children who were diagnosed as having adhd were invited to complete the parent s form of csi-4 questionnaire ( 23 ) . if the parent and teacher forms matched , then the student underwent the final screening stage which was a face - to - face interview with the parents . this interview was conducted to confirm the adhd diagnosis according to dsm - iv - text revised diagnostic criteria . forty children with adhd were matched with 40 children without adhd by age and gender . children in both groups performed the draw - a - person ( dap ) and draw - a - family ( daf ) tests in a silent environment . they were provided with a white a4 paper , a black pencil and an eraser and were asked to draw a person . the time taken to draw a person then they were asked about the gender of the drawn person . after finishing the test , the children were given another a4 paper and were told to imagine a family and then draw that family . if any of the emotional problems or the incompatibility variables such as deleting the important components of the body ( such as hand or leg ) , simplifying the head or body , distortion and lack of proportion ( such as inappropriate arm size ) were shown , score of 1 was given , and if not , the score of 0 was given , except for score 1 to 9 for the weak quality of the drawing and sexual differentiation if any of the daf variables were presented , score of 1 was given , if not , the score of 0 was given . impulsivity and non- impulsivity indices have 13 variables separately ; for example , if the signs which were suggestive of aggressiveness such as ( knife , gun , blood ) , deleting important parts of the body for impulsivity index and shading , emphasis on the eyes and mouth for non- impulsivity index were detected , the score of 1 was given ; for the existence of two components or more , the score of 2 was given , and if not , then the score of 0 was given . the mean scores of impulsiveness , non - impulsiveness , indices in the dap and daf were compared between the two groups using the independent t - tests . the mean scores of impulsiveness , non - impulsiveness , indices in the dap and daf were compared between the two groups using the independent t - tests . the maximum and minimum total scores of incompatibility and emotional problem indices in the adhd group were 23 and 12 , respectively , with a mean ( sd ) of 19.79 ( 2.94 ) . the corresponding figures for the normal group were 22 and 3 , respectively , with a mean ( sd ) of 12.11(4.74 ) ( p<0.001 , table 1 ) . the maximum and minimum total scores of impulsiveness in the adhd group were 21 and 7 , respectively , with a mean ( sd ) of 12.31 ( 1.84 ) . the corresponding figures for the normal group were 10 and 2 , respectively , with a mean ( sd ) of 5.63 ( 2 ) ( p<0.001 , table 2 ) . the maximum and minimum total scores of non - impulsiveness in the adhd group were 11 and 1 , respectively , with a mean ( sd ) of 5.26 ( 2.29 ) . the corresponding figures for the normal group were 16 and 6 , respectively , with a mean ( sd ) of 10.36 ( 2.33 ) ( p<0.001 , table 3 ) . the maximum and minimum total scores of daf indices in the adhd group were 10 and 0 , respectively , with a mean ( sd ) of 5.89 ( 2.13 ) . the corresponding figures for the normal group were 7 and 60 , respectively , with a mean ( sd ) of 2.88 ( 2.13 ) ( p<0.001 , table 4 ) . table 5 demonstrates the mean total scores of the four variables and their comparison between the two groups . therefore , the accurate diagnosis of children with adhd in its primary stages is of prime importance . so far , various methods have been used to evaluate and diagnose this disorder such as related questionnaires , clinical interviews with parents and teachers and monitoring the child in different situations . we used the dap test to evaluate and identify students with adhd , and we also used the daf test to assess the interpersonal relationship of the children with adhd and their families . some studies have shown a correlation between the indices of the dap test and children s verbal intelligence score ( 4 , 24 ) . this test has been used as an initial screening tool for evaluating intelligence ; however , it should not replace the standard intelligence tests ( 2 ) . naglieri and colleagues used the dap test for the initial screening and assessment of emotional disorders and found a significant difference between the drawings of exceptional and normal students ( 5 ) . a significant relationship existed between the conflict indices in the dap test and mental pathologies ( 6 ) . to assess a conflict in a child , groth - marnat has suggested to determine 9 indices in the dap test ( 2 ) . we found a significant difference between the case and control groups with respect to conflict indices in the dap test , with a mean of 19.79(2.94 ) in adhd and 12.11 ( 4.74 ) in normal children . in two other studies , using the indices of the dap test that assessed impulsivity , the researchers found that people with adhd draw a part of the person out of the border of the page ( 3 , 4 ) . drawing items such as zipper , pocket , or belt shows a tendency to control impulses , and the lack of consistency and organization in the image and drawing , thin shoulders and wide arms depicts a tendency to be impulsive ( 3 ) . currently , oas s study ( 7 ) is considered as the most successful formulation for evaluating impulsivity ( 2 ) . oas showed that impulsivity indices in the dap test are higher in impulsive children than non - impulsive children . consistently , in our study , the comparison of these indices between the two groups was highly significant . impulsiveness scores were 12.31(1.84 ) , 5.63(2 ) in adhd and normal children , and non - impulsiveness scores were 5.26(2.29 ) , 10.36(2.33 ) in adhd and normal children . zaback and colleagues ( 1994 ) found that in normal individuals , 92% of men and 64% of women draw their same sex first in the dap test ( 16 ) . in a study of 1500 people , leibowitz ( 1999 ) observed that 78% of normal people draw the image of the same sex while 81% of homosexuals draw the picture of their opposite sex first ( 3 ) . rachel ( 1999 ) found that the dap results differed significantly in the four general indices of the facial , eye , hand and arm and genital organs in adults who were sexually abused in childhood compared with those who were not ( 17 ) . the results of rachel s study were highly effective in discovering and identifying adults who had been sexually abused in childhood , and they suggested that the dap test be used as a clinical tool . moreover , rachel and coworkers ( 2000 ) used the dap test to predict aggressive behaviors in prisoners ( 18 ) . the results showed that the eyebrows , shoulder , hair , mustache and eye indices significantly differed in aggressive prisoners compared to non - aggressive ones . these recent ( 16 , 3 , 17 , 18 ) studies are other samples for dap test usage in psychological problem detection . human figure drawings were found to be a time / cost - effective , sensitive and culturally appropriate means for measuring emotional well - being in debiasie , s study ( 25 ) . there were significant differences between depressive patients and normal individuals in daf test variables ( 14 ) . also , comparing the results of this test between children from divorced and non - divorced families revealed significant differences ( 15 ) . in our study , daf scores were 5.89(2.13 ) , 2.88(2.13 ) in adhd and normal children , respectively . if children with adhd are not recognized at the right time , their disorder might be left untreated and their performance will weaken . moreover , it would lead to personality disorders , delinquency , drug addiction , alcohol use and depression in adulthood . hyperactivity , immaturity in social behavior , impulsiveness and interference with teacher s educational strategies severely affect the learning of these children . we aimed to introduce the dap and daf tests as primary screening tests . the simplicity and applicability of these tests are beneficial in the primary detection of high risk children at schools . detected children can be referred to physicians , their parents can be interviewed and the rest of the diagnosis and treatment process can begin . using a set of methods and techniques for diagnosis leads to better diagnosis of the disorder and significant differences were found between normal children and adhd children using these two drawing tests . the rate of impulsivity and emotional problems indices in adhd children s drawing was markedly more common than that of the typically developing children . this suggests the need for further assessment to screen adhd . | objectiveattention deficit hyperactivity disorder ( adhd ) is the most common behavioral problem during childhood and in school - aged children .
various projection drawings have been designed for assessing children s personality and psychological disorders including the tests of draw a person ( dap ) and draw a family ( daf ) .
we aimed to compare the differences between typically developing children and children with adhd using these tests.methodsin this case - control study , all the 9 - 10 year - old boy students studying at the third and fourth grades were enrolled from schools in the 2nd educational district of shiraz , south of iran .
eighty students were then selected and enrolled into the adhd group and the control group .
the diagnostic and statistical manual of mental disorders , 4th edition- text revised ( dsm - iv - tr ) , and the child symptoms inventory were used to diagnose the children with adhd .
we evaluated and analyzed impulsiveness , non - impulsiveness , emotional problems and incompatibility indices in the dap and daf tests in each group.resultsa significant difference was found in the indices of incompatibility and emotional problems , impulsiveness , non - impulsiveness and daf between typically developing children and those with adhd .
the mean ( sd ) total scores of the above mentioned indices in the adhd group were 19.79(2.94 ) , 12.31(1.84 ) , 5.26(2.29 ) and 5.89(2.13 ) , respectively ( p<0.001 ) .
the corresponding figures for these indices in the normal group were 12.11(4.74 ) , 5.63(2 ) , 10.36 ( 2.33 ) and 2.88(2.13 ) , respectively ( p<0.001).conclusionsignificant differences were obtained between the control group and children with adhd using these two drawing tests .
the rate of impulsivity and emotional problems indices in drawings of children with adhd was markedly more common than those of the typically developing children .
this suggests the need for further assessment to screen adhd . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
prior
to the evolution of protein - based enzymes , primitive cells
are thought to have relied on rna enzymes for catalysis . the hypothesis of such an early , rna - dominated stage in the evolution
of life is supported by the continued existence of ribozymes in modern
biology , and especially by the observation that protein
synthesis is catalyzed by the rna component of the large ribosomal
subunit . in addition to this evidence ,
ribozymes obtained by in vitro directed evolution
can catalyze a wide variety of reactions , encompassing all of the
basic classes of transformations necessary for protocell metabolism
and reproduction . however , the origins of rna - based
protocells remain unclear , and numerous problems must be solved to
establish an understanding of how prebiotic chemical systems gave
rise to protocells containing both replicating genomic rnas and functional
ribozymes derived from those genomic rnas . one of the central
problems faced by the earliest cells is likely
to have been the slow and inefficient nature of primordial rna replication .
indeed , prior to the evolution of a sophisticated rna replicase ribozyme ,
protocells may have had to rely on the ambient chemical environment
to drive non - enzymatic rna replication . efforts to demonstrate prebiotically
plausible rna replication chemistry date back some four decades , but despite recent progress , several obstacles remain . at present , non - enzymatic rna polymerization
allows for the partial copying of short rna templates . in model studies , the growing rna primer strand is elongated by addition of ribonucleotides
activated with a good leaving group ( e.g. , 2-methylimidazole
or hydroxyazabenzotriazole ) . in this
paper , we assume that the unsolved obstacles to non - enzymatic
rna replication reflect our current lack of understanding , and are
not insurmountable hurdles . however , we also expect that primitive ,
chemically driven rna replication was only marginally sufficient for
the emergence of protocells bearing small replicating fragments of
rna . we then ask : given such a situation , how could protocells bearing
selectively advantageous , heritable ribozyme activities have emerged ? ribozymes with a kcat of greater than
1 min are generally at least 30 nucleotides in
length and often 50100 nucleotides long ; the few known ribozymes
with a kcat of greater than 1 s are over 100 nucleotides in length . generating rnas of 50100 nucleotides in length
by non - enzymatic template - directed polymerization seems like a severe
challenge . fortunately , several previous reports have shown that such
ribozymes can be reconstituted from shorter oligonucleotides by their
assembly into a noncovalent complex that retains good catalytic activity . the minimal length of such oligonucleotides reflects the need for
stable base - paired stems as the driving force for complex assembly . if each oligonucleotide is anchored in the complex by two or three
stems of 56 nucleotides , with interspersed sequences of similar
length forming binding or catalytic sites , then a minimal fragment
length of 1525 nucleotides would be reasonable . we , therefore ,
hypothesize that primitive cells contained a set of genomic oligonucleotides
in this size range , all being replicated by non - enzymatic chemistry ,
with some of these oligonucleotides assembling into complexes with
useful catalytic activities . if it were possible for even shorter
genomic oligonucleotides to generate functional ribozyme complexes ,
the constraints on rna replication chemistry would be correspondingly
diminished . here we show that functional ribozymes can be reconstituted
from
oligonucleotides that have been further shortened by 3-truncation . in this scenario , the 3-truncated oligonucleotides assemble
into unstable and inactive precursor complexes that are held together
by very short unstable terminal stems . non - enzymatic template - directed
extension of the oligonucleotide 3-termini , which act as primers ,
lengthens the initially short and unstable base - paired stems , generating
stable complexes with good catalytic activity . such extended oligonucleotides
could be removed from the pool of replicating rna fragments , as they
would be locked into stable catalytic complexes . non - extended genomic
fragments would be easier to replicate , owing to both the comparative
ease of copying short rna sequences and the fact that they were not
sequestered in stable complexes . this simple mechanism substantially
relaxes the requirements for rna replication systems , and it could
have facilitated the transition from prebiotic chemical systems to
evolving living cells . we studied an aptamer and
a cis - acting ribozyme
that could each be reconstituted by the assembly of two oligonucleotides
into a functional complex , held together in part by terminal base - paired
stems . we also studied a second , trans - acting ribozyme
consisting of a single oligonucleotide , the active form of which required
the formation of a base - paired stem between its 3-terminus
and an internal sequence . we then modified the structure of each of
these oligonucleotides by shortening the 3-end by 3 or 4 nucleotides ;
as a result , the 3-truncated oligonucleotides were unable
to assemble into functionally active complexes . however , these shortened
oligonucleotides could assemble into transiently stable complexes ,
in which the oligonucleotide 3-ends formed one or two base - pairs
in the nascent stem . configuration , in which the 3-end could be extended by non - enzymatic
polymerization , thus generating an extended and , therefore , more stable
base - paired stem . in each case , we were able to observe the recovery
of ligand binding or catalytic activity following non - enzymatic template - directed
primer extension ( figure 1 ) . ( a ) 3-truncated oligonucleotides assemble into
an unstable , inactive structure . after extending one or more 3-ends
on a template derived from another region of the truncated functional
rna , the full - length functional rna folds into a stable and active
aptamer or catalyst . ( b d ) schematic diagrams of the aptamer
and two ribozyme systems used in this study . we
chose to work with this aptamer for an initial proof - of - principle
of the hypothesis that 3-truncated oligonucleotides can self - assemble
into intermediate structures that can serve as templates for the reconstitution
of functional rnas . the free malachite green ligand has very low fluorescence ,
but its fluorescence increases by several orders of magnitude upon
binding to the rna aptamer . in addition ,
the aptamer and its mode of ligand binding have been studied extensively . nmr studies indicate that the terminal base - paired
stems form in the absence of ligand , but the central region of the
aptamer folds around its ligand , and it becomes ordered only upon
ligand binding . thus , it seemed likely that the ability of the aptamer
to fold and bind its ligand could be readily controlled by altering
the length of its terminal stems . additionally , this aptamer has been
previously deconstructed into two oligonucleotides that can assemble
into a functional complex . to demonstrate
aptamer stabilization by non - enzymatic template - directed rna synthesis ,
we modified the previously reported sequences of the two - fragment
malachite green aptamer . we first changed
the original design of the binary aptamer so that one of the base - paired
stems is a four nucleotide gggg / cccc stem ( figure 2 ) ; this aptamer retains full malachite green binding activity . however , if the 3-terminal gggg sequence is shortened to a
single g , there is no detectable binding of malachite green . if the
3-terminal gggg sequence is shortened to two or three guanosine
nucleotides , malachite green binding is reduced , but not eliminated
( figure s1 ) . in the presence of the activated
monomer guanosine 5-monophosphate 2-methylimidazolide
( 2-meimpg ) , non - enzymatic extension of the single 3-terminal
g on the cccc template regenerates the gggg / cccc stem and results
in a stable , functional aptamer ( figure 3 and figure s1 ) . after a 24 h room temperature incubation
of 3-truncated and template strands with 2-meimpg , the measured
malachite green binding was comparable to that of the aptamer assembled
from two full - length oligonucleotides ( figure 2c ) . this experiment shows that inactive functional rnas , made from
oligonucleotides that are truncated at the 3-end , can be converted
to a functional form by non - enzymatic template - directed rna synthesis . ( a ) structure and sequence of the malachite
green aptamer before and after primer extension . ( b ) one molecule of
malachite green binds to each fully assembled aptamer . ( c ) fluorescence
of malachite green in the presence of aptamer rna at different concentrations
of mg . blue squares , unextended 3-truncated strand
plus template strand ; blue triangles , 3-truncated strand plus
template after 24 h of rna primer extension with 2-meimpg ; green squares ,
3-truncated strand after 24 h extension with 2-meimpg and
template strand . each sample contained 0.25 m tris - hcl ph 8.0 , 0.15
m nacl , 2 m malachite green , 1 m each strand of the
aptamer . having established
that a functional
rna aptamer can be generated by non - enzymatic extension of the 3-end
of one oligonucleotide on a template region of a second oligonucleotide ,
we investigated the possibility of generating an active ribozyme from
two truncated oligonucleotides , each of which could be extended on
the other . we chose the hammerhead ribozyme , which cleaves a substrate
strand at a defined cleavage site , for
these experiments . we modified a previously reported minimized consensus
sequence so that the ribozyme could be reconstituted from two strands ,
referred to as top and bottom . these strands form a complex held together by
two stems flanking the catalytic loop region ( figure 3 ) . ( a ) structure and sequence of the hammerhead
ribozyme before and after primer extension . the ribozyme is assembled
from top and bottom strands ; each of the strands contains a template
region ( blue ) and a primer extension region ( green ) . the 5-end of the top strand is
fluorescently labeled with cy3 for page analysis . each panel shows two
parallel reactions : samples incubated for 24 h with and without 2-meimpg
( i.e. , with or without primer extension ) . the sequences above the
gel panels show starting material in each sample : two hammerhead ribozyme
strands , each as either unextended 3-truncated or full - length
strand . each sample
contained 0.25 m tris - hcl ph 8 , 0.15 m nacl , 2.5 m each rna
strand , 50 mm 2-meimpg , 50 mm mgcl2 . 2-meimpg - containing
lanes exhibit several minor bands , which are believed to be derived
from 2-meim - induced rna degradation . the top strand , which contains
the cleavage site , was labeled at
its 5-end with cy3 . ribozyme activity was measured by fluorescence - detected
polyacrylamide gel electrophoresis ( page ) gel separation of the full - length
fluorescently labeled top strand from the shorter , cleaved product .
when we mixed full - length top and bottom strands in equimolar proportions
and incubated the resulting complex at room temperature , ribozyme
activity was evident from the appearance of cleaved product ( 65% product
in the reaction incubated with 2-meimpg , figure 3 , and experimental section ) . full - length
top strand mixed with truncated bottom strand , and truncated top strand
mixed with full - length bottom strand , resulted in weak ribozyme activity
in both cases . incubation of the 3-truncated top and bottom
strands resulted in no ribozyme activity ( figure 3 ) . we then examined the effect of incubating the above oligonucleotide
mixtures with the activated monomer 2-meimpg . in the complexes assembled
with 3-truncated oligonucleotides , both strands can act simultaneously
as primer and template : the 3-end of the top strand can be
extended by up to 3 guanosine residues ( employing the 5-end
of the bottom strand as a template ) , while the 3-end of the
bottom strand can be extended by up to 4 guanosine residues ( employing
the 5-end of the top strand as a template ) . in all cases ,
incubation with 2-meimpg resulted in extension of the 3-ends
of the truncated oligonucleotides and reconstitution of ribozyme activity .
in primer extension reactions , if the top 3-truncated strand
was incubated with the bottom full - length strand , 64% cleavage yield
was obtained ; the top full - length strand incubated with the bottom
3-truncated strand resulted in 33% product , and if both top
and bottom strands were 3-truncated , 25% product was observed
( compared to only 5% background cutting activity by mixing both 3-truncated
strands without 2-meimpg ) ( figure 3 ) . 3-truncation
of the bottom strand is generally more deleterious than that of the
top , likely due to the additional base pair present in the complex
of the 3-truncated top strand with either bottom strand . encouraged by the above
results , we sought to examine whether a trans - acting
ribozyme ( i.e. , a ribozyme that catalyzes a reaction between two substrates
not covalently bound to the ribozyme ) could be activated in a similar
manner . we started with the minimized sequence of a diels alderase
ribozyme , which catalyzes a diels alder
cycloaddition reaction between a broad range of substrates with very good stereoselectivity . we redesigned the ribozyme , in this case composed
of a single oligonucleotide , to end with the sequence 5-ggg-3 ,
which could pair with the internal sequence 5-ccc-3
( figure 4a ) . we then constructed a set of truncated
versions ending in one or two 3-g residues , which we expected
to be less active than the full - length ribozyme . ( a ) structure and sequence of the
diels alderase ribozyme before and after primer extension . ( b ) the reaction between n - propylmaleimide and 9-hydroxymethylanthracene ,
catalyzed by the diels alder ribozyme . ( c ) time course of the
diels alder reaction with and without the ribozyme . red squares ,
full - length ribozyme ; orange circles , 3-truncated ribozyme
after 24 h extension with 2-meimpg ; green triangles , nonextended 3-truncated
ribozyme ; blue diamonds , uncatalyzed reaction . reaction conditions :
100 mm tris - hcl ph 7.5 , 100 mm mgcl2 , 300 mm nacl , 200
m 9-hydroxymethylanthracene , 1000 m n - propylmaleimide , 10 m rna . ( d ) product region of hplc traces of
samples stopped at time 0.5 h , corresponding to the reactions on graph
in panel c. detection wavelength , 260 nm ; product peak retention time ,
12.5 min . we followed the reaction between
the substrates n - propylmaleimide and 9-hydroxymethylanthracene
by hplc . the background ( i.e. , uncatalyzed ) reaction , in the absence
of the catalyst , resulted in conversion of 11% of the anthracene substrate
to product in 30 min , and 26% in 1 h. the truncated ribozyme ending
in a single 3-g did not result in any significant increase
in product formation , while the full - length ribozyme yielded 72% product
in 30 min and 88% in 1 h. a synthetic ribozyme variant ending in two
3-g residues had intermediate activity ( figure s2 ) . in order to assess the ability of non - enzymatic
primer - extension
to convert the inactive truncated ribozyme ending in a single 3-g
residue to an active ribozyme , we incubated the truncated ribozyme
with the activated monomer 2-meimpg for 24 h. the resulting extended
ribozyme was nearly as effective a catalyst as the full - length ribozyme ,
converting 65% of substrates in 30 min and 78% in 1 h ( figure 4 ) . our observations indicate
that functional ribozymes can be generated
from non - functional sets of short rna oligonucleotides by using non - enzymatic
template - directed rna synthesis to convert unstable and inactive oligonucleotide
complexes into stable active assemblies . we have used three separate
model systems to show that inactive sets of 3-truncated oligonucleotides
can be converted to active aptamers or ribozymes following incubation
with the activated monomer 2-meimpg . in each case , the 3-end
of one or more oligonucleotides was extended by non - enzymatic template - directed
primer extension , converting a very weakly base - paired stem ( generally
a single base - pair ) into a stable base - paired stem consisting of four
g : c pairs . as a result , we suggest that
the first ribozymes may have originated within
protocells in a similar three - step process . first , multiple short
rna fragments replicated within protocells by non - enzymatic template - directed
rna synthesis . these rnas would have been generated in higher - yield
than full - length rnas , owing both to their lowered propensity to fold
and the higher efficiency of copying short rnas non - enzymatically . third , non - enzymatic rna copying chemistry
( potentially the same chemistry that generated the initial fragments )
extended the 3-ends of the ribozyme precursor oligonucleotides ,
using other regions of rna in the transient complex as templates ,
yielding fully functional stable ribozyme complexes . as a result of
such a scenario , short , nonfunctional rnas could afford a protocell
vesicle fitness , given their ability to assemble into transient complexes
and undergo subsequent primer extension to elaborate these complexes
into full - length functional rnas . as we have recently demonstrated
non - enzymatic primer extension inside fatty acid vesicles , such a
scenario seems highly plausible . additional
support for our hypothesis is provided by recent results demonstrating
that fe can substitute for mg in functional
rnas , suggesting that the low solubility of mg in the
presence of certain counterions , such as phosphate , may not have been
as problematic as anticipated , given the high availability of soluble
iron prior to the great oxidation event . while our system incorporates only 2-meimpg , the presence of the
other three nucleotide monomers 2-meimpa , 2-meimpc , and 2-meimpu does
not impair aptamer or ribozyme reconstitution ( figures s3 and s4 ) , demonstrating that even prebiotically
plausible levels of nonenzymatic replication fidelity allow for functional
rna reconstitution via this mechanism . given the enhanced thermal
stability of the products of primer
extension ( figure s5 ) and increases in
aptamer and ribozyme activity with increasing stem length ( figures s1 and s2 ) , it is likely that these rnas
develop function as a result of stabilization of critical structural
stems . it is also possible that some of the enhanced function observed
in the rnas occurs as a result of these lengthened homopolymeric stems
directing the rna to assume the correct fold . both such results would
occur as a direct result of the extension of truncated stems . our observations have significant implications with regard to two
aspects of the evolution of functional rnas in early cells . first ,
our results suggest a natural way in which genomic rna fragments ,
which must be replicated to enable heritability , can be shorter than
the rnas that actually assemble into functional complexes . multiple
previous examples have shown that aptamers and ribozymes can often
be reconstituted from relatively short oligonucleotides . this alone shows that primitive rna replication systems , whether
chemical or ribozyme - catalyzed , need only be sufficient to copy fairly
short pieces of rna . however , since primordial non - enzymatic replication
systems are expected to be slow and inefficient , and , in fact , no
robust copying system has yet been demonstrated experimentally , it
remains useful to consider scenarios in which the length of genomic
rnas can be further minimized . our results suggest that genomic rna
fragments could be at least 34 nucleotides shorter than functional
rna fragments , further ameliorating the difficulty associated with
primitive rna replication . thus , the process we describe for synthesis
of functional rnas relaxes the requirements for prebiotic rna copying
efficiency . the second aspect of primitive rna replication that
is facilitated
by the scenario outlined above has to do with the conflict between
the requirements for a good template as opposed to those of a good
catalyst . optimal templates should be completely unfolded , with no
internal structure to impede the synthesis of a complementary strand . similarly , a good template should not be sequestered within a stable
complex with other oligonucleotides . in contrast , a good ribozyme
must have a stable folded structure , and if the ribozyme is generated
through the association of two or more oligonucleotides , the resulting
complex should also be a stable structure . this conflict is partially
resolved through the strategy of assembling functional complexes from
sets of relatively unstructured oligonucleotides ; however , in order
to allow for uncomplexed templates and functional complexes to co - exist ,
the affinity of the oligonucleotides for each other must be quite
weak . such a loose association could make it difficult to form a structurally
well - defined complex , although this deserves closer experimental attention .
nonetheless , our observations suggest that nonenzymatic primer - extension
may provide a natural way to allow for the co - existence of short unstructured
templates , and slightly longer derivative oligonucleotides that form
stable , functional complexes . we have previously suggested that randomly
positioned 25 linkages , generated as a consequence
of non - enzymatic rna replication , could also contribute to the distinction
between good templates and good ribozymes . we suggest that the effects of 25 linkages
could act synergistically with the effects of non - enzymatic primer - extension
to produce distinct sets of genomic and functional oligonucleotides
in primitive cells . the reaction conditions we have employed generate
predominantly 35 linkages , but the small proportion
of 25 linkages produced could have been beneficial
in this regard . nucleotide 2-methylimidazolides
( 2-meimpn , where n = g , a , c , u ) were synthesized as previously described . the product of the diels alder reaction used
in our studies
( 3 ) was synthesized , for use as a standard , by refluxing
0.625 g ( 3 mmol ) of 9-hydroxymethylanthracene ( 1 ) and 0.390 g ( 2.8 mmol ) of n - propylmaleimide ( 2 ) in 15 ml of dry toluene for 14 h. the product was purified
by silica gel chromatography with 1:1 hexane : ethyl acetate , and it
was obtained as a yellow solid in 71% yield . h nmr ( 400
mhz , cdcl3 ) : 7.27 ( d , 2h ) , 7.36 ( d , 2h ) , 7.24 ( m ,
4h ) , 5.29 ( s , 1h ) , 5.11 ( m , 2h ) , 4.75 ( s , 1h ) , 3.32 ( m , 2h ) , 3.08
( m , 2h ) , 1.63 ( s , 1h ) , 0.86 ( m , 2h ) , 0.495 ( t , 3h ) . c
nmr ( 100 mhz , cdcl3 ) : 177.34 , 177.06 , 142.42 , 142.40 ,
139.57 , 139.24 , 127.15 , 127.11 , 126.85 , 126.76 , 125.50 , 124.27 , 123.35 ,
122.74 , 60.90 , 49.56 , 48.01 , 46.55 , 45.86 , 40.36 , 20.65 , 11.15 . non - enzymatic
rna polymerization was initiated by addition of activated
rna monomer(s ) to the mixture of 3-truncated and template
oligonucleotides . all reaction mixtures were incubated at
room temperature for specified times , desalted using ziptip c18 pipet tips ( millipore ) , and analyzed by tbe the binding of malachite green
to the aptamer was measured with a spectramax geminiem fluorescence
plate reader ( molecular devices ) with excitation wavelength 610 nm
and emission wavelength 670 nm . the malachite green stock solution
was mixed with rna top and bottom strands to the final concentration
of 2 m malachite green and 1 m of each strand of the
aptamer in 0.25 m tris - hcl ph 8.0 , 0.15 m nacl . in rna primer
extension reactions , the rna strands were mixed with 2-meimpg to final
concentrations of 50 mm 2-meimpg and 5 m each rna strand ( i.e. ,
5 m of the rna complex ) in 0.25 m tris - hcl ph 8.0 , 0.15 m nacl ,
50 mm mgcl2 . after the reaction samples were eluted
from the tip with 1:1 acetonitrile : water , solvent was removed on a
speedvac vacuum concentrator , and the rna was dissolved in water . the concentration of the sample was measured using a nanodrop spectrophotometer ,
adjusted to 2 m of the rna complex , and analyzed as shown above . hammerhead ribozyme reactions were
initiated by mixing both rna strands to a final concentration of 2.5
m each strand , in 0.25 m tris - hcl ph 8.0 , 0.15 m nacl , 50 mm
mgcl2 . reactions were incubated at room temperature for 24
h. after the reaction , samples were desalted using ziptip c18 pipet tips ( millipore ) and analyzed by tbe the reaction yield was calculated as the percentage of the product
band intensity relative to the total intensity of all bands on the
gel . the diels alderase
reaction was initiated by mixing 9-hydroxymethylanthracene
and n - propylmaleimide to final concentrations of
200 m 9-hydroxymethylanthracene and 1000 m n - propylmaleimide , in100 mm tris - hcl ph 7.5 , 100 mm mgcl2 , and 300 mm nacl . the diels alderase ribozyme - catalyzed
reactions were performed in the presence of 10 m rna . in rna primer extension reactions , the rna strands were mixed with 2-meimpg
to final concentrations of 50 mm 2-meimpg and 5 m each rna
strand in 0.25 m tris - hcl ph 8.0 , 0.15 m nacl , 50 mm mgcl2 . reactions were incubated at room temperature for 24 h. after the
reaction , samples were desalted using ziptip c18 pipet
tips ( millipore ) . samples were eluted from the tip with 1:1 acetonitrile : water ,
solvent was removed on a speedvac vacuum concentrator , and the rna
was dissolved in water . the concentration of the sample was measured
using a nanodrop spectrophotometer , the concentration was adjusted
to 10 m of the rna complex , and this solution was subjected
to the above ribozyme reaction conditions . the reactions were
incubated at room temperature for the reported amount of time and
analyzed on an agilent 1100 analytical hplc with a varian microsorb - mv
100 - 8 c18 250 4.6 mm column with a gradient of solvent
a : 0.1% tfa in h2o , and solvent : b 0.1% tfa in acetonitrile ,
with uv detection at 260 nm . | the earliest genomic rnas had to
be short enough for efficient
replication , while simultaneously serving as unfolded templates and
effective ribozymes .
a partial solution to this paradox may lie in
the fact that many functional rnas can self - assemble from multiple
fragments .
therefore , in early evolution , genomic rna fragments could
have been significantly shorter than unimolecular functional rnas .
here
, we show that unstable , nonfunctional complexes assembled from
even shorter 3-truncated oligonucleotides can be stabilized
and gain function via non - enzymatic primer extension .
such short rnas
could act as good templates due to their minimal length and complex - forming
capacity , while their minimal length would facilitate replication
by relatively inefficient polymerization reactions .
these rnas could
also assemble into nascent functional rnas and undergo conversion
to catalytically active forms , by the same polymerization chemistry
used for replication that generated the original short rnas .
such
phenomena could have substantially relaxed requirements for copying
efficiency in early nonenzymatic replication systems . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
colorectal carcinoma represents a global health burden as the most common cancer of the digestive tract . it is the third most frequent cancer diagnosed in males and the second in females . preoperative status of tumor invasion depth is important for the selection of endoscopic treatment or surgical resection . in addition , stratification based on stage is also important for selecting patients to the established neoadjuvant radiochemotherapy for rectal cancer and promising neoadjuvant chemotherapy for patients with colon cancer . in rectal cancer , transluminal endoscopic microsurgery has shown to be an effective and safe procedure for selected patients . the value of transrectal ultrasound ( trus ) for the staging of rectal cancer has been investigated in numerous studies . thus , trus has become the method of choice for locoregional t - staging of rectal cancer while n - staging needs further refinement in diagnostic criteria and endoscopic ultrasound ( eus ) technology to improve diagnostic accuracy , we aimed to systematically review the literature assessing the value of eus - based staging of malignant colonic neoplasms compared to histological stage . the review was conducted according to the prisma guidelines ( prospero registration number : crd42015016013 ) . literature search was performed in june 2015 in pubmed ( 19462015 ) , embase ( 19802015 ) , web of science ( 19002015 ) , and the cochrane library ( 19722015 ) [ figure 1 ] . a search was performed using the following mesh terms : [ colon cancer ] , [ colonic neoplasms ] , [ colon neoplasms ] , [ colonic cancer ] and [ endoscopic ultrasound ] , [ ultrasonography ] using the boolean operators or / and . two authors individually assessed all abstracts found in the primary search ( mlm , ig ) . search was manually performed from the reference lists of included studies . finally , all included studies were crosschecked in web of science under citations . flowchart on the search strategy data regarding study characteristics , diagnostic methods , and accuracies of t- and n - stages with histology as controls were extracted . studies with combined accuracies for rectum and colon were only included if the colonic data could be separated from the rectum data . authors of studies with mixed data where separation was not obvious were contacted . for each separate study , characteristics such as demography , study design , inclusion and exclusion criteria , patient numbers , sample size calculations , and endpoints were evaluated and are presented in table 1 . data on endoscopic equipment , operator experience , blinding , measure categorization , adverse events , missing data , and treatment and comparisons to other diagnostic modalities are presented in table 2 . studied data are referred to without interpretation . schematic setup summarizing the study characteristics and design including inclusion and exclusion criteria , demography , number of patients , sample size , and endpoints for the different studies summary of the diagnostic methods for the different studies with emphasis on endoscopes and operators , blinding , classification systems , adverse events , missing data , treatment of the patients , and comparison of endoscopic ultrasound to different diagnostic modalities there is no consensus on the assessment of the quality of clinical studies lacking a control arm ; however , we chose to include studies based on inclusion criteria and completeness of data as well as to assess the risk of bias in each individual study . the guidelines from the ( stard ) the standards for reporting of diagnostic accuracy initiative have been used to evaluate the completeness of reporting in studies on diagnostic accuracy [ tables 1 and 2 ] , as well as to assess bias . pooled estimates of sensitivity and specificity were calculated using the rutter and gatsonis hierarchical summary receiver operating characteristic ( hsroc ) model . possibly due to the limited number of studies and high proportions of studies having a specificity at 1 for the t2 tumors , the random effect model failed to fit . assumptions for using the rutter and gatsonis hsroc model might be violated as the t2s have two thresholds , both toward t3 and t1 . in this case , a fixed effect model was used for estimating sensitivity and specificity . copenhagen : the nordic cochrane centre , the cochrane collaboration , 2014 ) and used for drawing the figures . calculations of pooled sensitivity and specificity were performed using the sas macro metadas 1.3 ( user guide version 1.3 . 2010 july ; available from : http://srdta.cochrane.org/ref ) on sas version 9.4 ( sas institute inc . , 2012 , cary , nc , usa ) . pooled estimates of sensitivity and specificity were calculated using the rutter and gatsonis hierarchical summary receiver operating characteristic ( hsroc ) model . possibly due to the limited number of studies and high proportions of studies having a specificity at 1 for the t2 tumors , the random effect model failed to fit . assumptions for using the rutter and gatsonis hsroc model might be violated as the t2s have two thresholds , both toward t3 and t1 . in this case , a fixed effect model was used for estimating sensitivity and specificity . copenhagen : the nordic cochrane centre , the cochrane collaboration , 2014 ) and used for drawing the figures . calculations of pooled sensitivity and specificity were performed using the sas macro metadas 1.3 ( user guide version 1.3 . 2010 july ; available from : http://srdta.cochrane.org/ref ) on sas version 9.4 ( sas institute inc . , 2012 , cary , nc , usa ) . pooled estimates of sensitivity and specificity were calculated using the rutter and gatsonis hierarchical summary receiver operating characteristic ( hsroc ) model . possibly due to the limited number of studies and high proportions of studies having a specificity at 1 for the t2 tumors , the random effect model failed to fit . assumptions for using the rutter and gatsonis hsroc model might be violated as the t2s have two thresholds , both toward t3 and t1 . in this case , a fixed effect model was used for estimating sensitivity and specificity . copenhagen : the nordic cochrane centre , the cochrane collaboration , 2014 ) and used for drawing the figures . calculations of pooled sensitivity and specificity were performed using the sas macro metadas 1.3 ( user guide version 1.3 . 2010 july ; available from : http://srdta.cochrane.org/ref ) on sas version 9.4 ( sas institute inc . , 2012 , cary , nc , usa ) . the initial search proposed 747 articles , with 58 excluded as duplicates , 638 irrelevant records , and 51 assessed for full - text eligibility . six potential studies were excluded from the study due to data on colonic cancers alone could not be provided . one author provided separate tumor node metastasis classified data for colonic tumors but only included adenomas and very early cancers and was excluded from this review . other authors of mixed data studies were unable to provide information on colon cancer patients alone or did not reply . the total number of patients with colonic cancers evaluated for t - stage with eus and with surgical pathology at the gold standard was 208 . the study by haji et al . counted as two studies due to the same patient population none of the studies reported any complication due to the diagnostic procedures either with miniprobes or with radial transducers . some , however , had to give up tumor staging due to technical difficulties ; these were not included in our analysis . in terms of n - stage , most studies evaluated this parameter based on histology of the surgical resection specimen after laparoscopy or open surgery . the t- and n - stages from the six analyzed studies were as follows :
t1 : a summary of the studies for patients with t1 disease is shown in figure 2a . the pooled sensitivity was 0.90 ( 95% ci : 0.660.98 ) and the specificity was 0.98 ( 95% ci : 0.940.996)t2 : a summary of the studies for patients with t2 disease is shown in figure 2b . the pooled sensitivity was 0.67 ( 95% ci : 0.500.80 ) and the specificity was 0.96 ( 95% ci : 0.920.98)t3/t4 : a summary of the studies for patients with t3/t4 disease is shown in figure 2c . the pooled sensitivity was 0.97 ( 95% ci : 0.880.99 ) and the specificity was 0.83 ( 95% ci : 0.730.90)n : a summary of the studies for patients with n - positive disease is shown in figure 2d . the pooled sensitivity was 0.59 ( 95% ci : 0.310.82 ) and the specificity was 0.78 ( 95% ci : 0.680.86 ) . t1 : a summary of the studies for patients with t1 disease is shown in figure 2a . the pooled sensitivity was 0.90 ( 95% ci : 0.660.98 ) and the specificity was 0.98 ( 95% ci : 0.940.996 ) t2 : a summary of the studies for patients with t2 disease is shown in figure 2b . the pooled sensitivity and specificity the pooled sensitivity was 0.67 ( 95% ci : 0.500.80 ) and the specificity was 0.96 ( 95% ci : 0.920.98 ) t3/t4 : a summary of the studies for patients with t3/t4 disease is shown in figure 2c . the pooled sensitivity was 0.97 ( 95% ci : 0.880.99 ) and the specificity was 0.83 ( 95% ci : 0.730.90 ) n : a summary of the studies for patients with n - positive disease is shown in figure 2d . the pooled sensitivity was 0.59 ( 95% ci : 0.310.82 ) and the specificity was 0.78 ( 95% ci : 0.680.86 ) . forest plot with a cross table on included patients for diagnostic accuracy measurements for endoscopic ultrasound in comparison to histology for ( a ) t1 , ( b ) t2 , ( c ) t3/4 , and ( d ) lymph node positive patients . sensitivity and specificity with 95% confidence intervals the aim of this systematic review was to assess the diagnostic accuracy of eus for staging of malignant colonic tumors proximal to the rectum . six studies , evaluating 208 patients with colonic cancer , in terms of tumor stage compared to pathological stage , were identified and sensitivity and specificity on t- and n - stages were calculated . t - staging with eus of colonic and rectal tumors can be compared in terms of accuracies . for rectal tumors , eus in early tumors ( t1/2 ) has been considered the staging modality of choice . at present , the image modality of choice for staging of colonic cancer is a ct - scan . the study by haji et al . is a comparative study between the image modalities of ct and miniprobe eus . the eus was found to be significantly more accurate in the local staging of both early and advanced tumors . there is an ongoing prospective , randomized trial , evaluating neoadjuvant chemotherapy in locally advanced operable t3/t4 colonic tumors . neoadjuvant therapy may target micrometastatic disease earlier , downstage tumor , as well as reduce surgical tumor cell shedding . the accuracy of staging will become even more important , allowing quicker assignment to correct multimodal treatment . the results of this review have to be interpreted in the context of limitations of the calculations and the risk of bias in the systematic review and its sources . tables 1 and 2 illustrate some heterogeneity in study designs : the resection methods as well as the operator dependency using the eus technique . only a few of the studies mention blinding concerning pathologists and endoscopists , which may potentially lead to differentiated and nondifferentiated informational bias . this may increase the risk of type ii errors due to the lack of statistical power , especially considering the small numbers of patients in most studies . comparing different types of endoscopes and frequencies it is argued that staging by miniprobe is advantageous in staging of stenotic tumors because the miniprobe , which is passed through the biopsy channel of an ordinary colonoscope , is highly successful in passing stenotic lesions in contrast to conventional eus endoscopes . some studies emphasize that miniprobes are also useful for staging of small and flat lesions . miniprobes however using high ultrasonic frequency with low penetration depth will not be able to examine all layers of the colonic wall or lymph nodes . conventional eus may pose difficulties in obtaining cross - sectional images over lesions located over a colonic bend or in strictures . others argue that a radial echoendoscope can be considered a feasible staging instrument for colonic cancers in all sections of the colon . authors state following reasons for staging inaccuracy : ( 1 ) presence of inflammation in the subserosal layer as a reason for overstaging t1 to t2 and ( 2 ) understaging of t2 and t3 tumors primarily due to difficulties in distinguishing carcinomatous microinfiltration from inflammatory changes . this may be due to the fact that t4 tumors have a high risk of stenosis and are therefore difficult to evaluate by eus . eus in general is a standard procedure for staging of many gastrointestinal lesions , and an eus examination of the entire colon has been shown to be technically feasible and safe . due to the colorectal screening programs , an increasing number of tumors of the colon is found at earlier stages . this fact , combined with a population of increasing age with higher risks of comorbidity , puts clinicians in therapeutic dilemmas . for elderly comorbid patients who are questionable candidates for major surgery , eus could possibly open a new avenue for treatment decisions such as small local tumor resections , or in the near future , full - thickness endoscopic resection as a routine procedure , based on t - staging evaluation with exclusion of local lymph node metastases . the latter evaluation is a challenge at present as n - staging significantly impacts colonic cancer management and imaging methods used today are inaccurate . further studies should be undertaken in the future with larger numbers of patients to clarify whether eus will prove useful for staging of cancer of the colon . apart from therapeutic stratification to neoadjuvant chemoradiation , preoperative tn - staging of colorectal cancers is also of importance for prognostication . consequently , there is also at present interest in other imaging methods to additionally subclassify these cancers . evaluation of tumor vascularity by eus is such a method that may be useful both for prognostication and for assessment of the efficacy of antiangiogenic agents early in the course of therapy . imaging and evaluation of the blood flow velocity and direction can be carried out using doppler sonography . the feasibility of contrast - enhanced eus ( ce - eus ) examinations with second - generation microbubble contrast agents used as doppler signal enhancers was proven by several groups . evaluation of tumor perfusion can also be performed by low mechanical ce - eus . according to the european federation societies in ultrasound in medicine and biology guidelines , ce - eus can be utilized to assess early response to biologic therapy in tumors , such as metastatic gastrointestinal stromal tumor , renal cell carcinoma , and hepatocellular carcinoma . a similar approach has recently been described for quantitative assessment of tumor perfusion in colorectal cancer . very few studies have evaluated the use of eus for staging of malignant colonic neoplasms . accurate staging of colonic cancer proximal to the rectum is becoming increasingly important for treatment decisions in the setting of modern oncology and technical advancements in surgery and endoscopy . eus may become an important imaging modality in the determination of the therapeutic approach to patients with colon cancer ; however , further large multicenter trials are necessary before firm conclusions can be drawn , especially regarding evaluation of the n - stage . very few studies have evaluated the use of eus for staging of malignant colonic neoplasms . accurate staging of colonic cancer proximal to the rectum is becoming increasingly important for treatment decisions in the setting of modern oncology and technical advancements in surgery and endoscopy . in this study we found the pooled - staging sensitivity and specificity to be 0.90 and 0.98 for t1 tumors , 0.67 and 0.96 for t2 tumors , and 0.97 and 0.83 for t3/t4 tumors , respectively . concerning n+ disease , eus may become an important imaging modality in the determination of the therapeutic approach to patients with colon cancer ; however , further large multicenter trials are necessary before firm conclusions can be drawn , especially regarding evaluation of the n - stage . the study was partially funded by agnes and poul friis fund , astrid thaysens legat , axel muusfeldts fund , dansk medicinsk selskab , krista and viggo petersens fund , arvid nilssons fund , director jacob madsens and wife olga madsens fund , director svend espersens fund , lykfeldts fund , and the research councils of herlev and kge hospitals . the study was partially funded by agnes and poul friis fund , astrid thaysens legat , axel muusfeldts fund , dansk medicinsk selskab , krista and viggo petersens fund , arvid nilssons fund , director jacob madsens and wife olga madsens fund , director svend espersens fund , lykfeldts fund , and the research councils of herlev and kge hospitals . the study was partially funded by agnes and poul friis fund , astrid thaysens legat , axel muusfeldts fund , dansk medicinsk selskab , krista and viggo petersens fund , arvid nilssons fund , director jacob madsens and wife olga madsens fund , director svend espersens fund , lykfeldts fund , and the research councils of herlev and kge hospitals . | background and objectives : treatment of colonic cancer patients is highly dependent on the depth of tumor invasion ( t - stage ) as well as the extension of lymph node involvement ( n - stage ) .
we aimed to systematically review the accuracy of endoscopic ultrasound ( eus ) for staging of colonic cancer proximal to the rectum.patients and methods : men and women with colonic adenocarcinomas were included in the study .
eus staging was compared to histopathology as the gold standard .
outcome measures were t- and n - staging accuracies .
articles were searched in pubmed , web of science , the cochrane library , and embase.results:six studies were identified comparing eus staging of colonic cancer to histopathology . the pooled - staging sensitivity and specificity were 0.90 and 0.98 for t1 tumors , 0.67 and 0.96 for t2 tumors , and 0.97 and 0.83 for t3/t4 tumors , respectively .
sensitivity and specificity for n + disease were 0.59 and 0.78 , respectively.conclusions:eus is a feasible method for t - staging of cancers of the colon proximal to the rectum .
the accuracy of lymph node staging needs to be verified by prospective multicenter studies including larger patient populations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
walter dandy was the first research to describe radicular pain as an extradural mass associated syndrome in 19293 ) . mixter and barr , however , reported in 1934 that extruded disc materials cause sciatic pain and described the surgical approach11 ) . intervertebral disc degeneration18 ) , smoking , occupational
and sports - related physical activity , microtrauma , such as obesity ,
environmental factors covered by the different forms of
life12 ) , genetic factors1 ) , congenital defects of vertebral formation16 )
are involved in the etiology of lumbar disc herniation . the disc herniation depending on vertebral corpus developmental
anomaly is available only one case in english literature . a thirty - year - old female patient was admitted to our clinic with complaints of low back and right leg pain after a fall 3 weeks ago . physical examination was evaluated as normal and there were not any features identified in family history . the neurological examination was described as right straight leg raising test positive at 45 degrees , full muscle strength , norm active tendon reflexes , right s1 dermatome hypoesthesia . lumbar computed tomography ( ct ) was planned because s1 lumbar vertebra fracture was suspected in the lumbosacral graph ap / l ( fig . 1 ) based on history of trauma . lumbar magnetic resonance imaging ( mri ) was performed as fusion anomaly was detected but s1 vertebral corpus was not broken on the lumbar ct ( fig . l5-s1 right paramedian disc herniation was detected with the precipitation of trauma on the lumbar mri ( fig . development of the spinal canal due to congenital defects in the lumbar mr imaging was normal ( fig . the patient did not consent to surgical treatment and was referred to physical therapy clinic for conservative treatment . disc herniation occurs as a result of displacement of lumbar intervertebral disc annulus fibrosis and nucleus polyposis fibrosis in the vertebral canal7 ) . lumbar disc herniation can be symptomatic as a result of compression on the spinal nerves or roots . radicular symptoms occur due to generation of nerve root ischemia because of the direct mechanical effect of the herniated disc material , or with an inflammatory response and the effect of secondary edema and compression of vascular structures9 ) . in the etiology of the disease , cumulative microtrauma was held responsible and it is reported to be more frequently encountered in those driving motor vehicles , those with sedentary life style and in smokers12 ) . these anomalies are classified as disorders of formation , segmentation disorders and the composition of the two complex disorders5 ) . although the pathophysiology of spina bifida can be described , the embryological pathogenesis of vertebral corpus fusion anomaly is not fully known . mesoderm structure emerges after the second week of embryological development of the vertebrae that make up the spine . skeletal system develops from the para - axial mesoderm and the lateral plates ( somatic layer ) , and crista neuralis . paraaxial mesoderm creates a series of segmented tissue block known as the somite towards caudal from both sides of the neural tube , somatomeres and occipital region . the position of the sclerotome cells changes to surround the notochord and the spinal column in the fourth week of development . this positional change occurs with the differentiation of surrounding structures and not the active migration of those sclerotome cells13 ) . thus , the resulting mesenchymal column retains segmental traces by leaving less - dense areas containing intersegmental arteries of sclerotomies blocks . the caudal part of each sclerotome segment is heavily divided and collected in the ongoing stages of development . this division is so intense that it moves towards the environment intersegmental tissue and thus , a caudal part of the sclerotome is connected to cephalic part of another sclerotome . thus , vertebral body becomes intersegmental with the connection of intersegmental tissue and the pre - cartilaginous vertebra body10 ) . among sclerotomes that are poor in cell count in cranial and rich in cell count in caudal forms , the one that contains the poo r area forms the intervertebral disc , while the one that contains the rich area constitutes a section of vertebral body10 ) . these formation defects occurring in the embryological period cause anomalies regarding the vertebra and the disc10 ) . among congenital vertebral anomalies , double facet joint formation8 ) , the absence of pedicle17 ) , and absence of lumbar articular processes are reported ; however , vertebral corpus fusion defect as in our case was reported before only one time2 ) . butterfly vertebra is an uncommon congenital disorder characterized by a symmetrical fusion defect resulting in a sagittal cleft vertebra2 ) . intervertebral disc was filled due to the lack of development of annulus fibrosis in cavity associated with the defect of embryological development of the vertebral corpus and trauma as a result of weakness in our case and it caused the patient to develop lumbar disc hernia . therefore disc herniation could occur more easily in patients with vertebral body defect than in patients with normal vertebral body . congenital vertebra corpus fusion anomalies are typically asymptomatic . however , these anomalies incidentally detected during routine radiographic investigations and post - traumatic radiological evaluations6 ) . the specific radiological examinations usually include direct roentgenogram , ct and mri15 ) . in our case , vertebral fusion defects were determined with lumbar radiograph performed due to back pain after trauma . we thought that the defect occurred during the development in the embryological period of the vertebral body . however , that intervertebral disc was the weakest link as sufficient circular annulus fibrosis had not developed here . there were no other osseous pathologies such as spina bifida occulta , facet abnormalities , and/or pedicle anomaly in our case . disc herniation in patients with congenital bone deformities associated with congenital developmental defects are considered related problems in the spinal canal . on the level of the patient 's filum terminale , whether tethered cord syndrome should be investigated14 ) . there was not a congenital defect of development of the spinal canal in our case ( fig . 5 ) . presenting with similar symptoms , such as back and leg pain , especially adult clinical presentation patients with lumbar disc herniation group tethered cords can be confused with tethered cord . and these types of cases after trauma can be misevaluated and overlooked as congenital malformation without treatment . especially , patients with congenital malformations and an unusual clinic should be examined carefully with advanced radiological imaging techniques . | lumbar disc herniation is characterized with low back and leg pain resulting from the degenerated lumbar disc compressing the spinal nerve root .
the etiology of degenerative spine is related to age , smoking , microtrauma , obesity , disorders of familial collagen structure , occupational and sports - related physical activity .
however , disc herniations induced by congenital lumbar vertebral anomalies are rarely seen .
vertebral fusion defect is one of the causes of congenital anomalies .
the pathogenesis of embryological corpus vertebral fusion anomaly is not fully known . in this paper , a 30-year - old patient who had the complaints of low back and right leg pain after falling from a height
is presented .
she had right l5-s1 disc herniation that had developed on the basis of s1 vertebra corpus fusion anomaly in lumbar computed tomography .
this case has been discussed in the light of literature based on evaluations of lumbar computed tomography ( ct ) and magnetic resonance imaging ( mri ) .
this case is unique in that it is the first case with development of lumbar disc herniation associated with s1 vertebral corpus fusion anomaly .
congenital malformations with unusual clinical presentation after trauma should be evaluated through advanced radiological imaging techniques . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
rhabdomyolysis is a syndrome caused by leakage of large quantities of muscle cell contents including electrolytes , myoglobin and other sarcoplasmic proteins into the blood , following the necrosis of skeletal muscle . this manifests as limb weakness , myalgia and swelling , usually accompanied by gross pigmenturia without hematuria . acute renal failure ( arf ) is the most important complication of severe rhabdomyolysis , which results in worse prognosis of the patient [ 2 , 3 ] . in previous studies , the rates of arf secondary to rhabdomyolysis range from 42 to 50% in children [ 1 , 4 ] . there are various causes of acute rhabdomyolysis in childhood such as trauma , infection , connective tissue disease , ischemia - induced muscle necrosis , inflammation in muscle and exposure to drugs and/or toxins . , we describe a 15-year - old female with alveolar rhabdomyosarcoma who developed severe acute rhabdomyolysis shortly after chemotherapy . a previously healthy , 15-year - old japanese female presented with right nasal congestion and a right submandibular mass . on admission , physical examination revealed only a submandibular mass ( approximately 2 cm in diameter ) , which was solid , painless and immovable ; no other remarkable abnormalities were found . laboratory findings were all within normal limits : blood urea nitrogen ( bun ) 10 mg / dl , creatinine 0.58 mg / dl , aspartate aminotransferase ( ast ) 34 u / l , alanine aminotransferase ( alt ) 16 u / l , lactate dehydrogenase ( ldh ) 533 u / l , c - reactive protein ( crp ) 0.02 mg / dl , and creatine kinase ( ck ) 92 u / l . magnetic resonance imaging showed a mass in the right maxillary sinus with hyperintensity on t1- and t2-weighted images , which were enhanced by gadolinium . fdg - positron - emission tomography and bone and ga scintigraphy revealed multiple metastases in the right submandibular and bilateral cervical lymph nodes , and bones ( thoracic and lumbar vertebrae , right humerus and right sacroiliac region ) . histological examination showed monotonous proliferation of small round tumor cells , which were positive for desmin and hhf35 . these findings indicated a diagnosis of alveolar rhabdomyosarcoma ( stage 4 of the irs - v tnm staging classification , group iv of the irs clinical grouping classification ) . she received etoposide , cyclophosphamide , pirarubicin , vincristine and cisplatin as her first cycle of chemotherapy without any adverse effects . the second cycle of chemotherapy consisted of etoposide ( 100 mg / m intravenously for 5 days ) , ifosfamide ( 1,800 mg / m intravenously for 5 days ) , actinomycin - d ( 0.015 mg / kg intravenously for 5 days ) and vincristine ( 1.5 mg / m intravenously on days 1 and 8) . twenty - four hours after the end of chemotherapy , she developed a high fever , generalized myalgia and pigmenturia . she suffered from disturbance of consciousness , respiratory failure , arf , acute liver dysfunction and disseminated intravascular coagulation . the laboratory examination showed ; ck 114,268 u / l ( ck - mb 0.16% ) , bun 41 mg / dl , creatinine 3.01 mg / dl , total bilirubin 7.8 mg / dl , ast 1,187 u / l , alt 204 u / l , ldh 4,330 u / l , crp 22.13 mg / dl , fibrin degradation products 10.6 g / ml and d - dimer 3.1 g / ml . these findings were consistent with rhabdomyolysis . the estimated glomerular filtration rate ( egfr ) using serum creatinine was 27.1 ml / min/1.73 m. even after extubation , she remained in an oliguric state and was bedridden due to severe muscle atrophy . she was treated with continuous hemodiafiltration or continuous ambulatory peritoneal dialysis ( capd ) . in order to search for the cause of rhabdomyolysis , we performed magnetic resonance imaging , evoked electromyography , nerve conduction study , a muscle biopsy from the right musculus quadriceps femoris , bacterial cultures and additional laboratory tests . evoked electromyography and nerve conduction study showed no indication of neuropathy . there were basophilic muscle fibers , suggestive of regeneration of damaged fibers in the frozen sections . small angular fibers and a nuclear clamp were also seen . a mild endomysial mononuclear cell infiltrate was noted . these histological features of the muscle biopsy were compatible with the regenerative process of rhabdomyolysis . the final diagnosis was nontraumatic rhabdomyolysis , and anticancer drugs were thought to be responsible for rhabdomyolysis because other possible causes were unlikely . however , the particular causative drug was unclear . after discontinuing capd , the patient 's conditions improved slowly , and low - dose chemotherapy ( vincristine , actinomycin d and cyclophosphamide ) and local radiation therapy were started 5 months after the episode of rhabdomyolysis . her renal function has improved to the latest egfr of 96.5 ml / min/1.73 m , although it still takes some effort for her to walk and she continues to receive rehabilitation . the classic symptomatic triad of rhabdomyolysis includes myalgia , weakness and dark urine , although these findings may not always be consistent . the definitive diagnosis of rhabdomyolysis requires an elevation of ck levels to > 5 times the normal range in the absence of significant elevations of brain or cardiac ck fractions . . showed that the most common causes of rhabdomyolysis in adults included alcohol abuse ( 67% ) , recent soft tissue compression ( 39% ) and seizure activity ( 24% ) . ward also showed that trauma ( 38% ) , ischemia ( 14% ) and polymyositis ( 24% ) were major causes . however , mannix et al . showed that the risk factors for rhabdomyolysis in children are different than those in adults , including viral myositis ( 38.2% ) , trauma ( 25.7% ) , connective tissue disease ( 5.2% ) , drug overdose ( 4.2% ) , exercise ( 4.2% ) , metabolic disorder ( 3.7% ) , seizure ( 3.7% ) , sepsis ( 3.1% ) , drug reaction ( 2.1% ) and hypoxia ( 2.1% ) . most notably , viral myositis was the most common cause of rhabdomyolysis in patients < 9 years of age , whereas trauma and drugs were the major causes in children > 9 years of age and adults . as mentioned above this may be related to the fact that striated musculature , in contrast to other tissues such as liver and kidneys , exhibits little affinity for most drugs . in adults , there are several reports of rhabdomyolysis associated with anticancer drugs . the anticancer drugs and their combinations which cause rhabdomyolysis include et-743 , paclitaxel and gemcitabine , cytarabine , high - dose ifosfamide , carboplatin and etoposide , stem cell transplantation conditioning using thiotepa and melphalan , doxorubicin , cytarabine , thioguanine and vincristine , mitoxantrone and cyclophosphamide , pemetrexed , high - dose cyclophosphamide , vincristine , cytarabine and adriablastin , and 5-azacytidine ( table 1 ) . alkylating agents ( e.g. cyclophosphamide and ifosfamide ) , anthracyclines ( doxorubicin , etc . ) and anthracenediones ( mitoxantrone , etc . ) induce cardiac muscle damage , although skeletal muscle damage is uncommon [ 14 , 16 ] . anthracyclines and anthracenediones induce the generation of oxygen free radicals leading to cardiac muscle injury , although it is unclear why a similar phenomenon does not occur in skeletal muscle . taxanes may provoke myositis , sometimes in a severe form , and incidence reaches up to 2% when they are used in combination with gemcitabine . in most cases , common causes for rhabdomyolysis including trauma , infection , connective tissue disease , drug overdose , exercise , metabolic disorder , seizure and sepsis were either excluded or not evident . therefore , anticancer drugs were thought to be responsible for the development of rhabdomyolysis , although the particular causative agent is unclear . recently , common variants in slco1b1 were found to be strongly associated with an increased risk of statin - induced myopathy by a genome - wide study on the mechanism of drug - induced myopathy . to date , no studies on the genetic association between rhabdomyolysis and anticancer drugs have been reported . this disease is usually thought to be benign in children , with only a small risk of arf . however , rhabdomyolysis - induced arf is a very important complication in children as well . several mechanisms by which rhabdomyolysis leads to acute kidney injury ( aki ) are proposed , including direct renal tubular toxicity of myoglobin , intrarenal vasoconstriction and tubular obstruction , as well as secondary injury associated with volume depletion and renal ischemia . in addition , several adult studies have evaluated predictive factors of rhabdomyolysis - induced aki . these studies suggest that the presence of dehydration , metabolic acidosis with aciduria , oliguria , massive muscle damage , higher illness severity and systemic inflammatory response syndrome are predisposing factors for the development of aki with rhabdomyolysis . in our patient , some of the proposed mechanisms are far from impossible , but none of the predisposing factors were evident . rhabdomyolysis - induced aki / arf is potentially life - threatening and leads to the delay of chemotherapy . although the dose range of most anticancer drugs can be limited by renal function , many anticancer drugs can induce renal injury and rhabdomyolysis , which may lead to aki / arf . therefore , it is important that we be aware of the possibility of drug - induced rhabdomyolysis and make preemptive medical decisions to prevent rhabdomyolysis in high - risk patients during cancer chemotherapy . | this is the first paper to report the association of cancer chemotherapy with rhabdomyolysis in children .
a previously healthy , 15-year - old japanese female was diagnosed as having alveolar rhabdomyosarcoma .
she received the first cycle of multi - agent chemotherapy without any adverse effects .
however , she developed severe acute rhabdomyolysis shortly after the second cycle of multi - agent chemotherapy , which consisted of etoposide , ifosfamide , actinomycin - d and vincristine .
her condition deteriorated rapidly and she was treated with mechanical ventilation and fluid replacement . after further evaluation , anticancer drugs were thought to be responsible for the rhabdomyolysis . |
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epstein - barr virus ( ebv ) , a member of gamma herpes virus , infects most adults worldwide and persists throughout the lifetime of its host . the possibility of viral reactivation is a frequent concern for recipients of organ transplantation , especially its association with post - transplant lymphoproliferative disorder ( ptld ) solid organ transplantation ( sot ) or hematopoietic stem cell transplantation ( hsct ) recipients with ebv - positive early ptld have significantly higher viral load measured in blood than ptld negative patients ; high viral load levels often precede the clinical presentation of ptld . routine ebv viral load can provide an early diagnosis of ptld and support pre - emptive schema . on the other hand , a proportion of patients with high ebv viral loads may not exhibit ptld or a proportion of patients with low ebv viral loads may have ptld . so far , no report has been published regarding the temporary detection of ebv in urine of renal graft recipient . measuring the ebv load is essential to follow and diagnose ebv - associated diseases and to explore the pathogenesis of ebv infection . ebv dna can sometimes be detected in immunocompetent individuals and often in immunosuppressed patients without symptoms or clinical sequelae . this does not cause symptoms in the immunocompetent patient but serious disease can occur in immunosuppressed hosts . trigger points for intervention are poorly defined as natural history studies of ptld in high risk patients where clinicians are blinded because the implemented interventions have been limited . moreover , adult ptld patients have been observed to relapse in presence of persistently low vl . here , we report 4 cases of cadaver kidney transplantation patients who referred to our hospital post transplantation because of rising in their serum creatinine . we present unusual cases of urinary ebv shedding that were suspected to have bk virus or cmv infections . more case studies are needed to explore the clinical significance of such finding in renal transplant recipients . viral nucleic acids from plasma and urine specimens were extracted using an automated extractor , magnapure 96 ( roche , basel , switzerland ) , according to manufacturer s recommendation . the use of an internal control of each reaction tube excluded false negatives due to nonspecific inhibitors of the pcr enzymes . quantitative multiplex real time pcr ( qmrtpcr ) was carried out on 5 l of extracted materials using bce kit ( for the detection of bk virus , cmv and ebv , respectively ) ( fast - track diagnostics , luxembourg ) , according to manufacturer s instructions . each real time reaction incorporated at least three standards for analysis of quantification curves in each sample per run . if a positive laboratory test result was not compatible with the clinical picture of the patients , the assay was repeated by another technician on the same sample . a 44 years old woman who underwent kidney transplantation for diabetes and hypertension in post - transplant month ( ptm ) 14 was admitted for the suspicion of recurrent urinary tract infections ( uti ) since her transplantation . her immunosuppression consisted of cyclosporine ( 100 mg b.i.d . ) , mycophenolate mofetil ( 1000 mg b.i.d . ) and prednisone ( 5 mg daily ) . on routine follow - up , she had elevated serum creatinine ( 2.2 mg / dl ) . the blood culture was positive for e. coli ; hence she received imipenem 500 mg b.i.d . for 10 days . in qmrtpcr , ebv was positive in both plasma ( 3.510 u / ml ) and urine ( 4.210 u / ml ) ( table 1 ) . her follow up urine and plasma pcr analysis 3-months was positive only for cmv 510 u / ml in urine . a 47 years old woman with the known history of hypertension for > 15 years , was admitted in post - transplant month 10 due to high creatinine . her immunosuppression consisted of cyclosporine ( 75 mg b.i.d . ) , mycophenolate mofetil ( 500 mg b.i.d . ) and prednisone ( 10 mg daily ) . ultrasound and pelvic computerized tomography ( ct ) scan showed hydronephrosis and urine collections with septation . in qmrtpcr , cmv was positive in both plasma ( 3.910 u / ml ) and urine ( 410 u / ml ) ( table 1 ) . ebv was positive only in urine ( 3.210 u / ml ) ( table 1 ) . , e. coli was found ; therefore , she received ceftriaxone 1 g b.i.d . for 10 days for her uti . her follow up testing showed no ebv , cmv and bkv in her plasma and urine . a 54 years old woman with a known long history of diabetes and hypertension was admitted due to low back pain . her immunosuppression consisting of cyclosporine ( 75 mg b.i.d . ) , mycophenolate mofetil ( 500 mg tds ) and prednisone ( 7.5 mg daily ) . in ultrasonography , however , an ovarian tumor with a size of 27 mm was reported by ultrasonography . she was discharged with normal range of serum creatinine seven days after discontinuation of ganciclovir . her follow up testing showed no ebv , cmv and bkv in her plasma and urine . his immunosuppression consisting of tacrolimus ( 1 mg b.i.d . ) , mycophenolate mofetil ( 500 mg b.i.d . ) and prednisone ( 10 mg daily ) . 14 months following the transplantation , he had presented with repeated urinary tract infections and elevated serum creatinine concentrations . imagining studies showed reflux grade iv . upon urinary and blood culture testing , klebsiella were positive . his urine output dropped suddenly with a rise in creatinine , requiring hemodialysis three times weekly for two weeks . in qmrtpcr , cmv and ebv were positive in urine : 310 u / ml and 2.510 u / ml , respectively . ultimately , she was discharged with negative blood and urine cultures and normal serum creatinine . the present study includes four cases of cadaver kidney transplant recipients admitted to our center 9 to 20 ptm because of rise in their serum creatinine concentrations in three cases . all patients had end - stage renal disease ( ersd ) and all had a history of 1 - 2 years dialysis before their renal transplantation . to the best of our knowledge , this is the first report of asymptomatic ebv shedding in the urine of kidney transplant recipients . also , they all were received cmv antivirals for the first 3 months posttransplantation , including ganciclovir 1.25 mg / kg iv daily as induction for one month , which then switched to oral valganciclovir ( according to their serum creatinine levels , b.i.d ) . in cmv dnaemia cases , the level of viral load was below the cut off levels for anti - cmv treatment in our center ( 2000 u / ml ) . all patients were negative for ebv igm and were positive for ebv igg serology . for all cases , quantitative multiplex real time pcr for bkv , cmv and ebv in the urine and serum were carried out for the initial suspicion of bkvn . upon finding unusual test results , one advantage of this pcr method was its ability to identify the presence of two of herpes viruses plus bkv within the same sample at the same time . we found that all 4 patients had different levels of ebv in their urine , despite having no symptoms regarding the involvement of kidney or a systemic involvement like chronic hbv infection . however , to the best of our knowledge , this is the first report of asymptomatic urinary ebv shedding in immunodeficient individuals . the european best practice guidelines for renal transplantation and kidney disease : improving global outcomes transplant work group and other centers recommend testing for ebv nucleic acid in high - risk renal transplant patients every occasion soon after transplantation until a distinct period . in healthy transplant recipients , baseline viral loads tend to be higher than in normal immunocompetent hosts , both in whole blood and in plasma , and viral loads over time reflect the progression and resolution of ptld . detection of ebv in both healthy male and female genital secretions has been reported . however , the level of ebv in genital secretions was lower than in saliva . also , ebv has also been detected from spleen , intestines , pancreas and urinary bladder in autopsies specimen . it is not clear if b cells resided in kidneys could be infected by ebv and support virus replication . also , it is uncertain how this shedding is maintained , but the latent ebv load in urine might reflect a balance between removal of ebv infected cells due to the half - life of memory b cells and reinfection of new memory b cells during virus reactivation . nevertheless , in vitro evidence for direct infection and replication of ebv in cultured normal human epithelial cells has been reported previously . more investigations will unravel the exact mechanism of ebv shedding in urine and its relation to immunosuppression after kidney transplantation and perhaps occurrence of ptld . one of interesting finding in the present study was the presence of cmv dnaemia in 3 patients ( cases1 , 2 and 3 ) and cmv viruria in 2 patients ( cases 2 and 4 ) despite not being clinically significant . while asymptomatic shedding of cmv in the urine is not unusual in healthy ( immunocompetent ) subjects , coinfection of ebv with cmv or other viruses might be a risk factor for developing ptld . we appreciate that these patients did not present any symptoms that could be assigned as being co infected with both ebv and cmv in different body compartment . therefore , we believe more frequent testing is worth considering if these patients show any sign or symptoms regarding ptld due to coinfection . two of patients were positive for cmv in their urine 3 months after their discharge ; however , they were symptomfree . taken together , we believe that in transplant recipients , asymptomatic shedding of ebv finding in body fluids including urine in the absence of evidence for invasive disease ( virus found in tissues or in blood ) should be viewed with suspension as this hypothesis has been also raised by others generally for herpes viruses . in conclusion , we presented asymptomatic presence of ebv in the urine of kidney transplant recipients around the first post - transplantation year who admitted for uti and for the suspension of bkvn . prospective studies are required to answer many questions regarding the significance of this unusual finding in immunocompromized patients . | epstein - barr virus ( ebv ) is associated with a wide range of malignancies and complications like post - transplant lymphoproliferative disorder ( ptld ) . to suppress active ebv infection in transplant recipients , who are at a heightened risk of developing ptld , ebv dnaemia monitoring has been recommended . quantitative multiplex real time polymerase chain reaction ( qmrtpcr ) offered the advantage of detection of more than one target in the same sample .
we present four cases of kidney transplant recipient who were admitted for rising serum creatinine between 9 and 20 months post - transplant with a suspicion of bkv - associated nephropathy .
all but one patient had unusual sonography findings in their genitourinary tracts and were positive for urinary culture for bacteria . using a commercial qmrtpcr that could detect and quantitate bkv , ebv and cytomegalovirus simultaneously , all patients were positive for ebv in their urine for the levels between 2500 and 8108 u / ml .
none of the patients had any symptoms regarding this finding . on following up survey 3 month post discharge from hospital ,
all patients were negative for plasma and urine ebv .
absent of ebv dnaemia together with alternating phases of detectable ebv in urine might reflect the presence of functionally efficient central / effector memory t cells against ebv .
the significance of this finding in immunocompromized patients necessitates prospective longitudinal studies . |
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the 5 millennium development goal ( mdg ) aims at reducing maternal mortality by 75% by the year 2015 . according to the world health organization ( who ) developing countries accounted for 99% of these deaths of which three - fifths occurred in sub - saharan africa . maternal mortality remains a major public - health issue in developing countries . according to the who , 536,000 women die every year in the world from causes relating to pregnancy , childbirth , or postpartum . the majority of maternal deaths avoided if women had access to quality medical care during pregnancy . according to egypt demography and health survey , slightly more than one quarter of egyptian pregnant women do not receive antenatal care . however , among those who receive antenatal care only one - third of them received advised about signs of obstetric complications and where and when to seek medical assistance ( el - zanaty and way , 2009 ) . in egypt , maternal mortality ratio has declined dramatically from 174/100,000 live births in 1992 - 1993 to 67.6/100,000 live births in 2005 , a further decline to 44.6/100,000 was also reported by 2009 ( global health council , 2010 ; who , 2010 ) . great bodies of literature monitor to improve the quality of health care . in the developing countries , however , low interest in spite of overwhelming published evidence of low quality of care in these countries . in egypt , health services are of poor quality and the country has poor health status relative to other low - income countries . the government has focused on improving the quality of health services delivered to the population as a mean to attaining the mdgs . thus , patient satisfaction is an integral component of health services provided to the population . this situation raises the question of the quality of care offered in maternity health care . measurement of patient satisfaction plays an important role in raising accountability among health care providers . although system aspects such as cost , access , availability and waiting time related to patient satisfaction , they have been less important than the human aspect of medical care . antenatal care is regarded as a cornerstone of maternal health care and better evidence about what works and what does not work to reduce maternal mortality exists . many elements of antenatal care have no impact in reducing complications and maternal deaths , so the who guidelines are specific as regards the timing and content of antenatal care visits according to gestational age and the guidelines insist that only examinations and tests that have been proven to be beneficial should be performed . quality improvement ( qi ) is an integral part of the reform model in egypt . while the ministry of health and population has made significant changes in the past few years in development of a qi program , health facilities still face challenges in improving the quality of services in this area . clinical audit is a qi process that seeks to improve patient care and outcomes through systematic review of care against explicit criteria . aspects of the structure , processes and outcomes of care selected and systematically evaluated against explicit criteria . a cross - sectional primary health care ( phc ) study was conducted from january 2012 to april 2012 at urban suez , egypt , primary health centers according to national quality standards in egypt . auditing of quality of care through medical records randomly selected from the registry using an audit checklist modified from accreditation of phc facilities in egypt : program policies and proceduresassessing satisfaction of providers : a comprehensive sample using a questionnaire modified from trustees of dartmouth college , godfrey , nelson and bataldenassessing pregnant women satisfaction by a consecutive sample derived from pregnant women attending the phc , a questionnaire modified from patient satisfaction survey , the bureau of phc and patient satisfaction survey , new river health association . auditing of quality of care through medical records randomly selected from the registry using an audit checklist modified from accreditation of phc facilities in egypt : program policies and procedures assessing satisfaction of providers : a comprehensive sample using a questionnaire modified from trustees of dartmouth college , godfrey , nelson and batalden assessing pregnant women satisfaction by a consecutive sample derived from pregnant women attending the phc , a questionnaire modified from patient satisfaction survey , the bureau of phc and patient satisfaction survey , new river health association . data collected in the waiting area , for privacy . from records , we check if taking the client 's history , examination and investigation . likert scale used . the variables rated included ; satisfaction with access , waiting time , privacy , staff attitude , counseling , time with health worker , information given by health team . auditing of quality of care through medical records randomly selected from the registry using an audit checklist modified from accreditation of phc facilities in egypt : program policies and proceduresassessing satisfaction of providers : a comprehensive sample using a questionnaire modified from trustees of dartmouth college , godfrey , nelson and bataldenassessing pregnant women satisfaction by a consecutive sample derived from pregnant women attending the phc , a questionnaire modified from patient satisfaction survey , the bureau of phc and patient satisfaction survey , new river health association . auditing of quality of care through medical records randomly selected from the registry using an audit checklist modified from accreditation of phc facilities in egypt : program policies and procedures assessing satisfaction of providers : a comprehensive sample using a questionnaire modified from trustees of dartmouth college , godfrey , nelson and batalden assessing pregnant women satisfaction by a consecutive sample derived from pregnant women attending the phc , a questionnaire modified from patient satisfaction survey , the bureau of phc and patient satisfaction survey , new river health association . data collected in the waiting area , for privacy . from records , we check if taking the client 's history , examination and investigation . likert scale used . the variables rated included ; satisfaction with access , waiting time , privacy , staff attitude , counseling , time with health worker , information given by health team . table 1 shows pregnant women satisfaction with a significant difference in ( ease of the registration process and comfort of the waiting area ) ( p < 0.001 ) , there were a significant difference in waiting time p < 0.001 between centers and providers - clients relationship ( p = 0.007 ) . client satisfaction among participating women table 2 showing providers satisfaction , with a significant difference in physician respect ( p = 0.045 ) . health care providers satisfaction in different centers ( n=17 ) table 3 showed that the number of visits according to guidelines ( p < 0.001 , odds ratio [ or ] = 1.05 [ 1.03 - 1.07 ] ) , fetal movement ( p < 0.001 , or = 10.31 [ 3.25 - 32.73 ] ) , blood pressure ( p < 0.001 , or = 0.00 [ 0.00 - 0.005 ] ) , fetal lie ( after 32 weeks ) ( p < 0.001 , or = 3.34 [ 1.8 - 6.17 ] ) , engagement ( p = 0.017 , or = 1.39 [ 1.06 - 1.83 ] ) , fetal heart sound ( p < 0.001 , or = 5.57 [ 2.77 - 11.21 ] ) , urine examination ( p = 0.008 , or = 26.69 [ 2.36 - 302.1 ] ) and ultrasound ( p < 0.001 , or = 2.260 [ 1.49 - 3.45 ] ) . logistic regression analysis for best predictors for completeness regarding file auditing at periodic visits of antenatal care ( n=306 ) the total quality score of client satisfaction in urban health district was 79.2 23.5 with statistical difference between centers ( p < 0.001 ) . , 2008 where the respondents expressed a high level of overall satisfaction ( 81.4% ) with the care received . these results disagreed with a study in private health facilities in llorin , of nigeria by balogun 2007 where only 64% of women showed satisfaction with service they had . the difference could be explained by the difference of the study type and site that the current study was a cross - sectional study for pregnant women in public facilities while the other study was a retrospective study for women after delivery in private facilities where clients expectation may differ . the current study showed that the most dissatisfying items for clients were :
registration process and waiting area by 24%relation with nurses and medical assistant staff by 20%waiting time by 15.4% . registration process and waiting area by 24% relation with nurses and medical assistant staff by 20% waiting time by 15.4% . the total quality score of physicians satisfaction in the present study was 61.0% with no statistically significant difference between the centers ( p = 0.572 ) , which agreed with a study done in bahrain by sharaf et al . , 2008 where the study has revealed that primary care physicians in bahrain , were satisfied ( mean = 3.46 , sd = 0.67 ) according to job satisfaction survey . munawwara by al juhani and kishk , 2006 where the overall mean score of physicians satisfaction was 65.53% and agreed with a study at capital health region , kuwait by al - eisa et al . , nevertheless , these results disagreed with a study done in oman by taman , 2009 which showed that the overall staff satisfaction score with their profession was 79.44% . this difference could be due to the difference in sample size and characteristics as the current study include only 17 physician most of them recently graduated while other study included 390 staff with years of experience were more than 10 year . the current study showed that the most dissatisfied items for providers were :
suitability of the work place than it was 12 months ago by 64.7%the available tools , equipment and encouragement they had by 64.7%unrespectful treatment they had by 47.1% . suitability of the work place than it was 12 months ago by 64.7% the available tools , equipment and encouragement they had by 64.7% unrespectful treatment they had by 47.1% . the total quality score of file auditing was ( 76.5 5.6 ) with statistically significant difference between centers ( p < 0.001 ) which disagreed with a study in kingdom of saudi arabia by al - momen et al . , 2003 where the total assessment score approached was 71 out of 100 for primary health centers . the difference could be explained due to the difference in both sample size and sampling methods and might be due to the difference in both guidelines used as in the current study the egyptian guidelines was used , while in other studies , the policy of antenatal care formed by co - operation from department of obstetrics and family and community medicine in saudi arabia . the current study showed that the most deficient items in the file registration were :
recording of the immunization , of pregnant was ( 28.8%)recording of the fetal heart sound ( 18.3%)recording of the ultra sound ( 11.3%)recording of screening of diabetes in pregnant women ( 2.5%)recording of the diagnosis according to the clinical guideline ( 12.4%)referral of high risk pregnancies to specialist according to clinical guideline ( 4.1% ) . recording of the immunization , of pregnant was ( 28.8% ) recording of the fetal heart sound ( 18.3% ) recording of the ultra sound ( 11.3% ) recording of screening of diabetes in pregnant women ( 2.5% ) recording of the diagnosis according to the clinical guideline ( 12.4% ) referral of high risk pregnancies to specialist according to clinical guideline ( 4.1% ) . in the current study , showed that client satisfaction was higher in accredited centers than non - accredited center with a significant difference p = 0.002 . this agreed with a study in alexandria by gadallah et al . , 2010 where patient satisfaction was higher in accredited family health units compared to non - accredited units in all aspects : as cleanliness , doctors and nurses competency and attitude , waiting area and waiting time while provider satisfaction was higher in non - accredited center than accredited centers with insignificant difference p = 0.533 which disagreed with a study in alexandria by gadallah et al . , 2010 showed that providers in the reformed phcs were more satisfied , than providers in non - reformed phcs , regard availability of equipments , job satisfaction and income satisfaction . the difference explained that the current study involved only one non - accredited center while other study involved four non - accredited unites , also due to the difference in assessment methods as the current study involved only questionnaire while other study involved questionnaire and focus group discussion ( different study methodology ) . there was bias during the assessment of client satisfaction in the presence of health providers of the servicea lot of illiterate refused to share in the study , as they feel unsafe by sharing in something they can not readit was difficult for clients to understand likert scale and to select between different choicesregarding the auditing part , the problems of incompleteness and inaccuracy of the medical records constituted an obstacle in assessing the process of antenatal care . there was bias during the assessment of client satisfaction in the presence of health providers of the service a lot of illiterate refused to share in the study , as they feel unsafe by sharing in something they can not read it was difficult for clients to understand likert scale and to select between different choices regarding the auditing part , the problems of incompleteness and inaccuracy of the medical records constituted an obstacle in assessing the process of antenatal care . there was bias during the assessment of client satisfaction in the presence of health providers of the servicea lot of illiterate refused to share in the study , as they feel unsafe by sharing in something they can not readit was difficult for clients to understand likert scale and to select between different choicesregarding the auditing part , the problems of incompleteness and inaccuracy of the medical records constituted an obstacle in assessing the process of antenatal care . there was bias during the assessment of client satisfaction in the presence of health providers of the service a lot of illiterate refused to share in the study , as they feel unsafe by sharing in something they can not read it was difficult for clients to understand likert scale and to select between different choices regarding the auditing part , the problems of incompleteness and inaccuracy of the medical records constituted an obstacle in assessing the process of antenatal care . this study gives important baseline information used in intervention design and implementation of projects that seek to improve maternal health . major gaps exist in the staffing levels , adequacy of infection control facilities and provision of the full package of anc especially with regard to carrying out essential tests , counseling for risk factor recognition and birth preparedness . the erratic availability of drugs and supplies coupled with the long waiting hours are also major challenges . the district health teams should work with facility managers to ensure the availability and use of infection control practices . refresher training of health workers , assessment of workloads and recruitment of more health workers recommended ensured through investing more resources in procuring them and improving on the supply chain efficiency at all levels of procurement and supply . | introduction : the 5th millennium development goal aims at reducing maternal mortality by 75% by the year 2015 . according to the world health organization , there was an estimated 358,000 maternal deaths globally in 2008 .
developing countries accounted for 99% of these deaths of which three - fifths occurred in sub - saharan africa . in primary health care ( phc ) , quality of antenatal care is fundamental and critically affects service continuity .
nevertheless , medical research ignores the issue and it is lacking scientific inquiry , particularly in egypt.aim of the study : the aim of the following study is to assess the quality of antenatal care in urban suez governorate , egypt.materials and methods : a cross - sectional primary health care center ( phcc ) based study conducted at five phcc in urban suez , egypt . the total sample size collected from clients , physicians and medical records .
parameters assessed auditing of medical records , assessing provider and pregnant women satisfaction.results:nearly 97% of respondents were satisfied about the quality of antenatal care , while provider 's satisfaction was 61% and for file , auditing was 76.5 5.6.conclusion:the present study shows that client satisfaction , physicians satisfaction and auditing of medical record represent an idea about opportunities for improvement . |
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burn injury , one of the most common and devastating forms of trauma , is a major public health problem worldwide . burn wounds can easily become infected because the skin no longer acts as an effective physical barrier against microbes . while these bacteria rarely cause disease in healthy individuals , they may do so in immuno compromised patients , such as those with aids or cystic fibrosis , and in burns patients . the accurate identification of p. aeruginosa and detection of their susceptibility to antimicrobials are critical components of the management of burned patient . typical isolation media used in wound infections include blood agar and chocolate agar and selective agars such as mac- conkey agar and cetrimide - based media . although large numbers of p. aeruginosa isolates are often present in clinical samples from burns patients , their detection and precise identification can often be challenging . for example , other species of pseudomonas , as well as stenotrophomonas maltophilia , achromobacter xylosoxidans and ralstonia pickettii , have been shown to grow on cetrimide- based media , and may be indistinguishable from non - pigmented strains of p. aeruginosa . biochemical test kits such as api 20 ne are commonly used for identification ; however , this technique has been seen to display a high rate of misidentification of oxidase - positive gram - negative rods , including p. aeruginosa . in addition , testing requires the use of a pure bacterial subculture and a minimum incubation time of 48 h. hence , identification by means of this method requires at least 3 days . another limitation of the conventional culture technique is that p. aeruginosa can easily be mistaken for closely related gram - negative bacilli . the use of molecular techniques such as pcr could enable accurate and rapid identification of p. aeruginosa [ 6 , 7 ] . l and i lipoproteins are two outer membrane proteins of p. aeruginosa , and are responsible for the inherent resistance of the bacterium to antibiotics and antiseptics . as these proteins are found only in this organism , they could be used as a reliable marker for the rapid identification of p. aeruginosa in clinical samples [ 8 , 9 ] . in this study , we examined a technique for the rapid and precise identification of p. aeruginosa strains isolated from burns patients hospitalized in a main burns center in iran . the performance of this technique , which utilizes pcr amplification of i lipoprotein ( opri ) to detect the genus and l lipoprotein ( oprl ) to detectthe species of p. aeruginosa strains , was compared with that of phenotypic and routine biochemical identification used in laboratories . this study was carried out during a 5-month period , at a major center for the admission of burns patients in tehran , iran . samples were obtained from burn wounds by swabbing . as in the routine phonotype tests usually performed in clinical laboratories , we inoculated burn wound swabs onto several selective media for the isolation of p. aeruginosa , including blood agar , macconkey agar and muller hinton agar , and carried out incubation at 37c for 24 - 48 h. the isolates were presumptively identified by means of routine tests : colony morphology and pigment formation on selective medium , positive oxidase test , glucose fermentation , hydrolysis of gelatin and growth at 42c [ 10 , 11 ] . in order to minimize contamination and hence the possibility of false - positive results , all dna isolation procedures were carried out in a room physically separated both from that used to set up nucleic acid amplification reaction mixtures and from the post - pcr room . bacterial genomic dna was extracted from all phenotypically and biochemically tested strains , as well as from the reference strain p. aeruginosa atcc 27853 , by means of a boiling method . for this purpose , depending on colony size , three to six colonies were picked from bacterial plates and mixed into 0.25 ml dnase / rnase - free water in sterile 1.5 ml eppendorf tubes in order to obtain a turbid suspension of bacteria ( ~ 1 - 2 109 cells / ml ) . the cell suspensions were kept in a boiling water bath for 10 minutes to lyse the cells , and were then centrifuged at 10000 g at 4c for 10 minutes . finally , the supernatant was transferred , in sterile conditions , into another tube and used as a dna template . the primers used in this study are shown in table i. pcr amplification of i lipoprotein ( opri ) for the detection of pseudomonas genus and l lipoprotein ( oprl ) for the detection of p. aeruginosa species was performed on all phenotypically tested strains of p. aeruginosa . primers used in this study . in order to minimize contamination , all reaction mixtures were set up in a pcr room separate from that used for dna extraction and amplification and from the post - pcr room . pcr was completed in adapted pcr micro centrifuge tubes according to the thermocycler used . following optimization , reaction mixtures ( 25 l ) were set up as follows : 11 l dnase/ rnase - free water , 8 l 2x pcr master mix ( 1.5 mm mgcl2 , denmark ) , 0.5 l of each set of primers ( oprl or opri ) and 5 l of dna template . the reaction mixtures were subjected to the following empirically optimized thermal cycling parameters in a thermocycler ( senso- quest labcycler , germany ) : 94c for 5 min , followed by 30 cycles of 94c for 1 min , 55c for 1 min , 72c for 1 min , and a final extension at 72c for 10 min . positive ( p. aeruginosa atcc 27853 dna ) and multiple negative ( water ) amplification controls were included in every set of pcr reactions . following amplification , aliquots ( 10 l ) were removed from each reaction mixture and examined by means of electrophoresis ( 80 v , 45 min ) in gels composed of 1% agarose in tbe buffer ( 40 mm tris , 20 mm boric acid , 1 mm edta , ph 8.3 ) . gels were visualized under uv illumination by using a gel image analysis system ( uvitec , cambridge , united kingdom ) and all images were archived . where a band was visualized at the correct expected size for opri , the specimen was considered positive for pseudomonas genus ; if a band was visualized at the correct expected size for oprl loci , the specimen was considered positive for p. aeruginosa species . this study was carried out during a 5-month period , at a major center for the admission of burns patients in tehran , iran . samples were obtained from burn wounds by swabbing . as in the routine phonotype tests usually performed in clinical laboratories , we inoculated burn wound swabs onto several selective media for the isolation of p. aeruginosa , including blood agar , macconkey agar and muller hinton agar , and carried out incubation at 37c for 24 - 48 h. the isolates were presumptively identified by means of routine tests : colony morphology and pigment formation on selective medium , positive oxidase test , glucose fermentation , hydrolysis of gelatin and growth at 42c [ 10 , 11 ] . in order to minimize contamination and hence the possibility of false - positive results , all dna isolation procedures were carried out in a room physically separated both from that used to set up nucleic acid amplification reaction mixtures and from the post - pcr room . bacterial genomic dna was extracted from all phenotypically and biochemically tested strains , as well as from the reference strain p. aeruginosa atcc 27853 , by means of a boiling method . for this purpose , depending on colony size , three to six colonies were picked from bacterial plates and mixed into 0.25 ml dnase / rnase - free water in sterile 1.5 ml eppendorf tubes in order to obtain a turbid suspension of bacteria ( ~ 1 - 2 109 cells / ml ) . the cell suspensions were kept in a boiling water bath for 10 minutes to lyse the cells , and were then centrifuged at 10000 g at 4c for 10 minutes . finally , the supernatant was transferred , in sterile conditions , into another tube and used as a dna template . the primers used in this study are shown in table i. pcr amplification of i lipoprotein ( opri ) for the detection of pseudomonas genus and l lipoprotein ( oprl ) for the detection of p. aeruginosa species was performed on all phenotypically tested strains of p. aeruginosa . primers used in this study . in order to minimize contamination , all reaction mixtures were set up in a pcr room separate from that used for dna extraction and amplification and from the post - pcr room . pcr was completed in adapted pcr micro centrifuge tubes according to the thermocycler used . following optimization , reaction mixtures ( 25 l ) were set up as follows : 11 l dnase/ rnase - free water , 8 l 2x pcr master mix ( 1.5 mm mgcl2 , denmark ) , 0.5 l of each set of primers ( oprl or opri ) and 5 l of dna template . the reaction mixtures were subjected to the following empirically optimized thermal cycling parameters in a thermocycler ( senso- quest labcycler , germany ) : 94c for 5 min , followed by 30 cycles of 94c for 1 min , 55c for 1 min , 72c for 1 min , and a final extension at 72c for 10 min . positive ( p. aeruginosa atcc 27853 dna ) and multiple negative ( water ) amplification controls were included in every set of pcr reactions . following amplification , aliquots ( 10 l ) were removed from each reaction mixture and examined by means of electrophoresis ( 80 v , 45 min ) in gels composed of 1% agarose in tbe buffer ( 40 mm tris , 20 mm boric acid , 1 mm edta , ph 8.3 ) . gels were visualized under uv illumination by using a gel image analysis system ( uvitec , cambridge , united kingdom ) and all images were archived . where a band was visualized at the correct expected size for opri , the specimen was considered positive for pseudomonas genus ; if a band was visualized at the correct expected size for oprl loci , the specimen was considered positive for p. aeruginosa species . in order to minimize contamination and hence the possibility of false - positive results , all dna isolation procedures were carried out in a room physically separated both from that used to set up nucleic acid amplification reaction mixtures and from the post - pcr room . bacterial genomic dna was extracted from all phenotypically and biochemically tested strains , as well as from the reference strain p. aeruginosa atcc 27853 , by means of a boiling method . for this purpose , depending on colony size , three to six colonies were picked from bacterial plates and mixed into 0.25 ml dnase / rnase - free water in sterile 1.5 ml eppendorf tubes in order to obtain a turbid suspension of bacteria ( ~ 1 - 2 109 cells / ml ) . the cell suspensions were kept in a boiling water bath for 10 minutes to lyse the cells , and were then centrifuged at 10000 g at 4c for 10 minutes . finally , the supernatant was transferred , in sterile conditions , into another tube and used as a dna template . the primers used in this study are shown in table i. pcr amplification of i lipoprotein ( opri ) for the detection of pseudomonas genus and l lipoprotein ( oprl ) for the detection of p. aeruginosa species was performed on all phenotypically tested strains of p. aeruginosa . in order to minimize contamination , all reaction mixtures were set up in a pcr room separate from that used for dna extraction and amplification and from the post - pcr room . pcr was completed in adapted pcr micro centrifuge tubes according to the thermocycler used . following optimization , reaction mixtures ( 25 l ) were set up as follows : 11 l dnase/ rnase - free water , 8 l 2x pcr master mix ( 1.5 mm mgcl2 , denmark ) , 0.5 l of each set of primers ( oprl or opri ) and 5 l of dna template . the reaction mixtures were subjected to the following empirically optimized thermal cycling parameters in a thermocycler ( senso- quest labcycler , germany ) : 94c for 5 min , followed by 30 cycles of 94c for 1 min , 55c for 1 min , 72c for 1 min , and a final extension at 72c for 10 min . positive ( p. aeruginosa atcc 27853 dna ) and multiple negative ( water ) amplification controls were included in every set of pcr reactions . following amplification , aliquots ( 10 l ) were removed from each reaction mixture and examined by means of electrophoresis ( 80 v , 45 min ) in gels composed of 1% agarose in tbe buffer ( 40 mm tris , 20 mm boric acid , 1 mm edta , ph 8.3 ) . gels were visualized under uv illumination by using a gel image analysis system ( uvitec , cambridge , united kingdom ) and all images were archived . where a band was visualized at the correct expected size for opri , the specimen was considered positive for pseudomonas genus ; if a band was visualized at the correct expected size for oprl loci , the specimen was considered positive for p. aeruginosa species . following amplification , aliquots ( 10 l ) were removed from each reaction mixture and examined by means of electrophoresis ( 80 v , 45 min ) in gels composed of 1% agarose in tbe buffer ( 40 mm tris , 20 mm boric acid , 1 mm edta , ph 8.3 ) . gels were visualized under uv illumination by using a gel image analysis system ( uvitec , cambridge , united kingdom ) and all images were archived . where a band was visualized at the correct expected size for opri , the specimen was considered positive for pseudomonas genus ; if a band was visualized at the correct expected size for oprl loci , the specimen was considered positive for p. aeruginosa species . during the 5-month period , 491 bacterial samples recovered from burn wound infections were analyzed . the above - mentioned routine phenotype and biochemical tests enabled p. aeruginosa isolates to be recovered from 171 ( 34.8% ) patients . by contrast , molecular techniques detected only 133 ( 27.08% ) samples positive for p. aeruginosa species and 38 ( 7.73% ) samples positive for pseudomonas genus . pcr assays employing each primer pair yielded dna products of the predicted sizes ( fig . 1 and 2 ) . the opri and oprl amplicon genes were detected in all 133 p. aeruginosa isolates simultaneously . table ii shows the comparison of phenotype and biochemical testing with the molecular detection of p. aeruginosa in samples from burn wound infections . pcr amplification using pseudomonas genus - specific primers ( opr i gene ) , m : marker , line 1 : positive control , lines 2 , 3 , 4 : clinical isolates of pseudomonas genus , line 5 : negative control . pcr amplification using p. aeruginosa - specific primers ( opr l gene ) , m : marker , line 1 : positive control , lines 2 , 3 , 4 , 5 , 6 : clinical isolates of p. aeruginosa , line 7 : negative control . comparison of phenotype and biochemical tests with molecular detection of p. aeruginosa in samples from burn wound infections . pcr ( oprl)+ : strains confirmed as p. aeruginosa genus by pcr amplification of oprl . bacterial infections in burn wounds are common and are difficult to control . in recent decades , following the introduction of antibiotic therapy , p. aeruginosa has emerged as one of the most problematic gram - negative
bacteria in modern hospital settings . this organism is increasingly isolated as a nosocomial pathogen , and is responsible for high morbidity and mortality rates in burns patients , mechanically ventilated patients and those with cystic fibrosis [ 13 , 14 ] . infection by this bacterium is particularly problematic , since the organism is intrinsically resistant to many drug classes and is able to acquire resistance to all effective antimicrobial drugs . some studies carried out in iran have also indicated that infections caused by multi - drug resistant ( mdr ) p. aeruginosa are widespread in iranian hospitals [ 15 , 16 ] . it is therefore important to identify p. aeruginosa accurately and rapidly and to ascertain the susceptibility pattern of this organism ; this may avoid prolonged and sometimes unnecessary antibiotic treatments , which could select other antibiotic - resistant pathogens . these tests take a long time to perform and require extensive hands - on work by technicians , both for setup and for ongoing evaluation . various methods have been developed to identify p. aeruginosa species rapidly and accurately . according to our literature review , pcr has the potential to identify microbial species rapidly and precisely through the amplification of gene sequences unique to a particular organism . indeed , several pcr - based , dna probe methods have been developed to detect various pathogens in clinical samples and water and food samples . also in the case of p. aeruginosa , molecular methods have been reported to be superior to phenotypic methods in identifying p. aeruginosa species . the outer membrane proteins of p. aeruginosa play important roles in the interaction of the bacterium with the environment . in the present study , two pcr assays were performed individually for the molecular detection of two outer membrane lipoprotein genes , opri and oprl , in samples from burn wounds . according to the phenotype and biochemical tests carried out in this study , 171 ( 34.82% ) of 491 bacterial samples recovered however , while the results of our molecular tests on opri and oprl genes confirmed that many of these isolatesbelonged to the pseudomonas genus , only 133 ( 77.7% ) of them were confirmed as p. aeruginosa species . thus , there was nearly complete agreement between molecular and conventional phenotype and biochemical detection techniques with regard to the attribution of the pseudomonas genus , but not the p. aeruginosa species . this may account for the potential phenotype misidentification of p. aeruginosa which has been recently described . alternatively , discrepant results ( pcr- /biochemical+ ) may emerge in the case of true p. aeruginosa colonization , in that a false negative culture result may be due to sample overgrowth by other bacteria , or to the presence of non - cultivable organisms or auxotrophic mutations of the organism . indeed , it has been shown that opri is conserved among members of fluorescent pseudomonads [ 23 , 24 ] . de vos et al . designed a multiplex pcr assay based on opri and oprl genes for the molecular detection of p. aeruginosa , and showed that its specificity and sensitivity were 74 and 100% , respectively . also noted that all 268 of the p. aeruginosa strains that they detected contained the opri and oprl genes ( sensitivity = 100% , specificity = 80% ) . although our pcr and dna sequence analyses revealed some isolates that had been misidentified by phenotype testing , it must be said that our study was not designed to ascertain the frequency of misidentification of isolates from burn wound infections or to compare the relative accuracy of different phenotype identification systems . the isolates analyzed in this study constituted a biased set of atypical isolates that were difficult to identify . nevertheless , these isolates were well suited to providing a rigorous test of our pcr assays and represented strains for which molecular analysis would be expected to be most useful . our study also confirms that various non - aeruginosa pseudomonal species can occasionally be recovered from burn wound infection cultures . in this regard , genotype - based identification methods circumvent the problem of phenotype variation and provide more accurate species identification . it is important that primary diagnostic bacteriology methods have the ability to detect transient and early pseudomonas colonization in burns patients as soon as possible , so that : ( i ) aggressive antibiotic regimes may be reconsidered ; ( ii ) the patient can be managed optimally , with a view to avoiding early biofilm formation and chronic colonization with p. aeruginosa , and ( iii ) appropriate infection control precautions can be taken . | summarybackground.pseudomonas aeruginosa is an important lifethreatening nosocomial pathogen which plays a prominent role in wound infections in burns patients .
we designed this study to identify the isolates of p. aeruginosa recovered from burns patients at the genus and species levels by means of primers targeting opri and oprl genes.methods.during a 5-month period , wound samples were taken from burns patients and plated on macconkey agar .
all suspected colonies were screened for p. aeruginosa by means of a combination of phenotype tests .
specific primers for opri and oprl genes were then used for the molecular identification of colonies.results.during the 5-month period , bacterial isolates recovered from burn wound infections were analyzed .
phenotype identification tests identified 171 ( 34.8% ) p. aeruginosa isolates .
however , molecular techniques that used species - specific primers to detect the amplicon of the oprl gene confirmed the exact identification of p. aeruginosa in only 133 cases ; in the other isolates , the use of genus - specific primers detected the amplicon of the opri gene , which confirmed the identification of fluorescent pseudomonads.conclusions.this study indicates that molecular detection by means of an assay targeting the oprl gene is a useful technique for the rapid and precise detection of p. aeruginosa in burns patients .
in addition to phenotype testing , pcr detection should be carried out in order to promptly ascertain the best aggressive antibiotic therapy for p. aeruginosa infections , thereby significantly improving clinical outcomes . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
contrast - induced nephropathy ( cin ) is a common and serious complication of contrast media ( cm ) used in imaging studies and is the third leading cause of acute kidney injury in hospitalized patients . the incidence of cin is low in patients without cardiovascular risk factors and increases in patients with high cardiovascular risk . patients who develop cin after percutaneous coronary intervention are at an increased risk of both short- and long - term mortality [ 3 , 4 ] . therefore , it is important to identify high - risk patients to reduce the incidence of cin . chronic kidney disease ( ckd ) is one of the most important risk factors for cin . however , the mean reference renal artery diameter ( rvd ) was never tested prospectively to predict cin . in patients undergoing coronary angiography for ischemic heart disease , we detected that rvd and minimal luminal diameter ( mld ) are stronger determinants of ckd than the severity of renal artery stenosis . we hypothesized that rvd could be a predictor of cin , independently of major confounders . we took advantage of a very well phenotyped high cardiovascular risk population ( the ras - cad study [ 6 , 7 ] ; nct 01173666 ) to investigate these points . our aim was to look at the predictors of cin in patients undergoing coronary angiography for suspected coronary heart disease . the study conformed to the ethical guidelines of our institution , and informed consent was obtained from each participant . the primary outcome was the development of cin , defined as a relative increase in baseline serum creatinine ( scr ) > 25% within 1 week of contrast injection . patients were considered to be eligible for the study if they ( 1 ) were older than 18 years of age , ( 2 ) were referred for coronary angiography for ischemic heart disease , and ( 3 ) presented a stable baseline scr concentration . criteria for exclusion were ( 1 ) intravascular administration of an iodinated contrast medium ( cm ) within the previous month , ( 2 ) acute myocardial infarction , ( 3 ) pre - existing end - stage renal disease requiring dialysis , and ( 4 ) shock . the patients age , gender , and scr value were used to determine the estimated glomerular filtration rate ( gfr ) according to the formula developed and validated using data obtained from patients of the modification of diet in renal disease study : gfr ( ml / min/1.73 m ) = 186 ( scr ) age 0.742 ( if female ) 1.21 ( if black ) . patients with ef > 30% received 1 ml / kg / h of saline intravenously starting 24 h before and ending 24 h after the coronary angiography . patients with ef 30% received 0.5 ml / kg / h of saline intravenously 24 h before until 24 h after the coronary angiography . all patients with scr > 1.2 mg / dl received n - acetylcysteine ( 1,200 mg ) twice daily starting 24 h before and ending 24 h after the coronary angiography . scr was measured within 24 h prior to angiography and during the first week after the administration of cm . the iso - osmolar cm ( iocm ) iodixanol and the low - osmolar cm ( locm ) iobitridol , iopromide , iohexol , and iomeprol were used in this analysis . in this report , we look at the group effect of locm on cin development . to define the patency of the renal arteries , selective renal arteriography was performed at the end of the cardiac catheterization in the anterior - posterior and oblique projection with a hand injection of 48 ml of cm . after calibration with the outer diameter of the contrast - filled catheter was performed to provide a standard , the size of the renal arteries was measured . in stenotic vessels , rvd was measured before the stenosis or , when unavailable , after the post - stenotic dilatation . atherosclerotic renovascular disease ( arvd ) was defined as the presence of angiographically evident renal artery stenosis . the clinical characteristics of patients with and without cin were compared using analyses of variance for continuous variables and the test for categorical variables . multivariate logistic regression analysis was performed to identify independent predictors of cin and to estimate odds ratios ( or ) . risk factors that were significant in the univariate analysis ( p 0.20 ) were available for selection in the final model ; a backward stepwise method was used to select the best subset of independent predictors of cin to avoid overfitting of the data . three separate regression models were developed ( model 1 , 2 and 3 , respectively ) . the set of variables tested in model 1 included all the univariate predictors of cin ; the set of variables tested in model 2 included all the univariate predictors of cin tested in model 1 with the exception of rvd , and in model 3 , we forced the ckd into the model . the study conformed to the ethical guidelines of our institution , and informed consent was obtained from each participant . the primary outcome was the development of cin , defined as a relative increase in baseline serum creatinine ( scr ) > 25% within 1 week of contrast injection . patients were considered to be eligible for the study if they ( 1 ) were older than 18 years of age , ( 2 ) were referred for coronary angiography for ischemic heart disease , and ( 3 ) presented a stable baseline scr concentration . criteria for exclusion were ( 1 ) intravascular administration of an iodinated contrast medium ( cm ) within the previous month , ( 2 ) acute myocardial infarction , ( 3 ) pre - existing end - stage renal disease requiring dialysis , and ( 4 ) shock . the patients age , gender , and scr value were used to determine the estimated glomerular filtration rate ( gfr ) according to the formula developed and validated using data obtained from patients of the modification of diet in renal disease study : gfr ( ml / min/1.73 m ) = 186 ( scr ) age 0.742 ( if female ) 1.21 ( if black ) . patients with ef > 30% received 1 ml / kg / h of saline intravenously starting 24 h before and ending 24 h after the coronary angiography . patients with ef 30% received 0.5 ml / kg / h of saline intravenously 24 h before until 24 h after the coronary angiography . all patients with scr > 1.2 mg / dl received n - acetylcysteine ( 1,200 mg ) twice daily starting 24 h before and ending 24 h after the coronary angiography . scr was measured within 24 h prior to angiography and during the first week after the administration of cm . the iso - osmolar cm ( iocm ) iodixanol and the low - osmolar cm ( locm ) iobitridol , iopromide , iohexol , and iomeprol were used in this analysis . in this report to define the patency of the renal arteries , selective renal arteriography was performed at the end of the cardiac catheterization in the anterior - posterior and oblique projection with a hand injection of 48 ml of cm . after calibration with the outer diameter of the contrast - filled catheter was performed to provide a standard , the size of the renal arteries was measured . in stenotic vessels , rvd was measured before the stenosis or , when unavailable , after the post - stenotic dilatation . atherosclerotic renovascular disease ( arvd ) was defined as the presence of angiographically evident renal artery stenosis . the clinical characteristics of patients with and without cin were compared using analyses of variance for continuous variables and the test for categorical variables . multivariate logistic regression analysis was performed to identify independent predictors of cin and to estimate odds ratios ( or ) . risk factors that were significant in the univariate analysis ( p 0.20 ) were available for selection in the final model ; a backward stepwise method was used to select the best subset of independent predictors of cin to avoid overfitting of the data . three separate regression models were developed ( model 1 , 2 and 3 , respectively ) . the set of variables tested in model 1 included all the univariate predictors of cin ; the set of variables tested in model 2 included all the univariate predictors of cin tested in model 1 with the exception of rvd , and in model 3 , we forced the ckd into the model . overall , the mean age of the patients was 64.2 10.6 years , 74% were males , and baseline scr was 1.07 0.31 mg / dl . ckd was present in 57 patients ( 26% ) ; the incidence of cin was 22% . a total of 15 variables were associated ( p 0.20 ) with the development of cin and were selected to be studied in multivariate analysis . the selected correlates included demographics ( age and female gender ) , clinical characteristics ( pulse pressure , ckd , arvd , rvd , hypertension , and smoking ) , drug therapy ( -blockers , ace inhibitors / sartans , and nitrates ) , and several angiographic and/or procedural characteristics ( coronary artery disease , iocm administration , and cm dose ) . taking advantage of a logistic regression analysis for cin development , the influence of various correlates on the outcome was evaluated . cm dose , iocm , arvd , and rvd had the greatest effect on outcome and were identified as independent predictors of cin ( model 1 ; table 2 ) . when we developed a model excluding rvd from the set of variables studied by multivariate analysis , ckd was selected as an independent predictor of cin ( model 2 ; table 2 ) . therefore , we forced the variable ckd into the multivariate model ( model 3 ; table 2 ) . in the latter model , ckd did not reach a significant level ( or 1.49 , 95% ci 0.673.31 , p = 0.33 ) . the area under the roc curve for model 1 , 2 and 3 was 0.74 ( 95% ci 0.660.81 , p < 0.001 ) , 0.71 ( 95% ci 0.620.79 , p < 0.001 ) , and 0.74 ( 95% ci 0.660.81 , p < 0.001 ) , respectively . overall , the mean age of the patients was 64.2 10.6 years , 74% were males , and baseline scr was 1.07 0.31 mg / dl . ckd was present in 57 patients ( 26% ) ; the incidence of cin was 22% . a total of 15 variables were associated ( p 0.20 ) with the development of cin and were selected to be studied in multivariate analysis . the selected correlates included demographics ( age and female gender ) , clinical characteristics ( pulse pressure , ckd , arvd , rvd , hypertension , and smoking ) , drug therapy ( -blockers , ace inhibitors / sartans , and nitrates ) , and several angiographic and/or procedural characteristics ( coronary artery disease , iocm administration , and cm dose ) . taking advantage of a logistic regression analysis for cin development , the influence of various correlates on the outcome was evaluated . cm dose , iocm , arvd , and rvd had the greatest effect on outcome and were identified as independent predictors of cin ( model 1 ; table 2 ) . when we developed a model excluding rvd from the set of variables studied by multivariate analysis , ckd was selected as an independent predictor of cin ( model 2 ; table 2 ) . therefore , we forced the variable ckd into the multivariate model ( model 3 ; table 2 ) . in the latter model , ckd did not reach a significant level ( or 1.49 , 95% ci 0.673.31 , p = 0.33 ) . the area under the roc curve for model 1 , 2 and 3 was 0.74 ( 95% ci 0.660.81 , p < 0.001 ) , 0.71 ( 95% ci 0.620.79 , p < 0.001 ) , and 0.74 ( 95% ci 0.660.81 , p < 0.001 ) , respectively . for the first time , rvd , a factor associated with ckd , has been examined as a risk factor for cin . rvd was a stronger predictor of cin than ckd in patients at high cardiovascular risk undergoing coronary angiography for ischemic heart disease . for the first time , our data provide evidence that rvd is a better predictor of cin than ckd . in our study , prevalence of ckd was significantly higher in patients who developed cin than in those who did not . interestingly , when tested together with rvd , ckd was not selected as a predictor of cin . cm dose , iocm , arvd , and rvd were the most important factors associated with cin ( model 1 ; table 2 ) . due to its known impact on the development of cin , we tested ckd as a possible predictor of cin . when forced into the multivariate model ( model 3 ; table 2 ) , ckd does not reach a significant level ( p = 0.32 ) . therefore , we developed a model to test ckd as a determinant of cin excluding rvd from the set of variables studied with multivariate analysis ( model 2 ; table 2 ) . as expected , in model 2 , ckd was selected as a predictor of cin . therefore , our analysis confirmed that ckd is a predictor of cin but led to the conclusion that rvd is a better predictor of cin than ckd in patients at high cardiovascular risk . cin is classically attributed to vasoconstriction after iodinated cm injection [ 2 , 9 ] . our data confirm that the use of the iocm iodixanol is associated with a reduced risk of cin compared to locm . moreover , our results confirm that the risk of cin increases depending on cm dose , suggesting that coronary angiography in high - risk patients should be designed so that excessive volumes of cm are avoided ; thus , the iocm iodixanol should be preferred . , mld could represent the limiting factor for kidney blood flow , while in the absence of stenosis , rvd could be determined by blood flow through flow - mediated dilation . arterial diameter is dependent on several dynamic factors , among them the level of vascular tone on the short term and vascular remodeling on the long term . it is well known that long - term changes in local hemodynamic forces induce remodeling of arterial wall tissue , which is critical to vascular adaptation in the adult and to the progression of cardiovascular disease . sustained reductions in blood flow lead to flow - induced decreases in shear stress and inward arterial remodeling with a narrowing of lumen diameter . clinically significant arterial occlusive disease depressurizes the arterial tree downstream from the occlusion site and reduces the wall shear stress . , we detected that in patients with low rvd or low mld the or for ckd was markedly elevated . the association between rvd and ckd was confirmed in a fully adjusted model . in the present study , we extended the predictive power of rvd prospectively and reported that rvd is an independent predictor of renal function impairment after cm administration in high cardiovascular risk patients . to our knowledge , this is the first time that rvd was tested in a model for cin including arvd . we detected that cin incidence is increased in patients with arvd and that a reduction of mrac predisposes to a higher incidence of cin . the clinical implication of these data is the promising role of renal artery patency as a predictor of gfr reduction after cm administration . our cin prevention protocol , developed according to patients ef and baseline scr , consisted in hydration and n - acetylcysteine . several studies identified volume depletion as a risk factor for the development of cin ; as a result , fluid administration is frequently recommended [ 13 , 14 ] . however , patients with low ef are more prone to congestive cardiac failure than patients with normal ef . in these patients , n - acetylcysteine , a free radical scavenger with reported vasodilatory effects , has been reported in trials and in a recent meta - analysis to prevent cin [ 15 , 16 ] . a 3-fold reduction in the incidence of cin was reported in ckd patients undergoing elective coronary angiography following the administration of oral n - acetylcysteine ( 600 mg ) in addition to intravenous isotonic saline . recent studies suggest that higher doses of n - acetylcysteine may provide a significant protection against cin : treatment with 1,200 mg n - acetylcysteine given orally twice daily on the day before and the day of contrast exposure was associated with a lower risk of cin compared with 600 mg n - acetylcysteine . n - acetylcysteine side effects and drug interactions are very rare with continued use and are highly unlikely to result from the limited use for renal protection . therefore , we used n - acetylcysteine ( 1,200 mg twice daily ) in patients at higher risk of cin . the major limitation of this study was that the renal angiography was performed without 3d reconstruction of the renal artery . this may have led to an underestimation of the real prevalence and significance of arvd . another limitation of this study was that the locm iobitridol , iopromide , and iomeprol were analyzed together , assuming a comparable effect on outcome . in high cardiovascular risk patients undergoing coronary angiography for ischemic heart disease , rvd was a stronger independent predictor of cin than ckd . | introductionthe incidence of contrast - induced nephropathy ( cin ) increases in high cardiovascular risk patients . chronic kidney disease ( ckd )
is a known risk factor for cin development . in a previous report
, we demonstrated that the mean reference renal artery diameter ( rvd ) is an important determinant of ckd in patients undergoing coronary angiography for ischemic heart disease .
however , rvd was never tested as a predictor of cin .
aim : to look at the predictors of cin.methodsa total of 218 consecutive patients undergoing coronary and renal angiography were enrolled from the cohort of the ras - cad study ( nct 01173666 ) .
cin was defined as a relative increase in baseline serum creatinine 25% within 1 week of contrast administration.resultsthe incidence of cin was 22% . in a fully adjusted model , contrast medium dose ( 20 ml increase , or 1.12 , 95% ci 1.061.19 , p < 0.001 ) , iso - osmolar contrast media ( or 0.28 , 95% ci 0.090.99 , p < 0.05 ) , atherosclerotic renovascular disease ( or 2.69 , 95% ci 1.325.48 , p < 0.05 ) , and rvd ( 1 mm/1.73 m2 increase , or 0.59 , 95% ci 0.410.86 , p < 0.05 ) had the greatest effect on outcome and were identified as independent predictors of cin .
ckd was selected as a predictor of cin only in a model without rvd.conclusionsin patients undergoing coronary angiography for ischemic heart disease , rvd is a stronger predictor of cin than ckd . |
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in recent years , oral health in developed countries has improved , with the majority of people keeping more sound teeth for longer duration [ 17 ] . in particular , considerable interest has been directed at detecting caries at early stages , with the development of the international caries detection and assessment system ( icdas ) [ 8 , 9 ] and quantitative light - induced fluorescence ( qlf ) method [ 10 , 11 ] . in current daily dental practice and oral health care programs , the inhibition of initial tooth enamel demineralization and the promotion of remineralization are the most important targets [ 1214 ] . surface - reaction type prereacted glass - ionomer ( s - prg ) filler [ 15 , 16 ] has been reported to have biological efficacy in reducing dental plaque formation [ 17 , 18 ] , inhibition of dentin demineralization , fluoride release and recharge potential , and prevention of demineralization in surrounding orthodontic brackets . these efficacies might be due to the ability of s - prg filler to release various ion species ( fluoride , strontium , aluminum , sodium , etc . ) as well as its capacity as an acid buffer . s - prg filler can therefore be found in various dental products , such as composite resin , root canal sealer , orthodontic resin bonding systems , and denture base resin [ 19 , 2328 ] . we developed a novel ion - releasing tooth - coating material containing s - prg filler , which was developed for surface - coating sound teeth and which consists of a base liquid containing s - prg filler and an active liquid containing carboxylic and phosphonic adhesive monomers . in clinical practice , the dentist or dental hygienist would mix these two liquids , apply the mixture to the tooth surface with a brush , and polymerize it via light irradiation . to our knowledge , the inhibitory effect of an ion - releasing tooth - coating material that contains s - prg on the demineralization of tooth enamel has not been reported . here , in order to clear this point , we investigated various coating material applications on bovine tooth enamel using the quantitative light - induced fluorescence ( qlf ) method [ 2931 ] to longitudinally monitor the demineralization process and an electron probe microanalyzer ( epma ) for mineral mapping of demineralized enamel . extracted bovine incisor teeth were used as a source of enamel . using a core drill to excise bovine enamel specimens ( 5 mm in diameter ) , we obtained specimens with an enamel layer thickness of approximately 1.0 mm and a dentine layer thickness of approximately 2.0 mm . enamel surfaces were abraded using carbide paper of 600 to 1,500 grit and were then polished with gamma alumina polishing paste ( grain diameter , 0.05 m ) . ten samples from each enamel specimen group were painted with an acid - resistant nonfluorescent varnish ( control group ) , s - prg filler - containing tooth - coating material ( prg barrier coat ; shofu inc . , kyoto , japan ) , or non - s - prg filler - containing tooth - coating material ( shofu inc . ) , leaving an enamel window of approximately 2 2 mm ( coated area was approximately 15 mm in size and approximately 10 m in thickness ) . in separate bottles , specimens were then exposed to 14 ml of a demineralization solution for 96 hours at 37.0c . the demineralization solution contained 0.1 m lactic acid ( kishida chemical , osaka , japan ) and 0.2 g / l carboxymethyl cellulose sodium salt ( kishida chemical ) , was 50% saturated with hydroxyapatite ( nihon chemical , tokyo , japan ) , and was adjusted to a ph of 5.0 . images of specimens containing white spot lesions were acquired with a qlf - clin system ( inspektor research systems bv , amsterdam , the netherlands ) equipped with qlf.exe evaluation software ( version 2.00h ) to digitize and quantify the images . specimens were illuminated with violet - blue light ( = 390430 nm ) . a ccd camera with a yellow high - pass filter ( = 520 nm ) was fixed with a stand in order to provide optimal illumination of the specimen surface . quantitative results were obtained for the following parameters : mean fluorescence loss over the lesion ( % ) , area of the lesion ( mm ) , and total fluorescence loss over the lesion ( q in % mm ) . q is comparable to the total mineral loss from lesions , as measured via longitudinal microradiography . the three different coating materials remained on the tooth enamel surface throughout the 96 h test period . all analyses of digital images were conducted in the 2 2 mm area in the center of the enamel specimens . after 96 h , demineralized solution was subjected to analysis of ion concentration ( b , al , ca , p , si , and sr ) using an icp emission spectrometer ( icps-8000 ; shimadzu , kyoto , japan ) . analysis of ph and fluoride ion concentration was performed with an ion electrode ( ph : model 9102bnwp and f : model 9609bn ; orion research inc . , tisab iii ( orion research ) was added to the solution in order to obtain a constant background ionic strength for fluoride . demineralized enamel specimens were vertically sectioned with a low - speed diamond cutter and mounted on aluminum stubs . sectioned specimens were sputter - coated with a 300 gold layer using an ion coater ( ic-50 ; shimadzu ) and analyzed using a wavelength - dispersive x - ray spectroscopy electron probe microanalyzer with an image observation function ( sem - epma , epma1601 ; shimadzu ) . for morphological observation , the subsurface lesions of enamel specimens were analyzed under sem - epma at an accelerating voltage of 15 kv . chemical component bulk analysis and element mapping were carried out using sem - epma for the subsurface area ( 6070 m away from the interface ) . distributions of ca and f in the enamel were measured using the element line scan from the interface to the direction of inner enamel with an approximate range of 100 m . statistical calculations , analysis of variance ( anova ) followed by tukey 's all - pairwise - comparison test , were performed with software package spss version 11.0j for windows xp ( spss inc . , chicago , il , usa ) . the mean q for the three groups ( control group , non - s - prg filler group , and s - prg filler group ) , immersed in demineralized solution for 96 hours , is shown in figure 1 . mean q at 96 h was 148.1 38.9%mm for the control group , 130.0 21.2%mm for the non - s - prg filler group , and 0.8 0.4%mm for the s - prg filler group . the differences between the s - prg filler and the other two groups were statistically significant ( p < 0.001 ) . in the s - prg group , no signs of demineralization were observed in qlf digital images ( figure 2 ) . the ph values of the demineralized solution did not change after 96 h of immersion . mean ion concentrations in the demineralized solution , as assessed using icp emission spectrometry , are shown in table 1 . while no significant differences were noted between the control group and the non - s - prg filler group , five ions ( al , b , si , sr , and f ) were present at higher concentrations in the s - prg filler group than in the other two groups , with the concentration of f ion being particularly elevated . according to the qlf image , the ca and p of tooth enamel were dissolved in the demineralization solution in both the control and non - s - prg filler groups . concentrations of ca and p in solution may have therefore increased . however , the concentration of the ca and p in the demineralizing solution in both the control and non - s - prg filler groups showed the approximate value between s - prg filler group ( table 1 ) . plane - scanning analysis via epma is shown in figures 3(a ) and 3(b ) . as the images for the control and non - s - prg filler group were nearly identical , only the control and s - prg filler groups are shown . in both groups , a scarcity of b , si , and sr ions was observed in enamel specimens . results of line - scanning analysis for ca and f are shown in figures 4(a ) and 4(b ) . in the control group , concentrations of ca ions were lower on the subsurface of enamel specimens than sound enamel , and f ion concentrations were relatively low as well . in the s - prg group , however , no marked changes were noted in ca ion distribution , and f ions peaked on the surface of enamel specimens . mukai et al . reported that an all - in - one adhesive system containing s - prg filler was able to form protective layers and that it protected dentin against further demineralization in the case of secondary marginal dental caries . similarly reported that denture base resins containing s - prg filler have relatively good fluoride recharge and release capacities , which assist in preventing caries . most studies have investigated the preventative effects of dentin demineralization after restorative treatment . in our in vitro study , we investigated the inhibitory effects of a tooth - coating material that contained s - prg filler on the demineralization of bovine tooth enamel . it should be noted that bovine tooth enamel is demineralized more rapidly than human tooth enamel . however , many in vitro studies use bovine tooth [ 3840 ] , and it is well known that the human enamel has individual difference . we did not use human tooth enamel specimens given the difficulty in acquiring such specimens . based on qlf measurement , tooth - coating material containing s - prg filler protected the enamel surface against demineralization . in a previous study , apf - gel - treated enamel specimens also showed almost no demineralization . the s - prg filler - containing coating material blocked demineralization on the coated areas and inhibited demineralization of the surrounding areas . featherstone et al . reported that the continual presence of low concentrations of fluoride ( 0.10.5 ppm ) in whole saliva is critical in the inhibition of demineralization and promotion of remineralization . in the present study , the fluoride concentration in demineralized solution was 0.34 ppm , suggesting that the tooth - coating material containing s - prg filler might release ions and inhibit enamel demineralization . al , b , si , sr , and f ion concentrations in the demineralized solution of s - prg filler group were higher than in the other two groups . while ph values of the demineralizing solution did not change after 96 h of immersion , the elevated ion concentrations indicate that the demineralizing solution of s - prg filler group may be induced to behave differently during the demineralization process . of note , our in vitro study employed a static model to induce demineralization , which may have resulted in an overestimation of the effect of the coating due to a build - up of released ions in the demineralizing solution . the results of this study might not reflect the in vivo conditions in which there is constant salivary clearance . we also investigated the influence of ions released from s - prg filler using epma as the gold coating was anticipated to have an effect on quantitative analysis via scattering of electrons due to its high atomic number and density . line - scanning analysis showed that fluoride saturated the sound teeth surface in s - prg filler - treated specimens . high - dosage apf - gel application on the enamel surface produces a fluoride calcium layer on the tooth surface . rlla and gaard reported that reductions in ph from 7 to 5 or 4 led to increased solubility of calcium fluoride on the enamel surface into medium solution . this finding suggests that calcium fluoride on the enamel surface acts as a ph - controlled reservoir of fluoride . figure 4(b ) shows that in response to the s - prg coating material , f ions aggregated on the surface of the enamel specimens . s - prg filler - containing tooth - coating material also functions as a calcium fluoride - like substance on the tooth surface and may release low concentrations of fluoride ions around the coating material surface . most restorative treatments , such as resin fillings , lead to the growth of cariogenic bacteria . reported that the adherence of radiolabeled bacteria to the saliva - coated resin surface was significantly lower in s - prg filler - containing composite resin than in other filling materials . also reported that s - prg eluate suppresses streptococcal adherence and inhibits the protease and coaggregation activities of porphyromonas gingivalis . they suggested that the s - prg filler - containing material reduces dental plaque formation and bacterial adherence . in the present study , icp emission spectrometry showed that sr , b , and al were present in the demineralized solution . however , determining the mechanism of this interference will require further investigation . despite reports that the frequent use of low concentrations of fluoride agents , such as fluoride - containing toothpaste , is the most beneficial method of preventing demineralization , . found no evidence for the effects of fluoride agents on controlling caries in high - risk individuals . the ion - releasing potential of s - prg filler - containing tooth - coating material may contribute to the prevention of tooth enamel demineralization . | we compared the effect of a novel ion - releasing tooth - coating material that contained s - prg ( surface - reaction type prereacted glass - ionomer ) filler to that of non - s - prg filler and nail varnish on the demineralization of bovine enamel subsurface lesions .
the demineralization process of bovine enamel was examined using quantitative light - induced fluorescence ( qlf ) and electron probe microanalyzer ( epma ) measurement .
ion concentrations in demineralizing solution were measured using inductively coupled plasma atomic ( icp ) emission spectrometry and an ion electrode .
the nail varnish group and the non - s - prg filler group showed linear demineralization .
although the nail varnish group and the non - s - prg filler group showed linear demineralization , the s - prg filler group did not .
further , plane - scanning by epma analysis in the s - prg filler group showed no changes in ca ion distribution , and f ions showed peak levels on the surface of enamel specimens .
most ions in the demineralizing solution were present at higher concentrations in the s - prg filler group than in the other two groups . in conclusion , only the s - prg filler - containing tooth - coating material released ions and inhibited demineralization around the coating . |
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a study of the relationship between the pathologic stages of prostate cancer and the gleason score sum ( gss ) is important for refining prostate cancer diagnosis and treatment . among the antiapoptotic factors , bcl-2 , clusterin , heat shock protein ( hsp ) 27 , cellular - flice inhibitory protein ( c - flip ) , and glucose - related protein 78 ( grp78 ) hsp assists the folding , activation , trafficking , and transcriptional activities of most steroid receptors , including the androgen receptor ( ar ) . it also acts on cytochrome c or procaspase 3 to inhibit apoptosome formation and apoptosis . one study demonstrated that hsp expression inhibits apoptosis , and other studies have found that hsp27 is related to the acquisition of hormone resistance in the lymph node of prostate cancer cell line , a type of prostate cancer . c - flip , an important protein in prostate cancer , inhibits apoptosis mediated by fas within death - inducing signal complexes by inhibiting caspase-8 activity . c - flip is not expressed in normal bladder cells , and higher c - flip expression is associated with a more advanced disease stage . in addition , c - flip expression increases in stomach and gallbladder cancers . dual silencing decreases proliferation and enhances apoptosis of cancer cells in the laboratory and is technically feasible . because hsp27 , one of the small hsps , inhibits key effectors of the apoptotic pathway at the pre- and postmitochondria levels and because c - flip is regarded as a new therapeutic target for relevant cancers , we investigated these two factors to quantify their relationship with gss and pathologic state . in prostate cancer , hsp27 is correlated with pathologic stage , gleason score , lymph node metastasis , shorter the time until biochemical recurrence ( bcr ) , and poor clinical outcome . previous studies report separate correlations for hsp27 expression and c - flip expression to clinical features in cancers , including prostate cancer . however , there is little knowledge about the impact of simultaneous expression of these two proteins with the levels of correlation . according to a recent study by kim et al . , the ar is correlated with increased expression of cancerrelated survival markers , such as hsp27 , grp78 , clusterin , and c - flip , in prostate cancer . c - flip directly participates in c - jun n - terminal protein kinase ( jnk ) activation , a key activity that regulates cellular events , through mitogen - activated protein kinase kinase 7 ( mkk7 ) in a tumor necrosis factor--dependent manner . the interaction of c - flipl ( the long form of c - flip ) with mkk7 might selectively suppress jnk activation . hsp27 increases tumor activation though upstream serial signaling with mkk4 ( mek4 ) , which could result in hsp27-mediated increases in invasion with the phosphorylation of hsp27 . considering the emerging evidence about the functional role of mkk-4 , mkk-6 , and mkk-7 in prostate cancer and the fact that we could not assay the direct relationship of mkk with prostate cancer , we designed a pioneer experiment with hsp27 and c - flip that could hint at its role . moreover , there is little knowledge about how the simultaneous expression of hsp27 and c - flip relates to clinical variables . we used an immunohistochemical method to identify correlations between hsp27 and c - flip expression with gss and pathologic stage by analyzing tissues obtained after prostate cancer surgery . this study included 163 patients diagnosed with prostate cancer and benign prostate hyperplasia ( bph ) . between may 2004 and april 2010 , 83 patients had a radical prostatectomy ( n=81 ) and transurethral resection ( n=2 ) who could not under gone prostatectomy . as a control , we used samples from 80 patients who had undergone transurethral resection of the prostate to alleviate urinary symptoms from bph . patient age , tumor stage , tumor gss , presence or absence of lymph node metastasis , and presence or absence of distant organ metastasis were investigated . after the tissues were obtained from surgery or biopsy , they were fixed in 10% neutral buffer formalin and serial sections of 5 m in thickness were embedded in paraffin . these serial sections were attached to slides , dried , and then processed through deparaffin and hydrous procedures . the paraffin - embedded samples were then treated with 3% hydrogen peroxide for 10 minutes to prevent peroxidase activation . these were heated for 10 minutes in a pressure apparatus containing 10 mm citrate buffer ( ph 6.0 ) . after cleaning three times for 2 minutes with phosphate - buffered saline ( pbs ) to prevent abnormal binding of the background , they were pretreated with blocking solution for 10 minutes . a mouse monoclonal antibody to hsp27 was purchased from leica microsystems ( ncl - hsp27 leica microsystems newcastle ltd . , newcastle upon tyne , uk ) . for c - flip immunohistochemistry , polyclonal anti - c - flip antibody ( 1:50 dilution ; enzo life sciences , philadelphia , pa , usa ) was used . after adding the primary antibodies to hsp27 and c - flip , samples were treated for 1 hour at room temperature and then cleaned with tris buffer . samples were treated with biotinylated secondary antibody and streptavidin - hrp conjugate ( dako lsab kit , k0675 , carpinteria , ca , usa ) for 10 minutes each and then cleaned with tris buffer . louis , mo , usa ) color fixing and mayer 's hematoxylin contra staining were performed . slides were stained on an automated immunostainer ( dakocytomation , glostrup , denmark ) . as a negative control , the primary antibody was omitted and replaced with pbs . the immunohistochemical staining results of hsp27 and c - flip demonstrated the expression pattern in tumor cell cytosol . if a local reaction abnormality took place , it was judged as positive , and positive cell numbers were counted under 100 magnification microscopy . and after the procedure was repeated three times to compute an average , the results were analyzed by counting positive expression levels . positive cell numbers below 5% were denoted as + 1 , between 5%-50% as + 2 , and above 50% as + 3 ; this scale is referred to as the reaction score . the chi - square test was used to analyze hsp27- and c - flip - positive reaction rates , gss , and pathologic stage in prostate cancer cells . analysis of variance was used for correlation analysis of the average reaction scores of hsp27 and c - flip , gss , and pathologic stages . chi - square test was used to analyze the correlation between hsp27 and c - flip expression , gss , and pathologic stage . logistic regression analyses were performed to analyze the association of each clinical variable with expression of hsp27 and c - flip . the statistical software program pasw statistics ver . 18.0 ( spss inc . , this study included 163 patients diagnosed with prostate cancer and benign prostate hyperplasia ( bph ) . between may 2004 and april 2010 , 83 patients had a radical prostatectomy ( n=81 ) and transurethral resection ( n=2 ) who could not under gone prostatectomy . as a control , we used samples from 80 patients who had undergone transurethral resection of the prostate to alleviate urinary symptoms from bph . patient age , tumor stage , tumor gss , presence or absence of lymph node metastasis , and presence or absence of distant organ metastasis were investigated . after the tissues were obtained from surgery or biopsy , they were fixed in 10% neutral buffer formalin and serial sections of 5 m in thickness were embedded in paraffin . these serial sections were attached to slides , dried , and then processed through deparaffin and hydrous procedures . the paraffin - embedded samples were then treated with 3% hydrogen peroxide for 10 minutes to prevent peroxidase activation . these were heated for 10 minutes in a pressure apparatus containing 10 mm citrate buffer ( ph 6.0 ) . after cleaning three times for 2 minutes with phosphate - buffered saline ( pbs ) to prevent abnormal binding of the background , they were pretreated with blocking solution for 10 minutes . a mouse monoclonal antibody to hsp27 was purchased from leica microsystems ( ncl - hsp27 leica microsystems newcastle ltd . , newcastle upon tyne , uk ) . for c - flip immunohistochemistry , polyclonal anti - c - flip antibody ( 1:50 dilution ; enzo life sciences , philadelphia , pa , usa ) was used . after adding the primary antibodies to hsp27 and c - flip , samples were treated for 1 hour at room temperature and then cleaned with tris buffer . samples were treated with biotinylated secondary antibody and streptavidin - hrp conjugate ( dako lsab kit , k0675 , carpinteria , ca , usa ) for 10 minutes each and then cleaned with tris buffer . louis , mo , usa ) color fixing and mayer 's hematoxylin contra staining were performed . slides were stained on an automated immunostainer ( dakocytomation , glostrup , denmark ) . as a negative control , the primary antibody was omitted and replaced with pbs . the immunohistochemical staining results of hsp27 and c - flip demonstrated the expression pattern in tumor cell cytosol . if a local reaction abnormality took place , it was judged as positive , and positive cell numbers were counted under 100 magnification microscopy . and after the procedure was repeated three times to compute an average , the results were analyzed by counting positive expression levels . positive cell numbers below 5% were denoted as + 1 , between 5%-50% as + 2 , and above 50% as + 3 ; this scale is referred to as the reaction score . the chi - square test was used to analyze hsp27- and c - flip - positive reaction rates , gss , and pathologic stage in prostate cancer cells . analysis of variance was used for correlation analysis of the average reaction scores of hsp27 and c - flip , gss , and pathologic stages . chi - square test was used to analyze the correlation between hsp27 and c - flip expression , gss , and pathologic stage . logistic regression analyses were performed to analyze the association of each clinical variable with expression of hsp27 and c - flip . the average patient age was 69.587.52 , body mass index ( bmi ) was 26.46.54 kg / m , and prostate volume was 42.237.54 ml . two cases ( 2.41% ) were stage t1 , 44 cases ( 53.01% ) were t2 , 26 cases ( 31.33% ) were t3 , and 11 cases ( 13.25% ) were t4 . for the gss , three cases ( 3.61% ) had a gss of 4 points , four cases ( 4.82% ) had a gss of 5 points , eight cases ( 9.64% ) had a gss of 6 points , 25 cases ( 30.12% ) had a gss of 7 points , 16 cases ( 19.28% ) had a gss of 8 points , and 24 cases ( 28.92% ) had a gss of 9 points . lymph node metastasis was observed in nine cases ( 10.84% ) , and remote metastasis was observed in two cases ( 2.41% ) ( table 1 ) . the group of bph had 67.486.72 in age , bmi was 27.27.01 kg / m , and prostate volume was 51.478.59 ml . there was no hsp27 positive reaction in three cases with a gss of 4 points , in one case with a gss of five points ( 25% ) , in one case with a gss of six points ( 12.5% ) , in 15 cases with a gss of seven points ( 60% ) , and in all 16 cases with a gss of eight points . however , 23 of 24 cases with a gss of nine points ( 95.83% ) and all three cases with a gss of 10 points had hsp27-positive reactions ( p<0.001 ) . positive reactions were observed in one of two stage t1 cases ( 50% ) , 25 out of 44 t2 cases ( 56.82% ) , 23 out of 26 t3 cases ( 88.46% ) , and 10 out of 11 t4 cases ( 13.25% ) ( p=0.001 ) ( fig . a c - flip - positive reaction was not observed in three cases with a gss of 4 points and in four cases with a gss of 5 points . it was observed in three out of eight cases ( 37.5% ) with a gss of 6 points , 22 out of 25 cases ( 88% ) with a gss of 7 points , 15 out of 16 cases ( 93.75% ) with a gss of 8 points , all 24 cases with a gss of 9 points , and all three cases with a gss of 10 points ( p<0.001 ) ( fig . 2 ) . overall , reactions were observed in one of two t1 cases ( 50% ) , 29 out of 44 t2 cases ( 65.91% ) , all 26 t3 cases , and all 11 t4 cases ( p<0.001 ) ( table 2 ) . the averages in each hsp27 reaction range were 0.25 , 0.13 , 0.82 , 2.13 , 2.75 , and 3 with a gss of 4 , 5 , 6 , 7 , 8 , 9 , and 10 points , respectively ( p<0.001 ) , and 1 , 1.01 , 2.15 , and 2.64 in stages t1 , t2 , t3 , and t4 ( p<0.001 ) , respectively ( table 3 ) . in a multiple group post hoc comparison , the average hsp27 reaction score increased in proportion to gss between 4 and 10 points . hsp27 was expressed in all nine cases with lymph node metastasis and 26 of 74 cases ( 53% ) without lymph node metastasis . there was a statistically significant relationship between lymph node metastasis and the expression pattern of hsp27 ( p<0.001 ) . there was no correlation between the proportion of hsp27 positive reactions or reaction score to age . in logistic regression , hsp 27 expression was related with prostate volume , psa level , biopsy gleason score and percentage of maximum core involvement ( table 4 ) . the averages in each c - flip reaction range were 0 , 0 , 0.63 , 1.08 , 1.44 , 2.33 , and 3 with a gss of 4 , 5 , 6 , 7 , 8 , 9 , and 10 points ( p<0.001 ) , respectively , and 1 , 0.95 , 1.92 , and 2.36 with stages t1 , t2 , t3 , and t4 ( p<0.001 ) , respectively ( table 3 ) . in multiple group post hoc comparison , the c - flip average reaction score increased in proportion to gss between a gss of 4 and 10 points . c - flip was expressed in all nine cases with lymph node metastasis and 26 of 74 cases ( 53% ) without lymph node metastasis . there was a statistically significant relationship between lymph node metastasis and c - flip expression pattern ( p<0.001 ) . c - flip expression was not observed in the bph tissue selected as the control group . there was no significant relationship between the proportion of c - flip positive reactions or reaction score to age . in logistic regression , c - flip expression was also related with prostate volume , psa level , biopsy gleason score and percentage of maximum core involvement ( table 5 ) . the association between hsp27 , c - flip , and gss was shown significant difference ( p<0.001 ) . the association between hsp27 , c - flip , and pathologic stage was also observed statistically significant difference ( p=0.003 ) ( tables 6 , 7 ) . its production is initiated by various stressful stimuli including heat , ultraviolet irradiation , heavy metals , infection , inflammation , hypoxia , tissue damage , and tumors . hsp27 reacts to the cytochrome c / apaf-1/datp complex in the procaspase 9 pathway and partially inhibits connectivity between fas , ask1 , and daxx proteins . in addition , hsp27 inhibits cytochrome c secretion in thread granules of the procaspase 9 pathway . hsp27 also inhibits caspase 3 activation and apoptosome formation by reacting to cytochrome c and procaspase 3 . in this study , hsp initiates danger signals by reacting to antigenic chaperone peptides in tumor cells and by inducing immune reactions that protect tumor cells , as evidenced by formation of hsp - peptide complexes from isolated dead tumor cells by antigen - presenting cells . additionally , hsp aids in cytotoxic t - lymphocyte recognition by improving the ability to treat and transmit antigens in tumor cells . based on this , it is understood that hsp27 is expressed in prostate cancer cell lines . in this study , hsp27 expression differed depending on gss and pathologic stage . miyake et al . reported that while hsp27 , hsp70 , and hsp90 were present in prostate cancer cells , only expression of hsp27 was significantly correlated with gleason score and pathologic stage . similar to previous studies , this study also demonstrated the relationship between hsp27 with gss and pathologic stage . rocchi et al . reported that the increase in hsp27 protected cells from hormone - refractory prostate cancer development after hormone treatment . reported that viability significantly decreased in prostate cancer cell lines after 72 hours of irradiation and had decreased hsp27 expression compared to that of other prostate cancer cell lines . in the present study , hsp27 was expressed in prostate cancer . day et al . observed c - flip knockdown in michigan cancer foundation-7 breast cancer cells , reporting that the knockdown of c - flip with sirna transfection triggered spontaneous apoptosis and induced fas - associated death domain - mediated and d related-5-mediated apoptosis . they assessed c - flipl , not c - flips , as having a key role in preventing spontaneous death signaling and suggested c - flipl as a therapeutic target for breast cancer . similarly , on colorectal cancer cells , longley et al . reported that sirna targeting c - flipl synergistically enhanced chemotherapy - induced apoptosis . survival markers including hsp27 and c - flip could be important for clarifying the relationship of jnk activation with apoptosis in prostate cancer . in our study , hsp27 expression was correlated with gss and prostate cancer stage . more advanced pathologic stages corresponded to a higher incidence of c - flip presence and expression . c - flip is critical to prostate cancer progression because it inhibits apoptosis mediated by caspase-8 . in this study , c - flip was correlated with gss and pathologic stage , indicating that the level of c - flip expression plays a major role in cancer progress . activated cytoplasm is required for tumor cell survival , and c - flip plays an important role in inhibiting apoptosis . in prostate cell cytoplasm , the level of c - flip expression is correlated to proliferation and infiltration in vitro . c - flip contributes to the resistance of metastatic prostate cancer cells to docetaxel , tumor necrosis factor - related apoptosis - inducing ligand , and oxaliplatin . this indicates that c - flip has potential value as a resistance marker during medical treatment for metastatic prostate cancer . overexpression of c - flip is one feature of prostate cancer cells . in nude mice , c - flip causes androgen - independent growth of prostate cancer cells . in this study , c - flip was expressed in prostate cellular tissues . higher levels of c - flip expression were associated with higher gss and more advanced pathologic stage . in both hsp27 and c - flip , higher expression scores were associated with higher gss and more advanced pathologic stage . however , expression of the two proteins was not uniform in the same cell lines . compared to c - flip , hsp27 had a higher correlation with pathologic stage and gss . patients who could not undergo radical prostatectomy , including those with hormone - refractory prostate cancer , had higher hsp27 and c - flip expression because these patients underwent transurethral prostatectomy to alleviate urinary symptoms . these results predict that this group had a higher gss and more advanced pathologic stage than other cases . in addition to hsp27 and c - flip , there are many proteins related to prostate cancer , including ki-67 , p53 , ar , matrix metalloproteinase ( mmp)-2 , mmp-9 , vascular endothelial growth factor , aurora - a , bcl-2 , clusterin , hsp70 , and hsp90 . studies have investigated src tyrosine kinase and its relationship to hormone - refractory prostate cancer progression and bone metastasis , as well as signal transducer and activator of transcription 5a kinase and its relationship to hormone - refractory progression . two factors were selected as the most appropriate factors , considering our focus on the correlation between gss , pathologic stage , and expressed proteins . unlike other studies , this study investigated two factors at the same time to evaluate the correlations of hsp27 and c - flip expression to gss and pathologic stage in prostate cancer . additionally , this study investigated the correlation between the expression levels of the two proteins . however , there were limitations in verifying the correlation of hsp27 and c - flip expression to pathologic factors due to the small number of subjects . moreover , we could not verify the direct relationship between the mkk series and prostate cancer activity . further study is needed to have follow up and results and define the correlation of previously reported factors and their associations with gss and pathologic stage . therefore , an additional large - scale study is necessary to investigate the correlation between hsp27 , c - flip , and prostate cancer . higher gss and more advanced pathologic stage were associated with higher levels of hsp27 and c - flip expression . the expression of hsp27 and c - flip was correlated to gss and pathologic stage in prostate cancer . hsp27 and c - flip expression were shown significant related with gleason sum and pathologic stage at the same time . we recommend a future study about the role of hsp27 and c - flip in the initiation , development , and immune control of hormone - refractory prostate cancer . | purposeheat shock protein ( hsp ) 27 protects the cell by controlling apoptosis and immune reactions , and c - flip ( cellular - flice inhibitory protein ) inhibits apoptosis by inhibiting caspase-8 activity .
we investigated the relationship of hsp27 and c - flip expression to prostate - specific antigen , gleason score sum ( gss ) , and pathologic stage.materials and methodssamples from 163 patients between may 2004 and april 2010 were analyzed : 83 from patients that had underwent a radical prostatectomy , and 80 from those that underwent transurethral resection of the prostate to alleviate urinary symptoms from benign prostate hyperplasia .
c - flip and hsp27 expression were observed by immunohistochemistry staining .
samples with less than 5% expression - positive cells were scored as 1 , with 5%-50% were scored as 2 , and with more than 50% were scored as 3 .
local reactions were identified as 0.5 and evaluated.resultsboth the presence of hsp27 within the tumor and the number of cancer cells positive for hsp27 were significantly correlated to gss and pathologic stage ( p<0.001 , p=0.001 , p<0.001 , p<0.001 ) .
the same was true for c - flip expression ( p<0.001 ) .
gss was more highly correlated to hsp27 expression than to c - flip expression ( r=0.814 for hsp27 , r=0.776 for c - flip ) , as was pathologic stage ( r=0.592 for hsp27 , r=0.554 for c - flip).conclusionsin prostate cancer , higher gss and a more advanced pathologic stage were associated with a higher likelihood of having a hsp27-positive tumor and more hsp27-positive tumor cells .
hsp27 expression was correlated with gss and prostate cancer stage .
a more advanced pathologic stage corresponded to a higher likelihood of having a c - flip - positive tumor and more c - flip - positive tumor cells .
hsp27 expression had a higher correlation with prostate cancer stage and gss than c - flip expression did . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the greater trochanter ( gt ) is an important site for biomedical research1 , 2 . it
is also an important structure , giving the attachment architecture for tendons , enhancing
complex movement3 . furthermore , it
demonstrates topographical anatomy4 and
the location of the gt is used to calculate appropriate cane length5 . in the clinic , the location of the gt is commonly used by
manual therapists for diagnoses and treatment6 . as a bony landmark also ,
recognizing the migration of the gt from the original location can facilitate identification
of abnormal movement such as femoral anterior glide syndrome by manual therapists8 . despite the importance of gt within manual therapy , greater study of its measurement is
required . many previous studies of lower limbs have used computed tomography9 , 10 ,
magnetic resonance imaging11 or
x - rays12 . however , these methods have
several disadvantages : these are not cost and time - efficient and obtaining the data requires
special skills1 ; the data are obtained in
the supine position and are not appropriate for motion analysis13 ; computed tomography increases the risk of exposure to
radiation14 . three - dimensional motion capture systems do not have these disadvantages and are commonly
used to analyze movement . although the gt marker - based measurement ( gt - m ) system can be used
to determine the location of the gt by placing a reflective marker thereon15,16,17,18 ,
its location according to movement using this method has not been adequately investigated . also , few studies of other methods of examining gt using three - dimensional motion capture
systems have been conducted . thus , the purpose of this study was : ( 1 ) to develop a new measurement system that does not
use gt marker - based measurement ( no gt - m ) to calculate the location of the gt from the
coordinates of the other markers , ( 2 ) to compare the gt - m and no gt - m measurements . we
hypothesized that the two methods would produce significantly different results , with those
of no gt - m being more accurate . participants were excluded
if they had neurological or musculoskeletal problems within the past 12 months ; had a
history of surgery of the spine , pelvis or lower limbs ; performed hip extension less than
15 or had discomfort during trials . their age was 21.65 1.81 years ( mean , standard
deviation ) , height 168.80 7.38 cm and weight 60.95 7.69 kg . prior to the study , all
participants provided written informed consent and the study procedure was approved by the
inje university faculty of healthy sciences human ethics committee . participants were in the prone position with their legs straight and their heads positioned
on the midline . they were asked to perform a prone hip extension on their dominant side to
15 holding their knee extended . all individuals were considered right - leg dominant as they
used their right leg to kick a ball19 . prior to the test , subjects were instructed on the required posture and allowed to practice
for 5 min . they were asked to maintain the prone position and then perform the prone hip
extension , keeping each posture for 5 sec . oxford , uk ) was
used to measure three - dimensional hip kinematics with a sampling rate of 100 hz . when the
subjects were prone , one reflective marker was placed on the gt and four markers were placed
on their dominant upper leg to obtain the location of the gt during tasks . the coordinates
of markers were captured in the middle 3 sec of a task . to identify the location of the gt , the gt marker - based measurement ( gt - m ) and no gt
marker - based measurement ( no gt - m ) , were performed and compared . in the gt - m , the location
of the gt was determined from the migration of the coordinates of markers that were placed
during the prone position and prone hip extension . in the no gt - m method , the length from
the four reflective markers ( a , b , c , d ) on the upper leg to one reflective marker ( gt1 )
indicating the gt during the prone position was calculated to identify the location of the
gt . the length la between a ( a , b , c ) and gt1 ( x , y , z ) was calculated using eq .
1 : when the subjects extended their legs , the changes in the locations of the four markers on
an upper leg , a(aa , ba , ca ) , b(ab , bb , cb ) , c(ac , bc , cc ) d(ad , bd , cd ) ,
were used to calculate the change in the coordinates of gt , gt(x,y,z ) . the distances
between the four markers and gt1 , la , lb , lc , ld , respectively , are identical to the
distance between the four markers and gt during the prone hip extension because the upper
leg shank is a rigid body segment . according to this , the formula of gt is shown in eq .
2 : from this , the changed location of gt , gt can be calculated as shown in eq .
3 : to compare the two measurements , the gt migration distance measured using the two methods
and the length of the thigh were calculated by eq . the migration distance of the gt is
the distance between gt , the location of the gt during the prone and gt , measured using the
two methods during prone hip extension . the length of the thigh is the distance between the
lateral epicondyle and the gt that is a palpable length1 and it is measured during prone hip extension using the coordinates
of gt measured using the two methods . also , the original length , which is the actual length
from the lateral epicondyle to the gt , was measured using a measuring tape to compare the
two methods . chicago , il , usa ) to compare the two
measurements using a significance threshold of p<0.05 . the kolmogorov - smirnov test was
used to assess the normality of the distribution of variables . the migration distance of gt
was compared using the wilcoxon test and the coefficient of variation ( cv ) was calculated . bland - altman plots20 were used to show
visual agreement and compare the measurement methods using the length of the thigh . the
difference in thigh length was calculated with repeated one - way analysis of variance and
bonferroni corrections were used for specific pair - wise comparison . the mean values and coefficients of variation ( cv ) of the migration distance and leg length
are shown in table 1table 1.gt migration distance and thigh lengthgt migration distance gt - mno gt - mmean sd ( cm)1.09 0.944.33 0.76cv ( % ) 86.2417.55thigh lengthoriginal lengthgt - mno gt - mmean sd ( cm)40.33 0.6439.63 0.6740.44 0.6sd : standard deviation ; cv : coefficient of variation ; gt : greater trochanter ; gt - m :
greater trochanter marker - based measurement ; no gt - m : no greater trochanter
marker - based measurement . the migration distance of the gt during the prone to the prone hip extension
differed significantly between the two measurements ( p<0.001 ) , and the cv value was lower
for the no gt - m . there were significant differences in leg length between the original
length , gt - m and no gt - m ( f=28.163 , p<0.001 ) . there was not a significant difference
between the original length and no gt - m ( p=0.200 ) but the gt - m was not comparable for the
original length and no gt - m ( p<0.001 ) . the bland - altman plot showed that all observation
pairs were in a mean 1.96 sd difference between original length and no gt - m ( fig . 1.bland-altman plots of average versus difference between ( a ) original length and
greater trochanter marker - based measurement ( gt - m ) , ( b ) original length and no greater
trochanter marker - based measurement ( no gt - m ) , ( c ) gt - m and no gt - m . line ; short and
dashed lines : the mean 1.96 sd of the differences , long and dashed line : the average
of the difference , gray line : zero reference line . ) . furthermore , the limit of agreement for thigh length between the original length
and the no gt - m method was lower than that when the gt - m was compared with original length
and no gt - m . sd : standard deviation ; cv : coefficient of variation ; gt : greater trochanter ; gt - m :
greater trochanter marker - based measurement ; no gt - m : no greater trochanter
marker - based measurement bland - altman plots of average versus difference between ( a ) original length and
greater trochanter marker - based measurement ( gt - m ) , ( b ) original length and no greater
trochanter marker - based measurement ( no gt - m ) , ( c ) gt - m and no gt - m . line ; short and
dashed lines : the mean 1.96 sd of the differences , long and dashed line : the average
of the difference , gray line : zero reference line . in this study , we examined a non - marker - based measurement method ( no gt - m ) to evaluate the
location of the gt . three - dimensional motion capture systems are frequently used to analyze
movement and commonly use the movement of reflective markers placed on a bony landmark15,16,17,18 . in contrast , the no gt - m method involves calculating the coordinates of the gt from the
location of four markers placed on the thigh . it is different from the conventional method ,
in which markers are placed directly on the part of the body that is being examined , such as
gt marker - based measurement ( gt - m ) . in the study of hwang et al.21 , four markers were used as a reference landmark for
reorientation , and other markers were reoriented according to the reference landmark . although cephalometric landmarks were examined , several markers can be used as a reference
mark for reorientation . therefore , we obtained the changes in gt coordinates based on four
reference markers during prone hip extension , following determination of marker
location . we investigated the gt migration distance and the thigh length to compare the gt - m and no
gt - m measurement methods . although the data were captured during the same movement , this
study showed that the migration length of the gt using gt - m was significantly different to
that using the no gt - m method . this difference might be caused by movement of soft tissue
due to the gt marker being placed on the skin . the main limitation of body surface markers
is due to skin deformation depending on the body movement and sites to place a marker22 , 23 . furthermore , the displacement of the reflective marker placed on the
artefacts would be incorrect in sites closer to the hip joint24 . in the study of moriguchi et al.15 , the gt landmark resulted in greater discrepancies when measuring
the range of motion , although the findings were obtained during hip flexion . also , the coefficient of variation ( cv ) of the gt migration length was calculated to assess
the accuracy of the two methods ; the cv of no gt - m measurements was lower than that of the
gt - m method . the migration value was similar because all subjects were healthy without any
pain or difficulties8 . the precision of the gt migration
measurement is important for clinical tests and studies of the hip joint . hip joint pain is likely related to
accessory movements8 that are important
for a full range of motion27 . the
accessory movements of the hip joint are gliding , rolling , and spinning28 , and the qualities of the accessory movements are often
related to an adequately functioning limb29 . the anterior femoral glide syndrome suggested by sahrmann8 is one of the dysfunctional accessory
movements of the hip joint . anterior femoral glide syndrome can be evaluated by assessing
the extent to which the gt translates to the anterior during the prone hip extension
test8 that is frequently used as a
clinical tool30 , 31 and palpation of gt has the potential for error32 . thus , a study of gt displacement for
examination of hip joint problems is warranted . to determine which method is better , we compared the thigh lengths from the gt to the
lateral epicondyle , calculated using the two methods , as the thigh is a rigid body segment . the length using gt - m was significantly
different from the original length and that determined using the no gt - m method , suggesting
that the gt - m method is less accurate . also , the no gt - m method yielded a thigh length
similar to the original value ; however , the value determined using the gt - m was not in
agreement . obtaining accurate body landmarks is crucial because discrepancies could lead to
poor assessment of impairments and asymmetries . in this study , the no gt - m value differed
from the original length because the accuracy of the three - dimensional system is 12 mm in
the x and y axies and 46 mm in the z axis33 . therefore , further work should examine a greater range of movement and other patient groups . we also did not compare the actual location of the gt using the motion capture system . additional study is required to evaluate whether the location of the gt using these methods
is agreement with its actual location . | [ purpose ] the greater trochanter ( gt ) is an important structure in biomedical research ,
but the measurement methods require development .
this study presents data from a new
measurement method that does not use gt - marker - based measurement ( no gt - m ) in comparison
with gt - marker based measurement ( gt - m ) .
[ subjects and methods ] we recruited 20 healthy
subjects , who were asked to perform and maintain a prone position and then move to the
prone hip extension .
a motion capture system collected the kinematic data and the location
of the gt was calculated by two measurements . [ results ] gt migration distance differed
significantly between the two measurements and the coefficient of the variation value was
lower for the no gt - m method .
thigh lengths of the no gt - m method were comparable to the
original lengths .
there were significant differences between the gt - m and the other
methods .
[ conclusions ] these data suggest that the gt - m method yielded a lower precision
with a smaller gt migration distance . in the comparison of thigh length ,
the no gt - m
method was in close agreement with the original length .
we suggest that determining the
location of the gt using the no gt - m has greater accuracy than the gt - m method . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the mammalian epidermis is a constantly self - renewing protective barrier against external challenges and dehydration that is formed and maintained by basal progenitor cells with high proliferative potential . the epidermis arises from a single ectodermal layer with a stratification program initiated at approximately embryonic day 12.5 ( e12.5 ) . in the single layered developing epidermis the majority of divisions are symmetric ( scd ) , producing two basal cells , but at the onset of stratification the balance shifts toward asymmetric divisions ( acd ) , resulting in a basal and a more differentiated suprabasal cell ( lechler and fuchs , 2005 ; poulson and lechler , 2010 ; williams et al . , 2011 ) . the epidermal master regulator p63 controls epidermal stratification and differentiation , proliferative potential , and division orientation ( ferone et al . , 2013 ; koster and roop , 2007 ; lechler and fuchs , 2005 ; lefkimmiatis et al . , 2009 ; senoo et al . , 2007 ; truong and khavari , 2007 ) . however , how p63 is regulated to execute these different functions and if extracellular signals are required to couple division orientation to epidermal stratification is largely unclear . we previously identified insulin- and igf-1 receptor ( ir / igf-1r ) signaling in keratinocytes as key regulators of epidermal morphogenesis and proliferative potential as epidermal loss of ir , igf-1r , or both increasingly impaired stratification ( stachelscheid et al . , 2008 ) . insulin / igf-1 signaling - mediated inhibition of the foxo transcription family of forkhead proteins is important for the regulation of longevity , metabolism , and stem cell behavior ( calnan and brunet , 2008 ; tothova and gilliland , 2007 ) . foxos are phosphorylated by iis - activated pkb / akt , and this results in their export from the nucleus ( kloet and burgering , 2011 ) . here , we show that iis - mediated regulation of the forkhead transcription factor ( foxo ) controls p63-dependent transcription via a foxo dna - binding - independent interaction with p63 to couple cell cycle progression to spindle orientation and thereby drive stratification . to determine how iis regulates epidermal stratification , we initially focused on e16.5 mouse embryos carrying a k14-cre - mediated ( hafner et al . , 2004 ) epidermal ir and/or igf-1r deletion ( ir , igf-1r , or dko ) , the time point when the microscopic phenotype first becomes obvious . in control mice , the transition from e15.5 to e16.5 coincides with an expansion of the number of suprabasal layers . this expansion is reduced in the ir , more reduced in the igf-1r with dko mice being most affected . in the igf-1r and dko mice this is accompanied by less polarized basal cells ( stachelscheid et al . , surprisingly , short term in vivo labeling of brdu did not reveal a difference in the e16.5 epidermis whereas at e17.5 incorporation was significantly reduced in dko mice ( figures 1a and s1b ) , similar to what was observed using ki67 ( stachelscheid et al . , 2008 ) . as brdu is incorporated into the dna during s - phase , a late block in the cell cycle provides a possible explanation why the e16.5 morphological phenotype induced by loss of igf-1r / ir is not accompanied by changes in proliferation markers . in agreement , the number of anaphase spindles was significantly reduced in e16.5 epidermis of igf-1r and dko mice whereas the ir mice , which only have a mild defect in stratification , showed a slight , albeit nonsignificant , reduction ( figure 1b ) . as the expansion of suprabasal layers at e16.5 is at least in part driven by acd ( poulson and lechler , 2010 ; williams et al . , 2011 ) , we asked whether epidermal loss of ir / igf-1r affected scd and/or acd . ablation of ir , igf-1r , or both resulted in biased loss of acds most prominent in the epidermis of igf-1r and dko mice ( figures 1c , 1d , s1c , and s1d ) , in line with the severity of the stratification phenotype observed in these mice . thus , by preferentially promoting acd , iis couples the regulation of proliferative potential to the expansion of suprabasal differentiated layers . to determine at which cell cycle stage loss of iis - induced arrest , we performed cell cycle analysis . significantly more cells were in g2/m in igf-1r keratinocytes compared to control ( figure 2a ) . to determine if this was related to an arrest in mitosis , we counted the number of metaphase and anaphase spindles , as the mitotic checkpoint is activated between these two mitotic phases . this revealed an increase in number of metaphase spindles accompanied by a decrease in number of anaphase spindles either in igf-1r keratinocytes ( figure 2b ) or in vivo in e16.5 dko epidermis ( figure 2c ) . thus , loss of epidermal insulin / igf-1 signaling is associated with a mitotic checkpoint arrest . to ask if iis is required during mitosis , we synchronized human keratinocytes in s - phase and subsequently serum - starved these cells during late g2 and arrested cells in mitosis . release of the mitotic block combined with addition of either insulin / igf-1 or fetal calf serum ( fcs)-induced phosphorylation of akt ( figure 2d ) , indicating that iis is activated during mitosis , whereas no activation was observed in the absence of growth factors . more importantly , addition of insulin / igf-1 or the positive - control fcs was sufficient to release mitotic arrest and drive significantly more cells into g1 upon release of the block compared to serum - starved cells ( figure 2e ) . together , these results indicate that iis activity is directly required during mitosis . the transcription factor p63 controls proliferative potential , epidermal stratification , acd , and cell cycle progression of keratinocytes ( koster and roop , 2007 ; lechler and fuchs , 2005 ; lefkimmiatis et al . , 2009 ; senoo et al . , 2007 ; truong and khavari , 2007 ) . these processes are also affected by loss of epidermal iis ( figure 1 ) ( stachelscheid et al . , 2008 ) . regulation of p63 might thus provide a mechanism for how ir / igf-1r control epidermal morphogenesis . we therefore asked whether epidermal loss of iis altered the transcriptional activity of p63 . a significant increase in luciferase activity was observed in igf-1r keratinocytes compared to control upon transfection of a reporter that is repressed by p63 ( pg13-luc ) ( hermeking et al . , 1997 ; yang et al . , 1998 ) ( figure 3a ) , whereas the activity of the p63-transactivated reporter ( bds-2 ( 3x ) ) ( hermeking et al . , 1997 ; king et al . , 2003 ) was repressed in igf-1r keratinocytes ( figure 3b ) . importantly , overexpression of p63 in igf-1r keratinocytes reversed inhibition of the p63-transactivated reporter ( figure 3b ) . we next asked if these alterations in p63 transcriptional activity have functional consequences for p63 target gene expression . to obtain an unbiased overview , we performed gene expression analysis on rna isolated from newborn epidermis of control and dko mice and compared this to the gene expression data derived from either mouse keratinocytes in which p63 was knocked down ( della gatta et al . , 2008 ) or p63 e18.5 whole skin ( koster et al . , 2006 ) . this revealed a statistically highly significant overlap of the dko gene expression set with both p63-regulated sets ( figure 3c ) , the percentage of which was in a similar range as the overlap of the two p63 gene expression sets ( figure s2a ) . more importantly , a statistically significant enrichment for gene ontology terms related to epidermal development was only observed in the overlapping gene sets of dko with either the p63 kd keratinocytes ( 4-fold ) or the p63 e18.5 skin ( 7-fold ) but not in the nonoverlapping sets ( figures 3c and s2b ) . we next analyzed the expression of several genes that are regulated by both iis and p63 , most of which are also direct targets of p63 ( ferone et al . , 2012 ; kouwenhoven et al . , 2010 ; westfall et al . , 2003 ; yang et al . , we mostly focused on genes that have been implicated in epidermal differentiation and/or cell cycle regulation . several p63 repressed targets ( e.g. , 14 - 3 - 3 or runx2 ) were upregulated , whereas other targets ( e.g. , k15 or tgfbi ) are downregulated in the epidermis of both e16.5 igf-1r and newborn dko mice ( figures 3d and s2c ) . for 14 - 3 - 3 , this increase was further confirmed at the protein level ( figure s2f ) . surprisingly , the global gene expression analysis revealed that several members of the late envelope ( lce ) protein families and small proline rich ( sprr ) protein , which are part of the epidermal differentiation complex ( edc ) , were highly upregulated not only in the newborn dko but also in the different p63 knockout or knockdown gene expression sets ( della gatta et al . this is consistent with the complex function of p63 in balancing the promotion of epidermal stratification while inhibiting a subset of terminal differentiation genes or counteracting notch - induced differentiation ( koster and roop , 2007 ; nguyen et al . , 2006 ) . real - time pcr analysis confirmed the strong increase in lce3b , sprr2i , and sprr2f in the dko ( figure s2d ) . 3d ) , albeit that control expression levels were very low and sprr2i and sprr2f mrna levels were below detection at this developmental stage . other putative p63 targets , such as fgfr2 ( ferone et al . , 2012 ) were not affected by loss of igf-1r or ir / igf-1r either in vivo ( figures 3d and s2c ) or in vitro ( figure s2e ) . thus , epidermal iis signaling and p63 regulate the expression of an overlapping set of genes . if a decrease in p63 transcriptional activity is responsible for the altered expression of a range of p63 target genes in the epidermis of igf-1r or dko mice , then one would predict less p63 binding to p63 consensus elements in the endogenous promoters of these altered genes . based on previously identified p63-binding sites in promoters in human keratinocytes and cells ( kouwenhoven et al . , 2010 ; westfall et al . , 2003 ; yang et al . , 2006 ) we identified conserved p63 binding sites in promoters / enhancers of murine runx2 , stratifin ( sfn , encoding 14 - 3 - 3 ) , and cdkn1a and used the already known site in fgfr2 ( ferone et al . , 2012 ) as a control as its expression is not altered upon ir / igf-1r loss . we next asked whether p63 binding to these sites was changed upon loss of igf-1r . surprisingly , p63 binding to the p63 consensus site in these targets was similar in control and igf-1r keratinocytes in chromatin immunoprecipitation quantitative pcr ( chip - qpcr ) ( figure 3e ) , even though rna expression of these targets is altered . in agreement , loss of iis signaling did not induce significant changes in p63 expression in either e16.5 igf-1r ( figure 3f ) or in keratinocytes ( figure s2 g ) . taken together , these findings suggest that insulin / igf-1 regulate p63 transcriptional activity independent of p63 protein expression and promoter binding . the foxo family of forkhead transcription factors is one of the key targets through which iis exerts its biological effects . iis negatively controls the transcriptional activity of foxos through akt phosphorylation - dependent nuclear exclusion of foxos ( calnan and brunet , 2008 ) . foxos play central roles in longevity , stem cell regulation , metabolism , and tumor suppression ( accili and arden , 2004 ; partridge and brning , 2008 ) . more importantly , foxos were shown to regulate transcriptional activity of other proteins independent of binding to their consensus sites in promoters ( jensen et al . , 2011 ; nemoto et al . , thus , foxo transcription factors are potential downstream candidates to mediate iis regulation of genes also regulated by p63 . however , little is known on the expression of foxo members in the epidermis . real - time pcr analysis showed that all four members , foxo1 , foxo3 , foxo4 , and foxo6 , were expressed in e16.5 epidermis ( figure 4a ) , the time point when the phenotype induced by loss of iis signaling becomes obvious . western blot analysis for foxo1 and foxo3 also showed protein expression at e16.5 ( figure 4b ) and in newborn ( not shown ) as well as in primary keratinocytes ( figure s3a ) , whereas only a specific band could be detected for foxo4 in primary keratinocytes but not e16.5 epidermis , despite rna expression ( figure s3a ) . loss of igf-1r did not alter foxo1 and foxo3 expression levels in e16.5 embryos ( figures 4b and s3b ) . importantly , using western blot analysis , we detected less phospho - foxo1 in e16.5 igf-1r epidermis compared to control ( figures 4b and 4c ) , indicating a loss of iis - stimulated akt phosphorylation activity . this was associated with an increase in nuclear foxo1 protein ( figure s3c ) . most importantly , foxo - specific reporter assays showed increased foxo transcriptional activity in igf-1r keratinocytes ( figures 4d and s5a ) . together , these data suggest that loss of epidermal iis activates foxos . to examine whether foxo can regulate p63 , we transfected primary keratinocytes with either the p63-transactivated reporter or the p63-repressed reporter in the presence of either gfp , foxo1-wt , or of a foxo1 with mutated akt phosphorylation sites ( foxo1-ada ) resulting in a constitutively nuclear foxo that thus mimics loss of ir / igf-1r ( nakae et al . , 2000 ) . foxo1-wt was used as a control as this is excluded from the nucleus due to the presence of insulin / igf-1 in serum . foxo1-ada inhibited the p63-transactivated reporter ( figure 5a ) , whereas the activity of the p63-repressed reporter was upregulated by foxo1-ada ( figure 5b ) . mutation of the foxo dna binding domain ( foxo1-ada-dbd ) ( kitamura et al . , 2007 ) did not affect the ability of foxo1-ada to repress the p63-transactivated reporter in primary keratinocytes ( figure s4a ) . to rule out that foxo - ada can directly bind p63-binding sites foxo - ada - mediated repression required cotransfection with p63 ( figure s4b ) . to examine whether foxo directly interacts with p63 , we transfected cho cells with p63 and foxo1-ada and found that foxo1-ada precipitated p63 and vice versa , p63 precipitated foxo1-ada ( figure s4c ) , thus providing evidence that foxo1-ada interacts with p63 . more importantly , more endogenous foxo1 was immunoprecipitated by p63 from igf-1r than from control keratinocytes ( figure 5d ) . using k14-cre mice carrying a flox - stop - flox - foxo - gfp cassette under the control of the rosa26 promoter ( fukuda et al . , 2008 ) gfp immunoprecipitations showed an increased interaction of p63 with foxo1-gfp in the absence of igf-1r compared to control newborn epidermis ( figure 5e ) . thus , loss of iis results in nuclear translocation of foxos , where they interact with p63 to counteract p63 transcriptional activity . this would predict that loss of igf-1r would promote an association of foxos with p63 sitting on p63 binding sites present in endogenous promoters . as already shown , p63 binding to p63 consensus site containing promoters was not altered by iis loss ( figure 3e ) . foxo1 chip - qpcr experiments in control and igf-1r keratinocytes showed that upon loss of igf-1r foxo1 was enriched 2- to 3-fold on p63 consensus sites in the promoters of targets that showed altered expression upon loss of igf-1 ( e.g. , sfn or runx2 ) but not on p63 sites located in targets with unaffected expression , such as e.g. , fgfr2r ( figure 5f ) . in agreement , transfection of foxo1-ada-dbd dna binding mutant in control primary keratinocytes increased expression of these same targets , runx2 and 14 - 3 - 3 ( figure 5c ) , similar to in vivo loss of iis ( figure 3d ) . these results indicate that foxo interacts with p63 on p63-dna consensus sites to regulate expression of these p63 targets in a foxo - dna - binding - independent manner . most importantly , sirna mediated knockdown of foxo1 , -3 , and -4 but not a scrambled sirna reversed the expression of e.g. , 14 - 3 - 3 and runx2 in igf-1r primary keratinocytes ( figure 5 g ) , whereas these sirnas showed no effect in control ( figure s4d ) , indicating that the increased expression of these targets upon loss of igf-1r is a direct consequence of increased foxo activity . together , these findings identify a dna binding - independent function for foxos in the regulation of p63 activity . if foxo functionally counteracts p63 in vivo in a dna binding - independent manner , then expression of the constitutive nuclear foxo1-ada , but not a foxo1-dna binding - domain - only mutant ( foxo - dn ) ( nakae et al . , 2000 ) in the epidermis , should result in a skin phenotype that is similar to that caused by loss of p63 . in addition , foxo - dn should not be able to rescue the impaired stratification in igf-1r . we thus crossed mice that carry a flox - stop - flox cassette followed by either a foxo1-ada - ires - gfp or foxo1-dn - ires - gfp cassette in the rosa26 locus ( belgardt et al . 2013 ) to the keratin14-cre transgene ( hafner et al . , 2004 ) and in case of the foxo - dn also to igf-1r mice , to induce epidermal expression of the ada mutants ( foxo1-dn or foxo1-ada ) . foxo1-dn expression reversed foxo reporter activity induced by the loss of igf-1r in primary keratinocytes isolated from these mice ( figure s5a ) , showing that the foxo dna binding mutant was indeed a dominant negative toward foxo dna - binding - dependent transcriptional activity . expression of foxo1-dn did not affect epidermal morphogenesis ( figure 6a ) , as predicted based on our observations that the foxo1-ada dna binding mutant was still able to alter p63 reporter activity and gene expression . more importantly , foxo1-dn was unable to rescue stratification in igf-1r mice ( figure 6a ) . foxo1-ada was expressed in the epidermis already at e13.5 ( figures s5b and s5c ) and resulted in fragile , translucent skin and perinatal death ( figure 6b ) . histochemical analysis of newborn mice revealed a striking hypoplastic epidermis ( figure 6c ) , which strongly resembled that of p63 knockout mice ( mills et al . this phenotype became first apparent at e14.5 ( figure s5d ) accompanied by a failure to properly induce the suprabasal differentiation markers keratin10 and loricrin ( figure 6d ) , indicating that foxo1-ada interferes with proper initiation of stratification . tunel staining revealed no increase in apoptosis in the developing epidermis of k14-cre - foxo1-ada mice ( figure s6a ) , similar to what was observed in dko epidermis ( stachelscheid et al . , 2008 ) and in contrast , e16.5 foxo1-ada mice showed a strong reduction in the number of anaphase divisions in the epidermis ( figure 7a ) , further confirming that foxo is downstream of iis in the regulation of epidermal morphogenesis . this was even more pronounced than in dko epidermis ( figure 1b ) , thus reflecting the more severe phenotype . this was due to a biased loss of acds ( figures 7b and 7c ) , as was also observed in ir , igf-1r , dko ( figures 1c , 1d , and s1d ) , and p63 knockout mice ( lechler and fuchs , 2005 ) . most importantly , expression analysis revealed that foxo1-ada expression regulated a similar subset of p63 target genes in e16.5 epidermis ( figure 7d ) , most of which were also altered in e16.5 igf-1r epidermis ( figure 3d ) , whereas targets not altered by loss of iis ( e.g. , fgfr2 ) were again not changed . as expected , basal p63 localization was not obviously affected by epidermal foxo1-ada expression ( figure s6b ) . thus , insulin / igf-1-controlled foxos are crucial regulators of p63 , acd , and epidermal morphogenesis . our data demonstrate a role for iis signaling in foxo - dependent control of p63 , a key determinant of epidermal cell fate , to regulate acd and progression of mitosis during epidermal morphogenesis . this allows igf-1 , and to a much lesser extent insulin , to couple the maintenance of progenitors with high proliferative potential to suprabasal differentiation . we identify p63 as an interacting partner of foxos and show that foxo is a negative regulator of p63 transcriptional activity on a subset of targets important for epidermal stratification . this is based on the following observations : a constitutive nuclear foxo , foxo1-ada , inhibits transcription of a luciferase reporter that only contains p63 sites and only in the presence of p63 . more importantly , a point mutation that prevents dna binding , as shown by foxo reporter assays foxo1-ada-dbd , still inhibits p63-dependent reporters . last , epidermal expression of a mutant foxo1 that consists only of its dna binding domain did not obviously affect epidermal morphogenesis and , more importantly , was not able to rescue the phenotype caused by inactivation of igf-1 ( results not shown ) . this same foxo-1 mutant was shown to rescue loss of iis in the hypothalamus ( belgardt et al . , 2008 ) . in agreement with our findings , foxos can also regulate transcriptional activity of myc and p53 independent of binding to the consensus foxo recognition element ( jensen et al . the p63 transcription factor is crucial for the formation of stratifying epithelia such as epidermis and mutations in human p63 result in ectodermal dysplasia disorders ( koster and roop , 2007 ; vanbokhoven et al . , 2011 ) . , the dermal signals that regulate p63 have so far remained largely elusive . here , we identify p63 as a target of iis , which is necessary during epidermal morphogenesis to inhibit foxo activity , likely through nuclear export and thereby prevent foxo to negatively regulate p63 function . we find that loss of iis resulted in a decrease in akt - dependent foxo1 phosphorylation accompanied by an increase in nuclear foxo1 protein and , more importantly , increased foxo transcriptional activity as judged by reporter assays . as the foxo1-ada is readily detected in the nucleus ( figure s5c ) and expressed 5-fold more as endogenous foxo ( figure s5b ) , endogenous foxos are likely expressed at levels too low to be detected by immunostaining . thus far , only a few publications show nuclear translocation of endogenous foxos by immunohistochemistry and only upon fasting and not loss of iis ( kitamura et al . , 2006 ) . loss of p63 or epidermal expression of foxo1-ada phenotypically mimic each other and result in much more severe phenotype than combined epidermal loss of ir and igf-1r . although this in part may be explained by the developmental timing of deletion versus expression , this does suggest that other signals than iis control foxo and p63 in epidermal morphogenesis . potential candidates are tgf-superfamily members , as several tgf downstream transcription targets , some of which are also regulated by p63 , require the cooperative activity of smad4 and foxos in keratinocytes ( gomis et al . , 2006 ) . perturbed foxo - p63 interactions may also contribute to a range of common and rare skin diseases characterized by disturbed epidermal differentiation , such as psoriasis , ichthyosis , atopic dermatitis , and skin cancer . our data provide evidence for a model in which iis in keratinocytes controls epidermal morphogenesis through exclusion of foxo from the nucleus thereby releasing its inhibitory action on p63 . this in turn allows p63 to exert its transcriptional control of genes that regulate proliferative potential , cell cycle progression , and spindle orientation . our data suggest that 14 - 3 - 3 , an important regulator of epidermal differentiation , might be one of the key targets of ir / igf-1r / foxo / p63 axis . p63 promoter binding inhibits sfn transcription , which encodes 14 - 3 - 3 ( westfall et al . , this is relieved upon loss of insulin / igf-1 accompanied by increased foxo binding to the p63 site in the endogenous sfn promoter resulting in more rna and protein expression . in line with our findings , overexpression of 14 - 3 - 3 in murine epidermis results in a hypomorphic phenotype ( cianfarani et al . , 2011 ) , similar to the dko mice . it was also shown that 14 - 3 - 3 can induce g2/m arrest in cell culture ( reinhardt and yaffe , 2009 ) . although insulin / igfs and foxo have not yet been directly implicated in the regulation of mitosis and acd , akt , the upstream inhibitor of foxo , regulates acd in cancer cells ( dey - guha et al . , 2011 ) and combined loss of akt1 and akt2 results in less suprabasal layers similar to epidermal loss of ir and igf-1r ( peng et al . , 2003 ) . the small gtpase rac , previously shown to mediate iis regulation on epidermal morphogenesis likely upstream of akt ( stachelscheid et al . , 2008 ) , has also been implicated in control of acd ( halet and carroll , 2007 ; lu et al . , how insulin / igf - mediated regulation of foxo and p63 regulates acd and mitotic progression to control the balance between proliferation and differentiation will be an important question for the near future . epidermis - specific deletion of insulin receptor ( ir ) , igf-1-receptor ( igf-1r ) , or both using k14-cre - mediated deletion in mice have been described ( stachelscheid et al . , 2008 ) . conditional foxo knockin mice carrying a cacg promoter , a loxp - flanked stop cassette followed by the cdna of constitutively nuclear foxo1-ada or dominant negative foxo1-dn in the rosa26 locus ( belgardt et al . , 2008 ; sthr et al . , 2013 ) or a loxp - flanked stop cassette followed by foxo1-gfp in the rosa26 locus ( fukuda et al . , 2008 ) were crossed to k14-cre transgenic mice ( hafner et al . , 2004 ) to induce epidermal - specific expression . all mice are in c57bl/6 background and experiments were performed according to institutional guidelines and animal license of the state office north rhine - westphalia , germany . for histology , embryos or mouse skin were fixed in 4% pfa and embedded in paraffin . paraffin sections ( 5 m ) were deparaffinized and stained with hematoxylin and eosin ( h&e ) and imaged with an olympus bx51 microscope . for immunofluorescence , paraffin sections were deparaffinized , antigens were retrieved with buffer a , ug , or ag ( ems ) , and sections were blocked in pbs containing 5% normal goat serum and 0.1% triton x-100 . slides were incubated with primary antibody followed by washing and incubation with the appropriate secondary antibodies coupled to alexa 488 , alexa 594 , or cy3 ( invitrogen ) . nuclei were counterstained with dapi ( sigma ) and sections examined using olympus ix81 fluorescence or olympus fv1000 confocal microscopes . primary antibodies are listed in supplemental experimental procedures . to analyze the angle of divisions , the axis of divisions in e16.5 embryos was determined in anaphase / telophase cells using survivin staining as described by williams et al . the angle of division was determined by measuring the angle of the plane transecting two daughter cells relative to the plane of the basement membrane . the angles of divisions were quantified and angle orientation was plotted with oriana 4 ( kcs ) . the different divisions were then categorized as described with asymmetric divisions having an angle of 6090 , random 3060 , and symmetric 030 ( lechler and fuchs , 2005 ) . each of the total number of asymmetric , random , or symmetric divisions of the control were then set to 100% to compared the relative loss within each of the division categories to either knockouts or transgene . primary keratinocytes were isolated and cultured in minimal ca medium ( 50 m ca ) as described ( stachelscheid et al . , 2008 ) . for foxo1 and p63 overexpression , primary mouse keratinocytes were transfected using lipofectamine2000 ( invitrogen ) at a confluence of 70%90% according to manufacturer s protocol . for transfection 250 ng of the following plasmids were used : pcdna3-foxo1-ada , pcdna3-foxo1-ada - dbd , and pcmv - flag - foxo1 ( kitamura et al . , 2007 ) and p63 plasmids pcmv - fag-np63 ( ferone et al . , 2012 ) , pegfp-np63 , or pegfp empty vector as control . for luciferase reporter assays , cells were cotransfected with 25 ng ptk - renilla ( promega ) and 250 ng of one of the following p63 luciferase reporters : 250 ng of pgl3-bds-2 ( 3x ) and pg13-luc ( hermeking et al . , 1997 ; yang et al . , 1998 ) or the 6xdbe - fkhre foxo reporter ( potente et al . , 2005 ) . experiments were performed in triplicates and luciferase activity was determined 24 to 48 hr after transfection with the dual - luciferase reporter assay kit ( promega ) and a berthold tristar luminometer . cells were harvested by trypsin / edta treatment , washed in 5 ml pbs and resuspended in 3 ml pbs . for fixation , cells were incubated with 70% ethanol / pbs at 4c for minimum 2 hr and then centrifuged for 7 min at 1,000 rpm . the pellet was 2 washed with pbs , filtered with a 70 m cell strainer and resuspended in pbs containing 0.1% triton x-100 , rnase a ( 10 mg / ml ) and propidium iodide ( 2 mg / ml ; sigma ) . cells were stained at 37c for 15 min and analyzed by facs ( facscalibur , bd ) . to silence foxo gene expression , keratinocytes were transfected with on - target plus smartpool sirnas ( thermo fisher scientific ) targeting foxo1 , foxo3 , foxo4 , and foxo6 . subconfluent primary mouse keratinocytes were transfected using 50 nm of each smartpool or nontargeting control pool and lipofectamine2000 reagent ( invitrogen ) according to manufacturer s protocol . efficient knockdown was observed 48 hr posttransfection in rt - pcr and western blot analysis . for chip assays 3 10 primary mouse keratinocytes or newborn epidermis were crosslinked with 1% formaldehyde . lysates were subjected to sonication on ice to obtain dna fragments ranging from 200 to 1,000 bp in length . the supernatant was diluted for immunoprecipitation in ip buffer ( 150 mm nacl , 20 mm tris ph 8 , 2 mm edta ) and precleared with protein a beads ( roche ) . supernatant was incubated with 4 g antibody overnight at 4c , and beads were sequentially washed as previously described ( ferone et al . , 2012 ) . chromatin was eluted , dna was purified and analyzed by quantitative real - time pcr , or protein was analyzed by western blot . real - time pcr was performed using the sybr green pcr master mix ( applied biosystems ) in an abi stepone light cycler . for co - ip , cho cells were lysed in hepes buffer ( 50 mm hepes - koh ph 7.8 ; 140 mm nacl ; 1 mm edta ; 10% glycerol ; 0.25% triton x-100 ; 1% np-40 ) for 20 min at 4c , subsequently nuclei were disrupted by mild sonication and lysate after preclearing incubated with 4 g of antibody overnight . antibodies were precipitated with protein a / g ( roche ) beads and after washing in ip buffer , samples were analyzed by western blot . rna was extracted from keratinocytes and epidermis using trizol ( invitrogen ) and rneasy minikit ( qiagen ) . rna was reversely transcribed with quantitect reverse transcriptase ( qiagen ) and amplified using the taqman universal pcr master mix ( applied biosystems ) . calculations were performed by comparative cycle threshold ( ct ) method with data normalized relative to 18s and hprt1 . probes for target genes were ordered from taqman assay - on - demand kits ( applied biosystems ) . rna was isolated from control and dko epidermis ( n = 4 each ) and sent to the dna sciences core at the university of virginia for labeling , amplification , and hybridization to the affymetrix 430 - 2.0 platform . for global gene expression analysis , significantly regulated genes sets ( p < 0.05 ) from the different microarrays were analyzed for overlap using venny tool by oliveros ( http://bioinfogp.cnb.csic.es/tools/venny/index.html ) . overlapping and nonoverlapping gene sets were then annotated using david functional annotation tool ( dennis et al . , 2003 ) . epidermis was separated from dermis and dissociated with a mixermill homogenizer and lysed in 1% sds lysis buffer . lysates were separated on sds - page gels ( novex ) , transferred to a pvdf membrane , blocked in 5% western blot blocking solution ( roche ) and incubated with primary antibodies followed by incubation with the appropriate horseradish peroxidase coupled secondary antibodies and detected using ecl ( thermo fisher scientific ) . data were analyzed for statistical significance using two - tailed unpaired student s t test unless otherwise stated . relative loss of acd , random divisions , and scd were tested using one - way anova in prism 5 ( graphpad ) . the asterisks shown in graphs correspond to the p values as stated in the figure legends . the results were presented as the average of at least three independent experiments unless otherwise stated in the legends and arrow bars indicate sd or sem . | summarythe multilayered epidermis is established through a stratification program , which is accompanied by a shift from symmetric toward asymmetric divisions ( acd ) , a process under tight control of the transcription factor p63 .
however , the physiological signals regulating p63 activity in epidermal morphogenesis remain ill defined . here , we reveal a role for insulin / igf-1 signaling ( iis ) in the regulation of p63 activity .
loss of epidermal iis leads to a biased loss of acd , resulting in impaired stratification . upon loss of iis
, foxo transcription factors are retained in the nucleus , where they bind and inhibit p63-regulated transcription .
this is reversed by small interfering rna - mediated knockdown of foxos . accordingly ,
transgenic expression of a constitutive nuclear foxo variant in the epidermis abrogates acd and inhibits p63-regulated transcription and stratification .
collectively , the present study reveals a critical role for iis - dependent control of p63 activity in coordination of acd and stratification during epithelial morphogenesis . |
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centromeres were classically defined as the primary constriction of chromosomes a region where paired sister chromatids are held tightly together until the onset of anaphase . classic microscopic observations revealed that this was the region where mitotic chromosomes attached to the spindle microtubules . in the 1960s , electron microscopy first recognized that the surface of the centromere was the site of assembly of a button - like structure the kinetochore which formed the actual point of microtubule attachment ( luykx 1965 ; brinkley and stubblefield 1966 ; jokelainen 1967 ) . in subsequent years , studies by a great many laboratories have developed a still emergent picture of the kinetochore as one of the most wonderfully complex supramolecular structures found in cells ( maiato et al . 2004 ; cheeseman and desai 2008 ; hori and fukagawa 2012 ; westhorpe and straight 2013 ) . for years , there was an ongoing debate about the distinction ( if any ) between centromeres and kinetochores . both terms were initially proposed to describe essentially the same aspect of chromosome structure and function , so this seemed liked a semantic discussion . however , in the post - genomic era of cloned and sequenced genomes , a meaningful distinction has emerged . it is now generally accepted that the underlying dna sequence that defines the primary constriction of mitotic chromosomes is the centromere , whereas the proteinaceous structure that assembles on the surface of the centromeric chromatin is the kinetochore . the histone h3 variant cenp - a is a specific protein marker for centromeres ( earnshaw and rothfield 1985 ; earnshaw et al . 2013 ) , whereas kinetochores are characterized by the presence of > 100 different proteins , the earliest described being cenp - c ( earnshaw and rothfield 1985 ; saitoh et al . studies of the centromere have been more limited , in part due to its complex repetitive substructure . in budding yeast , the point centromere ( pluta et al . 1995 ) is defined by a 125 bp dna sequence ( hegemann and fleig 1993 ) . in fission yeast and vertebrates , there is no single sequence that defines the centromere , with cenp - a being distributed across wide regions of dna . we therefore coined the term regional centromeres to describe most centromeres outside budding yeast ( pluta et al . 1995 ) . with the advent of increasingly sophisticated molecular approaches , and particularly deep sequencing , it is now possible to begin to define the centromeric dna in vertebrates by chip - seq in a few specialized instances . detailed cenp - a mapping is not possible for most metazoan centromeres because they are comprised of hugely redundant families of repetitive dna that have foiled efforts to create contiguous linear maps . 1997 ; barry et al . 1999 ; burrack and berman 2012 ) , chromosome 11 of the horse ( wade et al . 2009 ) , and chromosomes z , 5 , and 27 in chicken ( shang et al . all of those centromeres are assembled on unique sequence dna , so that it is possible to exactly map the extent of the cenp - a domain . even though the cenp - a domain can not be mapped precisely in humans , analysis of extended chromatin fibers has revealed that centromeres are comprised of intercalated domains of cenp - a chromatin and chromatin containing centromeric canonical histone h3 ( blower et al . 2002 ; sullivan and karpen 2004 ; ribeiro et al . 2010 ; sullivan et al . 2011 ) . this observation lead to a working model in which the centromeric chromatin formed an amphipathic - like solenoid , with the cenp - a - containing surface at the chromosome exterior abutting the kinetochore and a h3-containing surface facing the interior of the chromosome ( sullivan and karpen 2004 ) . more recent super - resolution mapping of various kinetochore protein pairs ( joglekar et al . 2009 ; wan et al . 2013 ) and observation of antibody - labeled chromatin fibers led to the proposal of a boustrophedon model of the kinetochore chromatin as multi - layered sinusoidally folded patch on the chromosome surface , rather than a helix embedded in it ( ribeiro et al . one parameter that is essential in establishing detailed models for the folding of the centromeric chromatin fiber in the inner kinetochore is the amount of dna in the assembled kinetochore . by this , we do not mean the amount of dna occupied by cenp - a , as determined by chip - seq , but instead the amount of dna that is folded into the three - dimensional functional mitotic kinetochore . this has not been measured , largely because the inner kinetochore is embedded in the centromeric chromatin , and can not be distinguished from the surrounding dna in compact mitotic chromosomes by conventional microscopy methods . the aim of the present study was to take advantage of a chicken dt40 condensin mutant in which kinetochores undergo excursions during which they are occasionally stretched considerable distances from the surface of the centromeric chromatin ( ribeiro et al . 2009 ) . under those circumstances , the kinetochore is spatially isolated away from the rest of the underlying centromeric chromatin . we have used quantitative fluorescence to estimate the amount of dna within the folded kinetochore domain as defined by cenp - h - gfp , expressed from its own promoter and exogenous cenp - a - gfp . this analysis revealed that dt40 kinetochores in their native conformation contain ~5060 kbp of dna , a number that is remarkably close to 50150 kbp size of the cenp - a domain measured in human neocentromeres ( barry et al . 1999 ; alonso et al . 2007 ) and the 41 6 kbp size of the cenp - a domain measured by chip - seq for 12 chicken centromeres and neocentromeres ( shang et al . thus , when kinetochores assemble on mitotic chromosomes , they do not incorporate significant amounts of pericentromeric dna surrounding the core cenp - a domain . the distribution of kinetochore proteins along the dna has been mapped at vertebrate neocentromeres by a number of methods ( alonso et al . 2007 ; shang et al . 2013 ; saffery et al . 2003 ; capozzi et al . 2008 ) , but it is not known how much dna is folded into a functional mitotic kinetochore . our published studies have established that kinetochores of smc2-depleted chicken dt40 cells are structurally and functionally normal ( ribeiro et al . it is , however , required to regulate the compliance ( stretchiness ) of the pericentromeric heterochromatin , which acts as a spring linking the two sister kinetochores ( ribeiro et al . therefore , because condensin - depleted kinetochores are spatially well resolved from the surface of the primary constriction when undergoing excursions 1a , b ) , they can be used to determine the dna content of a functioning kinetochore in situ by quantifying the dna within the compact cenp - h and cenp - a domains.fig . 1quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna . inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5 m ) quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna . inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5 m ) the strategy adopted here was to image metaphase kinetochores of smc2-depleted cells that were expressing either cenp - h - gfp or cenp - a - gfp as three - dimensional image stacks , identify the volume occupied by cenp - h and cenp - a , and then ask how much dna ( stained with dapi ) was present within that volume ( fig . , the dapi staining was measured relative to a standard present in the same image . cenp - h was used in these experiments because the single copy cenp - h gene on the chicken z chromosome had been labeled by knock - in of a gfp cdna ( fukagawa et al . thus , the fusion protein was expressed from its native promoter and was able to support life of the cells . in the case of gfp - cenp - a , the fusion protein was a transgene expressed in a stable cell line . other work in human ( foltz et al . 2013 ) cells has shown that there appears to be a homeostatic regulation of cenp - a levels , such that when exogenous cenp - a is expressed , levels of the endogenous protein fall , so that overall cenp - a levels remain similar ( foltz et al . furthermore , chip - seq analysis in chicken has shown that the size of cenp - a chromatin domains is not significantly increased following expression of exogenous cenp - a - gfp ( shang et al . , we initially used s. cerevisiae cells in g1 phase , but rapidly realized that the amounts of dna in chicken mitotic kinetochores were far smaller than this . we therefore switched to the use of three bacteriophages , namely : t4 ( genome size 168 kb ) , p1 ( 90 kb ) , and ( 48 kb ) to calibrate the dna quantification since their genome size is within the range of the dna amounts in the kinetochore . when these bacteriophages were fixed , stained with dapi , and mixed and imaged with the deltavision microscope ( fig . 2a ) , a linear correlation between dna amount and intensity was obtained ( fig . 2three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5 m ) . b standard curve of fluorescence intensity ( dapi y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5 m ) . b standard curve of fluorescence intensity ( dapi y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) this calibration allowed us to quantify the amount of dna in functional mitotic kinetochores . smc2 cells expressing tagged endogenous ( knock - in ) cenp - h - gfp or exogenous gfp - cenp - a were mixed with bacteriophages and processed for image acquisition . we looked for cells in which one or more kinetochores had stretched polewards , away from the body of its chromosome and where we could also acquire the three different bacteriophages in the same field of view . in each case , the gfp - labeled protein was used to define a volume envelope for the kinetochore , and the amount of dapi staining within that envelope was measured . standard curves determined for the bacteriophages in each experiment were used to calibrate the dna intensity values for the cenp - a or cenp - h - containing chromatin ( fig . it is worth emphasizing that these gfp - labeled kinetochores were functional at the time of fixation , as they were attached to microtubule bundles and exerting force towards the spindle poles . furthermore , it is important to note that while the chromatin beneath the kinetochore was abnormally stretched , the kinetochores themselves appeared structurally normal at both the light and electron microscope levels ( ribeiro et al . 2009).fig . 3
a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) . these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna . c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) . d summary table showing values obtained from the quantification done in c
a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) . these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna . c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) . d summary table showing values obtained from the quantification done in c
a measurement of 40 centromeres in each case revealed that compact cenp - a and cenp - h domains of functional smc2-depleted kinetochores contained an average of 61 and 54 kb of dna , respectively ( range28147 kb , fig . because the kinetochores measured come from a number of different dt40 chromosomes , the observed range of dna content could be in part due to size variation between kinetochores . however , a recent study in the fukagawa lab has revealed that all of the kinetochores in dt40 cells are of similar size when measured with a variety of kinetochore markers ( t. hori and t. fukagawa , personnal communication ) . importantly , their measurements included kinetochores assembled on repetitive centromeres as well as those on nonrepetitive centromeres and on both micro and macro chromosomes . thus , the most likely explanation for the variation is that because kinetochores are pulled away from the primary constriction to differing degrees , the amount of pericentromeric chromatin immediately adjacent to the kinetochore may vary . the distribution of kinetochore proteins along the dna has been mapped at vertebrate neocentromeres by a number of methods ( alonso et al . 2007 ; shang et al . 2013 ; saffery et al . 2003 ; capozzi et al . 2008 ) , but it is not known how much dna is folded into a functional mitotic kinetochore . our published studies have established that kinetochores of smc2-depleted chicken dt40 cells are structurally and functionally normal ( ribeiro et al . it is , however , required to regulate the compliance ( stretchiness ) of the pericentromeric heterochromatin , which acts as a spring linking the two sister kinetochores ( ribeiro et al . therefore , because condensin - depleted kinetochores are spatially well resolved from the surface of the primary constriction when undergoing excursions 1a , b ) , they can be used to determine the dna content of a functioning kinetochore in situ by quantifying the dna within the compact cenp - h and cenp - a domains.fig . 1quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna . inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5 m ) quantification of dna in a functional vertebrate kinetochore . a experimental design used to measure kinetochore dna content in smc2 cells and bacteriophages used as standards with known amounts of dna . inset shows two kinetochores labeled with cenp - h - gfp with trailing chromatin ( scale bar 5 m ) the strategy adopted here was to image metaphase kinetochores of smc2-depleted cells that were expressing either cenp - h - gfp or cenp - a - gfp as three - dimensional image stacks , identify the volume occupied by cenp - h and cenp - a , and then ask how much dna ( stained with dapi ) was present within that volume ( fig . , the dapi staining was measured relative to a standard present in the same image . cenp - h was used in these experiments because the single copy cenp - h gene on the chicken z chromosome had been labeled by knock - in of a gfp cdna ( fukagawa et al . thus , the fusion protein was expressed from its native promoter and was able to support life of the cells . in the case of gfp - cenp - a , the fusion protein was a transgene expressed in a stable cell line . other work in human ( foltz et al . 2013 ) cells has shown that there appears to be a homeostatic regulation of cenp - a levels , such that when exogenous cenp - a is expressed , levels of the endogenous protein fall , so that overall cenp - a levels remain similar ( foltz et al . furthermore , chip - seq analysis in chicken has shown that the size of cenp - a chromatin domains is not significantly increased following expression of exogenous cenp - a - gfp ( shang et al . , we initially used s. cerevisiae cells in g1 phase , but rapidly realized that the amounts of dna in chicken mitotic kinetochores were far smaller than this . we therefore switched to the use of three bacteriophages , namely : t4 ( genome size 168 kb ) , p1 ( 90 kb ) , and ( 48 kb ) to calibrate the dna quantification since their genome size is within the range of the dna amounts in the kinetochore . when these bacteriophages were fixed , stained with dapi , and mixed and imaged with the deltavision microscope ( fig . 2a ) , a linear correlation between dna amount and intensity was obtained ( fig . 2three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5 m ) . b standard curve of fluorescence intensity ( dapi y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) three bacteriophages used as standards for the determination of dna amount based on fluorescence intensity . a dapi fluorescence image of a mixture of bacteriophages lambda , p1 , and t4 in the same field of view ( scale bar 5 m ) . b standard curve of fluorescence intensity ( dapi y axis ) and dna amount in heads of bacteriophages lambda , p1 , and t4 ( x axis ) this calibration allowed us to quantify the amount of dna in functional mitotic kinetochores . smc2 cells expressing tagged endogenous ( knock - in ) cenp - h - gfp or exogenous gfp - cenp - a were mixed with bacteriophages and processed for image acquisition . we looked for cells in which one or more kinetochores had stretched polewards , away from the body of its chromosome and where we could also acquire the three different bacteriophages in the same field of view . in each case , the gfp - labeled protein was used to define a volume envelope for the kinetochore , and the amount of dapi staining within that envelope was measured . standard curves determined for the bacteriophages in each experiment were used to calibrate the dna intensity values for the cenp - a or cenp - h - containing chromatin ( fig . it is worth emphasizing that these gfp - labeled kinetochores were functional at the time of fixation , as they were attached to microtubule bundles and exerting force towards the spindle poles . furthermore , it is important to note that while the chromatin beneath the kinetochore was abnormally stretched , the kinetochores themselves appeared structurally normal at both the light and electron microscope levels ( ribeiro et al . 2009).fig . 3
a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) . these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna . c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) . d summary table showing values obtained from the quantification done in c
a , b standard curves of the fluorescence intensity of dapi - stained bacteriophages lambda , p1 , and t4 mixed with smc2 cells expressing gfp - cenp - a ( a ) or cenp - h - gfp ( b ) . these standard curves were used to plot the fluorescence intensity of the region of interest ( roi ) corresponding to each of the gfp - tagged proteins and determine the amount of dna . c distribution of the amounts of dna within cenp - a and cenp - h regions after plotting the values onto the standard curve obtained with the three internal bacteriophage references ( graphs a and b ) . d summary table showing values obtained from the quantification done in c
a measurement of 40 centromeres in each case revealed that compact cenp - a and cenp - h domains of functional smc2-depleted kinetochores contained an average of 61 and 54 kb of dna , respectively ( range28147 kb , fig . because the kinetochores measured come from a number of different dt40 chromosomes , the observed range of dna content could be in part due to size variation between kinetochores . however , a recent study in the fukagawa lab has revealed that all of the kinetochores in dt40 cells are of similar size when measured with a variety of kinetochore markers ( t. hori and t. fukagawa , personnal communication ) . importantly , their measurements included kinetochores assembled on repetitive centromeres as well as those on nonrepetitive centromeres and on both micro and macro chromosomes . thus , the most likely explanation for the variation is that because kinetochores are pulled away from the primary constriction to differing degrees , the amount of pericentromeric chromatin immediately adjacent to the kinetochore may vary . in order to build a structural model of the vertebrate kinetochore , we need to know not only the length of chromosomal dna occupied by cenp - a but also the way in which that dna is folded in order to build a three - dimensional structure capable of withstanding the pulling forces generated by spindle microtubules . the earliest model proposed was for one or more stretches of chromatin superhelix with cenp - a facing the chromosome exterior and histone h3 facing the chromosome interior ( sullivan and karpen 2004 ) . we later proposed a multilayered patch - like model in which the chromatin fibers are folded in sinusoidal boustrophedons ( ribeiro et al . importantly , neither of these models can exclude the possibility that stretches of chromatin not associated with cenp - a might be incorporated into the folded kinetochore - associated chromatin . we have here combined gene targeting with deconvolution microscopy to estimate the amount of dna in a fully functional vertebrate kinetochore . our data indicate that functional chicken kinetochores , defined by the three - dimensional volumes occupied by cenp - a and cenp - h , contain 58 23 kb of dna . it is important to note that this amount is an estimate , as we have used a single dna - binding dye ( dapi ) , which typically shows a preference for at - rich dna . nonetheless , because the amount of dna contained within each stretched centromere is influenced by the degree of stretching , which can not be readily controlled , the values measured in the present experiments show a remarkable similarity to the 41 6 kb span of chromosomal dna occupied by cenp - a on 12 newly generated chicken neocentromeres as measured by chip - seq ( shang et al . interestingly , the amount of dna in chicken kinetochores is close to values determined by chromatin immunoprecipitation ( chip ) for human neocentromeres , which range from 54 to 464 kb [ reviewed in ( marshall et al . based on the present data as well as previous studies from our laboratory , we propose that the kinetochore is assembled from a single contiguous chromatin segment in which alternating cenp - a and h3 domains are folded into a planar sinusoidal patch , or boustrophedon . we have previously argued that the boustrophedon is composed of several layers held together by structural crosslinks that depend upon cenp - c for their formation and stability ( ribeiro et al . present studies from our group aim to determine whether other kinetochore proteins are also required to stabilize the multilayered kinetochore structure and determine the number of nucleosomes per layer . in the future , other super - resolution microscopy approaches will hopefully combine with genome three - dimensional mapping approaches ( e.g. , 3c , 4c , etc . 2013 ) ) to permit the development of more accurate models for the folding of the chromatin fiber in centromeres . the smc2 conditional knockout cell line , smc2:cenp - h : gfp and smc2:gfp : cenp - a were cultured as previously described ( ribeiro et al . condensin was depleted from smc2 conditional knockout cells expressing cenp - h - gfp or gfp - cenp - a by 30-h exposure to doxycycline , following which , cells were fixed with paraformaldehyde at 4 % . t4 , fifty microliter of each phage were washed in sm buffer ( 5 m nacl , 1 m mgso4 , 1 m tris hcl ph 7.5 ) , fixed in 4 % pfa - sm for 5 min , and resuspended in 30 l of vectashield with 1.5 g / ml dapi for 20 min . for the quantification analysis , 2 l of the stained phages in vectashield were used to mount the slides with smc2 cells . this concentration was determined empirically to give a sufficient number of bacteriophages so that each field of view containing a kinetochore undergoing an excursion would have a few bacteriophages in the background . smc2 immunostaining image stacks were acquired using a microscope ( ix-70 ; olympus ) with a charge - coupled device camera ( ch350 or hq ; photometrics ) controlled by deltavision softworx ( applied precision , llc ) and a 100 s plan apochromat na 1.4 objective using a sedat filter set ( chroma technology corp . ) and running at rt . we selected for imaging metaphase aligned cells where kinetochores were clearly resolved from the rest of the chromatin ( images of 1,024 1,024 pixels in z - stacks with a 200 nm step size ) . in the same field of view , phages were also acquired and used to define a standard curve between florescence intensity and dna amount . all files were saved as tiff files and exported to photoshop ( adobe ) for final presentation . levels were adjusted similarly for each experimental dataset to lower nonspecific background haze using the standard photoshop adjust levels tool . after deconvolution , we measured the dapi content in the volume occupied by cenp - a or cenp - h . image planes where kinetochore excursions were observed were sum - quick projected . regions of interest ( roi ) covering the phage area were selected by hand , and sum intensity value was annotated . the same roi was moved between 5 and 10 pixels , and the intensity was annotated as background . the intensity values for all phages in each experiment were entered in graphpad prism ( graphpad software , inc . ca , usa ) where a xy graph was plotted and the best - fit linear regression calculated representing the linear standard of amount of dna versus intensity . for the smc2 cells , separated tiff files of the 488 and 528 channels were opened in image - pro plus ( media cybernetics , inc . , bethesda , usa ) . using the 528-nm image , a roi covering the area covered by the pulled centromere was selected . this roi was copied and loaded into the 488-nm file where the sum value of intensity was determined . again , the background value was determined by moving the roi 510 pixels away from the metaphase plate . the absolute amount of dna was calculated according to the equation of the previously determined best - fit linear regression . the line profile was drawn in imagej ( research national institute of mental health , bethesda , usa ) values exported to excel ( microsoft , redmond , wa ) and graphs of each channel superimposed . the smc2 conditional knockout cell line , smc2:cenp - h : gfp and smc2:gfp : cenp - a were cultured as previously described ( ribeiro et al . condensin was depleted from smc2 conditional knockout cells expressing cenp - h - gfp or gfp - cenp - a by 30-h exposure to doxycycline , following which , cells were fixed with paraformaldehyde at 4 % . fifty microliter of each phage were washed in sm buffer ( 5 m nacl , 1 m mgso4 , 1 m tris hcl ph 7.5 ) , fixed in 4 % pfa - sm for 5 min , and resuspended in 30 l of vectashield with 1.5 g / ml dapi for 20 min . for the quantification analysis , 2 l of the stained phages in vectashield were used to mount the slides with smc2 cells . this concentration was determined empirically to give a sufficient number of bacteriophages so that each field of view containing a kinetochore undergoing an excursion would have a few bacteriophages in the background . smc2 immunostaining image stacks were acquired using a microscope ( ix-70 ; olympus ) with a charge - coupled device camera ( ch350 or hq ; photometrics ) controlled by deltavision softworx ( applied precision , llc ) and a 100 s plan apochromat na 1.4 objective using a sedat filter set ( chroma technology corp . ) and running at rt . we selected for imaging metaphase aligned cells where kinetochores were clearly resolved from the rest of the chromatin ( images of 1,024 1,024 pixels in z - stacks with a 200 nm step size ) . in the same field of view , phages were also acquired and used to define a standard curve between florescence intensity and dna amount . all files were saved as tiff files and exported to photoshop ( adobe ) for final presentation . levels were adjusted similarly for each experimental dataset to lower nonspecific background haze using the standard photoshop adjust levels tool . after deconvolution , we measured the dapi content in the volume occupied by cenp - a or cenp - h . image planes where kinetochore excursions single planes where phages were observed were selected for each image acquired . for the phage , the area was defined in the dapi channel . regions of interest ( roi ) covering the phage area were selected by hand , and sum intensity value was annotated . the same roi was moved between 5 and 10 pixels , and the intensity was annotated as background . the intensity values for all phages in each experiment were entered in graphpad prism ( graphpad software , inc . ca , usa ) where a xy graph was plotted and the best - fit linear regression calculated representing the linear standard of amount of dna versus intensity . for the smc2 cells , separated tiff files of the 488 and 528 channels were opened in image - pro plus ( media cybernetics , inc . , bethesda , usa ) . using the 528-nm image , a roi covering the area covered by the pulled centromere was selected . this roi was copied and loaded into the 488-nm file where the sum value of intensity was determined . again , the background value was determined by moving the roi 510 pixels away from the metaphase plate . the absolute amount of dna was calculated according to the equation of the previously determined best - fit linear regression . the line profile was drawn in imagej ( research national institute of mental health , bethesda , usa ) values exported to excel ( microsoft , redmond , wa ) and graphs of each channel superimposed . | in order to understand the three - dimensional structure of the functional kinetochore in vertebrates , we require a complete list and stoichiometry for the protein components of the kinetochore , which can be provided by genetic and proteomic experiments .
we also need to know how the chromatin - containing cenp - a , which makes up the structural foundation for the kinetochore , is folded , and how much of that dna is involved in assembling the kinetochore . in this ms , we demonstrate that functioning metaphase kinetochores in chicken dt40 cells contain roughly 50 kb of dna , an amount that corresponds extremely closely to the length of chromosomal dna associated with cenp - a in chip - seq experiments .
thus , during kinetochore assembly , cenp - a chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin . |
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induction of anesthesia has been identified as one of the distressing aspects of pediatric surgery . it has been reported that anxiety levels of children remains high throughout the perioperative period , however , anxiety levels peak when the anesthesia care provider introduces the face mask and begins the induction of anesthesia . elevated children 's preoperative anxiety is associated with negative behaviors including separation anxiety , nightmares , aggression toward authorities , nocturnal enuresis , and eating disorders , and may increase postoperative analgesic requirements prolonging postoperative recovery process , which may be emotionally traumatic for children and their parents . hence , minimizing children 's as well as their parents surgical anxiety at the time of anesthetic induction may reduce adverse psychological and physiological outcomes , and is considered to be an important care , especially for advanced practice nurses . today both pharmacologic and nonpharmacologic interventions are available to reduce children 's preoperative anxiety during induction of anesthesia . in pharmacologic methods , large doses of sedatives and premedication are administered preoperatively that are regarded by many as undesirable because they may create unnecessary risks , are time - consuming , and may be associated with delayed emergence from anesthesia . therefore , currently there is great motivation towards nonpharmacological interventions aimed at reducing anxiety for children and their families , such as animated cartoons , video games , educational programs , hypnosis , clown doctors , as well as parental presence during induction of anesthesia ( ppia ) . among the abovementioned nonpharmacologic methods , ppia has been the most frequently studied intervention in recent years for reducing children 's preoperative anxiety . researches have shown that most parents prefer to be present during induction of anesthesia , believe that their presence make the anesthesiologist 's job easier , and consider it helpful to reduce their children 's preoperative anxiety . despite the benefits of ppia for both children and parents , clinical evidence does not support the routine use of this method during anesthesia induction , and there are differing views among health professionals on whether parents should be present at anesthesia induction . in addition , results of recent clinical trials , where parents were randomly assigned to be present or absent during anesthetic induction , have not been positive and no differences in children 's preoperative anxiety were observed between parental presence or absence groups . a recent cochrane review of nonpharmacological methods to reduce children 's preoperative anxiety showed that ppia did not reduce children 's preoperative anxiety compared with not having a parent present in 5 trials , and in 3 trials , no clear difference was observed in children 's preoperative anxiety whether a parent was present or not . in another review study , ppia alone was the least effective means of decreasing a child 's anxiety during the induction of anesthesia compared to premedication midazolam and behavioral distraction . in light of the differing opinions and varied and controversial research findings and because much of the research has been focusing on the effects of ppia only on children 's preoperative anxiety and very few emphasizing its effects on the parents , we decided to assess the effects of ppia on the preoperative anxiety of both the children and their parents . this study was a randomized clinical trial and was registered on the iranian registry of clinical trials ( irct ) with registration number irct2013100612830n2 . participants in this study included 60 children , aged 210 years , who underwent minor - medium elective surgical procedures with an indication of general anesthesia at busheher educational hospitals along with their parents from january 9 to april 16 2014 . inclusion criteria were having a parent present during the admission and hospitalization and american society of anesthesiologists ( asa ) physical status i and ii . children with privious surgeries and history of central nervous system diseases , psychiatric diseases , and neurological or cognitive impairments or diseases were excluded from the study . the sample size was computed based on a previous study using clinical trial formula and analysis of variance estimates . given a medium effect size ( i.e. , f = 0.25 ) , a power of 80% , and an alpha statistic of 0.05 , approximately 30 samples were calculated in each group . for collecting demoghraphic and clinical data , a researcher - made questionnaire was used including sex , age , birth order , parental education , kind of surgery , and parent who accompanied the children to the operating room ( or ) . modified - yale preoperative anxiety scale ( m - ypas ) was used to measure children 's preoperative anxiety . this observational behavioral scale was developed by kain et al . to measure the anxiety state of young children . it contains 27 items divided into 5 categories including activity , emotional expressivity , state of arousal , vocalization , and use of parents . each category receives a score on a scale of 4 ( 6 for vocalization ) according to the behavior of children . the m - ypas score ranges from 23100 , with higher scores indicating greater anxiety . this scale has good reliability and validity for measuring children 's preoperative anxiety in the preoperative holding area and during the induction of anesthesia . for assessing parents anxiety , this self - report anxiety instrument contains two separate subscales that measure trait ( baseline ) and state ( situational ) anxiety . state anxiety has been defined as an emotional status characterized by feelings of apprehension and tension with changes in the level of activity of the autonomous nervous system , which can alter overtime . trait anxiety is related to the stable aspects of the individual 's personality , which tend to generate anxiety . for the purpose of this study , each response is scored between 14 points ( 1 meaning not at all and 4 meaning very much ) . once a child met the study inclusion criteria , the researcher contacted the parents by telephone and asked about their interest in having their child participate in the study . if the parents were willing , the researcher arranged to meet the parent(s ) and child on the day of the surgery . approximately 60 min before the children 's surgery , the researcher met both the child and the parent(s ) to obtain parental written informed consent for the child 's participation in the study and to obtain the child 's verbal assent . after that , demographic data about the children and parents were obtained and they were randomly assigned based on surgical types ( ear , nose , and throat [ ent ] surgeries and nonent surgeries ) and age group ( 25 and 610 years ) to parent present group ( n = 30 ) and parent absent group ( n = 30 ) . a computer - generated list of random numbers was used by an individual outside the study to generate the allocation , which was concealed by the use of security envelopes . the children in the parent absent group ( control group ) were taken to the or alone , while those in the parent present group ( intervention group ) were taken to the or with one of their parent . the decision as to which parent would accompany the child was left to them . in this study , parents were not prepared beforehand for accompanying the child into the or . it was then explained to them that their presence in the or may be beneficial for their child , and explained how to accompany them in the or . after routine monitoring , all children 's noninvasive blood pressures , oxygen saturation values , and heart rates were recorded in both the groups . anesthesia was induced using 60% nitrous oxide in oxygen and sevoflurane 68% via a mask and vecuronium 0.1 mg / kg was administered to facilitate orotracheal intubation . when the anesthetic mask was placed on the children 's face ( induction ) , the children 's anxiety was assessed using the m - ypas by researcher . as soon as anesthesia was induced , parents in the intervention group were escorted to the waiting area and were asked to fill the stai in the presence of the researcher . quantitative variables were shown as mean and standard deviation , and qualitative variables were represented as number of frequency and their percentage . to examine differences in qualitative and quantitative variables among the two groups , we used chi - square and independent sample t - test , respectively . statistical analysis of data was done using the statistical package for the social sciences software version 18 ( spss , inc . the trial was approved by a research ethics board at busheher university of medical sciences ( busheher , iran ) , and written informed consents were obtained from all participants . this study was a randomized clinical trial and was registered on the iranian registry of clinical trials ( irct ) with registration number irct2013100612830n2 . participants in this study included 60 children , aged 210 years , who underwent minor - medium elective surgical procedures with an indication of general anesthesia at busheher educational hospitals along with their parents from january 9 to april 16 2014 . inclusion criteria were having a parent present during the admission and hospitalization and american society of anesthesiologists ( asa ) physical status i and ii . children with privious surgeries and history of central nervous system diseases , psychiatric diseases , and neurological or cognitive impairments or diseases were excluded from the study . the sample size was computed based on a previous study using clinical trial formula and analysis of variance estimates . given a medium effect size ( i.e. , f = 0.25 ) , a power of 80% , and an alpha statistic of 0.05 , approximately 30 samples were calculated in each group . for collecting demoghraphic and clinical data , a researcher - made questionnaire was used including sex , age , birth order , parental education , kind of surgery , and parent who accompanied the children to the operating room ( or ) . modified - yale preoperative anxiety scale ( m - ypas ) this observational behavioral scale was developed by kain et al . to measure the anxiety state of young children . it contains 27 items divided into 5 categories including activity , emotional expressivity , state of arousal , vocalization , and use of parents . each category receives a score on a scale of 4 ( 6 for vocalization ) according to the behavior of children . the m - ypas score ranges from 23100 , with higher scores indicating greater anxiety . this scale has good reliability and validity for measuring children 's preoperative anxiety in the preoperative holding area and during the induction of anesthesia . for assessing parents anxiety , spielberg state - trait anxiety inventory ( stai ) this self - report anxiety instrument contains two separate subscales that measure trait ( baseline ) and state ( situational ) anxiety . state anxiety has been defined as an emotional status characterized by feelings of apprehension and tension with changes in the level of activity of the autonomous nervous system , which can alter overtime . trait anxiety is related to the stable aspects of the individual 's personality , which tend to generate anxiety . for the purpose of this study , each response is scored between 14 points ( 1 meaning not at all and 4 meaning very much ) . once a child met the study inclusion criteria , the researcher contacted the parents by telephone and asked about their interest in having their child participate in the study . if the parents were willing , the researcher arranged to meet the parent(s ) and child on the day of the surgery . approximately 60 min before the children 's surgery , the researcher met both the child and the parent(s ) to obtain parental written informed consent for the child 's participation in the study and to obtain the child 's verbal assent . after that , demographic data about the children and parents were obtained and they were randomly assigned based on surgical types ( ear , nose , and throat [ ent ] surgeries and nonent surgeries ) and age group ( 25 and 610 years ) to parent present group ( n = 30 ) and parent absent group ( n = 30 ) . a computer - generated list of random numbers was used by an individual outside the study to generate the allocation , which was concealed by the use of security envelopes . the children in the parent absent group ( control group ) were taken to the or alone , while those in the parent present group ( intervention group ) were taken to the or with one of their parent . the decision as to which parent would accompany the child was left to them . in this study it was then explained to them that their presence in the or may be beneficial for their child , and explained how to accompany them in the or . after routine monitoring , all children 's noninvasive blood pressures , oxygen saturation values , and heart rates were recorded in both the groups . anesthesia was induced using 60% nitrous oxide in oxygen and sevoflurane 68% via a mask and vecuronium 0.1 mg / kg was administered to facilitate orotracheal intubation . when the anesthetic mask was placed on the children 's face ( induction ) , the children 's anxiety was assessed using the m - ypas by researcher . as soon as anesthesia was induced , parents in the intervention group were escorted to the waiting area and were asked to fill the stai in the presence of the researcher . quantitative variables were shown as mean and standard deviation , and qualitative variables were represented as number of frequency and their percentage . to examine differences in qualitative and quantitative variables among the two groups , we used chi - square and independent sample t - test , respectively . statistical analysis of data was done using the statistical package for the social sciences software version 18 ( spss , inc . the trial was approved by a research ethics board at busheher university of medical sciences ( busheher , iran ) , and written informed consents were obtained from all participants . of the 136 participants who were eligible to participate , 22 did not meet the inclusion criteria , 42 declined to participate , and 12 could not participate for other reasons ( e.g. , the or was running ahead of schedule ) . of the remaining 60 participants , 30 were randomly assigned to the control group and 30 were randomly assigned to the intervention group . of the 60 parents who participated , all adhered to the study protocol . hence , data from all 60 children and their parents were included in the analyses [ figure 1 ] . summary of participant 's follow up most children in both the intervention and control groups were females . mean and sd of children age in the intervention and control groups were 5.11 2.30 and 5.81 2.32 years , respectively . children in the control and intervention groups were similar with respect to their ages and baseline characteristics , and chi - square test showed no significant difference in this regard [ table 1 ] . there was also an even distribution of children across the four randomization groups ( i.e. , ages 25 : ent surgery ; ages 610 : ent surgery ; ages 25 : nonent surgery ; and ages 610 : nonent surgery ) , indicating that the sample was equally distributed across age groups and surgical types . among the ent groups , procedures consisted mainly of tonsillectomy ( n = 16 ) , adenoidectomy ( n = 9 ) , and myringotomy ( n = 5 ) . among the nonent groups , the most common procedures were hernia repairs ( n = 9 ) , circumcisions ( n = 11 ) and orchidopexy ( n = 10 ) . demographic and clinical characteristics of children in intervention and control groups regarding children 's anxiety subscales , chi - square test indicated no significant difference between the two groups in activity ( p = 0.601 ) , vocalization ( p = 0.632 ) , and emotional expression ( p = 0.612 ) subscales , whereas a signifficant diference was observed between the two groups in state of arousal ( p = 0.033 ) and use of parents ( p = 0.031 ) subscales [ table 2 ] . based on independent t - test , no significant difference was seen between children 's preoperative anxiety in the intervention and control groups ( p = 0.621 ) . moreover , results showed no significant difference between the intervention and control groups regarding state ( p = 0.056 ) , trait ( p = 0.826 ) , and total ( p = 0.208 ) parents anxiety [ table 3 ] . comparison of children 's preoperative anxiety ( modified - yale preoperative anxiety scale ) in intervention and control groups comparison of children 's and parents anxiety in intervention and control groups of the 136 participants who were eligible to participate , 22 did not meet the inclusion criteria , 42 declined to participate , and 12 could not participate for other reasons ( e.g. , the or was running ahead of schedule ) . of the remaining 60 participants , 30 were randomly assigned to the control group and 30 were randomly assigned to the intervention group . of the 60 parents who participated , all adhered to the study protocol . hence , data from all 60 children and their parents were included in the analyses [ figure 1 ] . most children in both the intervention and control groups were females . mean and sd of children age in the intervention and control groups were 5.11 2.30 and 5.81 2.32 years , respectively . children in the control and intervention groups were similar with respect to their ages and baseline characteristics , and chi - square test showed no significant difference in this regard [ table 1 ] . there was also an even distribution of children across the four randomization groups ( i.e. , ages 25 : ent surgery ; ages 610 : ent surgery ; ages 25 : nonent surgery ; and ages 610 : nonent surgery ) , indicating that the sample was equally distributed across age groups and surgical types . among the ent groups , procedures consisted mainly of tonsillectomy ( n = 16 ) , adenoidectomy ( n = 9 ) , and myringotomy ( n = 5 ) . among the nonent groups , the most common procedures were hernia repairs ( n = 9 ) , circumcisions ( n = 11 ) and orchidopexy ( n = 10 ) . demographic and clinical characteristics of children in intervention and control groups regarding children 's anxiety subscales , chi - square test indicated no significant difference between the two groups in activity ( p = 0.601 ) , vocalization ( p = 0.632 ) , and emotional expression ( p = 0.612 ) subscales , whereas a signifficant diference was observed between the two groups in state of arousal ( p = 0.033 ) and use of parents ( p = 0.031 ) subscales [ table 2 ] . based on independent t - test , no significant difference was seen between children 's preoperative anxiety in the intervention and control groups ( p = 0.621 ) . moreover , results showed no significant difference between the intervention and control groups regarding state ( p = 0.056 ) , trait ( p = 0.826 ) , and total ( p = 0.208 ) parents anxiety [ table 3 ] . comparison of children 's preoperative anxiety ( modified - yale preoperative anxiety scale ) in intervention and control groups comparison of children 's and parents anxiety in intervention and control groups despite the fact that most parents and children prefer to stay together during procedures such as induction of anesthesia , our results showed that the mean score of parents and children 's anxiety were not significantly different between the two studied groups . to date , a few randomized controlled trials have found ppia to be an effective anxiety - reducing intervention for children and their parents when compared with the parent absent group . consistent with our results , in a recent clinical trial study conducted by bailey et al . among 93 children aged 210 years , the effectiveness of ppia in reducing children 's preoperative anxiety was not improved by the intervention at the holding stage ( p = 0.15 ) , the point at which the family left the holding area ( p = 0.39 ) , the point that they entered the or ( p = 0.28 ) , and the point at which the anesthesia mask was introduced ( p = 1.3 ) . in a prospective trial done by kim et al . among 117 children aged 27 years scheduled for minor elective surgery , parental presence during induction of sevoflurane anesthesia caused no changes in the mypas scores from baseline to induction among the studied groups ( p = 0.049 ) . in another study performed by wright et al . among 61 children aged 36 years , no significant difference was observed between parental presence and absence groups in children 's anxiety during induction of anesthesia , whereas at the time - point when children was typically separated from parents , preoperative children 's anxiety was found to be significantly higher in the parental absence group than in the parental presence group . regarding parent 's anxiety during induction of anesthesia , investigations are scant . in agreement with our results , akinci et al . reported that maternal presence during induction had no effects in reducing the mother 's anxiety . in another study conducted by kain et al . among 80 parents and their children undergoing elective outpatient surgery , there were no significant differences in parents self - reported anxiety , as measured by the stai , between parental presence group and control group . first , all instructions that nursing staff , residents , and anesthesiologists gave to the parents during parents preparation and before parents being led into the or with their child may have impacted the results . to reduce this type of limitation , future studies should quantify the amount of time health care providers spend preparing families and/or measuring their understanding of the preparatory information they receive . second , all parents in this study were given the option to be present for their child 's anesthesia induction regardless of the level of their own or their child 's anxiety . moreover , when parents entered the or , they were often explained to sit on a chair besides their child and were told they were allowed to hold their child 's hand . these explanations may have directed the behaviors of the parents and may have impacted child 's anxiety during anesthetic induction . based on previous research , anxious parents do not benefit anxious children and they actually increase anxiety in calm children during anesthesia induction . in a study , kain et al . reported that presence of a calm parent benefits an anxious child during induction of anesthesia and the presence of an overly anxious parent has no benefit . similarly , messeri et al . have recently indicated that anxious parents increase the stress of anesthetic induction in children , even those who have been premeditated . it is suggested that parents were allowed to use their own coping strategies during the preoperative period . hence , future studies in this area are needed to clarify the effects of this intervention in pediatric populations . this study has adopted from msc dissertation of razie rasti - emad - abadi with no . 147/71/20 this study has adopted from msc dissertation of razie rasti - emad - abadi with no . 147/71/20 that was supported by bushehr university of medical sciences . | introduction : parental presence during induction of anesthesia ( ppia ) has been a controversial issue , with some studies showing its effects on reducing anxiety . hence , this study aimed to investigate the effects of ppia on preoperative anxiety of children as well as their parents.materials and methods : this clinical trial was conducted among 60 children aged 210 years and their parents .
children were randomly assigned to intervention ( n = 30 ) and control ( n = 30 ) groups . children in the control group were taken to the operating room ( or ) alone , while those in the intervention group were taken to the or with one of their parents . when the anesthetic mask was placed on the children 's face ( induction ) , the children 's preoperative anxiety in both groups was assessed using modified - yale preoperative anxiety scale ( m - ypas ) , and after that the parents in the intervention group were escorted to the waiting area .
parents anxiety in both the groups was measured by the spielberg state - trait anxiety inventory ( stai ) in the waiting area .
data were analyzed using descriptive and inferential ( independent t - test and chi - square test ) statistic methods through the statistical package for the social sciences version 18 software.results:results showed no significant difference between children 's anxiety in the intervention ( 70.83 ) and control ( 70.39 ) groups in the preanesthetic period .
in addition , no significant difference was seen between the intervention ( 79.23 ) and control ( 85.86 ) groups regarding total parents anxiety.conclusions:ppia was not successful in reducing the children 's preoperative anxiety as well as parents anxiety .
future studies in this area are needed to clarify the effects of this intervention in pediatric populations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
auditory neuropathy spectrum disorder ( ansd ) can be defined as abnormal function of the vestibulocochlear nerve viii cranial pair that generates loss of synchrony in the nerve conduction of the sound stimulus and is very probably related to a change in the myelination of these fibers.1
the aim of the study was to monitor the hearing handicap and the recognition threshold of sentences in quiet and in the presence of noise in a patient with ansd using a hearing aid ( ha ) . the treatment for ansd has not been fully established , including the use of an ha , a cochlear implant ( ci ) , and a brainstem implant.2 the use of an ha may be contraindicated in ansd because if outer hair cells are preserved , they may be damaged by amplification.3 in adults with ansd , an ha only improves sound detection by amplification , with a minimum benefit for discrimination.4 in other studies , however , good responses have been observed with the use of both ha and ci , with improved hearing and communication skills , the latter of which is one of the main complaints of these patients.5
the present case is notable because few cases of unilateral ansd have been reported in the literature.6 a 47-year - old woman reported bilateral hearing loss , worse in the right ear and the presence of tinnitus of the whistling and buzzing type in this same ear , along with marked speech discrimination difficulty in the presence of noise and discomfort in the presence of intense sounds . conventional pure tone audiometry and high - frequency audiometry revealed mean hearing thresholds ( 500 hz to 4 khz ) within normal limits , with a descending configuration in the left ear and moderate sensorineural hearing loss in the right ear ( figs . 1 and 2 ) .
pure tone audiometry . abbreviations : dbhl , decibels hearing levels ; o , air conduction , right ear ; x , air conduction , left ear ; < , bone conduction , right ear ; > , bone conduction , left ear .
high - frequency audiometry . abbreviations : dbhl , decibels hearing levels ; o , air conduction , right ear ; x , air conduction , left ear ; < , bone conduction , right ear ; > , bone conduction , left ear speech recognition threshold was 55-db hearing level ( dbhl ) in the right ear and 10 dbhl in the left ear . speech discrimination index score was 56% for monosyllables and 68% for disyllables in the right ear and 92% for monosyllables in the left ear . immittance testing revealed bilateral type a curve and absence of ipsilateral and contralateral acoustic reflexes at all frequencies . otoacoustic emissions ( oae ) testing documented the presence of transient oae in both ears . audiometry brainstem response ( abr ) testing showed morphology , absolute latency , and interwave latency within normal limits in the left ear . abnormal morphology was observed in the right ear at 100 dbhl , with the presence of only the v wave and prolonged absolute latency . the results suggested a change in the functioning of the auditory pathway up to the inferior colliculus on the right . nuclear magnetic resonance sequences for the visualization of the eighth pair and of the inner ear showed normal aspect , with no evidence of bilateral expansive lesions and no pathologic reinforcement . recognition threshold of sentences in silence ( rtss ) and in noise ( rtsn ) were obtained7 : for rtss , a 1.9-db increase was observed during the first month of ha adaptation and an improvement of 3.6 db and 2.1 db in the subsequent months compared with rtss before ha adaptation . for rtsn , there was a 5.6-db improvement in the first and second months after ha use and a 6.0-db improvement 3 months later . these results demonstrate a progressive improvement in the signal - to - noise ratio ( s / n ) , which progressed from + 3.66 to 2.4 db . sentences and noise were on a cd in independent channels and were presented using a cd player coupled to an audiometer . abbreviations : lrss , recognition threshold of sentences in silence ; lrsr , recognition threshold of sentences in noise ; % , percentage of correct sentences ; s / r , signal - to - noise ratio . the hearing handicap inventory for adults ( hhia ) was applied in the interview format before ha adaptation and after 3 months of ha use.8 the evaluation of perception of hearing handicap before ha adaptation demonstrated that all responses were attributed to the always option regarding both the emotional and social subscales , corresponding to a severe degree of perception ( 100 points ) . three months after ha adaptation , there was a 40-point reduction of hearing handicap , although perception continued to be of a severe degree . the patient had follow - up once a month for 4 months for guidance on listening skills , use of lipreading , and hearing aid use . the percentage rate of speech recognition , the combined results of abnormal abr , and the presence of transient oae in the right ear , together with the patient 's complaints , support the evidence of hearing loss due to retrocochlear , and not cochlear , changes.1
9 there was hearing loss with changes in speech recognition that limited both the affective - emotional and social life of the patient , who reported social isolation during anamnesis and in her responses to the hhia . although the perception of the hearing handicap continued to be strongly present 3 months after adaptation of the ha , the patient reported a clear improvement and was able to return to activities that she long had been unable to perform . however , she continued to report discomfort in the presence of intense sounds and noise . positive5
10 and negative3
4 reports have been published in the literature regarding the use of an ha in ansd . in the present case , the use of an ha was of significant benefit to the patient , with an important improvement of rtss , rtsn , and s / n ratio in a free field . we did not find any study using the sentence list test in portuguese to evaluate patients with ansd,7 and therefore we can not compare our findings . the positive result in the present case reflects the importance for an individual with ansd to understand speech in silence and in noise , as these tests permit an analysis of auditory skills within a context close to the daily auditory experiences.11
the lack of improvement in rtss 30 days after the use of the ha may be explained by acclimatization.8 in contrast , an important improvement in rtsn was observed as early as the first month of ha use . thus , we believe that ha use in this patient with ansd mainly contributed to the relief of the major complaint of affected individuals ( i.e. , auditory speech perception in a noisy environment).5
10
12
13
the present findings agree with previous reports that suggested that , if the use of the ha shows some benefit in free field tests , in speech perception tests , or in terms of the user 's self - evaluation , its use should be considered positive.5
10
12 on this basis , new strategies should be added or modified to improve speech comprehension and communication . after 3 months of ha use , the patient showed a significant reduction of hearing handicap and an improvement in rtss , rtsn , and s / n ratio . | introduction treatment for auditory neuropathy spectrum disorder ( ansd ) is not yet well established , including the use of hearing aids ( has ) .
not all patients diagnosed with asnd have access to has , and in some cases has are even contraindicated .
objective to monitor the hearing handicap and the recognition threshold of sentences in silence and in noise in a patient with asnd using an ha
.
resumed report a 47-year - old woman reported moderate sensorineural hearing loss in the right ear and high - frequency loss of 4 khz in the left ear , with bilateral otoacoustic emissions .
auditory brainstem response suggested changes in the functioning of the auditory pathway ( up to the inferior colliculus ) on the right .
an ha was indicated on the right .
the patient was tested within a 3-month period before the ha fitting with respect to recognition threshold of sentences in quiet and in noise and for handicap determination .
after ha use , she showed a 2.1-db improvement in the recognition threshold of sentences in silence , a 6.0-db improvement for recognition threshold of sentences in noise , and a rapid improvement of the signal - to - noise ratio from + 3.66 to 2.4 db when compared with the same tests before the fitting of the ha .
conclusion there was a reduction of the auditory handicap , although speech perception continued to be severely limited .
there was a significant improvement of the recognition threshold of sentences in silence and in noise and of the signal - to - noise ratio after 3 months of ha use . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
an estimated one - third of the world s population is currently infected with mycobacterium tuberculosis ( m. tuberculosis ) . iran is located in a tuberculosis ( tb ) endemic region in which there are 2 countries with a high prevalence of tb . the majority of people harbor the bacteria as a latent tuberculosis infection ( ltbi ) , and even though they do not show the symptoms of active tbi , they are potentially vulnerable to progression to tb disease . the tuberculin skin test ( tst ) for several years was the only way to diagnose ltbi . nonetheless , because the tst had low specificity and sensitivity and was difficult to perform , it was necessary that a more accurate method be devised . the m. tuberculosis genome was sequenced in the late 1990s and it later became clear that the section of the bcg genome was deleted . two antigens of this part , namely early secretory antigenic target-6 ( esat-6 ) and culture filtrate protein-10 ( cfp-10 ) , are good candidates for stimulating t cells in persons who are infected with m. tuberculosis in order to stimulate the production of interferon- ( ifn- ) and other cytokines such as interleukin-2 ( il-2 ) . ifn- is measured by methods such as enzyme - linked immunospot assay ( elispot ) and is drawn upon to invent new methods for diagnosing tbi . almost 30% of individuals in contact with patients suffering from active tbi exhibit some symptoms of this infection , and half of them will develop the disease in the first 2 years . previous studies have shown that ltbi is more prevalent in persons in close contact with patients suffering from active tbi . thus , the identification and treatment of individuals with ltbi can prevent the progression of the disease to active tbi . since the distinction between ltbi and active tbi is impossible , a test that could distinguish between ltbi and active tbi will be very useful . several antigens of m. tuberculosis in addition to esat-6 and cfp-10 are currently used for this purpose . l - alanine dehydrogenase ( aladh ) is one of these antigens , and it is involved in the metabolism of nitrogen and the adaptation of m. tuberculosis in anaerobic conditions . in a research performed in our laboratory , esat-6 , cfp-10 , and it has been shown that esat-6/cfp-10 fusion antigens are valuable for the diagnosis of active tbi . also , recent research has demonstrated that the analysis of il-2 might help to distinguish active tbi from ltbi . therefore , in the current study , we aimed to analyze ifn- and il-2 responses to aladh and esat-6/cfp-10 fusion antigens in the diagnosis of ltbi vs. active tbi . this exploratory study was performed in the iranian city of shiraz from 2014 to 2015 . they had a positive tst , confirmed by the examination of the sputum for acid - fast bacilli and/or culturing . all the blood samples were obtained from the tb center of shiraz university of medical sciences . the mean age was 33 years ( range=2159 y ) , and there were 16 male individuals in this group . the 2nd group ( n=33 ) comprised individuals with ltbi who were selected from the household contacts of the 1st group and had a positive tst with no clinical or radiographic evidence of active tbi . the mean age was 31 years ( range=2060 y ) , and there were 16 male individuals in this group . the 3rd group ( n=33 ) was made up of healthy controls with a negative tst and without known contact with patients suffering from m. tuberculosis . the mean age was 34 years ( range=1956 y ) , and there were 17 male individuals in this group . all the patients and normal subjects gave written informed consent ahead of participation in the study . demographic characteristics of the total study population tbi : tuberculosis infection ; ltbi : latent tuberculosis infection ; tst : tuberculin skin test the gene of the aladh protein was amplified by polymerase chain reaction ( pcr ) from m. tuberculosis h37rv chromosomal dna , which was obtained from razi vaccine institute and serum research institute in tehran . the 2 primers used were upper : 5 -cgg ggt acc atg cgc gtc ggt att c -3 and down : 5-ccc aag ctt aca ggc cag cac -3. pcr amplification was performed with 35 cycles at 94 c for 45 seconds , 66 c for 45 seconds , 72 c for 45 seconds , and 72 c for 10 minutes . the pcr product was ligated to the pet32a vector , which is commercially available from novagen . this vector permits the genes of interest to be fused to the thioredoxin fusion protein , trxa , for high levels of expression with good solubility and it also contains cleavable his - tag sequences for detection and purification . it was thereafter transferred into escherichia coli ( e. coli ) strain dh5 , which was grown in a luria bertani ( lb ) medium ( gibco life technologies pa , uk ) containing ampicillin ( gibco life technologies pa , uk ) ( 100 g / ml ) . the recombinant plasmids were purified from the positive clones , and the pcr constructs were sequenced to verify their integrity . e. coli bl21 was transformed by pet32a and plated on an lb solid medium . an overnight culture ( 600 ml ) of the resulting strain was used to inoculate 12 l of lb , and when it was in the mid - log phase ( absorbance 600 reached 0.7 ) , it was induced by isopropyl - d - thiogalacto - pyranoside ( iptg , 0.5 mm ) ( fermentas ab , vl , lithuania ) . the bacterial cells were harvested by centrifugation , and the cell pellets were frozen at -70 c . the expression of the recombinant protein was investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds page ) and western blotting . the cell pellets were re - suspended with a lysis buffer containing phosphate - buffered saline , 1 mm of edta ( ph=8.0 ) , and 1% ( v / v ) triton x-100 . the cell lysates were sonicated ( 30-s pulses at 20-s intervals for 6 times ) , and the suspension was centrifuged . page analysis was performed by utilizing mini format vertical electrophoresis ( bio - rad , hercules , ca , usa ) . accordingly , the sds page - separated proteins were electrotransferred into nitrocellulose membranes ( millipore , bedford , ma usa ) using a mini trans - blot cell ( bio - rad , hercules , ca , usa ) . the membranes were incubated with horseradish peroxidase - conjugated anti - poly - his antibody ( santa cruz , dallas , tx , usa ) . pet32a supports the his - tag cloning system ; therefore , the recombinant protein was purified by the ni - nta affinity column ( qiagen gmbh , hilden , germany ) under denaturing and native conditions . after the sample was loaded on the column and extensive washing steps were performed with 8-m urea ( sigma , st . louis , mo , usa ) ( ph=7.4 ) , elution by using a linear 10500 mm gradient of imidazole ( sigma , st . then , the his - tagged protein was dialyzed against phosphate - buffered saline for 48 hours , while the buffer was changed every 12 hours to remove the urea and imidazole . the bicinchoninic acid method was used to assay the protein concentration ( pierce , rockford , il , usa ) . ifn- and il-2 elispot assays were performed for the quantification of the responses of the t cells to recombinant aladh and esat-6/cfp-10 antigens , which were produced in our lab previously , by utilizing a commercially available elispt kit ( u - cytech , utrecht , netherlands ) . subsequently , the peripheral blood mononuclear cells ( pbmcs ) of the samples were separated with ficoll ( sigma , st . louis , mo , usa ) , and the number of the cells was quantified . multiscreen 96-well plates ( millipore , bedford , ma , usa ) were individually pre - coated with anti - ifn- and anti - il-2 antibodies ( 10 g / ml ) and incubated overnight at 4 c . the mycobacterial antigens ( 1 g / ml ) and pbmcs ( 210 cells / well ) were added to each well . louis , mo , usa ) as a positive control were applied and incubated for 24 hours for ifn- and 72 hours for il-2 at 37 c . the wells were washed , and biotinylated anti - ifn- , anti - il-2 , and -labeled goat anti - biotin antibodies were added separately . finally , the spots were revealed by activators i and ii , which display ifn- and il-2 secreting cells , followed by the quantification of the spots using a stereomicroscope . the number of the spots from the unstimulated wells was subtracted from the number in each well . the mean of the duplicates was applied , and all the results were expressed as the number of spot - forming cells ( sfc ) per million pbmcs . the results of the shapiro wilk test for all the groups showed that the variables did not have a normal distribution . accordingly , the differences between the groups were assessed with the kruskal wallis and mann whitney tests for nonparametric data analysis . the receiver operating characteristic ( roc ) curve was plotted in order to find out the optimum cutoff for the elispot assays of il-2 and ifn- responses in discriminating individuals with active tbi from those with ltbi and also to determine the sensitivity and the specificity of these assays . ethical approval for the study was granted by the research ethics committees of shiraz university of medical sciences , and informed consent was obtained from all the participants . this exploratory study was performed in the iranian city of shiraz from 2014 to 2015 . they had a positive tst , confirmed by the examination of the sputum for acid - fast bacilli and/or culturing . all the blood samples were obtained from the tb center of shiraz university of medical sciences . the mean age was 33 years ( range=2159 y ) , and there were 16 male individuals in this group . the 2nd group ( n=33 ) comprised individuals with ltbi who were selected from the household contacts of the 1st group and had a positive tst with no clinical or radiographic evidence of active tbi . the mean age was 31 years ( range=2060 y ) , and there were 16 male individuals in this group . the 3rd group ( n=33 ) was made up of healthy controls with a negative tst and without known contact with patients suffering from m. tuberculosis . the mean age was 34 years ( range=1956 y ) , and there were 17 male individuals in this group . all the patients and normal subjects gave written informed consent ahead of participation in the study . demographic characteristics of the total study population tbi : tuberculosis infection ; ltbi : latent tuberculosis infection ; tst : tuberculin skin test the gene of the aladh protein was amplified by polymerase chain reaction ( pcr ) from m. tuberculosis h37rv chromosomal dna , which was obtained from razi vaccine institute and serum research institute in tehran . the 2 primers used were upper : 5 -cgg ggt acc atg cgc gtc ggt att c -3 and down : 5-ccc aag ctt aca ggc cag cac -3. pcr amplification was performed with 35 cycles at 94 c for 45 seconds , 66 c for 45 seconds , 72 c for 45 seconds , and 72 c for 10 minutes . the pcr product was ligated to the pet32a vector , which is commercially available from novagen . this vector permits the genes of interest to be fused to the thioredoxin fusion protein , trxa , for high levels of expression with good solubility and it also contains cleavable his - tag sequences for detection and purification . it was thereafter transferred into escherichia coli ( e. coli ) strain dh5 , which was grown in a luria bertani ( lb ) medium ( gibco life technologies pa , uk ) containing ampicillin ( gibco life technologies pa , uk ) ( 100 g / ml ) . the recombinant plasmids were purified from the positive clones , and the pcr constructs were sequenced to verify their integrity . an overnight culture ( 600 ml ) of the resulting strain was used to inoculate 12 l of lb , and when it was in the mid - log phase ( absorbance 600 reached 0.7 ) , it was induced by isopropyl - d - thiogalacto - pyranoside ( iptg , 0.5 mm ) ( fermentas ab , vl , lithuania ) . the bacterial cells were harvested by centrifugation , and the cell pellets were frozen at -70 c . the expression of the recombinant protein was investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds page ) and western blotting . the cell pellets were re - suspended with a lysis buffer containing phosphate - buffered saline , 1 mm of edta ( ph=8.0 ) , and 1% ( v / v ) triton x-100 . the cell lysates were sonicated ( 30-s pulses at 20-s intervals for 6 times ) , and the suspension was centrifuged . page analysis was performed by utilizing mini format vertical electrophoresis ( bio - rad , hercules , ca , usa ) . accordingly , the sds page - separated proteins were electrotransferred into nitrocellulose membranes ( millipore , bedford , ma usa ) using a mini trans - blot cell ( bio - rad , hercules , ca , usa ) . the membranes were incubated with horseradish peroxidase - conjugated anti - poly - his antibody ( santa cruz , dallas , tx , usa ) . pet32a supports the his - tag cloning system ; therefore , the recombinant protein was purified by the ni - nta affinity column ( qiagen gmbh , hilden , germany ) under denaturing and native conditions . after the sample was loaded on the column and extensive washing steps were performed with 8-m urea ( sigma , st . louis , mo , usa ) ( ph=7.4 ) , elution by using a linear 10500 mm gradient of imidazole ( sigma , st . then , the his - tagged protein was dialyzed against phosphate - buffered saline for 48 hours , while the buffer was changed every 12 hours to remove the urea and imidazole . the bicinchoninic acid method was used to assay the protein concentration ( pierce , rockford , il , usa ) . ifn- and il-2 elispot assays were performed for the quantification of the responses of the t cells to recombinant aladh and esat-6/cfp-10 antigens , which were produced in our lab previously , by utilizing a commercially available elispt kit ( u - cytech , utrecht , netherlands ) . subsequently , the peripheral blood mononuclear cells ( pbmcs ) of the samples were separated with ficoll ( sigma , st . louis , mo , usa ) , and the number of the cells was quantified . multiscreen 96-well plates ( millipore , bedford , ma , usa ) were individually pre - coated with anti - ifn- and anti - il-2 antibodies ( 10 g / ml ) and incubated overnight at 4 c . the mycobacterial antigens ( 1 g / ml ) and pbmcs ( 210 cells / well ) were added to each well . the plain medium as a negative control and phytohemagglutinin ( sigma , st . louis , mo , usa ) as a positive control were applied and incubated for 24 hours for ifn- and 72 hours for il-2 at 37 c . the wells were washed , and biotinylated anti - ifn- , anti - il-2 , and -labeled goat anti - biotin antibodies were added separately . finally , the spots were revealed by activators i and ii , which display ifn- and il-2 secreting cells , followed by the quantification of the spots using a stereomicroscope . the number of the spots from the unstimulated wells was subtracted from the number in each well . the mean of the duplicates was applied , and all the results were expressed as the number of spot - forming cells ( sfc ) per million pbmcs . the results of the shapiro wilk test for all the groups showed that the variables did not have a normal distribution . the receiver operating characteristic ( roc ) curve was plotted in order to find out the optimum cutoff for the elispot assays of il-2 and ifn- responses in discriminating individuals with active tbi from those with ltbi and also to determine the sensitivity and the specificity of these assays . ethical approval for the study was granted by the research ethics committees of shiraz university of medical sciences , and informed consent was obtained from all the participants . the pcr product ( 1100 bp ) ( figure 1 ) was successfully cloned into the pet32a expression vector . nta column was used to purify the aladh protein , and sds page analysis was performed to indicate the molecular weight of the desired protein around 40 kda . in order to achieve a pure recombinant protein , we removed thioredoxin , trxa , by thrombin cleavage ( figure 2 ) . however , since this process reduces the yield of the protein , we utilized recombinant proteins with and without thrombin cleavage for elispot to observe the differences . the aladh his - tag protein was recognized by the mouse anti - his - tag monoclonal antibody using western blotting , as is shown in figure 3 . 100-bp marker ( lane 1 ) , agarose gel analysis for the l - alanine dehydrogenase ( aladh ) gene ( lane 2 ) . expression and purification of l - alanine dehydrogenase ( aladh ) antigen samples , ( lanes 15 ) resolved in a 12% ( w / v ) polyacrylamide gel , under reducing conditions followed by coomassie blue staining . escherichia coli ( e. coli ) extracts before isopropyl d - thiogalacto - pyranoside ( iptg ) induction ( lane 1 ) . e. coli extracts 4 hours after induction with 0.1 mm of iptg ( lane 2 ) . washed ni - nta resin ( lanes 3 and 4 ) . recombinant proteins purified by affinity chromatography using ni - nta resin with thrombin cleavage ( lane 5 ) . elispot assays were performed in the study groups for ifn- and il-2 responses to aladh and esat-6/cfp-10 fusion antigens . the responses of ifn- and il-2 to the aforementioned antigens were compared between the 3 groups . the elispot assays of ifn- responses to aladh ( p=0.18 ) and esat-6/cfp-10 ( p=0.81 ) and il-2 responses to esat-6/cfp-10 ( p=0.99 ) were not significantly different between ltbi and active tbi , while il-2 responses to aladh ( p=0.001 ) were different significantly . the roc curve analysis was performed , and the optimum cutoff for il-2 responses to aladh was 275 sfc and ltbi was identified with sensitivity of 75.8% and specificity of 78.8% . as is shown in figure 4 , the roc curve analysis demonstrated that il-2 responses to aladh were the most sensitive and specific indicator of ltbi ( area under the curve=0.820 ) , while the roc curve analysis for il-2 responses to esat-6/cfp-10 , ifn- responses to esat-6/cfp-10 , and ifn- responses to aladh showed much weaker responses . diagnostic performance of elispot results in active tb patients , ltbi and healthy control groups elispot : enzyme - linked immunospot assay ; tbi : tuberculosis infection ; ltbi : latent tuberculosis infection ; results are illustrated as medians and interquartile ranges of spot forming colonies per million peripheral blood mononuclear cells ( pbmcs ) a receiver operating characteristic ( roc ) plot is shown , illustrating sensitivity and specificity of the enzyme - linked immunospot ( elispot ) assays of interferon- ( ifn- ) and interleukin-2 ( il-2 ) responses to aladh and esat-6/cfp-10 fusion antigens in discriminating latent tuberculosis infection ( ltbi ) from active tbi . area under the roc curve was 0.820 for aladh il-2 elispot , 0.612 for aladh ifn- elispot , 0.494 for esat-6/cfp-10 il-2 elispot and 0.532 for esat-6/cfp-10 ifn- elispot . the pcr product ( 1100 bp ) ( figure 1 ) was successfully cloned into the pet32a expression vector . nta column was used to purify the aladh protein , and sds page analysis was performed to indicate the molecular weight of the desired protein around 40 kda . in order to achieve a pure recombinant protein , we removed thioredoxin , trxa , by thrombin cleavage ( figure 2 ) . however , since this process reduces the yield of the protein , we utilized recombinant proteins with and without thrombin cleavage for elispot to observe the differences . the aladh his - tag protein was recognized by the mouse anti - his - tag monoclonal antibody using western blotting , as is shown in figure 3 . 100-bp marker ( lane 1 ) , agarose gel analysis for the l - alanine dehydrogenase ( aladh ) gene ( lane 2 ) . expression and purification of l - alanine dehydrogenase ( aladh ) antigen samples , ( lanes 15 ) resolved in a 12% ( w / v ) polyacrylamide gel , under reducing conditions followed by coomassie blue staining . escherichia coli ( e. coli ) extracts before isopropyl d - thiogalacto - pyranoside ( iptg ) induction ( lane 1 ) . e. coli extracts 4 hours after induction with 0.1 mm of iptg ( lane 2 ) . washed ni - nta resin ( lanes 3 and 4 ) . recombinant proteins purified by affinity chromatography using ni - nta resin with thrombin cleavage ( lane 5 ) . elispot assays were performed in the study groups for ifn- and il-2 responses to aladh and esat-6/cfp-10 fusion antigens . the responses of ifn- and il-2 to the aforementioned antigens were compared between the 3 groups . the elispot assays of ifn- responses to aladh ( p=0.18 ) and esat-6/cfp-10 ( p=0.81 ) and il-2 responses to esat-6/cfp-10 ( p=0.99 ) were not significantly different between ltbi and active tbi , while il-2 responses to aladh ( p=0.001 ) were different significantly . the roc curve analysis was performed , and the optimum cutoff for il-2 responses to aladh was 275 sfc and ltbi was identified with sensitivity of 75.8% and specificity of 78.8% . as is shown in figure 4 , the roc curve analysis demonstrated that il-2 responses to aladh were the most sensitive and specific indicator of ltbi ( area under the curve=0.820 ) , while the roc curve analysis for il-2 responses to esat-6/cfp-10 , ifn- responses to esat-6/cfp-10 , and ifn- responses to aladh showed much weaker responses . diagnostic performance of elispot results in active tb patients , ltbi and healthy control groups elispot : enzyme - linked immunospot assay ; tbi : tuberculosis infection ; ltbi : latent tuberculosis infection ; results are illustrated as medians and interquartile ranges of spot forming colonies per million peripheral blood mononuclear cells ( pbmcs ) a receiver operating characteristic ( roc ) plot is shown , illustrating sensitivity and specificity of the enzyme - linked immunospot ( elispot ) assays of interferon- ( ifn- ) and interleukin-2 ( il-2 ) responses to aladh and esat-6/cfp-10 fusion antigens in discriminating latent tuberculosis infection ( ltbi ) from active tbi . il-2 elispot , 0.612 for aladh ifn- elispot , 0.494 for esat-6/cfp-10 il-2 elispot and 0.532 for esat-6/cfp-10 ifn- elispot . in the present study , we demonstrated that aladh might be a promising marker for distinguishing ltbi from active tbi . the aims of the present study were cloning , expressing , and purifying the aladh recombinant antigen in order to evaluate the diagnostic characteristics of aladh and esat-6/cfp-10 in the distinction between adults with ltbi and adults with active tbi . it has been found that cfp-10 and esat-6 antigens distinguish individuals with active tbi from healthy individuals with high diagnostic sensitivity and specificity , whereas they are inadequate for the discrimination of active tbi from ltbi . in our previous study , esat-6/cfp-10 fusion was used to detect active tbi with 100% sensitivity and 100% specificity . in the current study , the elispot assays of ifn- and il-2 responses to esat-6/cfp-10 between individuals with active tbi and healthy controls were significantly different , which is consistent with previous reports . nonetheless , the results were not significant considering the elispot assays of ifn- and il-2 responses to esat-6/cfp-10 and ifn- responses to aladh between the individuals with active tbi and those with ltbi . the sfc values of il-2 , induced by aladh , were significantly higher in ltbi than in active tbi , considering the number of individuals with ltbi - identified aladh compared with the patients with active tbi . since this protein is involved in the adaptation of m. tuberculosis in anaerobic conditions , it is expected that antigens are exposed to more t cells and as a result more cytokine responses occur in persons with ltbi . aladh may play a role in cell wall synthesis as l - alanine is an important constituent of the peptidoglycan layer . ( 2008 ) demonstrated that aladh could alter the expression profile for adaptation to a state of latent infection . the authors also showed that the conformation and crystal structure of aladh was changed from open to closed ternary forms in the phase of latent infection , which indicated the different host immune responses of ltbi . in a study conducted in italy by chiappini et al . ( 2012 ) , the antibody responses of 6 antigens in children with ltbi and active tbi were compared , and their data revealed that il-2 responses to the aladh antigen were significantly different between the children with active tbi and the healthy control group , whereas there was no difference between active tbi and ltbi in their study . the authors showed that aladh was not capable of discriminating active tbi from ltbi , which is in contrast to the results of our study , in which significant differences were observed between these 2 infections . the discrepancies between our findings may be due to actual differences in the immune responses between adults and children , which in turn indicates that the potency of aladh in the diagnosis of ltbi from active tbi is greater in our study . we also showed that il-2 responses to aladh were higher than ifn- responses to aladh in ltbi . it has been previously revealed that a higher response of il-2 in ltbi was achieved after 72 hours incubation time , whereas 24 hours incubation time for ifn- had the same response . thus , a prolonged incubation period seems to be essential to indicate an increased number of central memory t cells in adults with ltbi . accordingly , we used the aforesaid experimental conditions . given that il-2-positive elispot was related to ancient exposure , the increase in il-2-secreting memory t cells may explain the high response of il-2 compared with ifn- in our study . in the past few years , a large number of studies , systematic reviews , and meta - analyses have been conducted to evaluate elispot in distinguishing individuals with ltbi . for instance , hougardy et al . ( 2007 ) showed that heparin - binding - hemagglutinin antigen had higher sensitivity than esat-6 for the detection of ltbi . nevertheless , their study was carried out in a country with a low tb prevalence and as such can not be generalized to other communities . in another study , martinez et al . ( 2007 ) evaluated the cellular immune response of the erp antigen and demonstrated that ifn- responses were higher in patients with ltbi than in those with active tbi . be that as it may , erp is present in the other species of mycobacteria and vaccine strain mycobacterium bovis bcg . chen and colleagues ( 2009 ) conducted a study that examined the 6 antigens and found that ifn- responses to the rv1978 antigen were higher in individuals with ltbi than in those with active tbi , although this antigen was not sensitive enough . the roc curve analysis between the 2 antigens demonstrated that the elispot assay for il-2 responses to aladh had a greater discriminatory power . our data revealed that il-2 responses to the aladh antigen were significantly different between the individuals with ltbi and those who suffered from active tbi . as a result , the sequential usage of esat-6/cfp-10 and aladh elispot assays could be useful for the detection of tbi and ltbi . for instance , when these 2 tests are positive for a person , the result could be considered as ltbi . if il-2 responses to esat-6/cfp-10 are positive and the same responses to aladh are negative , the result may indicate a person with active tbi . when il-2 responses to both esat-6/cfp-10 and aladh are negative , the result may indicate an uninfected person . indeed , the only one currently available is for the detection of the later developmental stage of active tbi . the assumption of the diagnosis of ltbi in our study was based on a mixture of background exposure , which is an unavoidable limitation . therefore , the definition of ltbi is probabilistic , based on the duration of exposures and history of contact with a patient suffering from active tbi . second , the design of our study does not permit any valuation of conversion over time . in order to do that , we would need an additional assessment of the diagnostic value of esat-6/cfp-10 and aladh by elispot in a large - scale study . a diagnostic method that can specifically identify ltbi would allow more targeted therapy , which in turn could significantly reduce active tbi development . to that end , our study suggests that the elispot assay of il-2 responses to aladh can detect individuals with ltbi who have recently been infected . . however , further prospective studies are needed to monitor the elispot assay of il-2 responses to aladh among patients exposed to tbi . | background : discriminating latent tuberculosis infection ( ltbi ) from active tbi may be challenging .
the objective of this study was to produce the recombinant l - alanine dehydrogenase ( aladh ) antigen and evaluate individuals with ltbi , those with active tbi , and uninfected individuals by enzyme - linked immunospot assay ( elispot ) in order to distinguish ltbi from active tbi.methods:this exploratory study was performed in the iranian city of shiraz from 2014 to 2015 .
the study population ( n=99 ) was divided into 3 groups : individuals with newly diagnosed active tbi ( n=33 ) , their household contacts ( n=33 ) , and controls ( n=33 ) .
aladh was produced through pcr and cloning methods .
the diagnostic characteristics of aladh vs. esat-6/cfp-10 were evaluated in responses to interferon- ( ifn- ) and interleukin-2 ( il-2 ) with elispot .
differences between the groups were assessed with the kruskal
wallis and mann whitney tests for nonparametric data analysis .
the statistical analyses were performed with spss , version 16.results:ifn- responses to both esat-6/cfp-10 ( p=0.81 ) and aladh ( p=0.18 ) revealed that there were no significant differences between the individuals with ltbi and those with active tbi .
the same results were determined for il-2 responses to esat-6/cfp-10 between the 2 groups , while significantly higher il-2 responses to aladh were observed in ltbi than in active tbi . according to the roc curve analysis ,
a cutoff value of 275 sfc showed sensitivity of 75.8% and specificity of 78.8% for distinguishing ltbi from active tbi by il-2 responses to aladh.conclusion:the current study suggests that it may be possible to discriminate ltbi from active tbi by il-2 responses to aladh . |
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midlevel health workers ( mlhws ) are the mainstay of health service delivery in many low income countries . given their prominent role , supporting the performance of mlhws is a critical lever for strengthening health systems and redressing health inequalities in underserved areas . mlhws are defined as health care providers with formal accreditation but shorter training and a more restricted scope of practice than professionals . mlhw cadres share characteristics that make them particularly attractive for initiatives to improve access to care and mitigate health workforce deficits [ 2 , 3 ] . lower entry level education requirements make it easier to recruit locals who have potential to provide more culturally and linguistically appropriate care and may be easier to retain in rural placements . studies indicate that with adequate training and continued support , mlhws can provide care comparable to professionals . increasing deployment of mlhws can also be appealing as a lower cost alternative for improving coverage . despite their centrality in the health workforce , they frequently lack professional organizations , and national information systems often do not differentiate them from professionals , making it difficult to monitor their deployment and productivity . these factors contribute to their weak integration into the health system , which is manifested in lack of attention to management issues , training , support , career development , and progression [ 1 , 7 ] . as they often work in underserved remote areas , many face constraints which affect performance such as staffing shortages , lack of medicine , and equipment . the challenging situation that mlhws and other service providers face exemplifies the complexity of improving performance in resource - constrained settings . efforts have traditionally focused on enhancing health worker knowledge and skills , but there is increasing recognition that abilities alone are insufficient to achieve improvement in these challenging conditions . the 2006 world health report identified three broad categories of interconnected factors influencing performance : characteristics of the health workers , the health system , and the population served . performance is the result of a transactional process between health workers and their environments , as they engage with the conditions in the health system and community to provide health care according to their abilities , professional values , and personal goals . this study focuses on health workers ' engagement in the transactional process leading to performance , which we call practice . health workers can provide key insights into how these intersecting conditions influence their practice and how they respond to them . increased recognition of the crippling effect of health workforce deficits in health system effectiveness has led to a growing number of studies focused on health workers in low and middle income countries . the results of studies of motivation to work in rural areas and satisfaction in these positions highlight the importance of resource availability , management support , recognition , opportunities for advancement , as well as salary levels [ 1117 ] . fewer studies have focused on how health workers respond to these conditions as they engage in their work . in the face of challenging circumstances , examples highlighted in the literature include strategies such as moonlighting , pilfering drugs , and informal charging , which promote personal goals to the detriment of organizational goals and performance [ 1820 ] . only one recent study documenting positive coping strategies that contribute to performance could be found . while health worker issues have received growing attention , there is particular need for investigation of latin american contexts , as there are few studies of health workers from this region [ 2224 ] . an exploratory study was conducted to gain insight into the intersecting influence of health system and community conditions on the practice of auxiliary nurses , a mlhw cadre delivering health services in a rural province of guatemala . this paper explores auxiliary nurses ' motivation and satisfaction and examines their responses to the challenging working conditions they confront in their practice . the overall aim of the study is to generate strategies for improving performance that are appropriate for addressing local conditions and can build on existing strengths . in guatemala , the health system utilizes mlhws primarily in the form of auxiliary nurses ( an ) . they represent the largest cadre of health workers in the country with 18 ans per 10,000 inhabitants in 2006 , while professional nurses have a proportion of four per 10,000 and doctors , ten per 10,000 . training is one year in duration and ten years of formal schooling are required for entry to the program . though the majority of ans work in hospitals , they are also utilized as frontline health workers in primary and secondary care settings in rural communities where health needs are great . primary care services are delivered through two types of providers : public sector health posts and nongovernmental organizations ( ngos ) contracted by the government under the coverage extension program ( programa de extensin de cobertura , pec ) . health posts provide coverage for a population of around 2,000 inhabitants and typically employ one to two ans . in the pec , ngos are responsible for the delivery of a basic package of health services to a jurisdiction of about 10,000 inhabitants using a mobile team of health workers . the mobile team normally includes one to two ans , one doctor or nurse , and an educator . primary care activities in both health posts and pec include maternal and child health services , as well as attending consultations , providing educational talks , maintaining census , and epidemiological data for their catchment area . in order to gain support for health promotion activities , their work also involves communication with local leaders in the community development councils as well as training and coordination with community health volunteers . secondary care is provided in public sector health centers , most of which are open 24 hours and provide inpatient , emergency , and maternity care as well as outpatient consultations , and maternal and child health services . typically , health worker staff includes : a doctor or nurse who serves as district manager , one or more nurses who oversee the different departments of the health center and district health posts , a few additional doctors who work shifts , and around 15 ans . in many sites , one an ans staff the different departments in the health center , including pre- and postconsultation , family planning , inpatient , and emergency . in practice , a doctor or nurse may be not available to attend patients due to position vacancies , administrative duties or when many patients are waiting . in these cases , ans also attend patient consultations , emergencies , deliveries and manage inpatient care in secondary care settings . the province of alta verapaz in northern guatemala was selected as the site for this study because it offers rich opportunities to explore factors influencing an practice , with positive strategic efforts alongside great difficulties . challenges include one of the highest rates of maternal mortality in the country ( 207/100,000 live births ) as well as the highest rate of extreme poverty ( 38% ) [ 26 , 27 ] . the majority of the rural population is monolingual in the local indigenous language with little formal education . recent national policy to fortify health services in prioritized regions has led to scaling up health worker positions , with the creation of many new jobs in primary and secondary care services in alta verapaz since 2008 . in alta verapaz as in most parts of the country , scaling up of an positions was successful , with 97% of positions created filled , while there was 50% vacancy in the positions created for doctors and nurses . however , the newly created positions are with temporary contracts and wage delays have been common . the increased number of an positions in rural health services has been filled in part by graduates from an innovative distance education program based in the national school of nursing in coban , alta verapaz . this program aims to increase the availability of ans who are indigenous rural residents by making education more accessible with technology and more appropriate through an adapted curriculum focused on community health and intercultural care . field work was conducted from january to march , 2010 by the first author , who is a nurse by training and has established contacts with nursing schools in guatemala through previous work . the study was supported by a committee of local stakeholders from the national nursing school and the regional health office who reviewed the study protocol , interview guide , and consent form . the director of the regional health office approved the study and provided a letter to present in the districts . field work was conducted in five districts of alta verapaz , in sites one to four hours from the provincial capital . sampling was purposive to include ans working in both primary and secondary care as well as a balanced representation of gender and training backgrounds . a total sample of 14 ans was achieved through 11 one - on - one interviews and one group interview with three coworkers . in the case of the group interview , the participants were coworkers with similar background , and the group dynamic provided further insight into their experience of the work environment . characteristics of the participants are shown in table 1 . all ans interviewed were indigenous and spoke q'eqchi ' or poqomchi ' as well as spanish . the average age of participants was 29.5 years . interviews were carried out in the work setting , except for one conducted when the an was en route to work . participants were informed about the purpose and focus of the interview , the voluntary nature of participation , and the confidentiality of their responses . they were provided with a written description of the research project and written consent was obtained . this process helped establish positive rapport as the participants expressed appreciation of the researcher 's attention to their practice . interviews were conducted by the first author in spanish , and lasted from 20 to 45 minutes . topics in the interview guide included : motivation , their work and work environment , sources of satisfaction and frustration , sources of support , and suggestions to improve work . additionally , contextual information was gathered through observation of ans delivering services in health centers , health posts and pec settings , and conversation with supervisors and community members . field notes documenting observations and insights gained from local stakeholders were used to illustrate the context of findings from an interviews , and provided space for beginning analysis . a thematic analysis approach was used to interpret the data . analysis began with a review of the accuracy of transcriptions , followed by careful reading to become thoroughly familiar with the data set . a guide to the interview data was prepared by the first author introducing each an , describing impressions from the interview , and observation of practice if relevant , and initial observations on points of interest were discussed by all authors . the initial version of the theme index was based on the topics addressed in the interview guide . analytical observations were recorded after each interview coding , and the theme index was revised to incorporate topics that emerged repeatedly and did not fit in the original themes . the data were then sorted into the themes , and the first author created matrices summarizing the contribution of all participants for each theme . this permitted the research team to examine and discuss the range of perceptions and experiences within each theme and across work settings . work setting was used as the organizing category because important variations in ans ' conditions and perceptions across primary and secondary care were observed during data collection . variation across other categories such as gender and training background dynamics within and connections across themes were analyzed by seeking to characterize central tendencies and examining the consistency of patterns in the experiences and attitudes expressed by workers who shared particular traits . within the themes of motivation , satisfaction , challenges , and coping strategies , the central characteristics were community connectedness , sense of value of work , confronting resource constraints , effort of community work , and learning in practice , and these organize the presentation of results . participants ' accounts of their motivation to work as an an reflected a sense of connection to the people they were serving . all participants were from rural communities , though most did not work in their own village or town . however , they identified with others from rural communities , who in this province of guatemala primarily belong to the q'eqchi ' ethnic group . they expressed that being an an was a vocation that allowed them to help their people and provided them with an identity . secondary care ans were more likely to mention wanting to help their families and that they like working with people , while primary care ans were more likely to mention helping communities . one primary care an whose work involved travelling to distant communities with a mobile team stated : even though we receive pay that is not why we go we are helping people living in remote areas thank god , everything we have learned we are sharing with people at the community level ( in places where ) they have never had access to health services . ( participant 5 , pec ) even though we receive pay that is not why we go we are helping people living in remote areas thank god , everything we have learned we are sharing with people at the community level ( in places where ) they have never had access to health services . ( participant 5 , pec ) among both primary and secondary care ans , some expressions of motivation reflected the unique perspective a local health worker brings . the following quotes from two ans illustrate how they were motivated to become an an to address needs they had experienced themselves in their community , and also how their role provided them with identity : back then , we did n't know what nursing was two of my sisters died one post - partum and one who was pregnant . since we did n't know the warning signs , we just watched them there suffering for two or three days since we did n't have the knowledge at that time , my two sisters died . thank god , with the achievement of studying this career , it is very important in life . ( i am able to ) support the communities , help the people and children . ( participant 6 , health center)(i was motivated to become an an by ) necessity . they would leave on thursday , come in late on monday that was how i decided to study this , and also to have a career , to be somebody in life . ( participant 4 , health center ) back then , we did n't know what nursing was two of my sisters died one post - partum and one who was pregnant . since we did n't know the warning signs , we just watched them there suffering for two or three days since we did n't have the knowledge at that time , my two sisters died . thank god , with the achievement of studying this career , it is very important in life . ( i am able to ) support the communities , help the people and children . ( participant 6 , health center ) ( i was motivated to become an an by ) necessity . they would leave on thursday , come in late on monday that was how i decided to study this , and also to have a career , to be somebody in life . ( participant 4 , health center ) even after describing feeling frustrated by lack of resources in her work , an an in secondary care shared the following quote , which further illustrates how connectedness to the community as a local worker relates to motivation . interviewer : based in your experience until now , do you think you would like to continue working here?an : yes , especially since it 's my people ( mi pueblo ) . i think there is more trust between us than with another person who comes in ( participant 7 , health center ) interviewer : based in your experience until now , do you think you would like to continue working here ? an : yes , especially since it 's my people ( mi pueblo ) . i think there is more trust between us than with another person who comes in ( participant 7 , health center ) ans in primary and secondary care expressed that the nature of their work was a source of satisfaction because they were able to provide valuable services . primary care ans emphasized that community education and actions to prevent maternal mortality were important aspects of their work . participant 2 expressed a clear vision of how his work activities contributed to prevention : for me the most important activities are firstly the immunization program because we prevent several diseases with vaccines , nutritional supplements for the children , educational talks in pec , we have to prevent disease so that the secondary care facilities are not overcrowded . from the community level ( participant 2 , pec ) for me the most important activities are firstly the immunization program because we prevent several diseases with vaccines , nutritional supplements for the children , educational talks in pec , we have to prevent disease so that the secondary care facilities are not overcrowded . from the community level ( participant 2 , pec ) secondary care ans described that their work allowed them to help patients who came in very ill by resolving their problem or referring them . one an said the best feeling was knowing her knowledge , skills and abilities saved a life ( participant 8 , health center ) . they commented on the importance of prioritizing pregnant women because two lives were at stake , and they appreciated the beauty of receiving new life . in general , primary care ans expressed a stronger sense of satisfaction than those working in secondary care because their work allowed them more positive contact with communities through health promotion activities . primary care ans described how they derived satisfaction from being recognized in the community and feeling they had their support . it makes me happy that the people like me , they know me , i have their support working with people in the communities , being able to help my people , that is why i feel satisfied . ( participant 7 , health center ) i like what i do , being in contact with the people . it makes me happy that the people like me , they know me , i have their support working with people in the communities , being able to help my people , that is why i feel satisfied . ( participant 7 , health center ) as the work of the secondary care ans consisted in attending patients seeking services in the health center , they did not have the same opportunity for positive contact and community recognition . instead their ability to carry out their work and resolve the patient 's need was more strongly influenced by resource constraints . while all of the ans interviewed expressed desire to continue working in rural health settings , 12 of the 14 interviewed were working in positions with temporary contract . several expressed that their future depended on the government and they did not know whether their positions would be renewed due to uncertainty of continued funding . gender dynamics in satisfaction in different care settings merit further attention . in this sample higher satisfaction in the primary care role the only female an in the primary care group expressed that she would only work in pec a couple of more years due to the difficulties of travelling to the communities . though she was the only an currently employed in primary care who expressed desire to leave her position , there were four other female ans in secondary care who had worked previously in pec and left due to transportation difficulties . we fall into the error of only seeing the bad and we do n't realize the good that one does . ( participant 8 , health center )
ans described many challenging conditions which influenced their practice . however , they also shared ways they are actively coping with these conditions in their efforts to deliver services . in resource - poor settings , the constraints impeding optimal health service delivery often form the predominant impression , but as participant 8 pointed out , there were also positive aspects of an practice that merit attention . in secondary care settings , they described the human impact of not having the resources needed in critical situations , as well as their responses through taking initiative to resolve problems and greater attention to relationship with the patient . medicine shortages were common and played a large role in ans ' interaction with patients . they described the experience of giving patients prescriptions , instead of medicines , and how patients left unsatisfied . the ans empathized with the patient perspective and expressed understanding of the effort and investment required to travel to the health center , with the expectation that they would get help . the emotional impact of patients ' dissatisfaction played a strong role in their work experience , because as one participant described , the people are dissatisfied with you because you are the one who takes responsibility ( dar la cara ) with the people . ( participant 10 , health center ) secondary care ans also shared that the lack of resources limited their capacity to apply their knowledge and skills to resolve patient problems . in one health center , the an related the experience of not having oxygen available to provide adequate treatment for children who came in with advanced pneumonia : the truth is , many children have died on us . in the case of pneumonia , they have come in already seriously ill , complicated , and sometimes they ca n't breathe . they have died here because we haven't been able to do anything because there is no oxygen . if we had oxygen , we would quickly put it on and refer them , and that way they would make it to the hospital . ( participant 13 , health center ) the truth is , many children have died on us . in the case of pneumonia , they have come in already seriously ill , complicated , and sometimes they ca n't breathe . they have died here because we haven't been able to do anything because there is no oxygen . if we had oxygen , we would quickly put it on and refer them , and that way they would make it to the hospital . ( participant 13 , health center ) the ans related these constraints to their own frustration in their work because they were not able to resolve their patients ' need . for the most part however , their motivation was also reflected in examples of ans taking initiative to resolve problems : using their own resources to purchase small items for patients or the work setting , travelling to the regional health office to try to obtain supplies , or working with community leaders to solicit support from the municipality . ans also faced resource constraints of their own due to delayed wages which limited their ability to respond as they would have liked , because we too are struggling to see how we are going to make it . ( participant 12 , health center ) ans also responded to these conditions through greater attention to the patient - provider relationship . they described using increased effort to communicate and achieve understanding with patients and provide good care . their capacity to relate to the patients was strengthened by their shared language and understanding of their culture and experience . they expressed that it was important to call patients by their names , be respectful of their use of traditional medicines , and give priority to attending those who had travelled into town from the villages . the following quotes further illustrate how secondary care ans saw themselves actively responding to the situation through their capacity to attend to patients.interviewer : what makes you feel good in your work in spite of so many limitations?an : the communication with ( the patients ) , helping them understand , explaining the reason why you ca n't give them what they want . good communication and kindness to them , that 's what makes you say i did my job , i did well today . ( participant 8 , health center ) interviewer : what makes you feel good in your work in spite of so many limitations ? an : the communication with ( the patients ) , helping them understand , explaining the reason why you ca n't give them what they want . good communication and kindness to them , that 's what makes you say i did my job , i did well today . ( participant 8 , health center ) ans in primary care also faced conditions that complicated their work , including lack of cooperation from community leaders and transportation issues . as in secondary care , their responses reflected their motivation and unique capacity to relate to the population served . a challenge that was unique to the primary care setting was establishing and maintaining community participation in the roles and organizations which are designed to facilitate health services coordination with the community . one an described the initial difficulty of convincing people to participate in the community development council.during my career i have encountered many difficulties because since we began our work in pec there are people who do n't want to get involved in the community development council they used to say they could n't participate because they were n't paid for their day . but thank god , we have raised awareness so that they participate and get involved in health issues , without receiving an incentive it was really difficult but thank god now they participate . ( participant 2 , pec ) during my career i have encountered many difficulties because since we began our work in pec there are people who do n't want to get involved in the community development council they used to say they could n't participate because they were n't paid for their day . but thank god , we have raised awareness so that they participate and get involved in health issues , without receiving an incentive it was really difficult but thank god now they participate . ( participant 2 , pec ) another an described the difficulty of recruiting health promoters to support his work in the health center catchment area because they were aware that volunteers in nearby communities covered by the pec received benefits that the health center did not have the resources to offer.we do n't have the same resources ( as pec ) ( in this district ) part of the population is covered by ( a pec provider ) . the promoters who work with me do n't receive any incentives , while those with ( pec ) receive monthly training , t - shirts , boots , hats , backpacks , flashlights we do n't have any of that , but i have been able to maintain them , because i have known how to manage them , how to incentivize them in other ways . if not , this would be finished . ( participant 4 , health center ) we do n't have the same resources ( as pec ) ( in this district ) part of the population is covered by ( a pec provider ) . the promoters who work with me do n't receive any incentives , while those with ( pec ) receive monthly training , t - shirts , boots , hats , backpacks , flashlights we do n't have any of that , but i have been able to maintain them , because i have known how to manage them , how to incentivize them in other ways . if not , this would be finished . ( participant 4 , health center ) in both cases , the ans described how they dealt with the perceived lack of incentives among community members through their own capacities to raise consciousness about the value of community participation and manage volunteers effectively . it is noteworthy that two ans in a health center who had previous experience as health promoters also described how participating and sharing their nursing knowledge in their own communities was the most important way they could contribute to their progress . their active role in community leadership outside of work seemed to enhance their sense of value in their role as ans as well . primary care ans also faced difficulty in transportation , because much of their work was in community - based health promotion activities . in the pec program , but one an pointed out that there was not enough money for vehicle maintenance . in some cases , ans utilized their own motorcycle to reach the communities , and paid their own fuel costs . when no vehicle was available , the ans combined buses , rides , and walking to reach the communities where they were scheduled to provide services . out of pocket expenditures for transport were not reimbursed . however , while these difficulties were mentioned , most of the primary care ans did not seem to be demoralized by this aspect of their work . however , this pattern of greater satisfaction derived through primary care community work did not appear to hold for female ans interviewed . feedback from stakeholders also indicated that female ans from rural communities were often more reticent and could experience greater difficulty gaining recognition with community leaders , due to traditional gender roles particularly in remote areas . knowledge limitations constrained ans in certain areas of their work , such as diagnosing patients , knowing which treatment to prescribe , filling in paperwork correctly , and attending deliveries . however , they also described strategies to improve their knowledge through learning from other health professionals in practice . ans from all settings and training backgrounds expressed that education is theory , implying that in training they acquired knowledge from books but lacked opportunity to practice . they shared that during training in the clinical setting , there was often a large group of students and it was difficult to get hands - on experience . ans working in understaffed health centers sometimes mentioned that some of their work activities were outside of their scope of practice , but both ans and supervisors expressed that they should not leave patients unattended or send patients away because there was no doctor . given the one - year duration of training , shortage of health professionals , and limited time of experience of many of the newly contracted ans , the structural conditions contributing to this constraint were evident . several ans who were recent graduates of the distance education program expressed an attitude of learning in practice that seemed to help them cope with their knowledge limitations . one an shared that he always asked questions during clinical training . when he did not understand , he would other recent graduates working in a health center said that they had gotten where they were by not standing by passively . they put themselves in the action so they could learn and improve.that's why we have learned something in life if we were n't this way , we would n't have learned anything we have never let things pass , we always get ourselves in . ( participant 13 , health center ) that 's why we have learned something in life if we were n't this way , we would n't have learned anything we have never let things pass , we always get ourselves in . ( participant 13 , health center ) on the job , they had improved by learning with the doctors , taking advantage of opportunities to observe how they attend deliveries , and by receiving orientation about classes of disease and their treatment . one medical director stated that he trained the ans to do his job because he was only one person and sometimes he was not there or there were too many patients . a health center an recounted that their supervisor told them that if they were going to work well , they had to work as a team and help and support each other . other strategies to confront knowledge limitations included : seeking advice from more experienced ans and nurse supervisor in person and by phone and consulting the standards of practice manual . learning was described as a source of satisfaction in work and continued learning was frequently named as what was most needed to help them work more effectively in their role as an an . the findings of this study illustrate interconnections between motivation , satisfaction , and health workers ' practice in a rural latin american setting . the ans described constraints to service delivery common in low resource settings : shortages of medicines and equipment , travel to distant communities , difficulties of community participation , and knowledge limitations . the coping strategies they used to respond to these constraints in practice reflected their motivation through connection to the community they were serving and satisfaction from a sense of the value of their work . these responses provide insight into locally - developed strategies that strengthen the health worker 's relationship to the health system as well as the community . the health worker characteristic of community connectedness was central to motivation in this rural guatemalan setting . ans interviewed expressed desire to serve their people and address health needs of their community and families . the value of residing in the community was contrasted with the historical experience of health workers coming from outside . they describe professional ethos , religious conviction , and desire to help the poor as factors that should be built on in efforts to improve performance [ 12 , 30 , 31 ] . ans working in primary care settings described that their satisfaction with their work was reinforced by recognition from communities , particularly in their health promotion activities . the experience of the ans working in secondary care settings reflected more tension between satisfaction with the value of their work and frustration due to resource constraints that restricted their ability to resolve patient problems . but all ans interviewed reported that they planned to continue working in the health services , which is an indicator of satisfaction . this resonates with findings on brazilian health agents : when agents talked about why they liked their jobs , the subject of respect from clients and my community often dominated the conversation describe this as social embeddedness and contend that it affects health worker motivation to provide good service . the findings of this study provide further evidence of the far - reaching influence connectedness to the population served can have on practice . health worker motivation is typically seen in the literature through the human resource management ( hrm ) lens and defined as the willingness to exert and maintain an effort towards organizational goals . studies seek to identify determinants of health worker motivation so that performance management strategies may be customized [ 13 , 14 , 17 ] . in this view , the lack of health worker motivation is the problem impeding performance and enhancing their motivation is the solution . the findings of this study suggest that in some contexts motivation may not be the problem . instead , the perspectives shared by ans raise the question whether the health system organization is giving them the support they need to enable them to meet their goals arising through their own experience of health needs in their community . fritzen has also pointed out that performance management strategies are difficult to apply in public services of developing countries where essential pre - requisites , including availability of drugs , equipment , and transport as well as living wages for workers , are often missing . the ans ' motivation and satisfaction led them to utilize coping strategies that reflected that their personal goals were closely related to organizational goals . these strategies indicate directions for strengthening health workers ' relationship to the health system and the community . the displayed coping strategy of learning with colleagues in practice is similar to existing recommendations to support performance through promoting lifelong learning and team work in an enabling work environment . ans taking initiative to solve resource constraints also resembles the desired outcome of suggested strategies for preparing health workers to adapt to challenges through problem - based learning and providing conditions for agency [ 7 , 9 ] . these strategies indicate directions for strengthening health workers ' integration with the health system , which are in line with existing recommendations for bringing their practice in line with standards of care and enabling them to adapt to changing conditions . the coping strategies exhibited by ans of greater attention to the patient relationship and building collaborative relationships with community leaders indicate directions for strengthening health worker relationship to the community which have received little attention in the literature . these strategies built on ans ' shared cultural and linguistic background with the population served and illustrated how community connectedness translated to a unique capacity for responsiveness in their practice . a recent study of rural health workers in papua new guinea describes how , even when health workers are not locals , efforts to build engagement with the community contribute to motivation and performance through the increased acceptability of services . in order to strengthen the health worker 's position as a bridge between the community and the health system , greater emphasis is needed on community - oriented strategies for a more balanced approach to improving performance . these findings differ from other examples of coping strategies in the literature , which focus on health workers reacting to adverse conditions through actions that help them to attain personal goals that are not in line with organizational goals . it is possible that these findings are due to differences in the severity of the adversity in the conditions faced in this setting compared to others , or perhaps reflect the relatively short time of experience of many of the participants . but it also seems important that research on rural health care is usually conducted from a deficit - describing approach . lindelow & serneels justify their focus on performance problems and the exclusion of the positive experiences of health workers and users as the most direct method to understanding current issues and identifying potential solutions . positive coping mechanisms and existing strengths also deserve attention as they can provide insight into local strategies that can be built on in developing context - appropriate support . suggest focusing on workhood assets as a means of exploring health workers ' capacity to cope , adapt and actively respond to constraints to performance in resource - poor settings . the findings of this study also brought to light a gender difference among health workers in this setting . male ans were more prevalent in primary care in this setting , while female ans expressed that the burden of travel to distant communities was too great , particularly when they also had family obligations . this contrasts with other mlhw cadres such as the lady health workers in pakistan and health extension workers in ethiopia which exclusively enroll women to provide community level primary care . these countries ' utilization of female health workers is based on their acceptability and cultural sensitivity in women 's health issues . however , this study suggested that gender - related characteristics also posed a constraint for women in the primary care role , limiting their mobility . this study aimed to generate insight into the practice of mlhws in rural guatemala and strategies for supporting their performance through the perspective of front - line workers . however , the results should be considered in light of the following limitations . due to access issues and time limitations , the most remote workers were not represented in this sample . language barrier also may have limited full understanding in the interviews , as spanish was the second language of participants and the interviewer . analysis of variation across health worker characteristics focused primarily on differences between primary and secondary care settings , even though these differences also coincided with gender in this sample . this decision was based on the observation during field work that the primary care role offered greater opportunity for self - realization and positive community contact . it is possible that the importance of gender in explaining variation across work settings has been underestimated . the an cadre exhibits the characteristics that make mlhws a strategic focus for strengthening health systems . they play a prominent role in service delivery in rural areas of guatemala with great health needs . recruitment of rural residents is facilitated by low entry education requirement and distance education modalities . successful scaling up of an positions in priority areas of guatemala in 2008 contrasted with high vacancy rates in new positions for professionals . current international priorities emphasize efforts to improve rural health services through incentive - based strategies to attract and retain professionals who are not motivated to work in rural areas . given the difficulty of attracting professional to rural facilities , improving support for the health workers who are already there is a logical complementary step to strengthen performance of the health system . this study highlighted existing strengths of individuals from a mlhw cadre who were motivated by community connectedness and employed a range of coping strategies in their practice in the face of difficult circumstances . policies to build on these strengths should focus on mechanisms that make midlevel as well as professional health worker training accessible to rural residents , such as targeted recruitment , housing allowances for study , and distance education . continued support for mlhws in practice should provide them the opportunity to develop their capacity as problem solvers , strengthen their skills through mentoring relationships , and increase lines of communication with professionals for consultation . finally , the practice of health workers in rural areas should be strengthened through efforts to fortify their relationship to the community , such as collaborative work with leaders and volunteers and greater attention to the provider - patient relationship . these recommendations indicate actions to make effective and efficient use of existing human resources and enhance their capacity to deliver health services to underserved populations in rural guatemala and other low - resource settings . | background .
the performance of midlevel health workers is a critical lever for strengthening health systems and redressing inequalities in underserved areas .
auxiliary nurses form the largest cadre of health workers in guatemala . in rural settings ,
they provide essential services to vulnerable communities , and thus have great potential to address priority health needs .
this paper examines auxiliary nurses ' motivation and satisfaction , and the coping strategies they use to respond to challenges they confront in their practice . methods .
semistructured interviews were conducted with 14 auxiliary nurses delivering health services in alta verapaz , guatemala .
results .
community connectedness was central to motivation in this rural guatemalan setting .
participants were from rural communities and conveyed a sense of connection to the people they were serving through shared culture and their own experiences of health needs .
satisfaction was derived through recognition from the community and a sense of valuing their work .
auxiliary nurses described challenges commonly faced in low - resource settings .
findings indicated they were actively confronting these challenges through their own initiative .
conclusions . strategies to support the performance of midlevel health workers should focus on mechanisms to make training accessible to rural residents , support problem - solving in practice , and emphasize building relationships with communities served . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the
determination of the binding cleft , and preferably the binding mode ,
of lead compounds has become a fundamental step of structure - based
drug design . protein binding site locations for medium - to - high affinity
ligands can be pinpointed by crystallization methods , cryo - electron
microscopy ( cryo - em ) , photoaffinity labeling , or by nuclear magnetic resonance ( nmr ) chemical shift mapping and
intermolecular cross - relaxation experiments . for weakly interacting
systems , with affinity constants in the millimolar range , cocrystallization
is challenging , and cryo - em densities , which are based on averaging ,
become too weak to identify the position of the ligand . the nmr chemical
shift response is also population - averaged , with the inherent small
molar ratio of the bound state making the majority of the signal content
originate from the uncomplexed molecules . although low - affinity interactions
can be confirmed using a variety of techniques , e.g. , isothermal titration
calorimetry , surface plasmon resonance , or ligand - detected nmr techniques such as water - logsy and saturation transfer difference experiments , the spatial localization of protein interaction
sites for low - affinity ligands is still cumbersome . it is , thereto ,
commonly impeded by limiting molecular solubility and concomitant
unspecific binding events at high ligand concentrations . we
propose the applicability of ligand - transferred paramagnetic
nmr restraints for binding site identification of low - affinity drugs
and drug candidates . the interaction of the unpaired electrons of
a ligand - contained paramagnetic radical or lanthanoid ion with the
nuclear spins of a nearby protein results in extensive paramagnetic
alterations of conventional biomolecular nmr spectra already at a
low ligand concentration . the foremost manifestations are paramagnetic
relaxation enhancement ( pre ) , residual dipolar couplings ( rdc ) , and
pseudocontact shifts ( pcs ) , which can all be obtained
from the spectroscopic analysis of the differences in spin relaxation
rates , j - couplings , and chemical shifts , respectively ,
for nuclei interacting with a paramagnetic ion or a diamagnetic reference
ion . the presented methodology utilizes
paramagnetic ligand tagging to identify protein binding sites by the
covalent attachment of a paramagnetic lanthanoid ion complexing chelate
to the ligand of interest to induce pseudocontact shifts ( pcs ) on the interacting protein ( figure 1 ) . schematic illustration of the sensitivity gained
when using the
presented methodology . the arbitrary chemical shift of a protein nucleus ,
shown in the 1d nmr trace ( in gray ) , is altered when in fast equilibrium
with either a native or diamagnetically tagged ligand ( dark gray ,
m ) , or with a paramagnetically tagged ligand ( red ,
pcs ) . for weak affinity ligands , the protein nucleus
chemical shift change caused by a diamagnetic ligand ( m ) may be very small , whereas the pseudocontact shift induced
by a paramagnetic ligand ( pcs ) is reliably measurable . the location of the paramagnetic
ion and hence the area for the
binding site is obtained by fitting the position , relative rotation ,
and size of the paramagnetic susceptibility tensor to the pcs and the protein structural coordinates , according to eq 1,1where pcs is the measured pseudocontact
shift for any protein nuclear spin ,
xax and xrh are the axial and rhombic components of the magnetic
susceptibility tensor , whereas the angles and describe
its orientation with respect to the protein coordination frame , and r is the lanthanoid nuclear spin distance . contact shift contributions can be neglected
for protein nuclei due to the noncovalent nature of the interaction . lanthanoid metals have found long use
as relaxation agents and
chemical shift reagents in both small molecule and biomolecular systems . ions from the lanthanoid series ( ln ) have comparable
ionic radii and strongly coordinate with metal binding sites in proteins
or with chelating ligands . except la and lu , which are the commonly used diamagnetic references , all lanthanoid
ions are paramagnetic . the methods available for incorporation of
lanthanoid ions to invoke protein paramagnetic nmr restraints follow
three principles : ( i ) the lanthanoid ion locates directly to a pre - existing
protein metal binding site , ( ii ) a lanthanoid ion binding tag is covalently
engineered to a protein terminus or side chain , or ( iii ) a lanthanoid
ion complexing soluble ligand interacts specifically or nonspecifically
with a protein . direct metal ion lanthanoid
ion replacement and high affinity lanthanoid ion complexing peptides or chelating agents covalently anchored to the protein
will , in addition to influencing the nmr signals of the tagged protein
itself , induce paramagnetic effects on noncovalent interaction partners . this can be used to convey binding site information on protein protein or protein ligand interactions . a covalently protein - attached tag based
on an edta derivative was designed early on . tags with one protein anchor point were then succeeded
by more rigid tags and tags with dual anchoring points in order to restrict tag movements . although paramagnetic protein tagging may
be used to convey information on protein ligand complexation ,
the opposite approach , paramagnetic tagging of the ligand , provides
a valuable complement . hitherto it has almost exclusively been applied
to measurements of ligand induced protein pre effects to probe protein
solvent accessible areas or used in mri applications with gd chelates , with the exception of sparse examples of ligand
induced protein pcs used to resolve resonance overlap , to facilitate resonance assigning and to improve structure determination . have also elegantly demonstrated the use of a lanthanoid
tagged sugar moiety to confirm the previously reported m affinity
galectin-3lactose interaction . however , the full potential of ligand lanthanoid tagging as a route
to study protein ligand binding of varying affinity has not
yet been explored . herein , we demonstrate the applicability
of paramagnetic labeling
of a weakly binding pharmacon for identification of its binding site . the interaction of the anesthetic agent sevoflurane ( figure 2 ) and the calcium signaling
protein calmodulin was selected for validation of the methodology . this interaction is specific and localized to two hydrophobic binding
surfaces , one in each of the two calmodulin lobe domains . calmodulin has been found to associate with over 300 intracellular
targets , including several ion channels that have been shown to be
targeted by anesthetics . for example , calmodulin associates with the
ryanodine receptor with nanomolar affinity , and in doing so represses its activity . calmodulin is also known to affect inactivation in voltage - gated
sodium and calcium channels . mutations in the ryanodine
receptor gene in combination with sevoflurane may lead to the potentially
lethal condition of malignant hyperthermia . volatile anesthetics ,
such as sevoflurane have also been shown to perturb the activity of
the ligand - gated ion channels ( gaba)a and that of glycine
receptors , but the low affinities of these molecular recognition phenomena
have so far hindered their in - depth mechanistic investigations . consequently ,
the mode of anesthetic actions remains unresolved and a subject for
further studies , despite its direct clinical relevance . h nmr spectrum at 800 mhz of the dy complexed
sevoflurane analogue showing an unusually wide range of chemical shift
dispersion , ca . its binding constants are on
the limit of the applicability of conventional nmr techniques , i.e. , kd = 918 mm , and the low aqueous solubility
and high volatility of sevoflurane renders the detection of its protein
binding especially challenging , posing it as a particularly suitable
test system for validation of this methodology . for paramagnetic tagging ,
sevoflurane was attached to dota , 1,4,7,10-tetraazacyclododecane - n-n-n-n-tetraacetic acid , which
has high affinity toward lanthanoid ions . in order to cause the smallest possible electronic and structural
perturbation , a terminal fluorine of sevoflurane was substituted with
oxygen ( figure 2 , scheme s2 ) , the atom most similar in electronic
properties to fluorine . this modification retains the hydrogen bond
accepting ability and the electron - withdrawing character and simultaneously
allows the attachment of dota . to exclude a direct dota calmodulin
interaction , a truncated analogue in which sevoflurane was replaced
by a methyl functionality ( scheme s1 ) was
applied as a negative control . individual samples of the dota - bound
sevoflurane were complexed separately to each of the lanthanoid ions
la , eu , yb , or dy . intramolecular ligand pcs of up to 550 ppm were
measured after complexation to dy ( figure 2 ) . we therefore anticipated that binding
of the paramagnetically labeled drug to its protein binding site would
induce chemical shift changes larger in magnitude than those caused
by the magnetic susceptibility of the drug itself , due to the pseudocontact
shift phenomenon , see figure 1 . in addition to the increased sensitivity of detection , the
known pcs dependence on the distance to and on the
spatial localization of the paramagnetic lanthanoid is expected to
enable direct location of the binding site area . the lanthanoid
chelates ( dy , yb , eu , or la , the latter used as the diamagnetic
reference ) of the dota - bound sevoflurane ( scheme s2 ) as well as of the control ligand in which a methyl group
replaces the sevoflurane adduct ( scheme s1 ) are highly water - soluble . tight lanthanoid complexation is evident
from the h nmr spectrum of the dy complexed
sevoflurane analogue covering an impressive 850 ppm spectral width
( figure 2 ) despite
macrocycle conformational exchange , as expected from previous studies ,
visible as additional line broadening in the h nmr spectrum
of the la complexed sevoflurane analogue . for analysis of each calmodulin binding site independently , the
calmodulin n - lobe ( residues 178 ) and c - lobe ( 79148 )
were expressed and purified separately . it is well - established that
the individual calmodulin lobes retain the same properties as in the
full length protein . intact structural
integrities of the lobes were sustained after comparison of the lobes hn n chemical shifts to those
of native calmodulin under identical conditions . slight chemical shift
deviations as a result of the short 3/6 residue n - terminal sequence
expansion ( for both lobes , see materials and methods ) were found for the first n - terminal residues and additionally for
residues sequentially located to the flexible interlobe hinge region
in native calmodulin , expected from the sequence division being executed
at residue 78 . as the further analysis is highly dependent on the
correct structural coordinates , pcs for the terminal
residues ( 13 and 7586 ) were excluded . chemical shift
comparisons of free calmodulin and calmodulin with the la chelated sevoflurane analogue resulted in negligible differences
( figure 3 ) and ensured
that the diamagnetic ligand interaction did not invoke large overall
changes to the protein structural coordinates and that the available
calmodulin coordinates could be used to fit the magnetic susceptibility
tensor and location of the paramagnetic center to both lobes , respectively . amide hn and n and methyl hme and c pcs were
obtained from pairwise comparison of chemical shifts from h n / c - hsqc spectra of 0.1 mm u c , n labeled calmodulin lobes , respectively ,
titrated with either the la or the dy complexed
sevoflurane analogue at 0.05 , 0.1 , and 0.2 mm ; see figures 3 and 4 . resonances displaying the most extensive pcs broadened
beyond detection at 0.2 mm ligand concentration due to pre and ligand
induced chemical exchange line broadening . the optimal trade - off between the number of observable resonances
and size of the pcs were determined to be at a 0.1
mm ligand concentration , resulting in a maximum pcs of 0.24 ppm for met109 hme and c. the expected magnitude of the induced protein
pcs can be estimated from the pcs of the free ligand and the affinity constant . for low millimolar
affinity ligands with the current sample conditions , the h pcs obtained for the dy complex of the sevoflurane
analogue are 8.9 ppm and 6.5 ppm , for its ( ch2 ) and ch , respectively . consequently , protein nuclei in the
calmodulin ligand complex at near equidistance of the sevoflurane
protons to the paramagnetic center are expected to exhibit pcs on the order of 0.1 ppm depending on the respective relative
orientations of the magnetic susceptibility tensor , the ligand , and
the protein . the experimentally observed pcs , as
illustrated for hn nuclei in figure 4 , agree well with this assumption . the dy complexed sevoflurane analogue did not produce
measurable protein jch3 rdcs ,
due to the very small degree of alignment produced by the weak binding
of a flexible system and combined rdc dynamic averaging . ligands chelating
to eu and yb produced considerably smaller
induced protein pcs than the dy complex ,
which was alone found to be sufficient for the study . identical experiments
comprising the control ligand , with the dota - attached sevoflurane
being replaced by a methyl group , were run to exclude the possibility
of the transferred paramagnetic restraints originating from specific
interaction of the lanthanoid chelating cage with either of the calmodulin
lobes . the protein hn pcs recorded with the dy ( and la ) complexed
control ligand were negligible ( < 0.015 ppm ) for both lobes ,
see figure 4 . the few
minor but significant pcs seen for the control ligand
are over an order of magnitude smaller than the pcs observed for the dota - attached sevoflurane analogue ensuring that
the parameters fitted in table 1 truly originate from specific sevoflurane c
hsqc spectra
of calmodulin c - lobe titrated with the lanthanoid la / dy complexed sevoflurane analogues . c hsqc spectra of 0.1 mm calmodulin c - lobe ( black ) titrated
with 0.05 mm ( blue ) , 0.1 mm ( purple ) , and 0.2 mm ( red ) dy complexed sevoflurane analogue are shown . resonances transverse
along approximately parallel lines with the addition of the paramagnetic
ligand . some peaks are broadened beyond detection at the higher concentration
of the dy complexed sevoflurane analogue . the true pcs are calculated with respect to the chemical shifts detected
upon titration with 0.1 mm la complexed sevoflurane analogue
( gray ) . the sensitivity of the methodology is illustrated by the size
of the induced pseudocontact shift ( chemical shift differences for
calmodulin in the presence of 0.1 mm dy or la complexed sevoflurane analogue , spectra in magenta and gray , respectively )
as compared to the size of the chemical shift changes induced by the
nonparamagnetic ligand ( chemical shift difference for calmodulin in
the absence or presence of 0.1 mm la complexed sevoflurane
analogue , spectra in black and gray , respectively ) . the affinity constants for the tagged sevoflurane
analogue and
the control ligand for the respective calmodulin lobes were measured
using itc . given the limited solubility of sevoflurane , previous measurements
of the affinity were conducted using competition experiments , prone to larger errors , but the increased ligand
solubility upon tagging enabled high ligand concentrations and thus
the use of direct itc measurements . titrations of tagged sevoflurane
analogue into either the calmodulin n - lobe or c - lobe yielded heats
significantly higher than those observed for the tagged control ligand
( figure s11 ) . the tagged sevoflurane analogue
was observed to have comparable affinity to the two lobes of calmodulin
as sevoflurane itself ; thus a kd of 4.0
mm was detected for the n - lobe and 1.8 mm for the c - lobe . the residual heats for the tagged control ligand were featureless
and did not indicate any detectable binding to either lobe . amide proton pcs in ppm versus
sequence for 0.1 mm calmodulin lobes , consecutively
plotted , in the presence of 0.1 mm of either the sevoflurane ligand
( black , filled circles ) or control compound ( grey crosses ) complexed
to dy . measurements of pcs were also done by ligand
titrations to full - length calmodulin ( figure s10 ) . the signs and magnitudes of the pcs correlated
well with those obtained for the individual lobes ; however , the concomitant
binding of two molecules of paramagnetically labeled sevoflurane to
the protein prevented extraction of pcs induced
by each sevoflurane interaction independently . the pcs obtained for full - length calmodulin were used to verify the interaction ,
but physical lobe separation was necessary for a single magnetic susceptibility
tensor fit and quantitative evaluation . it should , however , be noted
that , although the pcs detected on full - length calmodulin
were not quantitatively evaluated , they qualitatively indicated ligand
binding at two distinct clefts . the location of the ligand - attached
lanthanoid in the protein ligand
complex can thus be derived from the experimental fit of the induced
protein pcs to one magnetic susceptibility tensor . a single magnetic susceptibility tensor is found for a paramagnetically
tagged ligand weakly interacting ( fast exchange ) with a protein even
in the event of ligand diastereomers ( as has been shown for the dota
derivatives ) as the induced protein pcs are motionally
averaged . the reliability
of the magnetic susceptibility tensor determination from mobile paramagnetic
tags has been thoroughly investigated . a sizable number of pcs were extracted for each
of the calmodulin lobes , 190 pcs for the n - lobe
and 162 pcs for the c - lobe . for each lobe , the lanthanoid
position and the parameters of the magnetic susceptibility tensor
were fitted simultaneously to the structural coordinates of calmodulin
( pdb i d 1x02(68 ) ) using numbat , and the values are reported in table 1 . the axial and rhombic
components of the magnetic susceptibility tensor are subject to scaling
with the protein occupancy . parameters were fitted with the numbat
software using the experimental pcs and the calmodulin structure with pdb i d 1x02 . errors estimated are reported as one standard deviation
( std ) based on 1000 times repeated fitting with random removal of
10% of the pcs in each run . the previously reported higher calmodulin c - lobe sevoflurane
affinity
is preserved as reflected in the larger magnitude pcs and fitted tensor xax and xrh for the c - lobe as compared with the n - lobe .
for both lobes , the fitted lanthanoid position is in close proximity
to one face of the lobe , see figure 5 , and allowed for the spatial localization of one single
binding area in each calmodulin lobe . the sevoflurane binding sites
both locate to the exposed , methionine - rich surface areas previously
shown to bind free sevoflurane . although the relative rotation of
the sevoflurane adduct with respect to the location of the lanthanoid
ion remains undisclosed , the true protein ligand binding interface
is restricted to lay within a radius equal to the maximum distance
from the lanthanoid ion to the sevoflurane adduct . this distance ,
around 14 , essentially sweeps out a spherical sector of possible
binding sites on the calmodulin surface area . indeed , the distance
from the lanthanoid ion to the methionine h protons
previously showing intermolecular hoesy cross peaks with free sevoflurane
is 1013 . calmodulin , n - lobe ( left ) and c - lobe ( right ) , surface representation
with the lanthanoid position represented by a black sphere . sevoflurane ,
as positioned previously from conventional nmr restraint methods , is indicated in yellow . atoms within 14
of the lanthanoid position are color coded red and represent the determined
binding surface . the presented methodology
allows for protein binding site localization
at low ligand concentration even for very weakly interacting compounds ,
in the event that the overall protein structure is preserved upon
complexation . paramagnetic tagging of sevoflurane resulted in sizable
induced protein pcs , adequate for determination
of magnetic susceptibility tensors in complexes with either calmodulin
lobe , despite the dynamic nature of the low affinity sevoflurane , the determination of the lanthanoid
coordinates positions the anesthetic part of the ligand to a calmodulin
surface area surrounding the previously determined binding site for
free sevoflurane , with the higher c - lobe to n - lobe sevoflurane affinity
preserved , illustrating that the synthetic modification did not significantly
alter the molecular recognition . itc affinity measurements further
confirmed comparable binding coefficients for the tagged sevoflurane
analogue and free sevoflurane , as well as qualitatively supported
the higher calmodulin c - lobe to n - lobe binding affinity . the
sensitivity of the method , as compared to conventional chemical
shift mapping , is illustrated by the similar calmodulin amide and
methyl chemical shift alterations obtained with 10 mm sevoflurane
and with 0.1 mm of the dy complexed sevoflurane analogue ,
at 0.10.2 mm protein concentrations . the negative control ligand with the sevoflurane moiety replaced
by a methyl group did not induce substantial pcs , indicating that dota itself does not interact with the protein . the pcs obtained for the paramagnetic - tagged sevoflurane
are indeed the result of the sevoflurane calmodulin interaction
and increase the sensitivity of detection by a factor of one hundred ,
as compared to conventional chemical shift titration . it should be
noted that h pcs induced by paramagnetically
tagged bioactive compounds are also manifested in h nmr
spectra of proteins devoid of isotopic enrichment and may therefore
be used for rapid screening . the absolute values of the pcs can be increased
by titration of more ligand to the protein sample , although to some
extent this is concomitant with increased line broadening . the magnitude
of the pcs could also be tuned by alternation of
the lanthanoid metal ion due to their relative difference in magnetic
susceptibility anisotropy . this provides an opportunity for increased
accuracy by combined fitting of pcs from a series
of alternating lanthanoid ions even in the event of only a few recordable
or assigned pcs . for the current study , an examination
of the data obtained using the paramagnetic lanthanoid dy , with la as reference at equimolar 0.1 mm protein and
ligand concentrations , gave sufficient pcs to corroborate
the analysis . ligands complexed to either eu or yb resulted in smaller protein pcs . due to
the sensitivity of the methodology pcs alterations
were reliably measured at this low , 1% , binding site occupancy
and physiologically relevant , low sevoflurane analogue concentrations ,
comparable to that applied in a previous investigation , could be used . attachment of sevoflurane to
the lanthanoid binding tag has also increased its solubility , permitting
the use of direct itc measurement to determine its binding constants ;
however , this did not substantially influence the affinities . the elucidation of the unknown binding site of a bioactive compound
by the current tagging approach requires synthetic modification , i.e. ,
attachment of a paramagnetic tag . this modification is chemically
straightforward , as shown here for sevoflurane , but may risk affecting ,
or in the worst case even prohibiting , the binding in case a functional
group involved in the protein recognition is modified . it should be
underlined that this , and the fact that the paramagnetic tag is comparable
in size to the ligand itself , by no means present risks greater than
those taken upon fluorescent labeling , which to date is carried out
on a routine basis . however , attachment of the paramagnetic label
at several different functionalities of the ligand may be necessary
along with control experiments , analogous to those in this study ,
to ensure that the paramagnetic tag does not bind with high affinity
to the protein . a paramagnetic lanthanoid complexing unit , such as dota , dota - m8 , edta , taha , dtpa , 4mtda 4mmdpa , 3mdpa , or 4mdpa , may be easily attached to virtually any pharmacon or pharmaceutical
lead compound by an ether linkage , as shown here for sevoflurane ,
or by simple modification of the phenolic anchoring point of the linker
to another suitable functional group and its subsequent attachment
using standard organic synthetic transformations . hence , connection
of the tag may be achieved via amidation or esterification , via click
chemistry type transformations such as
the [ 3 + 2 ] ( husigen reaction of alkynes and azides ) , [ 4 + 1 ] ( reaction of isonitriles and tetrazines ) and diels alder cycloadditions , via nucleophilic substitution , via
olefin metathesis or via the thiolyne
reaction , for example . this makes the
technique applicable to a very broad range of substances with the
greatest advantage of its use being expected for investigation of
leads having low affinity for their cellular targets , such as , e.g. ,
the epothilone anticancer natural product . a main limitation of the technique is the requirement of compatibility
of the functionalities of the small molecule ligand with the conditions
of lanthanoid complexation ( ph 56 , 60 c ) . moreover ,
attachment of a polar metal binding site may introduce additional
interactions to the protein that are not available for the drug or
lead compound itself , even though dehydration of a metal complexed
dota tag may pose a significant energetic barrier and therefore is
less likely . however , such a scenario can easily be detected by using
a control ligand , as in this study . the length and flexibility of
the linker connecting the pharmacon / lead and the paramagnetic ion
complexing unit defines the accuracy of binding site localization .
by careful attention to the length of the linker thus , a shorter
linkage , e.g. , via omission of the 1,4-substituted benzene linker
applied in this study , is expected to increase the pcs , whereas by its extension , e.g. , by incorporation of a biphenyl
unit , the line broadening due to pre
can be reduced . the obtained pcs provide useful experimental
input for computational docking into the experimentally established ,
secluded binding area or as a basis for mutagenesis experiments to
provide further insight into the pharmacon binding modes . the
presented tagging approach is best suited for systems with
a 1:1 binding stoichiometry as quantitative analysis of protein ligand
complexes of higher stoichiometries would require software packages
allowing for simultaneous fits of more than one magnetic susceptibility
tensor . however , ligand tagging may allow rapid qualitative screening
for multiple protein binding sites , as shown for titration of full - length
calmodulin with the dy labeled sevoflurane analogue clearly
indicated binding to both lobes with comparably higher c - lobe than
n - lobe affinity ( figure s10 ) . paramagnetic
ligand tagging to identify protein binding sites provides
an attractive alternative to the opposite approach of binding site
detection based on analysis of the nmr signals of the ligand interacting with a paramagnetically
labeled protein , with both techniques having their strengths and limitations .
using the latter , specular approach , the attachment of the paramagnetic
tag , due to the sheer size of the protein , is less likely to influence
the binding , and selective attachment of the lanthanoid probe to different
side chain positions may improve the accuracy of the location of the
binding cleft . this , on the other hand , commonly necessitates protein
site - directed mutagenesis . furthermore , detection of the binding of
low affinity ligands may be cumbersome as the observed paramagnetic
effect is small due to the low molar fraction of bound ligand . the
complementary approach of ligand tagging presented here allows straightforward
screening of a bioactive substance against a large number of protein
targets and does not necessitate protein engineering . complexation
of a series of different lanthanoids ( dy , yb , tb , er , etc . ) to the dota of the tagged - ligand
can provide several sets of pcs data for the same
protein ligand complex , with the
combination of these data expectably allowing determination of the
binding site with a higher precision . the main advantage of ligand
tagging is doubtlessly the great sensitivity enhancement of detection
as compared to conventional chemical shift mapping , which reached
approximately a factor of hundred in this first study . on the other
hand , obviously , modification of the structure of the ligand may affect
its binding , and therefore tag attachment at several positions of
the ligand may be necessary . even if the attachment is expected to
typically follow standard synthetic transformations , it may require
adjustment for each studied ligand . for a highly accurate determination
of the binding cleft development of a more rigid linker than that
used in this investigation will be necessary . overall , further exploration
of paramagnetic ligand tagging for identification of protein binding
sites , as a complementary tool to the alternative of protein tagging ,
should be expected . paramagnetic labeling
by a dota tag increases the solubility of
hydrophobic substances , whose binding site identification using conventional
techniques is often seriously limited by their low aqueous solubility .
the sustained paramagnetic sevoflurane analogue binding site affinities
and binding site locations for both calmodulin lobes indicate that
the tagging did not alter the molecular recognition . with the combination
of increased substance solubility and higher sensitivity , the described
ligand tagging approach offers binding cleft identification at lower
ligand concentrations , thereby minimizing the risk of unspecific protein
binding this approach should be particularly attractive for the pharmaceutical
industry for rapid screening of protein targets , especially for weakly
binding lead compounds . the method for preparation of the paramagnetic
control ligand is outlined in supplementary scheme s1 , whereas that of the paramagnetically ( dy ,
eu , yb ) and diamagnetically ( la ) labeled sevoflurane is in supplementary scheme s2 . native , full - length
human calmodulin
and the individual calmodulin lobes , n - lobe : residues 178 ,
and c - lobe : residues 79148 , were expressed and purified as
hexahistidine , maltose binding protein - tev protease cleavage site
constructs as described previously , with
the alteration that n - terminal ghwggm- and ghm- adducts to the native
n - lobe and c - lobe sequences , respectively , remained after cleavage . protein concentrations were determined using the calculated extinction
coefficient at 280 nm in the presence of 6 m guanidine . n / c hsqc spectra were almost
identical to full - length calmodulin except for the n - lobe terminal
residues ( 13 , 7578 ) and c - lobe n - terminal residues
7986 . the latter constitutes the flexible hinge linkage between
the two lobes in the full - length sequence , and the chemical shifts
were expected to change upon cleavage . the lobe assignments were corroborated
using hnca , hnca+ , hnco , and hnco+ sequential walks . samples containing either lobe at 0.1 mm in 100 mm kcl ,
0.22 mm nan3 , 0.1 mm 2,2-dimethyl-2-silapentane-5-sulfonic
acid , and 65 mm cacl2 were run with 5 l additions
of 2.2 mm sevoflurane analogue ligand or control ligand coordinating
to either la or dy . n hsqc and h c ct - hsqc spectra were performed at 25 c
with a tci probe on a bruker avance iii hd spectrometer operating
at 800 mhz h frequency . spectral widths of 12820 hz ( h ) , 3621 hz ( c ) , and 2351 hz ( n )
run with 1024 , 200 , and 170 complex points , respectively , were employed
for the titration experiments . all nmr data were processed using nmrpipe , and the spectral analysis was performed using
sparky ( t. d. goddard , d. g. kneller , ucsf ) . the 10 l ligand
additions , resulting in 0.1 mm ligand sample concentration , presented
the best signal - to - noise ratio for all peaks combined and were used
to extract the pcs . the pcs were
fitted to the calmodulin structure , pdb i d 1x02(68 ) using the
numbat software , in which the location
of the paramagnetic ion and the parameters of the magnetic susceptibility
tensor were fitted simultaneously . for
the calmodulin n - lobe and c - lobe , 190 and 162 pcs , the data were fitted with and without
the built - in racs correction ; the maximum racs correction were 0.001
and 0.004 ppm for the n - lobe and c - lobe , respectively . errors estimated
were based on 1000 times repeated fitting with random removal of 10%
of the pcs in each run . the calmodulin lobes were dialyzed
overnight against 150 mm kcl , 10 mm na - hepes ph 7.4 , 2 mm cacl2 . the eu complexed sevoflurane analogue and control
ligand were dissolved into the same buffer at a final concentration
of 10 mm . titrations consisted of 20 injections of 2 l ligand
at 10 mm into the cell containing 1 mm calmodulin lobe . the background
heats from dilution of the ligands were determined by titrating them
into buffer . experiments were performed at 25 c and a stirring
speed of 750 rpm on an itc200 instrument ( ge healthcare ) . the data
were processed using origin 7.0 and fit to a single - site fitting model
after background buffer subtraction . | transient
biomolecular interactions are the cornerstones of the
cellular machinery .
the identification of the binding sites for low
affinity molecular encounters is essential for the development of
high affinity pharmaceuticals from weakly binding leads but is hindered
by the lack of robust methodologies for characterization of weakly
binding complexes .
we introduce a paramagnetic ligand tagging approach
that enables localization of low affinity protein ligand binding
clefts by detection and analysis of intermolecular protein nmr pseudocontact
shifts , which are invoked by the covalent attachment of a paramagnetic
lanthanoid chelating tag to the ligand of interest . the methodology
is corroborated by identification of the low millimolar volatile anesthetic
interaction site of the calcium sensor protein calmodulin .
it presents
an efficient route to binding site localization for low affinity complexes
and is applicable to rapid screening of protein ligand systems
with varying binding affinity . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
population - based and autopsy studies report that the incidence of intracranial aneurysms ( ias ) may be as high as 10% and peaks in the sixth decade of life . subarachnoid hemorrhages ( sahs ) due to ruptured ias account for 10% and 40% of deaths before hospitalization and after a 1-month hospital stay , respectively . more than 35% of patients with sah develop major neurological deficits preceding hospital discharge , even if they demonstrated favorable glasgow coma scores [ 58 ] . although it is easier to detect unruptured ias with various imaging modalities , it is difficult to predict if or when they will rupture , which poses a dilemma for both patients and physicians [ 911 ] . acquired and hereditary risk factors contribute to the multifactorial etiology of unruptured ia , including sex , hypertension , atherosclerosis , alcohol consumption , and smoking . parameters utilized for rupture assessment include aneurysm size , shape , and location , in addition to angle and flow hemodynamics [ 1318 ] . cumulative arterial wall deterioration as a result of constant remodeling characterized by degeneration and inflammatory cell infiltration lead to ia rupture and subsequent sah . altered arterial wall elastic properties and hypertrophic remodeling might predispose ias to rupture [ 2123 ] . arterial wall elasticity and intima - media thickness can be estimated non - invasively via ultrasound to indirectly evaluate arterial wall strength [ 2426 ] . we hypothesized that hypertrophic intimal remodeling and impaired elastic properties detected in the right carotid artery might co - occur with ia rupture and thus may predict an impending ia rupture . a total of 49 patients consecutively admitted with a diagnosis of ia ( n=23 ) or ruptured ia ( n=26 ) between january and december 2010 were included in this study . study subjects were screened and removed from the study if they qualified for the following exclusion criteria : history of stroke , heart failure , severe heart valve disease , renal dysfunction , diabetes mellitus ( dm ) , and local or systemic acute infection . the study protocol was designed in accordance with the helsinki declaration and was approved by the institutional ethics committee . written informed consent was obtained from all participants . maximum blood pressure ( bpmax ) was the systolic bp and minimum blood pressure ( bpmin ) was the diastolic bp . blood pressure was measured from the right brachial artery with a sphygmomanometer ( omron hem 705cp , colson ) after a 10-minute resting period . heart rate and bp were measured just before ultrasound examination , and within the first 3 days following ia rupture to avoid falsely elevated bps from sah - induced vasospasm . ultrasound examinations were performed for patients with ruptured ias within the first 3 days of rupture to avoid sah induced vasospasm and following dsa . specifically , the aplio xg scanner equipped with a 10 mhz linear array transducer ( toshiba medical systems , tokyo , japan ) was used . a pulse repetition frequency of 3 khz with an automatic cutoff filter ranging from 1 to 3 khz was utilized . m - mode ultrasound was performed at a speed of 50 mm / sec . the right common carotid artery ( cca ) the transducer was positioned parallel to the cca such that the lumen s diameter was maximized in the longitudinal plane . maximum ( dmax ) and minimum ( dmin ) internal lumen diameters were measured at 1 to 2 cm proximal to the cca bifurcation in magnified m - mode during systole and diastole . intima - media thickness measurements were taken at this same location , but were derived in b - mode . arterial elastic properties , including distensibility , strain , stiffness index , and elastic modulus , were measured to determine the stress on the right cca wall during diastole and systole . strain was defined as the percent change in cca artery lumen diameter during systole and diastole . the following calculations were performed to determine the aforementioned measures : statistical package for the social sciences for windows ( spss ver . 18 , chicago , il , usa ) software was used to analyze all data . correlation analyses were performed with the spearman test . a p - value less than 0.05 was considered significant . the right cca internal diameter was measured by an expert , blinded observer ( a.d . ) . to evaluate inter - observer reliability , minimum and maximum cca internal diameter and distensibility were also calculated by a second observer ( m.i . ) . maximum blood pressure ( bpmax ) was the systolic bp and minimum blood pressure ( bpmin ) was the diastolic bp . blood pressure was measured from the right brachial artery with a sphygmomanometer ( omron hem 705cp , colson ) after a 10-minute resting period . heart rate and bp were measured just before ultrasound examination , and within the first 3 days following ia rupture to avoid falsely elevated bps from sah - induced vasospasm . ultrasound examinations were performed for patients with ruptured ias within the first 3 days of rupture to avoid sah induced vasospasm and following dsa . specifically , the aplio xg scanner equipped with a 10 mhz linear array transducer ( toshiba medical systems , tokyo , japan ) was used . a pulse repetition frequency of 3 khz with an automatic cutoff filter ranging from 1 to 3 khz was utilized . m - mode ultrasound was performed at a speed of 50 mm / sec . the right common carotid artery ( cca ) the transducer was positioned parallel to the cca such that the lumen s diameter was maximized in the longitudinal plane . maximum ( dmax ) and minimum ( dmin ) internal lumen diameters were measured at 1 to 2 cm proximal to the cca bifurcation in magnified m - mode during systole and diastole . intima - media thickness measurements were taken at this same location , but were derived in b - mode . arterial elastic properties , including distensibility , strain , stiffness index , and elastic modulus , were measured to determine the stress on the right cca wall during diastole and systole . strain was defined as the percent change in cca artery lumen diameter during systole and diastole . 18 , chicago , il , usa ) software was used to analyze all data . correlation analyses were performed with the spearman test . a p - value less than 0.05 was considered significant . the right cca internal diameter was measured by an expert , blinded observer ( a.d . ) . to evaluate inter - observer reliability , minimum and maximum cca internal diameter and distensibility were also calculated by a second observer ( m.i . ) . a comparison between unruptured ia and ruptured ia groups in terms of demographics and aneurysm characteristics is provided in table 1 . patients with unruptured ia had a mean age of 47.112.0 years , with ages that ranged from 26 to 68 years . patients with ruptured ia had a mean age of 48.611.5 years , ranging from 31 to 67 years . unruptured ia and ruptured ia characteristics included shape , size , angle , aspect ratio , and bottleneck factor . for the entire study population , unruptured ia and ruptured ia locations included the internal carotid artery ( ica ) in 14% of cases , the anterior communicating artery ( acoa ) in 20% of cases , the middle cerebral artery ( mca ) in 41% of cases , and the vertebral and basilar arteries ( v - b ) in 25% of of cases ( figures 1 and 2 ) . the unruptured ia group exhibited a decreased mean cca intima - media thickness at 0.520.12 cm vs. 0.610.13 cm for the ruptured ia group ( p=0.013 ) . however , the unruptured ia group exhibited increased mean cca lumen diameter change ( d ) at 0.70.1 cm as compared to 0.50.1 cm for the ruptured ia group ( p=0.04 ) ( figures 3 and 4 ) . mean cca stiffness index was greater for the ruptured ia group at 6.00.5 vs. 6.70.5 for the ia group ( p=0.044 ) . this was also true for the mean cca elastic modulus , as this measure was higher for the ruptured ia group at 0.90.3 compared to the ia group at 0.70.2 ( p=0.026 ) . mean cca strain was decreased for the ruptured ia group at 6.11.7 vs. 8.11.9 for the ia group ( p=0.013 ) . this same trend was observed for mean cca distensibility , as the ruptured ia group mean value was lower at 1.80.4 than the ia group at 2.30.5 ( p=0.024 ) . the risk of rupture per year for patients with asymptomatic ias has been reported at 0.05% . in population - based studies , , much research has been devoted to studying ias and the factors that facilitate rupture . reduced arterial elasticity was observed in patients with ruptured ias in a recent ex vivo study . patients with ia rupture exhibit decreased arterial wall distensibility compared to those with unruptured ias . this phenomenon is widely accepted , even though one ex vivo study asserted the opposite upon measuring arterial wall distensibility in the anterior cerebral , radial , and dorsalis pedis arteries . moreover , arterial distensibility measurements have been commonly performed on the cca . in agreement with most studies , therefore , we designed an in vivo study using measurements from the right cca , such that our outcomes might aid in advancing the clinical management of patients with unruptured ia . numerous studies have been performed that focused on parameters predicting rupture , such as aneurysm location , size , shape , and angle [ 1418 ] . ias with an irregular shape and multilobular contours are associated with increased focal stress and distensibility . elliptical ias are more prone to rupture than spherical ias . decreased bottleneck and aspect ratios are also correlated with increased rupture risk . however , once rupture has occurred , spherical aneurysms become more oval - shaped ; thus , an aneurysm s elliptical quality may not be distinctive of increased rupture risk . a recent study based on biomechanical models of rupture reported that rupture risk is associated with increased aneurysm size . the cutoff value for increased rupture risk was determined to be 10 mm ; however , the vast majority of ias reported in other studies range between 5 to 9 mm . because of the high prevalence of ias less than 10 mm in diameter , it is important to use criteria other than size to determine rupture risk . it has been postulated that rupture occurs in small - sized ias if they rapidly expand . however , surgical interventions are not indicated for small ias , since the rupture risk is relatively low . an experimental study focusing on biomechanical alterations between unruptured ias and ruptured ias showed that patients with greater arterial wall rigidity were more prone to ia rupture . rigidity can be determined by the elastic properties of the arterial wall tissue , and a reduction in elasticity significantly contributes to the pathobiology of ia rupture . it has been widely reported that arterial distensibility with comorbid vasculopathy predisposes patients to abdominal aortic aneurysm formation , dissection , and rupture . these studies asserted that aneurysmal rupture occurred when wall tension exceeded the strength limit of the artery wall . another study evaluating the aneurysmal expansion limit demonstrated a correlation between the extent of distensibility and rupture . increased arterial wall stiffness , loss of elasticity , and decreased perivascular support might be associated with vasculopathies resulting from aging , essential hypertension , diabetes mellitus , and vasculitis [ 23,31,4345 ] . intimal remodeling and arterial stiffness might be associated with defects in elastin , collagen , and extracellular matrix . specifically , defective collagen contributes to arterial wall weakness due to decreased wall distensibility , which facilitates ia rupture . hemodynamic studies also highlight parameters that modulate aneurysmal wall rigidity , growth , and rupture . diminished arterial wall elasticity has been associated with a large discrepancy between diastolic and systolic bp ( p ) , which contributes to increased arterial resistance . this phenomenon of continuous , increased mechanical loading augments ia wall stress , which facilitates ia fatigue and rupture . our results were in accordance with these data , as a high p was associated with ia rupture . overall , our results are in parallel with most in vivo studies performed on the extracranial arteries . we found that patients with ia rupture exhibit impaired cca elasticity , which is associated with hypertrophic remodeling not observed in unruptured ias . impaired distensibility might not be completely attributed to ia rupture , as vasculopathy may also contribute to ia rupture . our data suggest that decreased cca elasticity and increased intima - media measurements might be associated with an increased likelihood of ia rupture , which may be helpful in decision - making for ia clinical management . the study sample size was small , lacked a healthy control group , and no long - term follow - up was performed . the study sample size was small , lacked a healthy control group , and no long - term follow - up was performed . our findings demonstrate that patients with ruptured ias exhibit impaired cca wall distensibility and increased intima - media thickness , which suggests hypertrophic remodeling . thus , determining cca distensibility and intima - media thickness might be useful in determining whether an ia may rupture in the future . to better determine whether cca elastic properties predispose patients to ia rupture , a larger prospective study design might be more informative , as the development of rupture may be correlated with changes in these factors over time . it is our hope that developing a set of predictive parameters for ia rupture will guide clinical management so to prevent complications such as subarachnoid hemorrhage . | backgrounda risk factor assessment that reliably predicts whether patients are predisposed to intracranial aneurysm ( ia ) rupture has yet to be formulated . as such , the clinical management of unruptured ia remains unclear .
our aim was to determine whether impaired arterial distensibility and hypertrophic remodeling might be indicators of risk for ia rupture.material/methodsthe study population ( n=49 ) was selected from consecutive admissions for either unruptured ia ( n=23 ) or ruptured ia ( n=26 ) from january to december 2010 .
hemodynamic measures were taken from every patient , including systolic and diastolic blood pressure using a sphygmomanometer .
unruptured ia and ruptured ia characteristics , including aneurysmal shape , size , angle , aspect ratio , and bottleneck factor , were measured and calculated from transverse brain ct angiography images . with ultrasound ,
the right common carotid artery intima - media thickness was measured , as well as the lumen diameter during systole and diastole .
arterial wall strain , distensibility , stiffness index , and elastic modulus were calculated and compared between patients with unruptured ias and ruptured ias .
a p - value less than 0.05 was considered statistically significant.resultsgeneral demographic data did not differ between patients with unruptured ias and ruptured ias .
greater mean intima - media thickness ( p=0.013 ) , mean stiffness index ( p=0.044 ) , and mean elastic modulus ( p=0.026 ) were observed for patients with ruptured ias .
moreover , mean strain ( p=0.013 ) and mean distensibility ( p=0.024 ) were decreased in patients with ruptured ias.conclusionspatients with ruptured ias demonstrated decreased arterial distensibility and increased intima - media thickness at the level of the carotid arteries . by measuring these parameters via ultrasound , it may be possible to predict whether patients with existing ias might rupture and hemorrhage into the subarachnoid space . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
improving quality of care in the pediatric intensive care unit ( picu ) is an imperative in healthcare . quality improvement efforts are focused on care processes with the goal of eliminating errors and adverse events . the use of endotracheal intubation is routine in the care of critically ill children . unplanned extubation is the displacement or removal of the endotracheal tube at a time other than that specifically chosen for a planned extubation and is a serious adverse event [ 24 ] . previous investigations have shown that the rate of unplanned extubations in infants and children in the picu ranges from 0.114 to 4.36 per 100 ventilated days [ 5 , 6 ] . generally , 1.0 unplanned extubations per 100 ventilated days are considered within national standards acknowledging that all unplanned extubations are unacceptable [ 2 , 5 ] . unplanned extubation exposes the patient to morbidity and mortality over and above those associated with the patient 's underlying disease [ 6 , 7 ] . showed that an unplanned extubation prolongs time of intubation thereby increasing the patient 's exposure to hazards of airway intervention and mechanical ventilation . in their investigation , picu length of stay more than doubled after an unplanned extubation . it is more common to require reintubation after an unplanned extubation than after a planned extubation . in addition , emergent reintubation may be needed at a time when the patient has not been fasting , posing a risk of aspiration . moreover , reintubation may be needed when personnel available for the procedure have less experience and skill with emergency airway management in contrast to a reintubation that takes place after a planned extubation where appropriate staff is readily available [ 2 , 10 ] . in a multicenter study of risk factors and outcomes of extubation failures in the picu the failure rate after unplanned extubation was 37.5% but only 6.2% after a planned extubation . all unplanned extubations are unacceptable due to their potential for causing unnecessary harm to the patient . our impression was that there was a high rate of unplanned extubations in our picu . as a quality improvement effort , we prospectively determined the unplanned extubation rate in the picu as well as the contributing factors . based on these data , we developed a targeted intervention program hypothesizing that it would be able to decrease unplanned extubations . the study included all intubated patients in a 10-bed picu located in a general county teaching hospital . the picu is staffed by board certified pediatric critical care attendants , pediatric critical care fellows as well as pediatric and emergency department residents and interns . at night , care is provided by a senior pediatric resident ( pgy 2 or 3 ) and an intern . the fellow and attendant are available by phone and return to the hospital if needed . nurse - to - patient ratios are 1 : 1 or 1 : 2 depending on acuity . the choice of the particular agent and the dose is based upon the patient 's clinical requirements . after intubation , the endotracheal tube is secured with tape , a chest radiograph is performed , and the position of the endotracheal tube is adjusted , if indicated . for patients who are intubated before admission to the picu , a chest x - ray is obtained as soon as possible after arrival and the tube is adjusted if needed . although there is no standardized protocol for obtaining radiographs on intubated patients , they are often done on a daily basis . at the time of initiation of the project for the purpose of this study , an extubation was considered to be unplanned when the displacement or removal of the endotracheal tube occurred at a time other than that chosen for a planned extubation . reintubation was defined as the replacement of the endotracheal tube within 24 hours , regardless of whether the extubation was planned or unplanned . since both 8- and 12-hour shifts are utilized in the picu , time periods were arbitrarily defined as 06001200 , 12011800 , 18010000 , and 00010559 . data collected included patient 's age , weight , diagnosis , indication for intubation , size of endotracheal tube , and date and time of intubation and extubation . the data were collected by the physician responsible for the patient 's care while intubated . for patients who were intubated prior to arrival to the picu , the time of admission to the picu was documented as the time of intubation . if a patient was transferred to an outside institution or expired , the time of transfer or death was documented as the time of extubation . if the extubation was unplanned , the presumed cause was documented by the data collector . any questions about the cause of the unplanned extubation were discussed with the study investigator who made the final determination . if the patient required reintubation , a new data sheet was started . data gathered during the first time period , september 1 , 2000 through march 31 , 2001 , were analyzed , and the rate and causes of unplanned extubation were determined . a small task force comprised of physician , nursing staff , and respiratory therapy staff was formed to identify specific areas for intervention . the program was comprised of an education component including a didactic session to improve knowledge about sedation for the intubated patient and the complications of an unplanned extubation . this mandated that the endotracheal tube was to be secured by painting the endotracheal tube , upper lip , and cheek with skin adhesive . one piece of pink tape was used to secure the endotracheal tube by placing one end on the right cheek and drawing it across the top lip , pressing firmly for good adhesion . the tape was then wrapped around the tube for a minimum of two revolutions in a clockwise direction . excess tape was secured to the right cheek . using a second piece of tape on the left cheek , the procedure was repeated wrapping the tape in a spiral fashion up the tube and back down again . the security of application was tested by gently pulling the endotracheal tube up and away from the patient 's face . the tape on the endotracheal tube was required to be completely changed at least every 48 hours or when loose , grossly contaminated , or needed to be repositioned . a detailed procedure was written and a brief computer video was made to illustrate the proper procedure . all nurses and respiratory care practitioners were required to view the video . competency was demonstrated by correctly performing the proper taping of the endotracheal tube and stating the policy requirements . prior to this program , unplanned extubations were viewed as a routine part of picu care . after the implementation of this program , a zero tolerance attitude towards unplanned extubations was adopted . the effects were evaluated in a second data collection period , november 1 , 2001 through april 30 , 2002 . there were no changes in the use of noninvasive ventilation modalities during the two time periods . although extensive education about sedation of the intubated patient took place , no sedation protocols were instituted , and the medications continued to be prescribed by physicians . nursing staff could administer sedative drugs only with a physician order . in order to standardize the number of intubated days , ventilator days were calculated using the difference between the times of intubation and extubation in hours and minutes . ventilated days were only counted for those patients with an endotracheal tube ; ventilator days for patients with a tracheostomy were not collected or used in the calculations . data from the first and second periods were analyzed using mann - whitney utest , chi - squaretest , or fisher 's exact test , as appropriate . rates of unplanned extubation per 100 ventilated days for each month of the study were also determined . data are presented as medians ( 25th , 75th percentiles ) , except as noted . during the initial period , there were 68 intubations in 62 patients ( table 1 ) . patients were intubated for airway protection ( 50% ) , apnea ( 24% ) , and respiratory failure ( 19% ) . those patients intubated for airway protection included surgical patients but these data were not specifically gathered . there were 10 ( 14.7% ) unplanned extubations for a rate of 6.4 unplanned extubations per 100 ventilated days . of the ten unplanned extubations , reintubation was required in 2 ( 20% ) . one patient had two unplanned extubations . of the 10 unplanned extubations in the initial part of the study , five happened between 06001200 , two between 12011800 , two between 18010000 , and one between 00010559 . in the second time interval , inadequate patient sedation , poor taping where the endotracheal tube is not properly secured to the face or slips through the tape , improper position of the endotracheal tube either above the clavicles or at or below the carina , and unknown were the items most frequently cited as leading to an unplanned extubation ( table 2 ) . based on these findings , a targeted intervention program was developed to address these specific issues . the program was instituted in september 2001 and training was completed in october 2001 . following the intervention program , the patients were intubated for respiratory failure ( 49% ) , airway protection ( 36% ) , and apnea ( 8% ) . in the second period , there were two ( 3.4% ) unplanned extubations for 1.0 unplanned extubations per 100 ventilated days . when comparing the two time periods , age , weight , endotracheal tube size , and duration of intubation were similar ( p > .05 ) . there was no difference ( p > .05 ) in the use of cuffed endotracheal tubes in the first time period ( 32% of patients ) compared with that in the second period ( 42% ) . in addition , there were no changes in personnel or assignments in the two periods . however , there was a difference in the reasons for intubation between the two groups for respiratory failure and apnea . there was no apparent increase or decrease in the monthly rate of unplanned extubations prior to the institution of the intervention program ( table 3 ) . due to the low number of unplanned extubations ( n = 2 ) , there were insufficient data to perform process control . there was a significant decrease in both the number ( p = .03 ) and the rate ( p = .04 ) of unplanned extubations after the implementation of the quality improvement program . the ratio of the incidence rate of unplanned extubations before and after the intervention program was 0.15 with a 95% confidence interval of 0.040.59 . this indicates that the postintervention rate is not greater than 59% and not less than 4% of what it was in the preintervention period . the ultimate goal of every intervention is to improve the health and quality of life in all patients . the objective of this study was to improve the quality of care in our picu by reducing unplanned extubations . in order to accomplish this , we used the plan ( p ) do ( d ) study ( s ) act ( a ) model . , effective interventions should be aimed at specific features of a target group , and the healthcare problem must be quantifiable . in our case , the target group included all those responsible for the care of the intubated patient in the picu . the objective was to reduce the rate of unplanned extubations to a level within national benchmark standards . by using this approach the first stage of pdsa is the planning ( p ) stage in which there is analysis of the intended area of improvement . in this case , it was determined that the rate of unplanned extubations in the picu was well above national benchmark standards . at the same time that we determined the rate of unplanned extubations in , several factors that contribute to an unplanned extubation have been previously identified [ 1 , 10 , 13 ] . these include inadequate sedation , the use of neuromuscular blockade , improper use of restraints , improper tube position , inattentive support staff who dislodges the tube during routine care ( e.g. , obtaining a radiograph ) , inadequate taping of the endotracheal tube , patient - to - nurse ratios of greater than 1 : 1 , occurrence of a procedure or transport at the time of the unplanned extubation , and a lax attitude towards an unplanned extubation [ 1 , 10 , 13 ] . as we investigated the causes of unplanned extubation in the picu during the planning ( p ) stage , we found that three of the aforementioned factors contributed significantly to the high rate of unplanned extubations . therefore , the intervention program used in the do ( d ) phase focused on addressing these issues , and time and effort were not wasted on correcting factors that were not contributing to the problem in our unit . in other institutions , different factors may be operative and would need to be addressed in a program specific to that setting . in the study ( s ) phase , we recollected data to determine whether the changes achieved the desired results . using the targeted intervention program , we were able to reduce the unplanned extubation rate from 6.4 to 1.0 unplanned extubations , per 100 ventilated days . when examining the time of day in which the unplanned extubations occurred , the intervention reduced unplanned extubations in all time periods nonetheless , even after education about sedation of the intubated pediatric patient , inadequate sedation continued to be a contributing factor in unplanned extubations . clearly , improved sedation contributed to the decrease in unplanned extubations but since both unplanned extubations in the second time period were attributed to inadequate sedation , the education program was not completely effective . sedation assessment scales such as the ramsay scale , modified ramsay sedation protocol , and the comfort scale have been used in the assessment of sedation in intubated children as well as for guiding medication administration [ 10 , 1417 ] . given that inadequate sedation continued to be a factor , further examination and adoption of a sedation protocol may be helpful . since care in our picu does not include the routine use of physical restraints , these data were not examined . because our interventions were successful , we acted ( a ) on them by adopting them on a permanent basis . some would argue that with the small sample size , the improvement in rate of unplanned extubation was due to the hawthorne effect where performance improvement is attributed to the fact that performance is being studied and not actual quality improvement . because pdsa is a dynamic process , the rate of unplanned extubation will be reexamined at a later date to determine whether the level of improvement has been maintained . ideally , the statistical process control method would have been used to investigate trends in the rate of unplanned extubation prior to the implementation of the program . however , since there were only ten unplanned extubations in the first time period and two in the second period , this method could not be utilized . nonetheless , there was no indication that the rate of unplanned extubations had begun to decrease prior to the implementation of the program ( table 3 ) . they were carefully selected due to the seasonality of pediatric diseases such as respiratory syncytial virus . the six - month period when there were no data collection was to allow for this seasonality . the age , weight , size of endotracheal tube , and duration of intubation were not different in the groups . although there were differences in the reasons for intubation in the two groups , the differences likely would have biased the results towards a higher rate of unplanned extubation in the postintervention group since the patients intubated for respiratory failure would likely have more secretions and be more ill than those intubated for apnea . the similarity in the two groups leads us to believe that the decrease in the rate of unplanned extubation was due to our interventions and not due to differences in patient groups . in conclusion , we demonstrated that the rate of unplanned extubation in a picu can be decreased with a targeted intervention program tailored for the specific problems . this illustrates that efforts directed at improving quality of care should be based on the issues operative at that institution . by doing so , providers will be able to decrease the rate of unplanned extubations in their picu . | objective . to prospectively determine the rate of unplanned extubations and contributing factors and
determine whether a targeted intervention program would be successful in decreasing the rate of unplanned extubations .
design .
prospective , observational study . setting . a 10-bed pediatric intensive care unit ( picu ) .
patients .
all intubated pediatric patients during two time periods :
september 1 , 2000march 31 , 2001 and november 1 , 2001april 30 , 2002 . interventions . after determining the rate and causes of unplanned extubation
, a program was developed consisting of education and a formalized endotracheal tube taping policy .
data were then collected after implementation of the program .
measurements and main results . prior to the implementation of the program , there were 10 ( 14.7% ) unplanned extubations for a rate of 6.4 unplanned extubations per 100 ventilated days .
of the ten unplanned extubations , reintubation was required in 2 ( 20% ) .
inadequate sedation , poor taping , and improper position of the endotracheal tube were the items most frequently cited as causing an unplanned extubation .
following the program , there were two ( 3.4% ) unplanned extubations for 1.0 unplanned extubations per 100 ventilated days .
neither patient required reintubation .
there were no significant differences ( p > .05 ) in age , weight , endotracheal tube size , or duration of intubation in the two time periods .
however , there was a significant decrease in both the number ( p = .03 ) and the rate ( p = .04 ) of unplanned extubations after the implementation of the quality improvement program .
conclusions . the rate of unplanned extubation in a picu can be decreased with a quality improvement program that targets the institution 's specific needs . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
there are multiple reports of intercostal arterial bleeding resulting in abdominal wall hematomas and hemothorax that require embolization or even thoracotomy . to our knowledge however , this is the first description in the literature demonstrating hemoperitoneum from intercostal arterial bleeding requiring laparotomy . a 59-year - old male presented to our trauma center after sustaining a fall while transferring from a chair to wheelchair . his past medical history also included coronary artery disease , chronic kidney disease and a cerebral vascular accident . in the trauma bay , he reported left chest and left upper quadrant abdominal pain . on physical examination he was hemodynamically stable but tender to palpation over the left ninth and tenth ribs along the mid - axillary line . a computed tomography ( ct ) scan of the abdomen and pelvis with intravenous contrast demonstrated a large expanding left lateral chest wall hematoma , which appeared to be dissecting into the abdominal wall and peritoneum . 1 ) was associated with intraperitoneal blood in the perisplenic region and left paracolic gutter ( fig . 2 ) . a selective angiogram of multiple left intercostal arteries and the deep circumflex iliac branch of the left external iliac artery did not show any active contrast extravasation . the patient was admitted to the intensive care unit for close monitoring and administered idarucizumab ( praxbind ) for reversal of his anticoagulation . eight hours later , he developed worsening abdominal pain and became hemodynamically unstable with a systolic blood pressure of 80 mmhg . an exploratory laparotomy was then performed through a left subcostal incision . upon entry into the peritoneal cavity a significant hematoma in the lateral abdominal wall just under the lower ribs had caused a tear in the peritoneum leading to the intraperitoneal blood . active arterial bleeding could not be appreciated however a small amount of continuous bleeding from the left abdominal wall was controlled with suture plication . the patient received two units of packed red blood cells during the operation and was stable in the immediate postoperative setting . his post - operative course was otherwise unremarkable and he was discharged home after five days . figure 1:ct axial with intravenous contrast demonstrating a large left abdominal wall hematoma with active contrast extravasation from an intercostal artery . figure 2:ct coronal with intravenous contrast demonstrating intraperitoneal blood products within the perisplenic region and left paracolic gutter . ct axial with intravenous contrast demonstrating a large left abdominal wall hematoma with active contrast extravasation from an intercostal artery . ct coronal with intravenous contrast demonstrating intraperitoneal blood products within the perisplenic region and left paracolic gutter . blunt thoracic trauma is often associated with rib fractures that may disrupt nearby structures including intercostal and internal mammary vasculature . such injuries can be associated with hemothorax , pneumothorax , pulmonary contusions and the development of an extrapleural hematoma that has a reported incidence of 7.1% . in this report , clinically stable patients with normal hemodynamic parameters and radiographic features consistent with a non - expanding hematoma can be monitored in the intensive care unit with serial abdominal exams , laboratory studies , and reversal of coagulopathies . in this situation , the patient was on dabigatran etexilate which is an oral prodrug converted by a serum esterase to dabigatran , a potent , direct , competitive inhibitor of thrombin . idarucizumab was used to reverse our patient 's coagulopathy ; it is an fda - approved reversal agent for dabigatran and has shown to completely reverse the effects of dabigatran within minutes . idarucizumab is a monoclonal antibody fragment that binds dabigatran with an affinity that is 350 times greater than thrombin . if unavailable , vitamin k , prothrombin complex concentrate , or fresh frozen plasma can be used in an attempt to reverse anticoagulation . for patients presenting with rapidly expanding extrapleural hematomas , hemodynamic instability , or persistent bleeding from intercostal arterial branches , options for intervention include transcatheter arterial embolization ( tae ) or surgical exploration [ 1 , 4 ] . tae has been reported as a safe and reliable technique in the management of blunt intercostal arterial injury associated with clinical deterioration . in our patient , tae was undertaken shortly after his arrival to our trauma center but did not reveal an active source of bleeding . surgical exploration should be considered with failure of angioembolization or when interventional radiology is not available . strategies for operative exploration are dependent on the clinical scenario , hemodynamic stability , and the impact of associated injuries . in our case , a left - sided subcostal incision allowed access superiorly to the involved intercostal artery as well as inferiorly to evacuate the hematoma . surgical plication of the suspected area was conducted and the tear in the periotneum was repaired during our fascial closure . at the end of the operation satisfactory hemostasis was achieved and we did not feel the need to place a drain . in conclusion , immediate reversal of medically - induced coagulopathy is also indicated . in patients with hemodynamic instability , expanding hematoma , or active contrast extravasation that can not be embolized , | blunt trauma resulting in rib fractures can be associated with hemothorax , pneumothorax , pulmonary contusions or less frequently chest and abdominal wall hematomas .
our case describes the first report of hemoperitoneum secondary to intercostal arterial bleeding from blunt trauma in a patient on anticoagulation . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
most research studies thus far have tried to investigate the dysfunctions or pathology of families with the hope of shedding light on wrong issues related to families . in this regard , there are plenty of studies on family strength , rather than family dysfunction . in contrast to this line of research , which tries to investigate issues of family dysfunction , other research studies are carried out in the field of positive psychology to find out issues related to family strength . schumm ( 1985 ) proposed a multivariate model for measuring the strength of families ( 1 ) . on the basis of schumm s studies , a 20-item survey was designed to assess family strength characteristics that assessed time spent together , positive interaction / appreciation , open and empathetic communication / conflict resolution , commitment to relationship stability , commitment to relationship growth , and personal worth of self and others ( 2 ) . it was found that six interconnected elements of family strength may influence the marital satisfaction degree of wives and husbands . in addition , the strength of certain components may significantly predict strength in other components and characteristics ; intrinsic religiosity predicted worth , worth predicted commitment to stability and commitment to growth , these two commitments predicted communication / conflict resolution , communication / conflict resolution predicted positive interaction / appreciation , positive interaction / appreciation predicted time of being together , and strengths in almost all characteristics predicted marital satisfaction ( 3 ) . as discussed by researchers ( 1 , 4 ) , spiritual / religious aspects of lifestyle are an important element in strong families . research findings revealed that there exists a correlation between the positive effect of religious and spiritual variables and that of the positive outcomes in families and individuals ( 3 , 5 - 8 ) . spirituality has been studied for several decades , and the definition has been debated among researchers . a comprehensive conceptualization of one s spirituality is spiritual well - being because according to the definition of this construct , meaning and purpose in life is not dependent on a specific religious framework . there are two dimensions in spiritual well - being : one is related to a person s relationship with a greater power in a system of religious belief and the other is related to a sense of meaning and goal in life ( 9 ) . previous research reported that marital satisfaction is an important dependent variable of family strength characteristics ( 3 ) . on the other hand , research findings stressed that although low satisfaction is a sign of unstable marriage , this dissatisfaction does not necessarily end up with divorce ( 10 ) . dyadic adjustment seems to be a comprehensive conceptualization of quality of marriage since it can be a blanket term to cover either or both marital satisfaction ( which have a cognitive basis that involves a relationship of one 's circumstances to some standard ) and marital happiness ( which is based on an effective evaluation ) ( 11 ) . it is an indicator of rate of couple s adaptation in the relationship with each other and indicates general adaptation and consistency of couple s behavior in the marital relationship frame ( 12 ) . dyadic adjustment is not similar to marital satisfaction , couples that have adjustment , are not necessarily satisfied with their current situation ( 12 ) . also , according to previous studies , couples may express satisfaction regarding their marital relationship in spite of inconsistency , maladaptation , and maladjustment within their dyadic life ( 13 ) . dyadic adjustment is a process with consequences that can be identified with the rate of couple s problematic conflicts , interpersonal tensions , individual anxiety , marital satisfaction , coherence integrity , and collaboration about important problems ( 12 ) . as a common instrument in assessing relationship adjustments , the dyadic adjustment scale ( das ; spanier , 1976 ) is used as a means of measuring the marriage quality and similar dyads . it is also valuable for researchers and clinicians as it is short and at the same time multidimensional ( 14 ) . the revised dyadic adjustment scale ( 14 ) was developed according to problems with some of the subscales and individual items , and consists of three scales ; marital consensus , marital satisfaction and marital cohesion . as cultural values , beliefs and norms influence individuals view of themselves as well as the construction of love and intimacy ( 15 ) , using previous reported model of prediction - pattern among family strengths ( 3 ) , this study aimed to investigate the relationships among spiritual well - being , intervening variables ( family strengths ) , and the dependent variable , dyadic adjustment in an iranian married sample through structural equation modeling . in this study , spiritual well - being was considered as a predictor of family strengths and dyadic adjustment . personal worth of self and others was considered as a predictor of commitment to stability and commitment to growth . eventually , spiritual well - being and family strengths were considered as predictors of dyadic adjustment . in this correlational study , the proposed conceptual model was tested through structural equation modeling ( sem ) . the psychometric properties of the instruments were estimated through cronbach s coefficient ( 16 ) , test - retest reliability , and confirmatory factor analysis . relative chi - square statistic ( /df ) , goodness of fit index ( gfi ) , comparative fit index ( cfi ) , normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) were used to investigate the fit indices of the conceptual model . in an acceptable model , the nfi should be more than 0.90 , the gfi more than 0.90 , the cfi greater than 0.93 ( 17 ) , and the rmsea is less than 0.08 ( 18 ) and ideally less than 0.05 ( 19 ) . the upper confidence interval of the rmsea , however , should not be more than 0.08 ( 20 ) and the relative chi - square should be less than 2 or 3 ( 21 , 22 ) . two hundred and sixty - eight ( 171 females and 97 males ) married parent subjects were selected by convenience sampling from three universities ( university of mazandaran , islamic azad university - sari branch , sari agricultural sciences and natural resources university ) in mazandaran , iran , in the year of 2015 . they all had children ( 1 - 3 ; 1.19 0.42 ) and their average length of marriage was 7.04 years ( sd = 3.50 ; range = 3 - 27 years ) . lower that bachelor s degree educational level , having no children , and single parenthood , were the exclusion criteria of the study . before the administration of the instruments , the participants received a brief introduction about the nature of the research , ethical requirements for confidentiality and voluntary participation . in order to avoid influence of their spouse , the participants were asked to fill out the scales alone . only the subjects who completed the instruments the 20-item swbs ( 23 ) was developed to serve as a global psychological measure of one s perception of swb . the scale consists of two scales , the religious well - being scale ( rwbs ) ( 10 items ) , and the existential well - being scale ( ewbs ) ( 10 items ) . the rwb subscale assesses how one perceives the well - being of his or her spiritual life in relation to god . the ewb subscale is considered the social psychological dimension and assesses how well an individual is adjusted to themself , the community and surroundings . items are rated on a six - point likert scale from strongly agree to strongly disagree . responses to items 1 , 2 , 6 , 9 , 12 , 16 and 18 , are reverse scored . therefore , scores can range from 20 to 120 with higher scores indicating higher levels of swb ( 23 ) . the psychometric properties of the swbs were confirmed in various researches ( 8) . investigating psychometric properties of the farsi version of the swbs in an iranian population showed reasonable construct validity and internal consistency ( = 141.1 , df : 53 , gfi : 0.93 , agfi : 0.90 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; swbs = 0.87 , rwbs = 0.84 , ewbs = 0.84 ) ( 5 ) . the internal consistency coefficients ( cronbach s ) of the swbs and its subscales , namely religious well - being and existential well - being , in this study were found to be 0.88 , 0.80 and 0.83 , respectively ( n = 298 ) . this 20-item survey assessed the family strength variables of time together , positive interaction / appreciation , open and empathetic communication / conflict resolution , commitment to growth , commitment to stability , and personal worth of self and others ( 2 , 3 ) . items were rated on a five- point likert scale from strongly agree to strongly disagree . responses to items 8 , 13 , 15 , 19 and 20 were reverse scored ( 3 ) . investigating psychometric properties of the farsi version of the fss in an iranian population resulted in reasonable construct validity and internal consistency ( = 336.8 , df : 151 , gfi : 0.90 , agfi : 0.89 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; fss = 0.91 , personal worth of self and others = 0.70 , commitment to the relationship growth = 0.83 , commitment to the relationship stability = 0.60 , communication / conflict resolution = 0.87 , positive interaction / appreciation = 0.82 , time spent together = 0.68 ) ( 5 ) . in this study , the internal consistency coefficients ( cronbach s ) of the fss and its subscales , namely personal worth of self and others , commitment to relationship growth , commitment to relationship stability , communication / conflict resolution , positive interaction / appreciation , and time together , were found to be 0.89 , 0.72 , 0.83 , 0.64 , 0.85 , 0.70 , and 0.68 respectively ( n = 298 ) . the rdas , which consisted of 14 items was extracted from the 32-item dyadic adjustment scale ( das , spanier , 1976 ) and was used to assess the dyadic relationship quality ( 14 ) . this scale ( rdas ) shows the total adjustment score and has three subscales : dyadic consensus , dyadic satisfaction and dyadic cohesion ( 14 ) . to measure the amount of marital agreement between partners , dyadic consensus is used . the items in the dyadic consensus scale consist of five - point likert - scale ranging from zero ( always disagree ) to five ( always agree ) . as an example likewise , the items in this part also use a five - point likert - scale ranging from zero ( all the time ) to five ( never ) . one example from this subscale asks about how often do you and your partner quarrel ? two different likert - point scales are used for this subscale : for one item a five - point likert scale ( 0 = never , 4 = every day ) is used ( e.g. do you and your partner engage in common leisure interests ? ) and for the other three items a six - point likert scale ( 0 = never , 5 = more often ) is used ( e.g. have a stimulating exchange of ideas ) . the psychometric properties of the rdas ( reasonable construct validity and cronbach s from 0.80 to 0.90 ) were confirmed by previous research ( 12 ) . the persian version of the instrument was checked for construct validity and reliability , which showed reasonable construct validity and internal consistency ( cronbach s = 0.86 ) ( 12 ) . the internal consistency coefficients ( cronbach s ) of the rdas and its subscales , namely dyadic consensus , dyadic satisfaction and dyadic cohesion in this study were found to be 0.87 , 0.77 , 0.83 and 0.73 , respectively ( n = 298 ) . in this correlational study , the proposed conceptual model was tested through structural equation modeling ( sem ) . the psychometric properties of the instruments were estimated through cronbach s coefficient ( 16 ) , test - retest reliability , and confirmatory factor analysis . relative chi - square statistic ( /df ) , goodness of fit index ( gfi ) , comparative fit index ( cfi ) , normed fit index ( nfi ) , and the root mean square error of approximation ( rmsea ) were used to investigate the fit indices of the conceptual model . in an acceptable model , the nfi should be more than 0.90 , the gfi more than 0.90 , the cfi greater than 0.93 ( 17 ) , and the rmsea is less than 0.08 ( 18 ) and ideally less than 0.05 ( 19 ) . the upper confidence interval of the rmsea , however , should not be more than 0.08 ( 20 ) and the relative chi - square should be less than 2 or 3 ( 21 , 22 ) . two hundred and sixty - eight ( 171 females and 97 males ) married parent subjects were selected by convenience sampling from three universities ( university of mazandaran , islamic azad university - sari branch , sari agricultural sciences and natural resources university ) in mazandaran , iran , in the year of 2015 . they all had children ( 1 - 3 ; 1.19 0.42 ) and their average length of marriage was 7.04 years ( sd = 3.50 ; range = 3 - 27 years ) . lower that bachelor s degree educational level , having no children , and single parenthood , were the exclusion criteria of the study . before the administration of the instruments , the participants received a brief introduction about the nature of the research , ethical requirements for confidentiality and voluntary participation . in order to avoid influence of their spouse , the participants were asked to fill out the scales alone . only the subjects who completed the instruments the 20-item swbs ( 23 ) was developed to serve as a global psychological measure of one s perception of swb . the scale consists of two scales , the religious well - being scale ( rwbs ) ( 10 items ) , and the existential well - being scale ( ewbs ) ( 10 items ) . the rwb subscale assesses how one perceives the well - being of his or her spiritual life in relation to god . the ewb subscale is considered the social psychological dimension and assesses how well an individual is adjusted to themself , the community and surroundings . items are rated on a six - point likert scale from strongly agree to strongly disagree . responses to items 1 , 2 , 6 , 9 , 12 , 16 and 18 , are reverse scored . therefore , scores can range from 20 to 120 with higher scores indicating higher levels of swb ( 23 ) . the psychometric properties of the swbs were confirmed in various researches ( 8) . investigating psychometric properties of the farsi version of the swbs in an iranian population showed reasonable construct validity and internal consistency ( = 141.1 , df : 53 , gfi : 0.93 , agfi : 0.90 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; swbs = 0.87 , rwbs = 0.84 , ewbs = 0.84 ) ( 5 ) . the internal consistency coefficients ( cronbach s ) of the swbs and its subscales , namely religious well - being and existential well - being , in this study were found to be 0.88 , 0.80 and 0.83 , respectively ( n = 298 ) . this 20-item survey assessed the family strength variables of time together , positive interaction / appreciation , open and empathetic communication / conflict resolution , commitment to growth , commitment to stability , and personal worth of self and others ( 2 , 3 ) . items were rated on a five- point likert scale from strongly agree to strongly disagree . responses to items 8 , 13 , 15 , 19 and 20 were reverse scored ( 3 ) . investigating psychometric properties of the farsi version of the fss in an iranian population resulted in reasonable construct validity and internal consistency ( = 336.8 , df : 151 , gfi : 0.90 , agfi : 0.89 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; fss = 0.91 , personal worth of self and others = 0.70 , commitment to the relationship growth = 0.83 , commitment to the relationship stability = 0.60 , communication / conflict resolution = 0.87 , positive interaction / appreciation = 0.82 , time spent together = 0.68 ) ( 5 ) . in this study , the internal consistency coefficients ( cronbach s ) of the fss and its subscales , namely personal worth of self and others , commitment to relationship growth , commitment to relationship stability , communication / conflict resolution , positive interaction / appreciation , and time together , were found to be 0.89 , 0.72 , 0.83 , 0.64 , 0.85 , 0.70 , and 0.68 respectively ( n = 298 ) . the rdas , which consisted of 14 items was extracted from the 32-item dyadic adjustment scale ( das , spanier , 1976 ) and was used to assess the dyadic relationship quality ( 14 ) . this scale ( rdas ) shows the total adjustment score and has three subscales : dyadic consensus , dyadic satisfaction and dyadic cohesion ( 14 ) . to measure the amount of marital agreement between partners , dyadic consensus is used . the items in the dyadic consensus scale consist of five - point likert - scale ranging from zero ( always disagree ) to five ( always agree ) . as an example , one of the items in dyadic consensus asks about making major decisions . likewise , the items in this part also use a five - point likert - scale ranging from zero ( all the time ) to five ( never ) . one example from this subscale asks about how often do you and your partner quarrel ? two different likert - point scales are used for this subscale : for one item a five - point likert scale ( 0 = never , 4 = every day ) is used ( e.g. do you and your partner engage in common leisure interests ? ) and for the other three items a six - point likert scale ( 0 = never , 5 = more often ) is used ( e.g. have a stimulating exchange of ideas ) . the psychometric properties of the rdas ( reasonable construct validity and cronbach s from 0.80 to 0.90 ) were confirmed by previous research ( 12 ) . the persian version of the instrument was checked for construct validity and reliability , which showed reasonable construct validity and internal consistency ( cronbach s = 0.86 ) ( 12 ) . the internal consistency coefficients ( cronbach s ) of the rdas and its subscales , namely dyadic consensus , dyadic satisfaction and dyadic cohesion in this study were found to be 0.87 , 0.77 , 0.83 and 0.73 , respectively ( n = 298 ) . the 20-item swbs ( 23 ) was developed to serve as a global psychological measure of one s perception of swb . the scale consists of two scales , the religious well - being scale ( rwbs ) ( 10 items ) , and the existential well - being scale ( ewbs ) ( 10 items ) . the rwb subscale assesses how one perceives the well - being of his or her spiritual life in relation to god . the ewb subscale is considered the social psychological dimension and assesses how well an individual is adjusted to themself , the community and surroundings . items are rated on a six - point likert scale from strongly agree to strongly disagree . responses to items 1 , 2 , 6 , 9 , 12 , 16 and 18 , are reverse scored . therefore , scores can range from 20 to 120 with higher scores indicating higher levels of swb ( 23 ) . the psychometric properties of the swbs were confirmed in various researches ( 8) . investigating psychometric properties of the farsi version of the swbs in an iranian population showed reasonable construct validity and internal consistency ( = 141.1 , df : 53 , gfi : 0.93 , agfi : 0.90 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; swbs = 0.87 , rwbs = 0.84 , ewbs = 0.84 ) ( 5 ) . the internal consistency coefficients ( cronbach s ) of the swbs and its subscales , namely religious well - being and existential well - being , in this study were found to be 0.88 , 0.80 and 0.83 , respectively ( n = 298 ) . this 20-item survey assessed the family strength variables of time together , positive interaction / appreciation , open and empathetic communication / conflict resolution , commitment to growth , commitment to stability , and personal worth of self and others ( 2 , 3 ) . items were rated on a five- point likert scale from strongly agree to strongly disagree . responses to items 8 , 13 , 15 , 19 and 20 were reverse scored ( 3 ) . investigating psychometric properties of the farsi version of the fss in an iranian population resulted in reasonable construct validity and internal consistency ( = 336.8 , df : 151 , gfi : 0.90 , agfi : 0.89 , cfi = 0.93 , rmsea = 0.06 , cronbach s ; fss = 0.91 , personal worth of self and others = 0.70 , commitment to the relationship growth = 0.83 , commitment to the relationship stability = 0.60 , communication / conflict resolution = 0.87 , positive interaction / appreciation = 0.82 , time spent together = 0.68 ) ( 5 ) . in this study , the internal consistency coefficients ( cronbach s ) of the fss and its subscales , namely personal worth of self and others , commitment to relationship growth , commitment to relationship stability , communication / conflict resolution , positive interaction / appreciation , and time together , were found to be 0.89 , 0.72 , 0.83 , 0.64 , 0.85 , 0.70 , and 0.68 respectively ( n = 298 ) . the rdas , which consisted of 14 items was extracted from the 32-item dyadic adjustment scale ( das , spanier , 1976 ) and was used to assess the dyadic relationship quality ( 14 ) . this scale ( rdas ) shows the total adjustment score and has three subscales : dyadic consensus , dyadic satisfaction and dyadic cohesion ( 14 ) . to measure the amount of marital agreement between partners , dyadic consensus is used . the items in the dyadic consensus scale consist of five - point likert - scale ranging from zero ( always disagree ) to five ( always agree ) . as an example likewise , the items in this part also use a five - point likert - scale ranging from zero ( all the time ) to five ( never ) . one example from this subscale asks about how often do you and your partner quarrel ? two different likert - point scales are used for this subscale : for one item a five - point likert scale ( 0 = never , 4 = every day ) is used ( e.g. do you and your partner engage in common leisure interests ? ) and for the other three items a six - point likert scale ( 0 = never , 5 = more often ) is used ( e.g. have a stimulating exchange of ideas ) . the psychometric properties of the rdas ( reasonable construct validity and cronbach s from 0.80 to 0.90 ) were confirmed by previous research ( 12 ) . the persian version of the instrument was checked for construct validity and reliability , which showed reasonable construct validity and internal consistency ( cronbach s = 0.86 ) ( 12 ) . the internal consistency coefficients ( cronbach s ) of the rdas and its subscales , namely dyadic consensus , dyadic satisfaction and dyadic cohesion in this study were found to be 0.87 , 0.77 , 0.83 and 0.73 , respectively ( n = 298 ) . data analyses were performed using the spss 22.0 software , and amos 20.0 statistics packages . the age range of participants was 23 - 47 ( 31.07 4.37 years ) . the mean age for men was 32.89 years ( sd = 4.25 ) and for women was 30.05 years ( sd = 4.11 ) . in the case of the distribution of the educational level , 13% of the participants had phd , 50% had ma / msc , and 37% had ba . since d values were not distinctively apart ( 24 ) , the multivariate outliers were not a problem . the critical ratio of 2.05 for mardia s coefficient ( 4.27 ) proved the multivariate normality ( 25 ) . the model was therefore tested using maximum likelihood ( ml ) estimation and bootstrap procedure ( to assess mediation through examining the specific indirect effects within the model , ( 26 ) in amos.20 ) . table 1 shows the descriptive statistics , and the matrix of the relationships among the model variables , respectively . as shown in table 1 , the correlation coefficient between dyadic adjustment scales ( dyadic consensus , dyadic satisfaction , and dyadic cohesion ) and existential well - being ( r = 0.58 , 0.51 and 0.44 , respectively ) were higher compared to the correlations between each of them and religious well - being ( r = 0.31 , 0.22 and 0.21 , respectively ) . the correlation coefficients between both dyadic consensus and dyadic satisfaction and communication / conflict resolution ( r = 0.69 , and 0.60 , respectively ) were higher than the correlation coefficients of these scales and other family strengths . the correlation coefficient between dyadic cohesion and time spent together ( r = 0.52 ) was higher than the correlation coefficient of this scale and other family strengths . the correlation coefficients between both dyadic consensus and dyadic cohesion and commitment to stability ( r = 0.32 and 0.18 , respectively ) were lower than the correlation coefficients of these scales and other family strengths . the correlation coefficient between dyadic satisfaction and commitment to growth ( r = 0.30 ) was lower than the correlation coefficient of this scale and other family strengths . cs , commitment to stability ; cg , commitment to growth ; c / cr , communication / conflict resolution ; cfi , 0.98 ; dcon . , dyadic cohesion ; /df , 1.77 ; da , dyadic adjustment ; ewb , existential well - being ; gfi , 0.96 ; nli , 0.96 ; pwso , personal worth of self and others ; pi / a , positive interaction / appreciation ; p , 0.005 ; rmsea , 0.054 ; hoelter 's index = 253 ( 0.01 ) ; rwb , religious well - being ; swb , spiritual well - being ; tt , time spent together . results showed that the standardized direct effects of spiritual well - being on positive interaction / appreciation ( 0.13 , p = 0.09 ) , personal worth of self and others on commitment to stability ( 0.02 , p = 0.11 ) and dyadic adjustment ( 0.09 , p = 0.15 ) , commitment to stability on commitment to growth ( 0.03 , p = 0.13 ) , positive interaction / appreciation ( 0.03 , p = 0.18 ) , time together ( 0.08 , p = 0.10 ) and dyadic adjustment ( .06 , p = 0.20 ) , commitment to growth on positive interaction / appreciation ( 0.04 , p = 0.13 ) , time together ( 0.04 , p = 0.15 ) and dyadic adjustment ( 0.09 , p = 0.06 ) , were not significant . as shown in figure 1 , spiritual well - being has a direct effect on personal worth of self and others ( 0.69 , p < 0.01 ) , commitment to stability ( 0.42 , p < 0.01 ) , commitment to growth ( 0.28 , p < 0.01 ) , communication / conflict resolution ( 0.20 , p < 0.01 ) , time together ( 0.20 , p < 0.01 ) and dyadic adjustment ( 0.24 , p < 0.01 ) . personal worth of self and others had a direct effect on commitment to growth ( 0.33 , p < 0.01 ) , communication / conflict resolution ( 0.41 , p < 0.01 ) , positive interaction / appreciation ( 0.38 , p < 0.01 ) and time together ( 0.21 , p < 0.01 ) . commitment to stability and commitment to growth had a direct effect on communication / conflict resolution ( 0.15 and 0.18 , p < 0.01 , respectively ) . communication / conflict resolution had a direct effect on positive interaction / appreciation ( 0.39 , p < 0.01 ) , time together ( 0.23 , p < 0.01 ) and dyadic adjustment ( 0.34 , p < 0.01 ) . positive interaction / appreciation had a direct effect on time spent together ( 0.16 , p < 0.01 ) and dyadic adjustment ( 0.25 , p < 0.01 ) . time spent together had a direct effect on dyadic adjustment ( 0.27 , p < 0.01 ) . as shown in table 2 , spiritual well - being has indirect effects on commitment to growth through the mediating role of personal worth of self and others ( 0.23 , ci [ 0.11 , 0.36 ; p = 0.003 ] , standardized total effect = 0.51 ) , communication / conflict resolution through mediating roles of personal worth of self and others , commitment to stability , and commitment to growth ( 0.43 , ci [ 0.33 , 0.54 ; p = 0.002 ] , standardized total effect = 0.63 ) , positive interaction / appreciation through the mediating roles of personal worth of self and others , commitment to stability , commitment to growth , and communication / conflict resolution ( 0.51 , ci [ 0.41 , 0.59 ; p = .005 ] , standardized total effect = 0.51 ) , time spent together through the mediating roles of personal worth of self and others , commitment to stability , commitment to growth , communication / conflict resolution , and positive interaction / appreciation ( 0.38 , ci [ 0.28 , 0.49 ; p = 0.003 ] , standardized total effect = 0.58 ) , and dyadic adjustment through the mediating roles of personal worth of self and others , commitment to stability , commitment to growth , communication / conflict resolution , positive interaction / appreciation , and time spent together ( 0.50 , ci [ 0.41 , 0.63 ; p = .003 ] , standardized total effect = 0.74 ) . the results showed that personal worth of self and others had no effect on commitment to stability but had an indirect effect on communication / conflict resolution through the mediating role of commitment to growth ( 0.06 , ci [ 0.02 , 0.13 ; p = 0.003 ] , standardized total effect = 0.47 ) , positive interaction / appreciation through mediating roles of commitment to growth and communication / conflict resolution ( 0.18 , ci [ 0.11 , 0.29 ; p = .002 ] , standardized total effect = 0.56 ) , time spent together through the mediating roles of commitment to growth , communication / conflict resolution , and positive interaction / appreciation ( 0.20 , ci [ 0.11 , 0.32 ; p = 0.003 ] , standardized total effect = 0.41 ) , and dyadic adjustment through the mediating roles of commitment to growth , communication / conflict resolution , positive interaction / appreciation , and time spent together ( 0.41 , ci [ 0.29 , 0.51 ; p = .003 ] , standardized total effect = 0.41 ) . commitment to stability had an indirect effect on positive interaction / appreciation through the mediating role of communication / conflict resolution ( 0.06 , ci [ 0.02 , 0.10 ; p = 0.003 ] , standardized total effect = 0.06 ) , time spent together through mediating roles of communication / conflict resolution and positive interaction / appreciation ( 0.04 , ci [ 0.01 , 0.09 ; p = 0.003 ] , standardized total effect = 0.04 ) , and dyadic adjustment through the mediating roles of communication / conflict resolution , positive interaction / appreciation , and time spent together ( 0.08 , ci [ 0.03 , 0.13 ; p = .004 ] , standardized total effect = 0.08 ) . commitment to growth had an indirect effect on positive interaction / appreciation through the mediating role of communication / conflict resolution ( 0.07 , ci [ 0.02 , 0.12 ; p = 0.007 ] , standardized total effect = 0.07 ) , time spent together through the mediating roles of communication / conflict resolution and positive interaction / appreciation ( 0.05 , ci [ 0.02 , 0.11 ; p = 0.004 ] , standardized total effect = 0.05 ) , and dyadic adjustment through the mediating roles of communication / conflict resolution , positive interaction / appreciation and time spent together ( 0.09 , ci [ 0.03 , 0.16 ; p = 0.005 ] , standardized total effect = 0.09 ) . communication / conflict resolution had an indirect effect on time spent together through the mediating role of positive interaction / appreciation ( 0.07 , ci [ 0.02 , 0.12 ; p = 0.004 ] , standardized total effect = 0.30 ) , and dyadic adjustment through the mediating roles of positive interaction / appreciation and time spent together ( 0.18 , ci [ 0.11 , 0.26 ; p = 0.003 ] , standardized total effect = 0.52 ) . positive interaction / appreciation had an indirect effect on dyadic adjustment through the mediating role of time spent together ( 0.04 , ci [ 0.02 , 0.09 ; p = 0.003 ] , standardized total effect = 0.29 ) . the purpose of the present study was to test a structural model of hypothesized relationships between spiritual well - being , intervening variables of personal worth of self and others , commitment to relationship stability , commitment to relationship growth , positive interaction / appreciation , communication / conflict resolution , time spent together , and the dependent variable of dyadic adjustment . the results confirmed that spiritual well - being can be a strong indicator of personal worth of self and others . as mentioned in the literature , intrinsic religiosity was found to be a strong indicator of personal worth of self and others ( 3 ) . it has also been reported that intrinsic religiosity is an attribute of a person , who has religious experiences and engages in internal / external gains and as a member of that religious community , he / she gains some internal beliefs of personal worth ( 3 ) . as spiritual well - being includes both existential and religious aspects of spirituality ( 9 ) , this result can confirm the feeling of personal worth of self and others in a dyadic relationship through spiritual well - being . according to the results of the present study , spiritual well - being has a direct effect on personal worth of self and others , commitment to stability , commitment to growth , communication / conflict resolution , time together , and dyadic adjustment , and also has a positive effect on positive interaction / appreciation through the mediating role of personal worth of self and others , commitment to stability , commitment to growth , and communication / conflict resolution , indirectly . the results showed that spiritual well - being has an indirect effect on commitment to stability through the mediating role of personal worth of self and others , communication / conflict resolution through the mediating role of personal worth of self and others , commitment to stability , and commitment to growth , time together through the mediating role of personal worth of self and others , commitment to stability , commitment to growth , communication / conflict resolution , and positive interaction/ appreciation , and dyadic adjustment through the mediating role of personal worth of self and others , commitment to stability , commitment to growth , communication / conflict resolution , positive interaction / appreciation , and time together . inconsistent with previous findings ( 3 ) , the results of the present study revealed that personal worth of self and others had no effect on a person s level of commitment to the relational stability of their marriage . however , as shown in figure 1 and table 2 , personal worth of self and others had a positive effect on a couple s commitment to relationship growth . since personal worth of self and others include three areas of worth , namely , self - worth , valuing their spouse and feeling valued by a spouse , this result seems to state that if a person feels personal worth , feels good about his / her spouse , and believes his / her spouse values him / her , he / she may be more motivated to grow the quality of his / her marriage . according to the results of this study , personal worth of self and others has a positive direct effect on commitment to growth , communication / conflict resolution , positive interaction / appreciation , time spent together and dyadic adjustment . as shown in figure 1 and table 2 , personal worth of self and others had a positive total effect on dyadic adjustment through the mediating role of commitment to growth , communication / conflict resolution , positive interaction / appreciation , and time spent together , indirectly . the results of the present study also indicated that both commitment to stability and commitments to growth have a positive effect on communication / conflict resolution as noted by some researchers ( 3 ) . strong commitment and sense of worth positively affect a couple 's ability to negotiate and resolve areas of conflict . as shown in figure 1 and table 2 , both commitment to stability and commitment to growth had a positive effect on positive interaction / appreciation , time together and dyadic adjustment , indirectly . the results also confirmed the positive effect of communication / conflict resolution on positive interaction / appreciation , time together and dyadic adjustment . the positive effect of communication / conflict resolution on positive interaction / appreciation , confirms that strength in the areas of worth , commitment , and communication / conflict resolution can affect the quality of couple s interaction . according to the results of the present study , communication / conflict resolution can affect time spent together through positive interaction / appreciation , and dyadic adjustment through positive interaction / appreciation and time spent together . it seems , through feeling personal worth of self and others and commitment to the relationship , couples may be more motivated to communicate based on mutual openness , honesty , respect and kindness . the results also indicated that couples level of time spent together can increase when they are strong in the previous intervening variables . generally , the findings revealed that high spiritual well - being , valuing each other , commitment to each other and communicating well as well as enjoying and spending time together all increase couple s degrees of dyadic adjustment . these results consisted of a previous hierarchical model of hypothesized relationships between intervening variables of intrinsic religiosity , personal worth of self and others , commitment to relationship stability , commitment to relationship growth , positive interaction / appreciation , communication / conflict resolution , and time spent together , and the dependent variable of marital satisfaction ( 3 ) . the statistical population and non - equal sample size of male and female participants are the limitations of the present study . of course , more studies are needed with different measures and in different populations ( e.g. different socioeconomic levels , different cultures and sub - cultures , investigating the conceptual model based on gender and family life cycle difference ) to provide a comprehensive theoretical explanation . | objectivesthe aim of the present study was to test a structural model of hypothesized relationships between spiritual well - being , intervening variables of personal worth of self and others , commitment to relationship stability , commitment to relationship growth , positive interaction / appreciation , communication / conflict resolution , time spent together , and , the dependent variable , dyadic adjustment.materials and methodstwo hundred and sixty eight ( 171 females and 97 males ) married parent subjects were selected by convenience sampling from three universities in mazandaran , iran , to take part in this study .
they were all volunteers and were not paid and their age range was 23 to 47 ( 31.07 4.37 years ) .
all participants were asked to complete the spiritual well - being scale ( swbs ) , family strengths scale ( fss ) and revised dyadic adjustment scale ( rdas).resultsthe results from structural equation modeling confirmed a hierarchy for the development of family strengths , and indicated that spiritual well - being and strength in most characteristics affected dyadic adjustment , positively ( p < 0.05).conclusionscouples level of dyadic adjustment is increased when they have higher spiritual well - being , value each other , have commitment to each other , communicate well , enjoy being with each other , and spend time together . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the idea of fabricating
synthetic molecular machines has fascinated a whole generation of supramolecular chemists . molecular
switches , which undergo mechanical motion in response to chemical , photochemical , or electrochemical stimuli , represent excellent precursors for
molecular machines . especially , mechanically interlocked molecules such as rotaxanes have been investigated thoroughly
in this respect . in solution , molecular switches are randomly
oriented , which makes the implementation of directional processes
difficult . the still challenging integration of molecular switches
and machines into larger ordered arrays and functional devices will
help solve this problem . inspired by nature s use of membranes
to generate order , chemists can employ soft interfaces such as langmuir blodgett
films or hard interfaces such as metal surfaces as alternatives to membranes to fabricate ordered arrays of synthetic
molecular machines . the use of metal surfaces is particularly appealing ,
as the generation of self - assembled monolayers , layer - by - layer self - assembly , and the fabrication
of surface - bound metal organic frameworks ( surmofs ) are well understood . several seminal steps in this direction have
been made , e.g. , leigh s monolayers
of axle - fluorinated , light - switchable rotaxanes moving diiodomethane
droplets uphill , rapenne s unidirectionally
rotating surface - bound piano - stool rotors , feringa s surface - anchored overcrowded
double bond motors , or stoddart s
electrochemically switched cantilever - bending molecular - muscle
rotaxanes . in the latter example , the finding
that a cantilever can be bent , when the rotaxanes are switched , suggests
that the rotaxanes deposited on the cantilever operate in a directional
way . this aspect , however , has so far not been further investigated
in greater detail . the aim of the present study is to provide
evidence for a coupled motion of switchable interlocked molecules . the term coupled motion as we use it here refers to switching whole
arrays of rotaxane shuttles that are aligned in a well - ordered state
before switching and are again aligned in another well - ordered state
after switching . for such motion , lateral order and the alignment
of the deposited molecules are essential , and multilayers may enhance
the achievable effects . therefore , our study also aims to fabricate
well - ordered multilayers of rotaxanes , in which a coupled motion can
be observed . here we describe novel ordered mono- and multilayers
of chemically switchable amide rotaxanes on gold using either a click
chemistry approach to covalently attach them to an azide - terminated
self - assembled monolayer ( sam ) or a coordination - chemistry - based layer - by - layer
self - assembly strategy . the rotaxane layers reversibly switch
in a coupled manner between two differently ordered layer structures . the present study examines the importance of ordered arrays for coupled
molecular motions in stimulus - responsive shuttle rotaxanes . starting from suitably functionalized tetralactam macrocycles mcn , a templated
one - step rotaxane synthesis has been achieved by efficiently trapping
diketopiperazine 1 inside the wheels to form hydrogen - bonded
pseudorotaxanes protn ( scheme 1 ; for experimental details , see the supporting information ) . the sterically demanding
tritylphenyl stopper groups have been attached to the axle alkynes
by a cu - catalyzed click reaction with 2 . for surface deposition , alkyne
and terpyridine side chains have been introduced by sonogashira and suzuki cross - coupling
reactions with ( trimethylsilyl)acetylene and boronic acid pinacol
ester 3 , respectively . after trimethylsilyl ( tms ) deprotection , rot3 is ready to be clicked covalently to an azide - functionalized
surface . rot6 can be used for the deposition by metal - coordination - based
layer - by - layer self - assembly . top : the axle threading reaction
is templated by hydrogen - bond - mediated formation of pseudorotaxane protn , to which the two tritylphenyl azide
stoppers are then clicked . bottom : the acetylene and
terpyridine side chains are attached to the iodo - substituted precursor
rotaxanes by sonogashira and suzuki cross - coupling reactions , respectively . evidence for rotaxane formation comes from significant
upfield shifts ( figure 1a , b ) observed for the
diketopiperazine protons h ( = 1.01
ppm ) and h ( = 0.84 ppm ) , which
are located inside the wheel s cavity and experience the anisotropy
of the surrounding aromatic rings . also , the triazole proton h is affected by the presence of the wheel ; the corresponding
signal shifts to higher field by = 0.33 ppm . bottom : partial h nmr spectra ( 4 mm , cdcl3 ,
298 k ) of ( a ) free , stoppered axle , ( b ) rotaxane rot6 before addition of 1 equiv of tetrabutylammonium chloride ( ( tba)cl ) ,
( c ) rotaxane rot6 after addition of 1 equiv of ( tba)cl ,
and ( d ) rot6 after chloride removal by precipitation
of nacl with 1 equiv of sodium tetrakis(3,5-bis(trifluoromethyl)phenyl)borate
( nabar4 ) . the large signal
shifts upon chloride addition and removal agree with those expected
for the wheel translating from the central diketopiperazine station
to one of the triazoles . the slight peak broadening and minor shift
differences in spectrum d compared to spectrum b are due to the polarity
change caused by the added salts . to
gain insight into the switching process before transfer to the surface ,
a h nmr study of the chloride - induced axle movement of rot6 has been performed in cdcl3 ( figure 1b d ) . clearly , chloride addition causes significant
signal shifts , providing evidence for a wheel translocation from the
central diketopiperazine to a triazole station , where the wheel binds
mainly through c the downfield shifts of the signals ( i ) for the amide nh protons
h ( = 0.70 ppm ) reflecting the change in
hydrogen - bonding partner , ( ii ) for the triazole proton h ( = 0.44 ppm ) , which moves deeper into the wheel ,
but at the same time interacts with the chloride anion , and ( iii )
for the diketopiperazine proton h ( = 0.14
ppm ) , which moves somewhat away from the center of the wheel cavity ,
are most indicative . also , the isophthaloyl diamide protons h and h and almost all other signals are clearly
affected by the axle movement . as only one set of nmr signals is observed
for either one , the wheel s shuttling motion between the two
triazoles and a concerted chloride flip / axle rotation both proceed
fast on the nmr time scale . removal of the chloride anion by precipitation
with nabar yields the same spectrum as obtained prior
to the switching cycle with the exception of the additional signals
for the bar anions . consequently , the stimulus - induced
structural changes in the rotaxane can be fully reversed . analogous
results have also been obtained with the diiodo - substituted precursor
rotaxane rot5 ( supporting information , figure s14 ) . for
the deposition of a monolayer of rot3 ( figure 2a ) , an azide - functionalized self - assembled monolayer
( sam ) was generated by reacting a hcl - cleaned gold substrate with
a 1 mm ethanol solution of bis(11-azidoundecyl ) disulfide ( aud ) for 24 h. subsequently , the acetylene - functionalized
rotaxane rot3 was attached covalently through a click
reaction catalyzed by the cu / n - heterocyclic carbene complex cat . this catalyst exhibits excellent
activity in dichloromethane , a solvent that needs to be used because
of the limited solubility of the rotaxane in other solvents . ( a ) monolayers of
covalently fixed rot3 are made by first depositing an
azide - terminated sam of aud from ethanol solution onto
a hcl - cleaned gold surface . ( b ) on a pst sam , first pd ions are deposited from acetonitrile
and mediate the connection to the first layer of rot6 deposited from dichloromethane solution . subsequently , this monolayer
can be converted into multilayers by an alternating deposition of
fe ions and rot6 . similarly , a 1:3 mixed
monolayer of tdt and dt can serve as a templating
sam with fe ions connecting the next layer to it . figure 2b summarizes the formation of mono- and multilayers of rot6 by coordination - based layer - by - layer self - assembly on a templating ,
rigid pyridine - functionalized sam of ( e)-4-(pyridin-4-yl)stilbenethiol
( pst ) . a 10 min metal ion deposition
from a 1 mm acetonitrile solution of [ pd(ncch3)4](bf4)2 was followed by the deposition of a
monolayer of the rotaxane from a 1 mm dichloromethane solution of rot6 which was given 24 h to assemble into an ordered monolayer
array . as this rot6 monolayer is again terminated by
terpyridyl side chains , multilayers can be fabricated following this
procedure repeatedly , now using fe ions to connect the
two terpyridines of adjacent rotaxanes . alternating deposition steps
with fe(bf4)26h2o as the metal
ion source and rot6 thus led to a growing multilayer . alternatively , a mixed template sam prepared from a 1:3 mixture
of terpyridine - terminated dodecanethiol ( tdt ) and decanethiol
( dt ) ( figure 2b ) can be used as
the basis for multilayer growth ( supporting information , figures s24s27 ) . this ratio
is the optimum for metal deposition , and phase segregation of the
two components was ruled out earlier . the dt decyl chain is shorter by two carbon atoms as
compared to the tdt dodecyl chain , resulting in well - ordered
alkyl chains , yet flexible , unordered terpyridine groups that are
exposed on top of the sam . the fabrication
of the tdt / dt - supported rot6 multilayer follows the same procedure as that deposited on pst . as this sam bears terpyridine groups as the anchors for
the next layer , fe is also used as the first metal ion
layer . to allow imaging of the deposition process , not only
fully covered surfaces but also surfaces structured laterally by microcontact
printing ( cp ) have been produced . these surfaces
have the advantage that atomic force microscopy ( afm ) can be used
to determine layer thicknesses by comparing the heights of the growing
multilayer with the constant height of the areas passivated by a simple
alkanethiol . poly(dimethylsiloxane ) stamps patterned with 10 m
wide dots spaced at 5 m distances were inked with octadecanethiol
( odt ) and then brought into contact with a freshly cleaned
gold surface . afterward , the space between the dots was backfilled
with pst , followed by formation of the rot6 multilayer as described above . the characterization of the surface films
is achieved by combining data from x - ray photoelectron spectroscopy
( xps ) , transmission uv / vis spectroscopy , afm , time - of - flight secondary
ion mass spectrometric ( tof - sims ) imaging , and near - edge x - ray absorption
fine structure ( nexafs ) spectroscopy . ( a ) a comparison of the high - resolution n 1s
xp spectra ( excitation energy : 500 ev ) of the aud sam
before and after click deposition of rot3 reveals conversion
of the azide ( green signals ) to the triazole ( red ) superimposed by
the amide and diketopiperazine n atoms of the rotaxane ( blue ) . ( b )
xps c 1s / au 4f7/2 ratios increase with each deposited rot6 layer . ( c ) transmission uv / vis spectra of 010 rot6 layers on pst / semitransparent gold . ( d )
relative increase of the c 1s / au 4f7/2 ratios upon deposition
of rot6 on different templating sams ( pst vs tdt / dt , 1:3 ) . afm images and height profiles of micropatterned surfaces functionalized
( a ) with odt dots between unfilled voids , ( b ) with odt dots and voids backfilled with pst , and ( c )
with odt dots and voids backfilled with pst and one rot6 layer . ( d ) tof - sims images of the surface
backfilled with pst and rot6 . left : secondary
ion intensities of cxhy ions indicative of odt alkyl chains . right : secondary ion intensities of cn and c3n ions indicative of the presence of pyridines . in line with earlier results , the aud sam exhibits three signals
in the high - resolution n 1s xp spectrum ( figure 3a ) which can be assigned to the three azide nitrogen atoms . after
the click reaction with rot3 , three signals appear shifted
to those binding energies expected for the triazole nitrogen atoms . in addition , a more intense signal for the secondary amides of the
macrocycle and a smaller signal for the tertiary amides of the diketopiperazine
axle appear . multilayer growth of rot6 on the pst sam can be followed by transmission
uv / vis , when the multilayer is deposited on a semitransparent gold
surface ( figure 3c ) . all bands observed in
the solution spectrum of the ( rot4)2fe control complex are also found for the rotaxanes deposited
on the surface : the metal - to - ligand charge transfer ( mlct ) band at
ca . 560 nm , the ligand - centered band at ca . 340 nm , and the *
transition of the aromatic rings at ca . in addition , the surface
plasmon band appears at around 470 nm and , as expected , shifts slightly
to higher wavelengths with increasing layer thickness . a plot of the
intensity at 270 nm over the rotaxane layer number clearly demonstrates
a linear growth from which we conclude that the same amount of rot6 is added in each rotaxane deposition step . the carbon / gold ratio extracted
from the c 1s and au 4f7/2 xps data ( figure 3b ) increases with each deposition of the rotaxane , because
the number of carbon atoms in the organic layer increases and the
gold signal is more attenuated at the same time caused by inelastic
scattering of the photoelectrons emitted from the gold surface at
the organic layers above . a comparison of the xps c 1s / au 4f7/2 ratios of the rot6 single and triple layers on pst with the
same layers on tdt / dt ( 1:3 ) reveals significantly
more rotaxane deposited in each layer on the pst sam
( figure 3d ) . this is in marked contrast to
the formation of densely packed multilayers of mc3 on tdt / dt . the introduction
of the axle with its two bulky stopper groups increases the size of
rotaxane rot6 significantly compared to that of mc3 . thus , it does not match the grid of the terpyridine anchor
points on the tdt / dt monolayer as nicely
as mc3 . the smaller terminal pyridines of the pst sam offer a finer grid of coordination sites that allows rot6 to
assemble laterally more easily and to form layers of higher density . when a micropatterned surface was examined by afm directly after
deposition of the odt dots , a clear contrast between
the odt - passivated dots and the void space between them
was observed , which translates into a layer thickness of the odt monolayer of 1.94 nm ( figure 4a ) .
after the space between the passivated dots is backfilled with one
layer of rot6 on pst , the contrast reverses
as the rotaxane layer is now higher than the odt layer ( figure 4b ) . the thickness of one rotaxane layer can be determined
from figure 4b ( voids backfilled with pst alone ) and figure 4c ( voids backfilled
with pst and rot6 ) to be ca . this value
nicely corresponds to thicknesses of 1.51.8 nm determined
earlier for layers of macrocycle mc3 . as the fe fe distance calculated for mc3 and rot6 oligomers is ca . 3.5 nm , the tilt angle between
the rotaxane wheel and the surface can be estimated to be ca . 30. the distribution of odt and rot6 on the micropatterned surface was also imaged by tof - sims . a 25 25 m area of the surface was scanned
with 256 256 pixel resolution by a focused beam of bi3 ions using the novel collimated burst alignment
mode for high lateral as well as high mass resolution . in the positive mode , the cxhy secondary ions characteristic
for the odt alkyl chains preferentially originate from
the dot areas ( figure 4d , left ) . instead , the
areas covered by the pst - based multilayer of rot6 do not contain any linear alkyl chains and produce only a very small
cxhy ion count . vice versa , cn and c3n secondary ions in the negative mode confirm the presence
of pyridines and thus of rot6 in the areas between the odt dots ( figure 4d , right ) . notably ,
these ions are absent in the odt areas , indicating an
excellent specificity of binding rot6 to the pst areas . contact angle measurements upon repeated chloride addition and
removal indicate a strong and reversible change of polarity of the
rotaxane multilayers which is tentatively attributed to on - surface
chloride - mediated switching ( figure 5a ) . when
the aud rot3 monolayer and a pst rot6 multilayer are treated with a
1 mm dichloromethane ( dcm ) solution of tetrabutylammonium chloride
( ( tba)cl ) and then immersed for 10 min in pure dichloromethane to
remove residual salt , the contact angles drop by almost 20.
this indicates a significantly higher polarity after chloride addition . removal of the chloride with nabar4 ( 1 mm ,
dcm ) and subsequent washing with dcm , water , and dcm again to remove
salt residues bring the contact angles back to the initial values ,
and switching can be repeated . one might argue that the deposition
of a salt is expected to increase polarity , even if it is not specifically
bound and does not switch the rotaxane . therefore , a macrocycle mc3 multilayer was used as a control , and this exhibited much
smaller contact angle changes ( ca . furthermore , the contact
angle changes were not affected significantly when the less lipophilic
tetramethylammonium chloride ( ( tma)cl ) was applied . investigation of the
on - surface switching via contact angle measurements for the aud rot3 monolayer and pst and tdt / dt ( 1:3)rot6 multilayers ( 20 layers ) over five switching steps . for
comparison , an mc3 multilayer ( 20 layers ) is examined . these results are an indication
of , but certainly not strict evidence for , on - surface rotaxane switching . angle - resolved nexafs experiments provide evidence for stimulus - induced
structural changes within the surface : as synchrotron light is linearly
polarized , linear dichroism effects are observed when the deposited
molecules have a preferred orientation . the transition dipole moment vectors for the excitation of core
electrons into the * ( aromatic carbons ) or c h * orbitals
( aliphatic carbons ) line up with the electric field vector of the
incident light differently at different angles , and angle - dependent
intensities for the * and c h * resonances result . before
we discuss the nexafs results shown in figures 6 and 7 , let us briefly mention the following
limitation : it would be very desirable to be able to analyze the nexafs
data in much greater detail to correlate the linear dichroism effects
with the details of the structures and the orientation of the rotaxanes
on the surface . while this is certainly possible for simpler self - assembled
monolayers , let us take the * resonance
as an example , as this is the most important one in our paper : the
linear dichroism depends on the orientation of aromatic rings in the
mono- and multilayers . the macrocyclic wheels are rather rigid , but
contain aromatic rings in many different orientations , even when the
macrocycles are fixed in a certain orientation . if one includes the
axle with the trityl stoppers , additional orientations of aromatic
rings come into play . consequently , many different dichroism effects
finally are superimposed , resulting in a net effect , which is typically
not very large as we have experienced several times in the past for
other surfaces . despite this limitation , the observation of such a linear dichroism
clearly indicates the rotaxanes to be ordered into arrays with preferred
orientations . angle - resolved nexafs spectra of ( a ) a rot3 monolayer on aud , ( b ) a rot6 monolayer on pst ( left , ( tba)cl ; right ,
( tma)cl ) , and ( c ) a rot6 monolayer on tdt / dt ( 1:3 ) . in each case , the nexafs results
obtained for the pristine layer , that after chloride addition , and
that after chloride removal with nabar4 are
shown ( top to bottom ) . red and black lines represent the nexafs spectra
obtained at 30 and 90 angles of the incident synchrotron
light beam , respectively . similar to the results in figure 7 ,
the nexafs results obtained for the pristine layer , that after chloride
addition , and that after chloride removal with nabar4 are shown ( top to bottom ) . red and black lines represent
the nexafs spectra obtained at 30 and 90 angles of the
incident synchrotron light beam , respectively . the pristine monolayer
of rot3 on aud ( figure 6a ) exhibits a linear dichroism on the * resonance after chloride addition , this angle dependence vanished almost completely .
consequently , either the switched rotaxanes may be disordered after
switching or the dichroism effects almost exactly cancel . chloride
binding to the rotaxane thus induces a structural change , while the
question of whether this is accompanied by a loss of orientation can not
clearly be answered . as neither the underlying aud alkyl
chains nor the tetrabutylammonium counterions contain systems
and thus do not affect the * resonance , the structural change
must have occurred within the rotaxane structure , and we ascribe it
to the chloride - induced switching of the rotaxane . interestingly ,
chloride removal with nabar4 leads back to
angle - resolved nexafs spectra very similar to the initial ones . consequently ,
switching the rotaxanes back into the chloride - free state yields an
ordered structure similar to the initial one . the reversibility of
the structural changes strongly supports rotaxane switching to be
the origin of the linear dichroism changes . the monolayer of rot6 on pst ( figure 6b )
again exhibits a linear dichroism , indicating a preferred orientation
of the rotaxanes . in contrast to the rot3 monolayer ,
the linear dichroism became even more pronounced upon chloride addition . clearly , this surface can be switched between two different
structures , which both contain the rotaxanes in a preferred orientation .
as a control , the pst the results are virtually identical indicating
that the counterion is not the primary origin of the structural changes . in marked contrast , the tdt / dt rot6 monolayer exhibits only negligible linear dichroism effects
before and after switching ( figure 6c ) . this
observation is interesting as this surface was found to be less densely
packed ( see above , figure 3d ) . even if the
individual , nonordered rotaxanes deposited on the tdt / dt sam undergo chloride - induced switching , no coupled
axle movement is observed that transfers an ordered structure into
another ordered structure . in turn , we conclude the reversibly switchable
transition of one ordered layer structure of rot6 on pst into a second , yet different ordered structure as a clear
indication that the densely packed rot6 layer on pst switches in a coupled manner . finally , the triple
layer of rot6 on pst again exhibits linear
dichroism effects on the * resonance that change with chloride
addition and removal ( figure 7 ) . the *
resonance of the initial triple layer is more intense at a 30
angle of the incident light . after chloride addition , the more intense
* resonance appears at 90 instead . rot6 monolayer is consequently transferable to multilayers of rot6 on
pst as well . in principle , one might envisage another mechanism
by which the transition between two ordered surfaces could be accomplished
at least for those surfaces that are fabricated using the metal coordination
approach : the rotaxanes on the surface might dissociate from the surface ,
switch , and redeposit in a different packing . this scenario can , however ,
be ruled out on the basis of the following arguments : ( i ) in those
cases in which the metal ion coordination is clearly reversible ( e.g. ,
when zn(ii ) is used to connect two terpyridines ) , no continuous layer
growth is observed . ( ii ) the mono- and
multilayers of macrocycles have been tested extensively , even against
alkaline edta solutions for stability , and have not shown any degradation
overnight . this quite high stability
agrees well with our assumption of densely packed surfaces in which
lateral interactions between the rotaxanes help keep them in place . amide rotaxanes that exhibit axle translocation
in the wheel between two different stations in response to the presence
of chloride have been synthesized and effectively deposited on gold
substrates . two approaches yielded monolayers : covalent attachment
through a click reaction and a coordination - chemistry - based approach . the latter monolayer formed the basis for multilayer fabrication leading
to up to 20 rotaxane layers . chloride - induced axle movement
has successfully been transferred from the solution to the surface . linear dichroism effects observed in angle - resolved nexafs experiments
not only provide evidence for oriented surface - deposited rotaxanes ,
but clearly demonstrate switching of single and triple layers between
two different layer structures . a number of control experiments and
the comparison of different layers allow us to ascribe the structural
changes to the rotaxane switching rather than other potential sources
for structural changes . this study represents a structural examination
of ordered rotaxane multilayers on a solid support that exhibits coupled
transitions between two different structures by stimulus - induced mechanical
motion . in particular , the almost complete absence of linear dichroism
effects in the switching experiments performed with the tdt / dt - based surfaces shows how important order is for
a structurally well - defined switching between two different orientations .
a dense packing of the rotaxanes under study in well - ordered layers
results in limited degrees of freedom for the axles of each rotaxane
through spatial constraints caused by the next neighbors . a perfectly
simultaneous switching of all rotaxanes in a domain is unlikely , because
the overall barrier would be expected to be very high . therefore ,
we propose a nucleation / growth mechanism for the switching process :
the switching of the first rotaxane is quite unfavorable , as the unswitched
state packs well , whereas the switched one does not in an environment
of unswitched neighbors . once the nucleus of a small ensemble of switched rotaxanes
has formed , neighboring rotaxanes will switch faster , as the switched
state now packs well when embedded within switched neighbors . this
leads to a quicker growth of the switched domain . a detailed
understanding of the factors that govern these processes will have
strong implications for the future integration of molecular machines
into larger devices . also , the conversion of chemical processes on
the molecular scale into macroscopic effects may be envisaged on the
basis of our study , if one , for example , considers volume changes
caused by rotaxane switches tilting up in a coupled fashion . | interfaces provide the structural
basis for function as , for example , encountered in nature in the membrane - embedded
photosystem or in technology in solar cells .
synthetic functional
multilayers of molecules cooperating in a coupled manner can be fabricated
on surfaces through layer - by - layer self - assembly .
ordered arrays of
stimulus - responsive rotaxanes undergoing well - controlled axle shuttling
are excellent candidates for coupled mechanical motion .
such stimulus - responsive
surfaces may help integrate synthetic molecular machines in larger
systems exhibiting even macroscopic effects or generating mechanical
work from chemical energy through cooperative action .
the present
work demonstrates the successful deposition of ordered mono- and multilayers
of chemically switchable rotaxanes on gold surfaces .
rotaxane mono-
and multilayers are shown to reversibly switch in a coupled manner
between two ordered states as revealed by linear dichroism effects
in angle - resolved nexafs spectra .
such a concerted switching process
is observed only when the surfaces are well packed , while less densely
packed surfaces lacking lateral order do not exhibit such effects . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
after a stroke event , only 5 to 20% of patients demonstrate complete functional recovery ,
while 80% reveal a variable degree of impairment and inability to attend to daily life
activities1 . pain , spasticity , joint
constraint , and skin or vascular damages are typical consequences observed in stroke
survivors with upper limb disability and represent a paramount rehabilitation challenge
either in the subacute and chronic phase2 , 3 . in relation to the increasing prevalence of
stroke cases in an aging societies , there is a compelling need for innovative rehabilitation
strategies . one of the most promising approaches intended to recover upper limb function after stroke ,
is robotic rehabilitation ( rr)4 . the effectiveness of robotic devices for the treatment and rehabilitation of poststroke
upper limb disorders is recognized by the scientific community5,6,7 . understanding of the regulatory principles of clinical ,
neurophysiological , and biomechanical adaptations of each of the available means of rr is
the key to achieving the best outcomes when treating the various and specific upper limb
disorders affecting stroke patients5,6,7 . many researchers have tried to identify the concurrent and reciprocal influence of all of
the different factors that lead to the final results of the treatments8,9,10,11 . study of the biological
and functional adaptations during and after rr in this population would be of particular
interest in light of their intrinsic correlation with postural modulation of the upper limb
and in relation to neuroplasticity harnessing , even in chronic poststroke patients12 . with the aim of contributing to post - stroke functional recovery of the hand , a newly
designed rr device named gloreha has recently been introduced in the field of
rehabilitation . this new device consists of a soft exoskeleton similar to a glove that
envelops the wrist and fingers of the paretic hand with velcro attachments and straps . passive mobilization of the fingers is provided by a hydraulic system that generates force
transmitted through semirigid cables . the main innovation of this new rr device is that it
enables well calibrated sequential movement of each single finger while the patient is
observing the action induced by the device in a synchronous modality . different exercises
and ranges of motion can be selected through the dedicated software . at present , the biological and functional adaptations of the upper limb structures and
tissues induced by the gloreha glove in patients with poststroke hemiparesis have not been
fully explored . studies are needed to determine the changes in local tissue blood flow and
oxygen supply , the degree of spasticity , and the patients perception of heaviness and
stiffness during and after passive hand mobilization with the gloreha glove . seven consecutive patients , 2 women and 5 men , from 18 to 70 years old who presented
poststroke hemiparesis in the nondominant hand were recruited from the waiting list of
physical therapy department . the causes of stroke were ischemia in 3 patients and hemorrhage
in 4 patients ; all patients had a left - sided lesion . diagnosis was performed by computed
tomography ( ct ) or magnetic resonance imaging ( mri ) scan . the study was limited to patients
in the chronic phase of stroke ( more than 6 months after the onset of acute injury)13 . the inclusion criteria included preserved
cognitive capacities according to age ( mini - mental state examination score > 24)14 , primary ischemic or hemorrhagic stroke ,
mild to moderate paresis of the upper limb , and absence of pain on movement . the exclusion
criteria included subjects that had a spasticity score of greater than three on the modified
ashworth scale ( mas)15 , a pain score
greater than four on the visual analogue scale ( vas)16 , presence of finger flexion contracture , de quervain s
tenosynovitis , and degenerative or non - degenerative neurological conditions in which pain
perception could be altered17 . informed
consent was obtained from all participants , and all procedures were conducted according to
the declaration of helsinki . each patient received 10 sessions with the gloreha device over a period of 3 weeks . the
sessions were performed at the same time of day ( morning ) , although circadian patterns
modulating neural and vascular functions can be profoundly altered and highly variable in
stroke patients18 . just before and
immediately after the first and last sessions of treatment , upper limb muscle strength ,
spasticity , and pain have been detected . muscle perfusion of the flexor carpi was measured
during the first and last sessions of treatment . each patient received treatment with the gloreha exoskeleton device for passive
mobilization of the affected hand after the therapist performed passive range of motion
therapy for the hand during a one - hour session . the rr treatment session was half an hour
and consisted of the following : isolated ( mobilization ( the 2nd to 5th fingers are mobilized simultaneously , while the
thumb was mobilized individually ) . the fingers of the patients were hooked to individual
thimbles connected through a nylon thread to a device fixed on the glove that interfaced
with a hybrid system ( compressed air and oil ) causing passive flexion - extension movement of
the fingers . evaluation of the patient s response to therapy was monitored by use of the following
assessment tools : a probe of near - infrared spectroscopy ( nirs ) for noninvasive measurements
of the forearm regional blood supply was placed on the flexor carpi belly for determination
of the muscle tissue oxygenation level and regional blood supply19 , the mas was used to measure spasticity in the shoulder ,
elbow , wrist , and fingers , and motor function was assessed using the motricity index section
for upper limb evaluation ( mi)20 . raters
reviewed charts to gather the self - reported levels of the subjects ( visual analogue scale of
0100 ) for wrist - finger , elbow , and shoulder heaviness and stiffness21 . a nimo system ( nirox optoelectronics , brescia , italy ) was
employed to study local muscle oxygenation and perfusion dynamics . to study the changes in
local blood flow and tissue oxygenation induced by passive mobilization of the hand through
the gloreha system , the profiles of total hemoglobin ( thb ) and tissue oxygenation index
( toi , related to concentration of o2hb ) were gathered . with reference to baseline
( average value during a 30 second of rest just before the intervention with the gloreha
device ) , the values corresponding to the largest variation in thb and toi dynamics ( average
value of the delta calculated for each passive movement ) during robot - assisted mobilization
were quantified . care was provided to place the probe in the same position during the first
and last treatment sessions . , armonk , ny , usa ) . the
normality of the distribution was examined by using the kolmogorov - smirnov test . a one - way
analysis of variance ( anova ) with repeated measurements and the bonferroni test was used as
a post hoc test to evaluate statistical significance . within - group cohen considers an effect size greater than 0.8 as
large , around 0.5 as moderate , and less than 0.2 as small . for all the data of the study , p
values lower than 0.05 were considered significant . seven patients with poststroke hemiparesis , 2 women and 5 men , aged 51 to 69 years ( mean ,
60.1 ; sd , 6.2 years ) , were included in this study . the anthropometric and clinical
characteristics of the subjects included in the present case series are listed in table 1table 1.characteristics of the impaired subjectspatientsagegendertype of strokeimpaired armtime since stroke155femaleischemicleft8 years , 5 months258maleischemicleft7 months359maleischemicleft7 years , 11 months467maleischemicleft1 year , 3 months568malehemorrhagicleft1 year663malehemorrhagicleft2 years , 5 months751femaleischemicleft8 months . no subjects dropped out during the different phases of the study , and no
adverse effects were induced by the treatment . none of the subjects began any drug therapy
during the course of the study . after the 10 sessions , a small
within - group effect size ( d<0.2 ) was found between before and after treatment ( table 2table 2.mean ( sd ) for outcome at all study visits , mean ( sd ) difference within
groupoutcomegroupdifference within groupsday 0day 21day 21 minus day 0beforeafter(n=7)(n=7)(n=7)nirstoi68.5 ( 12.3)71.2 ( 10.8)2.6 ( 0.09)thb95.4 ( 5.1)101.5 ( 4.3)6.1 * ( 0.03)maselbow1.3 ( 0.5)0.7 ( 0.5)0.6 * ( 0.03)wrist1.7 ( 0.9)0.6 ( 0.5)1.1 * ( 0.005)fingers1.4 ( 1.0)0.3 ( 0.5)0.3 * ( 0.01)supination1.0 ( 0.6)0.3 ( 0.5)0.7 * ( 0.047)mishoulder16.4 ( 6.1)16.4 ( 6.1)0.0elbow17.4 ( 9.1)18.7 ( 6.6)1.3 ( 0.4)pinch14.7 ( 6.4)16.4 ( 7.2)1.7 ( 0.2)nirs : near - infrared spectroscopy ; mas : modified ashworth scale ; mi : motricity
index . * significantly different within the same group , p<0.05 ( 95% confidence
interval ) ) . nirs : near - infrared spectroscopy ; mas : modified ashworth scale ; mi : motricity
index . * significantly different within the same group , p<0.05 ( 95% confidence
interval ) regarding the results of spasticity measured with the mas , a significant interaction was
found for time in the extension elbow , wrist , and fingers was found , and supination of the
forearm ( f=8.0 , p=0.03 ; f=19.2 , p=0.005 ; f=11.294 , p=0.015 ; and p=0.047 , respectively ) . in
addition , there were significant differences between before and after treatment ( all ,
p=0.03 ) . also , a small within - group effect sizes ( d<0.2 ) was found between before and
after treatment ( table 2 ) . regarding abduction of the shoulder , flexion of the elbow , and key pinch as assessed by the
mi , there was no significant interaction for time in the shoulder ( f = errors ; p = errors ) ,
elbow ( f=1.0 ; p=0.4 ) , and pinch strength ( f=2.077 ; p=0.2 ) ( table 2 ) . a significant time interaction ( f=11.413 ; p=0.02 ) the post hoc analysis revealed significant
differences between the 10 sessions ( p=0.02 ) . for subject who reported perceptions of elbow and wrist - fingers heaviness , there was no
significant finding for time ( f=2.4 , p=0.2 , and f=3571 , p=0.1 , respectively ) . we also found a significant change with time ( f=21.833 , p=0.03 ) for subject who perceived
wrist - finger stiffness . the post hoc analysis revealed significant differences between the
10 sessions ( p=0.003 ) . for subjects who reported perceptions of shoulder and elbow
stiffness , there was no significant finding for time ( f=5.426 , p=0.59 , and f=4.395 , p=0.08 ,
respectively ) ( table 3table 3.mean ( sd ) for outcome at all study visits , mean ( sd ) difference within
groupoutcomegroupdifference within groupsday 0day 21day 21 minus day 0beforeafter(n=7)(n=7)(n=7)perception of heavinessshoulder45.0 ( 33.0)20.0 ( 17.1)25.0 * ( 0.015)elbow28.6 ( 28.0)12.9 ( 9.1)15.7 ( 0.2)wrist - fingers34.3 ( 39.9)9.3 ( 14.3)25.0 ( 0.1)perception of stiffnessshoulder27.9 ( 30.5)7.1 ( 12.5)20.7 ( 0.06)elbow20.7 ( 27.5)5.7 ( 9.8)15.0 ( 0.08)wrist - fingers47.1 ( 28.1)19.3 ( 22.4)27.8 * ( 0.003)*significantly different within the same group , p<0.05 ( 95% confidence
interval ) ) . * significantly different within the same group , p<0.05 ( 95% confidence
interval ) this novel study found that specific robot - assisted mobilization ( gloreha ) of paretic hand
and wrist decreased spasticity after treatment ( 10 sessions ) in subjects with poststroke
hemiparesis . skeletal muscle thb in the flexor carpi muscles of the forearm increased during
the intervention , and major deviation from baseline was found for the levels of thb ,
suggesting a beneficial effect of robot - assisted therapy on patients presenting stroke in
terms of improved regional blood flow during the passive movements , and favored washout of
the catabolites accumulating in the tissues of the poorly active upper limb . it is known that nirs detects variations in oxygenated and deoxygenated hemoglobin amounts
at the microcirculation level and that it is therefore able to specifically monitor muscle
capillary supply . improved local perfusion changes represent a fundamental mechanical factor
( shear stress ) for angiogenesis at the capillary level , suggesting remarkable implications
for muscle tissue functions and circulatory homeostasis22 , 23 . muscle disuse due to
hemiparesis may significantly impair this balance , leading to oxygen supply / uptake mismatch :
if less oxygen is available than required , effort intolerance and early onset of fatigue as
well as impairments in muscle functions will inevitably occur . this study is in agreement with randomized controlled trial of sale et al.24 that found that robotic rehabilitation
produced positive results in the recovery of hand function following stroke . the innovative
robotic rehabilitation device ( gloreha idrogenet ) intervention spasticity in the subjects ,
and all subjects reported a perceived improvement in hand stiffness . this can translate into
improved functional use of the hand for completion of activities of daily living . the sample size in the present study was small , but it was sufficient to determine
significance . however , since this pathology is often
accompanied by depression and neurodegenerative disorders and we excluded patients with
neurodegenerative and non - neurodegenerative disorders , there was a number of important
patients with poststroke hemiparesis that were excluded from the study . we are aware that we
only examined the mid - short - term effects of arm joints mobilization directed at post stroke
hemiparesis . therefore , we can not affirm that the positive results would remain over time . finally , the study was not a randomized controlled trial , and the assessor of the outcomes
was not blinded . the present work provides novel evidence that robotic assistance of the hand induced
changes in local muscle blood flow , diminished spasticity , and decreased subject - reported
symptoms of heaviness and stiffness in subjects with post stroke hemiparesis . | [ purpose ] the purpose of this case series was to determine the effects of robot - assisted
hand rehabilitation with a gloreha device on skeletal muscle perfusion , spasticity , and
motor function in subjects with poststroke hemiparesis .
[ subjects and methods ] seven
patients , 2 women and 5 men ( mean sd age : 60.5 6.3 years ) , with hemiparesis ( > 6
months poststroke ) , received passive mobilization of the hand with a gloreha ( idrogenet ,
italy ) , device ( 30 min per day ; 3 sessions a week for 3 weeks ) .
the outcome measures were
the total hemoglobin profiles and tissue oxygenation index ( toi ) in the muscle tissue
evaluated through near - infrared spectroscopy .
the motricity index and modified ashworth
scale for upper limb muscles were used to assess mobility of the upper extremity .
[ results ] robotic assistance reduced spasticity after the intervention by 68.6% in the
upper limb . the motricity index was unchanged in these patients after treatment . regarding
changes in muscle perfusion
, significant improvements were found in total hemoglobin .
there were significant differences between the pre- and posttreatment modified ashworth
scale .
[ conclusion ] the present work provides novel evidence that robotic assistance of
the hand induced changes in local muscle blood flow and oxygen supply , diminished
spasticity , and decreased subject - reported symptoms of heaviness and stiffness in subjects
with post - stroke hemiparesis . |
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the use of recombinant activated protein c ( rapc ) is one of the hottest topics in septic shock therapy . the pivotal phase 3 placebo - controlled protein c worldwide evaluation in severe sepsis ( prowess ) clinical trial demonstrated a 19.4% relative risk reduction in 28-day mortality ( 6.1% absolute risk reduction ) with an increased risk ( 3.5% versus 2.0% ) of serious bleeding events compared with placebo . two recent and important articles have highlighted the role of apc as a protective endothelial drug and as a cyto - protective drug . beneficial effects of rapc in the prowess study were thought to be related to a reduction in coagulation and , to a lesser extent , to a reduction in inflammatory response to sepsis . post - prowess investigations have been associated with a myriad of cellular or animal studies demonstrating that rapc , through reactions mediated by endothelial protein c receptor and the effector receptor , protease activated receptor-1 , acts directly on cells to exert multiple cytoprotective effects including : down regulation of pro - inflammatory gene expression ; anti - inflammatory activities ; anti - apoptotic activity ; and protection of endothelial barrier function . sepsis , per se , may induce phenotypic modulations of the endothelium through direct or indirect interaction of the endothelial layer with components of the bacterial wall , inducing a myriad of host - derived factors from endothelial cells . phenotypic modifications include changes in pro - coagulant and proadhesive properties , increased endothelial permeability , endothelial cell apoptosis and changes in vasomotor properties ; the last of these is crucial since vasoplegia is directly related to septic shock mortality . recent animal and human data have suggested that rapc may improve both vascular and myocardial dysfunction and vascular reactivity to catecholamine during endotoxin and/or septic challenge . experimental evidence supports a role of apc in maintaining the integrity of the endothelium through both direct and indirect mechanisms . nevertheless , the results of in vivo studies are less explicit . in a retrospective study of septic shock in humans , monnet and colleagues demonstrated that apc infusion was associated with a decrease in the amount of delivered norepinephrine . wiel and colleagues demonstrated in a rabbit model of endotoxin induced shock that apc decreased aorta endothelial injury . by contrast , in a lung model of endotoxin induced inflammation , robriquet and colleagues demonstrated a trend to an increased vascular permeability using high doses of human apc . this last result was in sharp contrast with the results obtained by nick and colleagues in a human model of pulmonary endotoxin administration . apc appears to improve mortality in septic shock with a high apache 2 score and is potentially detrimental in severe sepsis . in rats , apc markedly decreased tumour necrosis factor concentrations whereas they remained unchanged in either human septic shock or endotoxemia . the question arises , therefore , as to whether it is truly possible to reconcile all these discrepancies ? clearly , in cellular and animal models , rapc has been given either as a pre - treatment or concurrent with septic challenge . this mode of administration favours the anti - inflammatory effects of rapc , which are particularly efficient in murine models in protecting the endothelium from cytokine - mediated apoptosis or upregulation of endothelial adhesion molecules . thus , studies using post - injury treatment are needed in models that mimic septic shock , such as experimental pneumonia or peritonitis treated by antibiotics and volume resuscitation , and where the effects of rapc would be investigated 16 to 24 hours after septic challenge . if we can demonstrate the efficiency of rapc to protect or to reduce endothelium injury in these conditions , we can ultimately postulate that rapc is also a therapeutic drug for the endothelium . if rapc is efficient when infused in the early phase of septic challenge , we thus need to treat our patients earlier . at least two studies suggest that treatment with rapc within 24 hours may carry a larger survival advantage for patients with severe sepsis , compared with those treated more than 24 hours after organ dysfunction . interventions directed at specific endpoints , when initiated early in the ' golden hours ' of a patient 's condition , seem to be promising . the beneficial effects of earlier administration of rapc to appropriate patients may fit into this paradigm . extensive in vivo and in vitro studies have focused on the cytoprotective effects of apc and most authors agree that its anticoagulant and cytoprotective effects are mediated by distinct apc structural features . positively charged residues in surface loops in the apc protease domain have been identified as participating in the anticoagulant activity but not in cellular effects . hence , variants have been designed with greater anti - apoptotic activity and reduced anticoagulant activity relative to wild - type apc . whether these genetically engineered variants actually provide superior pharmacological properties such investigations may allow the design of therapeutic apc variants with decreased anticoagulant activity to reduce the risk of bleeding on the one hand , but also with normal cytoprotective properties in order to retain full beneficial effects on sepsis outcome . prowess = protein c worldwide evaluation in severe sepsis ; rapc = recombinant activated protein c. | endothelium dysfunction is one of the hallmarks of sepsis .
looney and mattay , in the previous issue of critical care , highlight the role of activated protein c ( apc ) as a protective endothelial drug in septic situations . nevertheless , the results of in vivo studies are less explicit and it remains uncertain whether these properties are relevant in human septic shock . before considering recombinant apc ( rapc ) as a therapeutic drug for the endothelium
, we have to demonstrate its efficiency to protect or to reduce endothelium injury when infused a long time after the septic challenge .
nevertheless , if rapc is efficient when infused in the early phase of septic challenge , we thus need to treat our patients earlier . at the least
, genetically engineered variants have been designed with greater anti - apoptotic activity and reduced anticoagulant activity relative to wild - type apc .
further studies are needed to demonstrate the usefulness of these variants in septic shock therapy . |
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germinal matrix - intraventricular hemorrhage ( gm - ivh ) is one of the most common complications in the premature infant . second , survival rate for smaller premature infants increases consistently . therefore , there are many previous reports in order to identify risk factors associated with development of gm - ivh and to establish effective strategies . perinatal risk factors such as a low birth weight and low gestational age , vaginal delivery , intrauterine infection , low apgar score , sepsis have been proposed as associated with the pathogenesis of gm - ivh16 ) . therefore , the purpose of this study is to identify the risk factors associated with the development of gm - ivh and the relationship of the severity of disease and prematurity . a total of 168 premature neonates whose birth weight 1500 g or gestational age 34 weeks were examined by cranial ultrasound ( cus ) for detection of gm - ivh among the babies admitted between january 2011 and december 2012 in our medical center neonatal intensive care unit . patient 's data were collected retrospectively such as sex , gestational age , birth weight , delivery method , disease diagnosis and its grade , initial blood glucose , sodium levels and presence of comorbidities such as respiratory distress syndrome ( rds ) , patent ductus arteriosus ( pda ) and anemia . and maternal data were collected retrospectively such as maternal age , gravidity , presence of preeclampsia and premature rupture of membrane ( prom ) . cus was performed for all neonates in this study in the first week of life for detection of ivh and its grading were done . if a neonate had gm - ivh , then serial cus would be done weekly . all subjects were divided into two groups - gm - ivh group and non - ivh groups according to presence of gm - ivh . gm - ivh group was subdivided by grading system proposed by papile et al.19 ) ( table 1 ) . grade i and ii group were considered as mild gm - ivh group , grade iii and iv group were as severe gm - ivh group . statistical analysis was performed by using spss version 21.0 ( spss , chicago , il , usa ) . to compare the continuous variables , the student 's t - test was used . logistic regression analysis was used to find the independent association between development of gm - ivh and explanatory variables . variables that were p0.20 as determined by univariate analysis were entered into multivariate logistic regression . the odds ratios ( ors ) with a 95% confidence intervals ( cis ) were calculated . a total of 168 premature neonates whose birth weight 1500 g or gestational age 34 weeks were examined by cranial ultrasound ( cus ) for detection of gm - ivh among the babies admitted between january 2011 and december 2012 in our medical center neonatal intensive care unit . patient 's data were collected retrospectively such as sex , gestational age , birth weight , delivery method , disease diagnosis and its grade , initial blood glucose , sodium levels and presence of comorbidities such as respiratory distress syndrome ( rds ) , patent ductus arteriosus ( pda ) and anemia . and maternal data were collected retrospectively such as maternal age , gravidity , presence of preeclampsia and premature rupture of membrane ( prom ) . cus was performed for all neonates in this study in the first week of life for detection of ivh and its grading were done . if a neonate had gm - ivh , then serial cus would be done weekly . if not , follow up cus was taken after 2 weeks . all subjects were divided into two groups - gm - ivh group and non - ivh groups according to presence of gm - ivh . gm - ivh group was subdivided by grading system proposed by papile et al.19 ) ( table 1 ) . grade i and ii group were considered as mild gm - ivh group , grade iii and iv group were as severe gm - ivh group . statistical analysis was performed by using spss version 21.0 ( spss , chicago , il , usa ) . to compare the continuous variables , logistic regression analysis was used to find the independent association between development of gm - ivh and explanatory variables . variables that were p0.20 as determined by univariate analysis were entered into multivariate logistic regression . the odds ratios ( ors ) with a 95% confidence intervals ( cis ) were calculated . infants born from mother with multigravida were 44 ( 26.19% ) . mean maternal age was 32 years . the mothers who had past history were 31 cases ( 18.45% ) for preeclampsia and 44 cases ( 26.19% ) for prom . 34 babies ( 20.24% ) had pda , 95 babies ( 56.55% ) with rds , 67 babies ( 39.88% ) with anemia . among 168 subjects , gm - ivh was diagnosed in 31 cases ( 18.45% ) ; 15 cases ( 48.39% ) with grade 1 , 9 cases ( 29.03% ) with grade 2 , 2 cases ( 6.45% ) with grade 3 , 5 cases ( 16.13% ) with grade 4 . in univariate analysis , gestational age , birth weight , delivery method , presence of prom and level of sodium and glucose were statistically meaningful factors ( p<0.05 ) . to operate multivariate analysis , gestational age , birth weight , delivery method , presence of prom , pda , rds , anemia and level of sodium and glucose were selected . only two factors - gestational age and presence of pda were statistically meaningful in multivariate logistic regression ( p<0.05 ) . infants born from mother with multigravida were 44 ( 26.19% ) . mean maternal age was 32 years . the mothers who had past history were 31 cases ( 18.45% ) for preeclampsia and 44 cases ( 26.19% ) for prom . 34 babies ( 20.24% ) had pda , 95 babies ( 56.55% ) with rds , 67 babies ( 39.88% ) with anemia . among 168 subjects , gm - ivh was diagnosed in 31 cases ( 18.45% ) ; 15 cases ( 48.39% ) with grade 1 , 9 cases ( 29.03% ) with grade 2 , 2 cases ( 6.45% ) with grade 3 , 5 cases ( 16.13% ) with grade 4 . in univariate analysis , gestational age , birth weight , delivery method , presence of prom and level of sodium and glucose were statistically meaningful factors ( p<0.05 ) . to operate multivariate analysis , gestational age , birth weight , delivery method , presence of prom , pda , rds , anemia and level of sodium and glucose were selected . only two factors - gestational age and presence of pda were statistically meaningful in multivariate logistic regression ( p<0.05 ) . gm - ivh is rare in full - term babies11 ) , but more common in preterm babies and it leaves significant sequelae such as cerebral palsy and mental retardation . therefore , it is very important to find the risk factors associated with development of gm - ivh and prepare for them . these contain gender , lack of antenatal steroids , low apgar score , prom , intrauterine infection , vaginal delivery , in vitro fertilization , mechanical ventilation , a large pda , rds and transfusion of blood products7,12,14,16,18,25,26 ) . however , in our study , there was no significant difference in gender , birth weight , delivery method , preeclampsia , prom , anemia . the only two factors , gestational age and presence of pda , were statistically meaningful risk factors for development of gm - ivh . this result could be understood easily because gestational age reflects fetal maturity more accurately than birth weight . as several reports 15,21,23 ) and our results has shown , it is certain that younger gestational age is related with higher risk for severe gm - ivh . if children were born before the 32 weeks of gestation , the subependymal region is equipped with a tight net of capillaries that are mainly supplied by the heubner 's artery . after the 32 weeks into pregnancy , the germinal matrix involutes and the vessels will be differentiated10 ) . preterm infants are quite helpless against cerebrovascular injury due to a unique constellation of pathophysiological factors . maintain cerebral perfusion pressure is more difficult due to normally reduced hypotension and low cardiac output for the new borns to adjst extrauterine life , especially in the first day of life . in addition , intrinsic cerebral vasoreactivity and autoregulatory mechanisms are poorly developed in the immature brain . as decreasing gestational age although there is a objection20,21,24 ) , some retrospective studies reported that caesarean delivery does not improve neonatal survival of very low birth weight infants but decreases gm - ivh occurrence2,4,17,27 ) . the germinal matrix is a transient neural cell proliferative zone with poor developed vasculature and located on the head of caudate nucleus . the resistance to ischemic injury is weaker and autoregulation range of blood pressure is lower as decreasing gestational age . certain situation like presence of pda or normal vaginal delivery could result in hypoperfusion - reperfusion state . several possible factors are suggested cardiorespiratory system ( hypotension , pda , hypoperfusion - reperfusion pattern , hypercarpnia , hypoglycemia , hypernatremia ) , hematologic ( anemia , thrombocytopenia ) , immunologic response , impared cerebral reactivity , immature anatomy , eventhough they are not proven yet1,5,7,10,12,13,14,16,18,22,25,26 ) . another reason why the hemorrhage occurs in this region , beside the massive vascularization , is the fact that the area around the caudate nucleus represents a border zone between ventriculopetal and fugal blood supply3 ) . furthermore , the endothelium of the vessels located in the brain of premature newborns is more sensitive to hypoxemia . this means that the cerebral blood flow is directly influenced by changes of the systemic blood pressure . hence hypertension , as well as hypotension , will lead directly to changes in the cerebral blood flow4,6,8 ) . other triggers that can cause an ich are rupture of the alveolars , rds , pneumothorax and artificial ventilation of the new born . endotracheal intubation with positive pressure ventilation increases central venous pressure , which can lead to episodic poor cerebral perfusion . but , in the present study , high risk pregnancy situations such as preeclampsia , prom and rds were not related to the development of gm - ivh . so we could n't sort all the cases by gestational age and estimate odds ratio by decreasing gestational age . second is a retrospective study from a single institution , although this also might be an advantage due to consistency of practice and expertise . despite of several limitations , our study revealed that presence of pda and gestational age were the important risk factors associated with development of gm - ivh . how the severity of disese relates to gestational age will be necessary to study with larger population in multicenter . | objectivethe purpose of this study is to identify the risk factors associated with the development of germinal matrix - intraventricular hemorrhage ( gm - ivh ) and the relationship of the severity of disease and prematurity.methodsa total of 168 premature neonates whose birth weight 1500 g or gestational age 34 weeks were examined by cranial ultrasound ( cus ) for detection of gm - ivh among the babies admitted between january 2011 and december 2012 in our medical center neonatal intensive care unit .
the babies were divided into two groups : gm - ivh and non - ivh .
clinical presentations , precipitating factors of the patients and maternal factors were analyzed.resultsin univariate analysis , gestational age , birth weight , delivery method , presence of premature rupture of membrane ( prom ) and level of sodium and glucose were statistically meaningful factors ( p<0.05 ) .
but only two factors , gestational age and presence of patent ductus arteriosus ( pda ) were statistically meaningful in multivariate logistic regression ( p<0.05 ) .
delivery method [ normal vaginal delivery ( nvd ) to caeserean section ] was borderline significant ( p<0.10).conclusionpresence of pda and gestational age were the important risk factors associated with development of gm - ivh . |
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platyopuntias such as opuntia basilaris , opuntia ficus - indica , and opuntia stricta are the most intensely studied cacti ( nobel , 1988 ; osmond et al . , they are classic examples of plants that exhibit crassulacean acid metabolism ( cam ) , a water - conserving mode of photosynthetic co2 assimilation which is characterized by the uptake of co2 at night when the driving forces for transpirational water loss are low ( neales et al . , 1968 ; winter et al . , 2005 ) the cam cycle is considered to be constitutively expressed , i.e. cam is the principal pathway of carbon acquisition in mature cladodes , irrespective of seasonal and day - to - day variation of environmental conditions in the native habitat . indeed , in situ studies of platyopuntias growing in arid semi - desert environments demonstrate net co2 uptake predominantly at night and little or no co2 uptake in the light . under more mesic conditions , significant co2 uptake may occur during the early morning or in the late afternoon , but the bulk of co2 is still absorbed in the dark ( gerwick and williams , 1978 ; osmond et al . in contrast , in many cam species from other families c3 photosynthetic co2 uptake may equal or exceed co2 uptake in the dark ( holtum and winter , 1999 ; winter and holtum , 2002 ) . in some species , co2 may be overwhelmingly fixed by the c3 pathway and cam is only detectable as a small nocturnal increase in tissue acidity ( silvera et al . , 2005 ) . another photosynthetic phenotype is exhibited by highly flexible species which may operate in either the c3 mode when unstressed , or in the cam mode when challenged by drought or salinity stress ( facultative cam ) ( winter and holtum , 2005 , 2007 ; lttge , 2006 ; winter et al . , 2008 ) . before displaying their full potential for cam , young plants and young leaf or photosynthetic stem tissue of species with constitutive cam may exhibit a strong c3 component of net co2 uptake which decreases as plants and tissues mature . in o. ficus - indica and hylocereus monocanthus , new photosynthetic stem segments which develop on mature stems may rapidly shift from daily net co2 loss to co2 gain via cam during the sink - to - source transition with little interim use of c3 photosynthesis ( wang et al . , 1998 ; winter and holtum , 2002 ) , but these observations do not necessarily apply to autonomous cactus seedlings which fully rely upon co2 uptake from the atmosphere . measurements of titratable acidity indicate the presence of cam in 1-d - old cotyledons of six species of columnar cacti ( hernndez - gonzlez and briones villarreal , 2007 ) , but the relative contributions of day and night co2 fixation to total carbon gain have not yet been assessed in young developing cactus seedlings ( acevedo et al . , 1983 ; pimienta - barrios et al . , 2005 ) . here cam and c3 photosynthesis during early growth of the platyopuntia o. elatior were quantified by continuously monitoring net co2 exchange of seedlings for up to 83 day in addition , long - term co2 exchange and titratable acidity were used to determine the extent to which seedlings of o. elatior up - regulate cam in response to drought stress , i.e. are capable of expressing cam facultatively . if such up - regulation occurs , the current perception of strictly constitutive cam in platyopuntias would require reassessment . recent studies with young cladodes of o. ficus - indica developing on mother cladodes were indeed suggestive of a facultative component of cam expression ( winter et al . , 2008 ) , but interpretation of the gas - exchange data was complicated by the contribution to carbon gain by mother cladodes and by possible stress - related reduction in mitochondrial respiration in the young tissues . furthermore , supporting information on nocturnal increases in acidity in stressed and non - stressed tissues was lacking . here it is demonstrated unambiguously that seedlings of o. elatior can exhibit c3 photosynthesis and facultative cam . seeds collected from wild plants growing in sarigua national park , republic of panama , were germinated in moist jiffy pellets ( hummert international , earth city , mo , usa ; fig . 1a ) . the seedlings ( plus pellets ) were transferred to terracotta pots ( 1.6 l or 3.7 l ) or plastic pots ( 0.5 l ) containing potting mix ( cactus , palm and citrus soil ; miracle - gro lawn products , marysville , oh , usa ) and osmocote plus fertilizer ( scotts - sierra horticultural products , marysville , oh , usa ) . cotyledons attached to the plants were enclosed in a perspex cuvette ( internal dimensions of either 111110 cm or 202015 cm ) by passing the hypocotyl carrying the two cotyledons through a 1-cm diameter hole in the cuvette base and sealing the hypocotyl cuvette interface with a non - porous synthetic rubber sealant ( terostat vii ; henkel - teroson , heidelberg , germany ) . ( a ) a seedling with cotyledons , incipient cladode , and remains of the seed coat , and ( b ) a 5-cm tall cladode with cotyledons at the base inside a gas - exchange cuvette . throughout this report , plant age is defined as the number of days following the unfolding of cotyledons . the gas - exchange cuvette was located inside a controlled - environment chamber ( environmental growth chambers , chagrin falls , oh , usa ) operating under 12-h light ( 28 c)/12-h dark ( 22 c ) cycles . ambient air was drawn from a 1 m container placed on an eight - storey building to dampen short - term fluctuations in [ co2 ] , and passed through the cuvette at a flow - rate of 2.3 l min . in one experiment , the cuvette was supplied at a flow rate of 1.26 l min , with air containing 400 ppm co2 generated by a mass - flow controlled co2/co2-free air mixing unit ( walz gmbh , effeltrich , germany ) . the dew - point of the air entering the cuvette was 19 c or 20 c . net co2 exchange of the cotyledons and the first cladode was measured in a flow - through gas - exchange system consisting of walz components and a li-6252 co2 analyser ( li - cor , lincoln , ne , usa ) ( holtum and winter , 2003 ) . seedlings were grown in plastic pots inside a controlled environment chamber under conditions similar to those used in the gas - exchange experiments . cotyledons and cladodes were harvested at the end of the light and dark periods and were frozen in liquid nitrogen . organic acids were extracted by sequentially boiling samples in 50% ethanol and in water , each for 5 min . extracts were cooled to room temperature and titrated with 25 mm naoh to ph 6.5 . seeds collected from wild plants growing in sarigua national park , republic of panama , were germinated in moist jiffy pellets ( hummert international , earth city , mo , usa ; fig . 1a ) . the seedlings ( plus pellets ) were transferred to terracotta pots ( 1.6 l or 3.7 l ) or plastic pots ( 0.5 l ) containing potting mix ( cactus , palm and citrus soil ; miracle - gro lawn products , marysville , oh , usa ) and osmocote plus fertilizer ( scotts - sierra horticultural products , marysville , oh , usa ) . cotyledons attached to the plants were enclosed in a perspex cuvette ( internal dimensions of either 111110 cm or 202015 cm ) by passing the hypocotyl carrying the two cotyledons through a 1-cm diameter hole in the cuvette base and sealing the hypocotyl cuvette interface with a non - porous synthetic rubber sealant ( terostat vii ; henkel - teroson , heidelberg , germany ) . ( a ) a seedling with cotyledons , incipient cladode , and remains of the seed coat , and ( b ) a 5-cm tall cladode with cotyledons at the base inside a gas - exchange cuvette . throughout this report , plant age is defined as the number of days following the unfolding of cotyledons . the gas - exchange cuvette was located inside a controlled - environment chamber ( environmental growth chambers , chagrin falls , oh , usa ) operating under 12-h light ( 28 c)/12-h dark ( 22 c ) cycles . ambient air was drawn from a 1 m container placed on an eight - storey building to dampen short - term fluctuations in [ co2 ] , and passed through the cuvette at a flow - rate of 2.3 l min . in one experiment , the cuvette was supplied at a flow rate of 1.26 l min , with air containing 400 ppm co2 generated by a mass - flow controlled co2/co2-free air mixing unit ( walz gmbh , effeltrich , germany ) . the dew - point of the air entering the cuvette was 19 c or 20 c . net co2 exchange of the cotyledons and the first cladode was measured in a flow - through gas - exchange system consisting of walz components and a li-6252 co2 analyser ( li - cor , lincoln , ne , usa ) ( holtum and winter , 2003 ) . seedlings were grown in plastic pots inside a controlled environment chamber under conditions similar to those used in the gas - exchange experiments . cotyledons and cladodes were harvested at the end of the light and dark periods and were frozen in liquid nitrogen . organic acids were extracted by sequentially boiling samples in 50% ethanol and in water , each for 5 min . extracts were cooled to room temperature and titrated with 25 mm naoh to ph 6.5 . in well - watered 7-d - old seedlings consisting of only the cotyledons ( fig . 1a ) , net co2 uptake was restricted to the light and occurred at a more or less constant rate ( fig . net co2 uptake was not discernable in the dark . if present , it was below the limits of resolution of the gas - exchange system . in the smallest cotyledons studied ( fresh weight < 0.08 g per plant ) , titratable acidities at the end of the light and dark did not differ significantly ( fig . light dark patterns of net co2 exchange during early development of well - watered o. elatior seedlings . cladode height was 0 , < 1 , 5 , 10 , and 15 cm on days 7 , 14 , 37 , 54 , and 64 , respectively . relationship between developmental stage of o. elatior and titratable acidity in cotyledons at the end of the light ( open circles ) and at the end of the dark ( closed circles ) . there was no significant difference between titratable acidity at the end of light and dark for cotyledons with fresh weights of < 0.08 g ( two - sample t - test , p>0.05 ) . for cotyledons with fresh weights of 0.08 g the titratable acidity at the end of the dark period was significantly greater than that at the end of the light period ( two - sample t - test , p<0.01 ) . lines are linear regressions . as cotyledons matured and the first cladode developed , co2 exchange rates increased in the light and the dark ( fig . the proportional contribution of dark co2 fixation to daily carbon gain increased progressively but only exceeded the level of carbon gain in the light in plants greater than 10 cm tall . the time taken to reach this stage of development varied somewhat between individuals ( fig . net co2 balance in the dark ( closed symbols ) and in the light ( open symbols ) during the early growth of two well - watered seedlings of o. elatior . when watering was stopped for 14- to 16-d intervals at different stages of the development of the emerging cladode , carbon gain in the light decreased during the drying cycle but the daily increase in dark co2 fixation accelerated ( fig . this positive effect of stress on dark co2 fixation slowed and eventually ceased as water limitation intensified . upon rewatering , effect of drought stress on net co2 balance of 12-h dark ( closed symbols ) and 12-h light ( open symbols ) periods during the early growth of o. elatior . three experiments are shown , ( a ) , ( b ) , and ( c ) , in which watering was withheld during different stages of development . shaded areas indicate periods without irrigation and values over arrows are cladode height in cm . the stimulation of dark co2 fixation during water stress was accompanied by increased nocturnal acidification in both cotyledons and cladodes ( table 1 ) . on a whole - organ basis , the increase in acidification was 1.3-fold in cotyledons and 1.6-fold in the cladodes . at the stage of development investigated , the cladodes constituted the bulk of plant mass and contributed the most to nocturnal acid storage . the stress - related stimulation of acidification in cladodes as shown in table 1 on a whole - organ basis is even greater on an area or fresh weight basis as cladode size was marginally smaller in the water - stressed plants ( table 2 ) . effect of drought stress on titratable acidity in cotyledons and cladodes of o. elatior seedlings values are means sd of five replicates . end of light 18:00 ; end of dark 06:00 ; , nocturnal accumulation of h. effect of drought stress on size of seedlings of o. elatior values are means sd of 10 replicates except for the value indicated by an asterisk , for which n=8 . this research highlights three features of photosynthetic carbon assimilation previously not fully recognized in platyopuntoid cacti : ( i ) o. elatior is a c3 plant immediately after germination , ( ii ) c3 photosynthesis remains the principal pathway of carbon acquisition for several weeks in well - watered plants up to 10 cm tall , and ( iii ) during this early phase of development seedlings can display a facultative component of cam when drought stressed . the contribution of c3 photosynthesis was quantified by direct measurement of daily co2 uptake in addition to measurements of titratable acidity . in earlier studies of seedlings of cacti , co2 exchange was not measured and the operation of c3 or cam photosynthesis was simply inferred from the presence ( hernndez - gonzlez and briones villarreal , 2007 ) or absence ( altesor et al . , 1992 ; loza - cornejo et al . , 2003 ) of fluctuations of titratable acidity , which , although an indicator of cam , provide no information about rates of c3 photosynthesis or respiration . in the absence of measurements of co2 exchange it is not possible to determine the point at which photosynthetic co2 uptake surpasses respiratory co2 loss and carbon gain becomes positive during early development . the drought - stress - induced up - regulation of net co2 fixation in the dark reflects a true facultative stimulation of the cam pathway , as drought stress also leads to an increase in the nocturnal accumulation of organic acids . previously , a facultative component of cam was postulated for young tissues of o. ficus - indica developing on mother cladodes ( winter et al . , 2008 ) . it could not be ruled out that in these previous experiments , the transient acceleration of net co2 fixation in the dark in response to drought stress was the result of a transient reduction in the efflux of respiratory co2 , as water deficit is known to reduce mitochondrial respiration ( e.g. fig . 7 in winter there can now be little doubt that a facultative component of cam expression is present in cactus seedlings . remarkably , on most days during the development of well - watered o. elatior there was continuous uptake of co2 throughout the 24-h cycle , albeit at varying rates that reflected the four phases of cam gas exchange . this feature of uninterrupted daytime and night - time co2 uptake has previously been highlighted as a speciality of some tropical woody species of clusia ( lttge , 2006 ) . the implications of continuous 24-h co2 uptake for biomass accumulation warrant further study ( borland et al . , 2009 ; holtum et al . , there have been no in situ studies of photosynthetic performance of cactus seedlings under tropical mesic conditions . it is conceivable that the expression of c3 photosynthesis in o. elatior shortly after germination assists establishment at relatively high growth rates when water is still available . the facultative cam component can speed up the development of constitutive cam and aid survival if the availability of water is rapidly reduced . thus , in the field , the contribution of c3 photosynthesis to net carbon gain in seedlings may not endure as long as observed here and the shift to nocturnal co2 uptake may be accelerated . under well - watered laboratory conditions these growth rates substantially exceed those reported for seedlings of desert cacti in the field ( despain et al . , 1970 ; jordan and nobel , 1981 ; nobel , 1988 ) for which the initial c3cam shift is probably extremely short compared with that in cacti under more mesic conditions . similarly when reproduction is vegetative , as is not uncommon in cacti ( nobel , 1988 ) , the supply of carbon to new growth by the c3 phase is largely replaced with carbon supplied from the parent , thus shortening the initial c3 phase ( winter and holtum , 2002 ) . this study documents in detail the change from c3 to cam photosynthesis during the early development of seedlings of a tropical cam cactus . in the context of the evolution and functional significance of cam , the observations support the argument that constitutive and facultative cam are merely extremes on a continuum that ranges from expression of cam that is fully controlled by ontogeny to the full control of cam expression by drought stress , and that facultative and constitutive cam are not mutually exclusive . | immediately after unfolding , cotyledons of the tropical platyopuntoid cactus , opuntia elatior mill . , exhibited a c3-type diel co2 exchange pattern characterized by net co2 uptake in the light .
significant nocturnal increases in titratable acidity typical of crassulacean acid metabolism ( cam ) were not detected at this early developmental stage . as cotyledons matured and
the first cladode ( flattened stem ) developed , features of cam were observed and the magnitude of cam increased .
nonetheless , in well - watered seedlings up to 10 cm tall , c3 photosynthetic co2 fixation in the light remained the major pathway of carbon fixation .
reduced soil water availability led to an up - regulation of net dark co2 fixation and greater nocturnal increases in tissue acidity , consistent with facultative cam .
these observations demonstrate that c3 photosynthesis , drought - stress - related facultative cam , and developmentally controlled constitutive cam can all contribute to the early growth of o. elatior .
the strong c3 component and facultative cam features expressed in young o. elatior contrast with mature plants in which obligate cam is the major pathway of carbon acquisition . |
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chronic hepatitis b virus ( hbv ) infection affects an estimated 370 million people worldwide . [ figure 1 ] . chronic hepatitis b insidiously causes liver damage over years and decades without any warning symptoms or signs . up to 2535% of infected individuals eventually die due to liver failure and hepatocellular carcinoma ( hcc ) induced by hbv . screening those individuals at risk of acquiring hepatitis b , and universal vaccination for prevention of acquisition , would help in limiting the spread and public health repercussions of the virus , especially in countries where this virus is more prevalent . worldwide prevalence of hepatitis b although many new antiviral therapies have been developed for the management of hepatitis b , they still do not offer the possibility of cure . when starting treatment for hbv , it is important to evaluate the potential benefits of therapy in the context of risk , side effects , resistance development and cost , most individuals who begin oral suppressive therapy will be indefinitely treated . continuous suppression of hbv replication in asymptomatic individuals with advanced liver disease prolongs life , decreases need for liver transplantation , and potentially reduces the risk for hcc . in this clinical review , we present a practical approach to prevention of and screening for hbv , the natural history of hbv , as well as its management . areas of the world with low - prevalence of hbv carriers ( 0.12% of the population ) include canada , the united states , western europe , australia and new zealand . areas of the world with the highest prevalence ( 1020% ) include southeast asia , china and sub - saharan africa . this large variation in carrier rates can be explained by differences in the routes of transmission and age of infection . in low - prevalence areas , infection is mainly acquired in adulthood through sexual and parenteral exposure , resulting in a low risk of chronicity ( 510% ) . in areas of higher prevalence , infection also takes place through intra - familial spread in the perinatal period ( 90% ) or in childhood ( 20% ) , resulting in a high risk of chronicity . hepatitis b virus transmission is associated with increasing viral loads and is more infectious than either hepatitis c or hiv , with a transmission rate of up to 30% being reported . hbv exposure can occur in the following situations : sexual contact ( partner infected with hbv , multiple sexual partners , men who have sex with men ) , parenteral contact ( injection drug users , hemodialysis , healthcare workers ) , household contact ( infected parents / siblings , shared personal hygiene items like toothbrushes , razor blades , nail clippers ) . these select populations should be routinely screened by testing for hepatitis b surface antigen ( hbsag ) , antibody against hepatitis b surface antigen ( anti - hbs ) , and antibody against hepatitis b core antigen ( anti - hbc ) . anti - hbs antibodies , present as a vestige of exposure to previous hbv infection or induced through vaccination , protect against the establishment of infection by binding and neutralizing the virus before it can infect the liver . those negative for all three markers are at risk for hbv infection and should be vaccinated if they remain at high risk of exposure . those who are positive for anti - hbs alone were likely vaccinated in the past and are still immune . those positive for anti - hbc and anti - hbs have already been exposed to hbv in the past and , therefore , do not require vaccination . acute hbv infection is clinically symptomatic in only 30% ( icteric hepatitis ) , whereas the other 70% have subclinical or anicteric hepatitis . the incubation period lasts between 1 and 4 months , and may be followed by a serum sickness - like syndrome with associated jaundice , right upper quadrant pain , nausea and anorexia . those with underlying liver disease are more likely to be symptomatic during acute hbv infection . while 95% of immune - competent adults can clear the infection within 6 months of exposure , the other 5% go on to develop life - long chronic infection . varying degrees of immune control can be achieved through a combination of virus - specific t - cell responses and neutralizing antibodies from virus - specific b - cell responses . acute hbv infection is treated symptomatically , as pharmacotherapy has not been effective in either decreasing the risk of chronic infection or the 1% risk of fulminant hepatic failure . there is a dynamic relationship between the hbv and the immune response in chronic hbv infection . to date , four phases have been described : immune tolerance , immune clearance ( hepatitis b e - antigen [ hbeag]-positive hepatitis b ) , inactive carrier , and reactivation ( hbeag - negative chronic hepatitis b ) . interpretation of hepatitis b serology and liver enzymes the immune tolerance phase is usually found in younger individuals under the age of 30 . the immune system does not recognize or clear the hbv and viral loads are extremely high ( often > 8 logs iu / ml ) . serum alanine transaminase ( alt)/aspartate transaminase ( ast ) levels remain normal and with the lack of immune damage the liver is normal - appearing on biopsy . antiviral therapy may help bring the viral load down to these individuals , but these young people are at low risk of developing complications during this phase of the disease , antiviral therapy is not usually offered unless there are special circumstances ( i.e. 3 trimester of pregnancy ) . this phase of immune clearance can be as short as a few months , to as long as years or even decades . the immune system attempts to suppress the virus , with the viral load decreasing to between 10 and 10 iu / ml ( 1 thousand to 100 million iu / ml ) . hepatocytes harboring hbv and those in the surrounding hepatic parenchyma are damaged in this immune attack , leading to the clinical observation of hepatitis . patients will have elevated serum alt / ast levels and evidence of inflammation on liver biopsy . antiviral therapy at this stage may help prevent significant liver injury , especially if alt / ast levels are very high or if this phase is prolonged over several years . the viral load is suppressed to < 10 iu / ml , hbeag is negative and there is no further damage to the liver . however , the virus does persist within the hepatocyte and can reactivate if the immune system is weakened later on in life . reactivation may occur in the setting of immune suppression , such as chemotherapy , use of biological agents for rheumatoid arthritis or crohn 's disease ( anti - tumor necrosis factor agents ) , transplantation , and even high - dose steroid boluses . patients in this phase may already have developed cirrhosis during the preceding viral phases and have normal alt despite this . antiviral therapy is not indicated at this stage , given that hbv is not replicating . hbv dna levels may vary between 10 and 10 iu / ml , with associated increases in alt / ast and liver injury . liver failure is much more likely to occur in the setting of advanced liver fibrosis or cirrhosis , although this has become less common in this age of effective antiviral therapy . both cirrhotic and noncirrhotic patients with chronic hbv can develop hcc , which is the third leading cause of cancer death worldwide has a median 5-year survival of 8.9% . long - term survival is possible if liver masses are detected early when they are small . therefore , screening ultrasounds are performed every 6 months in those at risk [ table 2 ] . the role of alpha fetoprotein in hcc screening is controversial , given its poor sensitivity and specificity . it can be elevated during active hepatitis and pregnancy ( false positive ) , and low in patients with already diagnosed hcc . a number of variables need to be considered in the approach to hepatitis b management . serum alt / ast elevation can occur due to various causes of liver disease , including hbv , hepatitis c , fatty liver , autoimmune hepatitis , idiosyncratic drug reactions , etc . , if hbsag is positive in this context , the elevated transaminases are most likely due to hbv , especially if the viral load is over 10 iu / ml . if the hbv viral load is below this level , the clinician should consider that there is an alternate etiology for the elevated liver enzymes . a positive hbsag with normal transaminases indicate that the patient is probably immune - tolerant ( especially if younger than 30 with a high viral load ) or an inactive carrier ( viral load < 10 ) . vaccination for hepatitis b has been available since 1982 , which has effectively reduced the incidence of acute viral hepatitis b. there has in turn been a decline in the sequelae of chronic hepatitis b , and a dramatic decrease in hepatitis d infections given its replicative dependence on hbv machinery . currently , universal immunization of children is being practiced in countries around the world . in the adult population , physicians should identify those patients at high risk ( as delineated above ) , screen and vaccinate them if anti - hbs is negative . pregnant women with active hbv replication should commence antiviral therapy during the 3 trimester in order to prevent vertical transmission . the baby should be administered hepatitis b immunoglobulin and vaccinated at birth to prevent the establishment of infection . recent evidence points to likely father - to - child transmission of hbv , making the case for universal vaccination . with respect to patients with established chronic liver disease , both hepatitis a and b vaccination are advised unless they are already immune . the immunogenicity of the 3-dose hbv vaccine is decreased in all chronic liver diseases except for fatty liver and in liver transplant patients . the pneumococcal vaccine is also strongly recommended , given evidence of heightened risk of pneumococcal infection particularly among patients with advanced liver disease , although the efficacy of vaccination is somewhat decreased in this population , the influenza vaccine should be administered to any patient with a high - risk medical condition , including chronic liver disease and postliver transplant . antiviral therapy is considered for patients in the chronic hbeag - positive and chronic hbeag - negative phases ( the second and fourth phases of hbv infection ) , which are characterized by abnormal alt / ast levels and viral loads persistently over 1 10 iu / ml . for example , a patient in the immune clearance phase may successfully control hepatitis b and transition to the inactive phase within the next few months , thereby not requiring antiviral therapy . however , another patient in the immune clearance phase with very high alt levels is likely sustaining heavy liver damage , and is a candidate for antiviral therapy . hepatitis b may be treated using two different approaches : immune modulation and viral suppression . interferon is the only example of an immune modulator in hbv therapy , as it can induce entry into the inactive carrier phase , thereby effectively controlling hbv . interferon therapy results in the loss of hbeag , detection of anti - hbe , sustained low to undetectable hbv viral load and normalization of alt levels occurs in 2030% of patients . this successful treatment outcome is associated with decreased risk of fibrosis progression and hcc , as well as increased survival . interferon therapy involves regular injections with many side effects , particularly flu - like symptoms , and therefore not well tolerated . interferon has been used successfully even in patients with advanced liver fibrosis , but is contra - indicated in decompensated cirrhosis ( i.e. those who have ascites , hepatic encephalopathy , variceal bleeding , jaundice ) . however , interferon is still an option for younger patients given its higher efficacy . patients may also avoid the need for life - long treatment with potentially teratogenic antivirals during the reproductive age . nucleoside or nucleotide analogue antivirals are used in the viral suppression approach to hbv therapy . these medications have daily oral dosing and minimal side effects , which makes them an attractive therapeutic option . given that these agents are cleared by the kidneys , their dosage must be decreased in the context of a glomerular filtration rate below 50 ml / min . the virus relapses in most patients if therapy is stopped , which means that it must be continued for years ( perhaps even life - long ) . however , life - long use is rendered difficult by the high cost of medications , issues with compliance and potential teratogenicity for those patients of childbearing age . lamivudine is now a second - line medication , given the high risk of resistance over time . in general , drug - resistance mutations for one nucleoside analogue confer cross - resistance or significantly increased risk of resistance to all other nucleoside analogues . these variants can fortunately still be successfully treated with the nucleotide analogues . given the complex nature of hepatitis b treatment , the decision to start one agent versus another is the best left to an expert in liver diseases . the understanding of the natural history and management of chronic hbv has come a long way over the last few decades . prevention of acquisition through universal vaccination and screening for hbv in individuals at risk are the ideal public health strategies that have been put into place in many countries worldwide . there is a greater role for prevention of hepatic cirrhosis and hcc , given our ability to influence the natural history of hbv through antivirals . the above strategies will hopefully translate into improved clinical outcomes and lessen the economic impact of this chronic infectious disease . | chronic hepatitis b virus ( hbv ) infection affects an estimated 370 million people worldwide .
hbv is endemic throughout the world , and insidiously causes liver damage over years and decades without any warning symptoms or signs .
up to 2535% of infected individuals eventually die due to complications of liver cirrhosis and hepatocellular carcinoma ( hcc ) induced by hbv . screening those individuals at risk of acquiring hepatitis b , and universal vaccination for prevention , would help in limiting the spread and public health repercussions of the virus .
although many new antiviral therapies have been developed for the management of hepatitis b , they still do not offer the possibility of cure .
most individuals who begin oral suppressive therapy will be indefinitely treated . continuous suppression of hbv replication in individuals with advanced liver disease prolongs life , decreases the need for liver transplantation , and potentially reduces the risk for hcc . in this clinical review
, we present a practical approach to prevention of hbv , its natural history and life cycle , as well as its management . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
to understand the precise mechanism of transcriptional regulation of a gene , it is essential to identify and analyze its transcriptional start sites ( tsss ) , which are located in the vicinity of its potential promoter regions . several genome - wide studies , including ours , have identified multiple tsss or their corresponding alternative promoters of each gene ( 13 ) . it is even unknown whether they represent no more than intrinsic biological errors or cloning artifacts . to understand biological relevance of those divergent tsss , we have developed dbtss , datebase of transcriptional start sites , and continued its updates since 2001 ( 4 ) . dbtss provides tss information , which was determined using our cap - site detection method , oligo - capping ( 5 ) . to obtain the genome - wide data of tss information , rna sequences immediately downstream of the tsss are sequenced with an illumina massively parallel sequencing platform as tss tags . on the other hand , a large number of systematic epigenomic studies are underway , aiming at comprehensive understanding of chromatin conditions in the genome . recently , a group of the encode project published a chromatic map of nine representative cell types from the chip - seq analyses of nine chromatin markers for various types of histone modifications ( 8) . integration of such kind of data with the tss data would be useful for both studies . in fact , we too have generated similar types of chip - seq data for the studies of individual cells as listed in our web site . similarly , rna - seq data from several subcellular components , such as nucleus , cytoplasm , and polysome fractions , are useful to further characterize the tss data . meanwhile , human genome re - sequencing projects including exome sequencing projects have been also massively accumulating single - nucleotide variation ( snv ) data ( 9,10 ) although understanding their biological consequences is still difficult . since we believe that many of genetic disorders should be attributed to malfunctions in transcriptional regulations , the link between snps and the transcriptional information based on tsss , histone modification status , and transcripts must be established . in this report , we report three main progresses in dbtss . first , we expanded our tss - seq data by 3-fold , so that a major part of human adult and embryonic tissues are covered . second , we added various types of transcriptomics data that can be useful to further interpret tss information . for example , we integrated our original rna - seq data of subcellular - fractionated rnas ( 11 ) as well as the chip - seq data of histone modifications , rna polymerase ii and several transcriptional regulatory factors ( 1214 ) in cultured cell lines to collectively understand the relationship between tss , transcript dynamics and epigenetic factors . some of the external epigenomic data , such as those by the encode project ( 8) , are also added . third , we associated our tss data with snv data to identify snv candidates that may be responsible for disorders of transcriptional regulation . we believe that such integration provides in - depth biological insight of divergent tsss in vivo . in this update , we added new tss data that were derived from our tss - seq experiment . now dbtss contains 418 146 632 tss tags , collected from 28 tissues or cell types , including 16 kinds of human adult tissues , 5 kinds of human fetal tissues and 7 kinds of cultured cells ( table 1 ) . these tss tags were clustered into subgroups with 500-bp bins to define tss clusters ( tscs ) as putative promoter unit ( see ref . 15 for more detail ) . as a default viewer setting , we adopted tscs with the expression levels higher than 5 ppm ( particles or tags per million ; > 5 ppm tscs ) , which exists from 11 300 to 36 718 in varying cell types ( table 2 ) . in several cell lines , we collected tss - seq information under different experimental conditions such as before or after the stimulation by cytokines or hypoxic shocks . also , a total of 70 386 438 tss - seq tag data from several developmental stages and cultured cells are included in mice . users can search tsss where different expressions are observed between different tissue types or culture conditions ( figure 1b ) . ( a ) users can use a refseq i d for the simplest search ( red box in the figure ) . search condition window . in this case , users can search tsss that are overexpressed after il4 stimulation by 2-folds , with their expression level higher than 5 ppm , showing h3k4me3 signals , and having nearby dbsnp data . ( c ) users can search tsss around a given snp or any genomic position ( upper window ) . snps that are neighboring with known genes can be sought , too ( bottom window ) . table 1.statistics of tss - seq datacategoryno . of cell types or tissuesno . of total conditionno . of tss - seqhuman adult tissues1620138 864 978human fetal tisssues5541 744 136human cell lines723237 537 518human total2848418 146 632mouse embrio1438 897 846mouse cell lines3331 488 592mouse total4770 386 438total3255488 533 070
table 2.tscs corresponding to ncbi refseq genes and snp informationtss - seq tagstotal tscstscs in refseqtsc > 5 ppmoverlap refseq ( > 5 ppm)db_snp ( > 5 ppm)jpt / yri / chs / ceu ( > 5 ppm)hek29320 686 169193 140137 51811 33810 23411 2853930/5471/4391/3476ramos31 022 974371 759239 30811 455922711 4184036/5433/4340/3513beas2b98 761 770708 912440 30227 62820 38672202993/3979/3194/2167dld148 580 850462 724272 17119 94116 96519 8787084/9735/7731/6211mcf715 785 949172 834120 69511 79010 38311 7434094/5645/4432/3584tig318 780 087198 129144 62211 89310 51211 8474186/5857/4674/3711hela3 919 71999 24174 71911 300971011 2624187/5787/4705/3734adult tissue138 864 9781 496 409911 87236 71826 02350 67416 874/22 996/18 163/14 927fetal tissue41 744 136822 577572 94132 53326 77332 38610 400/14 192/11 088/9260samples : category of samples , tss - seq tags : tag number in each category , total tscs : observed tsc number , tscs in refseq : tscs overlapping with the refseq transcribed region ( including their 50 k bp upstream region ) , tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm , overlap refseq ( 5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region , db_snp ( > 5 ppm) : number of tscs which contain snps in dbsnp , jpt / yri / chs / ceu ( > 5 ppm ) : number of tscs which contain ethnic snps ( jpt : japanese in tokyo , ceu : utah residents with northern and western european ancestry from the ceph collection , chs : chinese in singapore , and yri : yoruba in ibadan ) ( a ) users can use a refseq i d for the simplest search ( red box in the figure ) . in this case , users can search tsss that are overexpressed after il4 stimulation by 2-folds , with their expression level higher than 5 ppm , showing h3k4me3 signals , and having nearby dbsnp data . ( c ) users can search tsss around a given snp or any genomic position ( upper window ) . snps that are neighboring with known genes can be sought , too ( bottom window ) . statistics of tss - seq data tscs corresponding to ncbi refseq genes and snp information samples : category of samples , tss - seq tags : tag number in each category , total tscs : observed tsc number , tscs in refseq : tscs overlapping with the refseq transcribed region ( including their 50 k bp upstream region ) , tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm , overlap refseq ( 5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region , db_snp ( > 5 ppm) : number of tscs which contain snps in dbsnp , jpt / yri / chs / ceu ( > 5 ppm ) : number of tscs which contain ethnic snps ( jpt : japanese in tokyo , ceu : utah residents with northern and western european ancestry from the ceph collection , chs : chinese in singapore , and yri : yoruba in ibadan ) . in addition to the tss - seq data , we also included rna - seq tags in the data of cultured cells . for example , in dld-1 cell data , rna - seq data from total rna , nuclear , cytoplasm , polysome , and the argonaute complex - associated fractions are stored ( 15 ) . users can search and browse the transcriptional levels of each gene in each fraction to understand the dynamics of its transcripts . to further understand the relationship between tsss and chromatin states of the surrounding regions , we integrated chip - seq data of histone modifications : h3k4me3 and h3ac for active chromatin markers and h3k27me3 for a silent chromatin marker . data of the binding sites of rna polymerase ii and several transcription factors , such as stat6 in ramos and beads2b cells and hif1a in dld-1 cells , are also included . particularly , we added the above - mentioned data of chromatin states in nine human cell lines ( 8) . in addition to the dbsnp data , we obtained exome data for four ethnic groups from the ncbi site ( ftp://ftp.ncbi.nlm.nih.gov/1000genomes/ftp ) and generated snvs uniquely called for these ethnic groups . we called 687 881 jpt ( japanese in tokyo ) , 646 522 ceu ( utah residents with northern and western european ancestry from the ceph collection ) 578 299 chs ( chinese in singapore ) and 1 120 302 yri ( yoruba in ibadan ) snvs from 73 , 64 , 90 and 82 individual 's exome data , respectively . also , we added genome - wide association studies ( gwas ) data which connect 5800 snvs with clinical information ( 16 ) . we integrated the internal and external tss - seq , rna - seq , chip - seq and snv data with each other . table 2 ( and the statistics page of our web site ) represents the statistics of each data and the overlap between them . it should be noted that all of the active markers do not always overlap . in some cases , active chromatin markers these cases may be interesting targets for future analysis of potentially stepwise transcriptional regulations . from a population genetic point of view , it may be also intriguing to further characterize tsss which are observed in an ethnic group - specific manner with solid supports from chip - seq and rna - seq data in cultured cells . for example , users can use a refseq i d for the simplest search ( figure 1a ) . users can narrow down the targets by considering expression levels within a particular cell types or fold expression changes between different cell types ( figure 2a ) . in the main viewer , users can recalculate tag counts by varying genomic regions ( figure 2b and c ) . users can also consider overlap of the chip - seq data and rna - seq data with the tss - seq data ( figure 2a ) . particularly , using the snp search function , users can input an snv in a particular category and search tsss in its surrounding region . conversely , users can search whether there are any tsss that have snvs of various categories in its surrounding regions ( figures 1b and 2d as search results ) . ( a ) overview of tss - seq and chip - seq for nm_013293 , transformer 2 alpha homolog . there are three major putative alternative promoters ( ap4 , ap10 and ap15 ) in dld1 cells . the expression of ap10 under the normoxia condition ( 21% ) is relatively low compared with that under the hypoxia condition ( 1% ) . using check boxes , users can also check the tss - seq and chip - seq results in other tissues . ( b ) function of recalculating tag counts by specifying desired genomic regions . in this case , 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed . an ethnic snp ( chs ) and two dbsnps ( rs11523571 and rs41273990 ) are also found in this region . searching chr1:159680868 based on the input window ( figure 1c ) also leads to a similar result . ( a ) overview of tss - seq and chip - seq for nm_013293 , transformer 2 alpha homolog . there are three major putative alternative promoters ( ap4 , ap10 and ap15 ) in dld1 cells . the expression of ap10 under the normoxia condition ( 21% ) is relatively low compared with that under the hypoxia condition ( 1% ) . using check boxes , users can also check the tss - seq and chip - seq results in other tissues . ( b ) function of recalculating tag counts by specifying desired genomic regions . in this case , 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed . an ethnic snp ( chs ) and two dbsnps ( rs11523571 and rs41273990 ) are also found in this region . searching chr1:159680868 based on the input window ( figure 1c ) also leads to a similar result . all of the raw data are freely and anonymously available from our download site at ftp://ftp.hgc.jp / pub / hgc / db / dbtss / dbtss_ver8. we expect more genome - wide data to become available not only for tss - seq data but also for rna - seq and chip - seq data from humans , mice and other model organisms in the near future . also , tens of thousands of snv data for different ethnic groups are being generated without further biological information except their potential disease associations . we believe that , by the integration of the data , our dbtss will continue to serve as a unique database for a wide variety of researchers : both for researchers analyzing transcriptional regulations of individual genes as well as for those analyzing comprehensive transcriptional regulatory networks from a systems biological point of view . in this update , we added new tss data that were derived from our tss - seq experiment . now dbtss contains 418 146 632 tss tags , collected from 28 tissues or cell types , including 16 kinds of human adult tissues , 5 kinds of human fetal tissues and 7 kinds of cultured cells ( table 1 ) . these tss tags were clustered into subgroups with 500-bp bins to define tss clusters ( tscs ) as putative promoter unit ( see ref . 15 for more detail ) . as a default viewer setting , we adopted tscs with the expression levels higher than 5 ppm ( particles or tags per million ; > 5 ppm tscs ) , which exists from 11 300 to 36 718 in varying cell types ( table 2 ) . in several cell lines , we collected tss - seq information under different experimental conditions such as before or after the stimulation by cytokines or hypoxic shocks . also , a total of 70 386 438 tss - seq tag data from several developmental stages and cultured cells are included in mice . users can search tsss where different expressions are observed between different tissue types or culture conditions ( figure 1b ) . ( a ) users can use a refseq i d for the simplest search ( red box in the figure ) . tss - seq detailed search , users will obtain the search condition window . in this case , users can search tsss that are overexpressed after il4 stimulation by 2-folds , with their expression level higher than 5 ppm , showing h3k4me3 signals , and having nearby dbsnp data . ( c ) users can search tsss around a given snp or any genomic position ( upper window ) . snps that are neighboring with known genes can be sought , too ( bottom window ) . table 1.statistics of tss - seq datacategoryno . of cell types or tissuesno . of total conditionno . of tss - seqhuman adult tissues1620138 864 978human fetal tisssues5541 744 136human cell lines723237 537 518human total2848418 146 632mouse embrio1438 897 846mouse cell lines3331 488 592mouse total4770 386 438total3255488 533 070
table 2.tscs corresponding to ncbi refseq genes and snp informationtss - seq tagstotal tscstscs in refseqtsc > 5 ppmoverlap refseq ( > 5 ppm)db_snp ( > 5 ppm)jpt / yri / chs / ceu ( > 5 ppm)hek29320 686 169193 140137 51811 33810 23411 2853930/5471/4391/3476ramos31 022 974371 759239 30811 455922711 4184036/5433/4340/3513beas2b98 761 770708 912440 30227 62820 38672202993/3979/3194/2167dld148 580 850462 724272 17119 94116 96519 8787084/9735/7731/6211mcf715 785 949172 834120 69511 79010 38311 7434094/5645/4432/3584tig318 780 087198 129144 62211 89310 51211 8474186/5857/4674/3711hela3 919 71999 24174 71911 300971011 2624187/5787/4705/3734adult tissue138 864 9781 496 409911 87236 71826 02350 67416 874/22 996/18 163/14 927fetal tissue41 744 136822 577572 94132 53326 77332 38610 400/14 192/11 088/9260samples : category of samples , tss - seq tags : tag number in each category , total tscs : observed tsc number , tscs in refseq : tscs overlapping with the refseq transcribed region ( including their 50 k bp upstream region ) , tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm , overlap refseq ( 5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region , db_snp ( > 5 ppm) : number of tscs which contain snps in dbsnp , jpt / yri / chs / ceu ( > 5 ppm ) : number of tscs which contain ethnic snps ( jpt : japanese in tokyo , ceu : utah residents with northern and western european ancestry from the ceph collection , chs : chinese in singapore , and yri : yoruba in ibadan ) . a ) users can use a refseq i d for the simplest search ( red box in the figure ) . tss - seq detailed search , users will obtain the search condition window . in this case , users can search tsss that are overexpressed after il4 stimulation by 2-folds , with their expression level higher than 5 ppm , showing h3k4me3 signals , and having nearby dbsnp data . ( c ) users can search tsss around a given snp or any genomic position ( upper window ) . snps that are neighboring with known genes can be sought , too ( bottom window ) . statistics of tss - seq data tscs corresponding to ncbi refseq genes and snp information samples : category of samples , tss - seq tags : tag number in each category , total tscs : observed tsc number , tscs in refseq : tscs overlapping with the refseq transcribed region ( including their 50 k bp upstream region ) , tsc > 5 ppm : number of tscs whose expression level is higher than 5 ppm , overlap refseq ( 5 ppm) : > 5ppm tscs which overlap with the refseq transcribed region , db_snp ( > 5 ppm) : number of tscs which contain snps in dbsnp , jpt / yri / chs / ceu ( > 5 ppm ) : number of tscs which contain ethnic snps ( jpt : japanese in tokyo , ceu : utah residents with northern and western european ancestry from the ceph collection , chs : chinese in singapore , and yri : yoruba in ibadan ) . in addition to the tss - seq data , we also included rna - seq tags in the data of cultured cells . for example , in dld-1 cell data , rna - seq data from total rna , nuclear , cytoplasm , polysome , and the argonaute complex - associated fractions are stored ( 15 ) . users can search and browse the transcriptional levels of each gene in each fraction to understand the dynamics of its transcripts . to further understand the relationship between tsss and chromatin states of the surrounding regions , we integrated chip - seq data of histone modifications : h3k4me3 and h3ac for active chromatin markers and h3k27me3 for a silent chromatin marker . data of the binding sites of rna polymerase ii and several transcription factors , such as stat6 in ramos and beads2b cells and hif1a in dld-1 cells , are also included . particularly , we added the above - mentioned data of chromatin states in nine human cell lines ( 8) . in addition to the dbsnp data , we obtained exome data for four ethnic groups from the ncbi site ( ftp://ftp.ncbi.nlm.nih.gov/1000genomes/ftp ) and generated snvs uniquely called for these ethnic groups . we called 687 881 jpt ( japanese in tokyo ) , 646 522 ceu ( utah residents with northern and western european ancestry from the ceph collection ) 578 299 chs ( chinese in singapore ) and 1 120 302 yri ( yoruba in ibadan ) snvs from 73 , 64 , 90 and 82 individual 's exome data , respectively . also , we added genome - wide association studies ( gwas ) data which connect 5800 snvs with clinical information ( 16 ) . we integrated the internal and external tss - seq , rna - seq , chip - seq and snv data with each other . table 2 ( and the statistics page of our web site ) represents the statistics of each data and the overlap between them . it should be noted that all of the active markers do not always overlap . in some cases , active chromatin markers these cases may be interesting targets for future analysis of potentially stepwise transcriptional regulations . from a population genetic point of view , it may be also intriguing to further characterize tsss which are observed in an ethnic group - specific manner with solid supports from chip - seq and rna - seq data in cultured cells . for example , users can use a refseq i d for the simplest search ( figure 1a ) . users can narrow down the targets by considering expression levels within a particular cell types or fold expression changes between different cell types ( figure 2a ) . in the main viewer , users can recalculate tag counts by varying genomic regions ( figure 2b and c ) . users can also consider overlap of the chip - seq data and rna - seq data with the tss - seq data ( figure 2a ) . snp search function , users can input an snv in a particular category and search tsss in its surrounding region . conversely , users can search whether there are any tsss that have snvs of various categories in its surrounding regions ( figures 1b and 2d as search results ) . ( a ) overview of tss - seq and chip - seq for nm_013293 , transformer 2 alpha homolog . there are three major putative alternative promoters ( ap4 , ap10 and ap15 ) in dld1 cells . the expression of ap10 under the normoxia condition ( 21% ) is relatively low compared with that under the hypoxia condition ( 1% ) . using check boxes , users can also check the tss - seq and chip - seq results in other tissues . ( b ) function of recalculating tag counts by specifying desired genomic regions . in this case , 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed . an ethnic snp ( chs ) and two dbsnps ( rs11523571 and rs41273990 ) are also found in this region . searching chr1:159680868 based on the input window ( figure 1c ) also leads to a similar result . ( a ) overview of tss - seq and chip - seq for nm_013293 , transformer 2 alpha homolog . there are three major putative alternative promoters ( ap4 , ap10 and ap15 ) in dld1 cells . the expression of ap10 under the normoxia condition ( 21% ) is relatively low compared with that under the hypoxia condition ( 1% ) . using check boxes , users can also check the tss - seq and chip - seq results in other tissues . ( b ) function of recalculating tag counts by specifying desired genomic regions . in this case , 1.5 ppm tsc specific to normoxia and 10.6 ppm tsc to hypoxia are observed . an ethnic snp ( chs ) and two dbsnps ( rs11523571 and rs41273990 ) are also found in this region . searching chr1:159680868 based on the input window ( figure 1c ) also leads to a similar result . all of the raw data are freely and anonymously available from our download site at ftp://ftp.hgc.jp / pub / hgc / db / dbtss / dbtss_ver8. we expect more genome - wide data to become available not only for tss - seq data but also for rna - seq and chip - seq data from humans , mice and other model organisms in the near future . also , tens of thousands of snv data for different ethnic groups are being generated without further biological information except their potential disease associations . we believe that , by the integration of the data , our dbtss will continue to serve as a unique database for a wide variety of researchers : both for researchers analyzing transcriptional regulations of individual genes as well as for those analyzing comprehensive transcriptional regulatory networks from a systems biological point of view . funding for open access charge : grant - in - aid for publication of scientific research results ; program for improvement of research environment for young researchers from the ministry of education , culture , sports , science and technology ( mext ) , japan . | to support transcriptional regulation studies , we have constructed dbtss ( database of transcriptional start sites ) , which contains exact positions of transcriptional start sites ( tsss ) , determined with our own technique named tss - seq , in the genomes of various species . in its latest version ,
dbtss covers the data of the majority of human adult and embryonic tissues : it now contains 418 million tss tag sequences from 28 tissues / cell cultures .
moreover , we integrated a series of our own transcriptomic data , such as the rna - seq data of subcellular - fractionated rnas as well as the chip - seq data of histone modifications and the binding of rna polymerase ii / several transcription factors in cultured cell lines into our original tss information .
we also included several external epigenomic data , such as the chromatin map of the encode project .
we further associated our tss information with public or original single - nucleotide variation ( snv ) data , in order to identify snvs in the regulatory regions .
these data can be browsed in our new viewer , which supports versatile search conditions of users .
we believe that our new dbtss will be an invaluable resource for interpreting the differential uses of tsss and for identifying human genetic variations that are associated with disordered transcriptional regulation .
dbtss can be accessed at http://dbtss.hgc.jp . |
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partial anomalous pulmonary venous connection ( papvc ) is relatively uncommon congenital anomaly and covers 0.51% of congenital heart diseases . the quite rare combination ( 35% ) of papvc with hypoplasia of the right lung and dextroposition of the heart is designated as scimitar syndrome . the term scimitar , referring to a turkish sword , was first used to describe the shape of the vein by nicholas halasz . in two - third of cases , the scimitar vein ( sv ) provides drainage for the entire right lung , but in others for only the lower portion . via sv pulmonary veins are discharged into the inferior or superior vena cava , or directly to right atrium . so a left - to - right shunt is established and this shunt result in long - standing right ventricular volume overload and induce increased risk of right ventricular failure . the diagnosis frequently can be established with a chest radiograph and heart doppler ultrasound . recognizing the clinical and radiographical features of this rare disease is important when dealing with patients presented with cardiac and pulmonary complaints . a 38-year - old man was referred to our hospital with abnormal findings on plain chest roentgenogram and he presented with complain of cough for 10 days . the patient appeared well on chest examination respiratory rate was 18/min and breath sound was normal . chest x - ray showed a sloping opacity at the right heart border [ figure 1a ] . the contrast - enhanced computed tomography ( ct ) demonstrated that the left pulmonary venous drainage was normal , but mild hypoplasia of right lung , mild dextroposition of heart , and left renal agenesis were detected [ figure 1b and c ] . the abnormal right pulmonary venous drainage from the right lung via a large characteristically curved anomalous vein draining into inferior vena cava at just superior to diaphragm [ figure 2a ] and resembling to the curved turkish sword scimitar [ figure 2b ] . artery blood gas analysis revealed ph of 7.44 , po2 of 72 , pco2 of 37 , and o2 saturation of 95% . while expected alveolar - arterial po2 gradient of the patient was 10.5 mmhg , the measured a - a gradient was 22.4 mmhg . transthoracic and transesophageal echocardiogram revealed an ejection fraction of 65% , systolic pulmonary artery pressure of 30 - 35 mmhg , slight ra and rv dilatation , and a one - fourth tricuspid regurgitation were also detected . ( a ) chest x - ray demonstrates a sloping opacity at the right heart border . ( b ) axial ct image shows mild hypoplastic right lung and mild dextroposition of heart . the arrow shows that hypoplastic right pulmonary veins do not flow to the left atrium ( c ) axial ct image shows the absence of left kidney ( a ) reconstructed coronal oblique image of contrast - enhanced computer tomography demonstrates that the scimitar vein was connected to the inferior vena cava . ( b ) the scimitar , which is a short , curved turkish sword based on chest roentgenogram and contrast - enhanced ct findings , scimitar syndrome was diagnosed and renal agenesis was detected . we investigated in literature but did not detect any case both with scimitar syndrome and renal agenesis . the sv must be present in scimitar syndrome , but rarely the only abnormal findings . other anomalies together with the sv are : abnormal right lung lobation and hypoplasia ( almost 100% , with widely varying degrees of hypoplasia ) ; dextroposition of the heart ; hypoplasia of the right pulmonary artery ( 60% ) ; systemic arterial blood supply to the right lower lung from the infradiaphragmatic aorta ( 60% ) ; asd of secundum type . in our case ; sv , mild hypoplasia of right lung , mild dextroposition of heart , and left renal agenesis were determined . most patients are symptomatic infants , but there are sparse reports of adults with scimitar syndrome . in 82% of adult patients , left to right shunt is less than 50% and in 77% of these patients pulmonary artery pressures are normal and in 23% are slightly elevated , and these patients lead a normal life span without surgical correction . however , in cases with shunt more than 50% , dyspnea and pulmonary artery hypertension ( pah ) develop . but his artery blood gas analysis showed some hypoxemia ( po2 of 72 ) and spirometric investigation showed mild restrictive ventilatory defect . the corrective surgery was not performed because of his refusal and the absence of both pah and symptoms . patients with scimitar syndrome generally undergo comprehensive evaluation with ct scans and cardiac angiography to characterize the anomalous vessels . recently cardiac magnetic resonance imaging has been proposed to assess the congenital abnormalities . in our case ; chest radiograph , contrast - enhanced ct , transthoracic and transesophageal echocardiogram were sufficient to diagnose and eliminate the other anomalies . the incidence of unilateral renal agenesis is about 1 in 1300 births and the prognosis is excellent . in renal agenesis this process starts at approximately 5 week and ceases at approximately 36 week of gestation . in normal lung development , primary blood supply is formed after the 7 week and pulmonary venous drainage to the left atrium is settled by 11 week . since both organogenesis take place around the same period , in our case , we thought that during fetal period there might be some factor effecting both cardiovascular and urinary system . we submitted the case because of being asymptomatic male adult patient which is seen quite rarely and for the first time to be determined scimitar syndrome and renal agenesis together . | partial pulmonary venous connection anomaly is relatively uncommon form of congenital heart diseases .
the quite rare combination of this anomaly with hypoplasia of the right lung and dextroposition of the heart is designated as scimitar syndrome .
most cases are presented in infantile period and adult presentation is exceedingly rare .
our patient , a 38-year - old man , was admitted to a doctor with flu - like complaint and because of abnormalities on chest x - ray he was sent to our clinic .
he did not have any chronic complaints such as shortness of breath and fatigue .
after investigation , scimitar syndrome was diagnosed .
left renal agenesis was determined with abdominal examination .
best of our knowledge in literature we did not detect any case both with scimitar syndrome and renal agenesis , and we wanted to report the asymptomatic adult scimitar syndrome case with left renal agenesis . |
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capecitabine ( xeloda ) is an oral fluoropyrimidine antimetabolite , which is converted by thymidine phosphorylase to 5-fluorouracil ( 5-fu ) . this enzyme is preferentially expressed by tumor cells rendering capecitabine highly selective for sensitive tumor cells . it is enlisted in the who list of essential medicines and is currently approved for early - stage and advanced colorectal cancer and metastatic breast cancer . cardiotoxicity with fluoropyrimidine - based treatment is occasionally observed , and could even be fatal [ 2 , 3 ] . although 5-fu is known to cause cardiotoxicity , capecitabine - induced cardiac side effects are poorly described in the literature . herein , we report a case of coronary vasospasm induced by capecitabine in a patient with no pre - existing coronary artery disease ( cad ) or risk factors . a 41-year - old female with metastatic breast cancer presented to the emergency department with chest pain of 1 day duration . the chest pain started gradually , and was relieved by rest and exacerbated by exertion . she denied shortness of breath , nausea , vomiting , diaphoresis , fever , syncope , heartburn , and peripheral edema . the patient did not have a history of cad or had known risk factors for cad . five years before the current event , she underwent mastectomy and axillary lymph node dissection for a stage iii ( pt2n3 ) breast cancer . the tumor was er+ , pr+ , her2 + , and she received adjuvant chemotherapy and trastuzumab , followed by tamoxifen . she was diagnosed with metastatic disease in the liver in 2014 , and was started on trastuzumab , pertuzumab , and docetaxel . fifteen months later , she had disease progression in the liver and was switched to trastuzumab - emtansine . nine months later , she progressed again , and therapy was changed to lapatinib and capecitabine ( 1,500 mg b.i.d . ) . electrocardiogram ( ecg ) testing showed normal sinus rhythm and no changes suggestive of coronary ischemia . ct angiography of the chest did not reveal any evidence of pulmonary embolism . a transthoracic echocardiogram documented normal cardiac function and a left ventricular ejection fraction of 60% . her chest pain resolved after aspirin and analgesics were administered upon admission in the er . a cardiology opinion was sought for possible angiography , but was not undertaken because of the patient 's improving condition . however , capecitabine and its parental compound may induce cardiotoxicity , depending on the dose , cardiac comorbidity and schedule of chemotherapy . capecitabine cardiotoxicity is thought to occur through the action of 5-fu on the endothelium resulting in production of endothelin-1 and subsequent coronary vasospasm . patients present with symptoms similar to variant angina , with chest pain at rest and may or may not have ecg changes of ischemia . reported exercise - induced global myocardial ischemia in a patient with normal coronary arteries and left ventricular function who was on capecitabine for recurrent breast cancer . with regard to onset of chest pain with capecitabine therapy , wijesinghe et al . reported an acute coronary syndrome in a patient who had been on capecitabine for only 2 days . reported a patient who experienced symptoms due to coronary vasospasm a few days after starting capecitabine . depending on the dose , cardiac side effects can occur within 24 h after taking the drug . as for our patient , the resolution of chest pain , normal ecg and troponins ruled out myocardial infarction and dysrhythmias . the recording of st segment changes on ecg depends on the presence of symptoms at the time of assessment . our patient 's chest pain subsided before ecg was recorded that could have possibly led to the absence of changes suggestive of coronary vasospasm . the normal echocardiogram excluded direct damage to the myocardium or coronary vessels by capecitabine metabolite . it was thought that a coronary angiography and a stress test are not necessary for her management , and therefore it was not obtained . the cardiotoxicity of fluoropyrimidines manifests as vasospasm , hypertension , ventricular arrhythmias , cardiogenic shock , and even cardiac arrest [ 2 , 5 , 10 ] . 5-fu - mediated cardiotoxicity can occur in patients with no pre - existing cad [ 5 , 6 , 7 , 12 ] . in summary , our patient had capecitabine - induced coronary vasospasm in the absence of pre - existing cad . | capecitabine , an oral prodrug of 5-fluorouracil ( 5-fu ) , is approved for early - stage and advanced colorectal cancer and metastatic breast cancer .
cardiotoxicity of 5-fu is well described in the literature .
however , cardiac adverse effects of capecitabine are poorly described .
we report a case of coronary vasospasm induced by capecitabine . a 41-year - old female with metastatic breast cancer presented with chest pain 3 days after starting capecitabine .
the chest pain was relieved by rest and exacerbated by exertion .
her physical examination was unremarkable except for a rapid heart rate of 100 bpm .
electrocardiogram test showed no acute ischemic changes .
troponin tests were negative .
ct angiography of the chest was negative for acute pulmonary embolism .
an echocardiogram showed a left ventricular ejection fraction of 60% without any wall motion abnormalities . the chest pain resolved with aspirin and analgesic use .
she was discharged following an inconclusive cardiac workup .
further use of capecitabine was discontinued . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
simulated distributions of light at the bfp were produced by propagating dipole emission fields using the appropriate green 's tensor . the mathematical formalism , including symmetry considerations for the current application , is provided in the supplementary information . we assumed a numerical aperture of 1.4 and emission wavelength of 686 nm , consistent with the peak emission of dcdhf - a-6 . 3 assume the indices of refraction of both the sample and imaging medium are matched to that of immersion oil ( n = 1.518 ) . 5 considers an emitter placed in water 1 m above a water - glass interface ( e. g. as in a cell imaging measurement ) , while supplementary fig . 6 corresponds to the molecule embedded at the bottom of a 30-nm layer of pmma , as in our first demonstration . to simulate the action of the y - phi mask we effectively multiplied the field computed at the bfp by the spatially - varying jones matrix j : j=[cossinsincos ] simulated images were produced by taking the appropriately scaled fourier transform of the field at the bfp ( details in supplementary information ) , with 100x magnification . high - resolution images ( 20-nm sampling ) were first produced then binned into pixels of side length 160 nm to match the conditions of our imaging setup . for simulated data we realized 10 noisy images for each data point . 3 . mean signal photons were scaled as necessary for polarization , relative detection efficiency ( e.g. ~0.7 , 0.8 , and 0.9 for d = 30 , 45 , and 60 , resp . ) , and relative pumping efficiency sind ( assuming a collimated , circularly polarized source ) . to compute the crlb of r and d we assumed poisson noise and constructed the fisher information ( fi ) matrix as outlined in previous work . the square roots of the diagonal elements of the fi matrix give the crlb for each parameter . to compute r we constructed a 22 fi matrix with parameters ( x , y ) ; to compute d the fi matrix was 33 with parameters ( x , y , d ) . this distinction was made because we noted that r actually worsens when d is included because off - diagonal elements appear in the fi matrix , representing a propensity to confuse the estimated parameters . we chose to report the crlb of r in this way since typical position estimators of the standard psf do n't fit d . post height , lattice constant , and ellipse diameters were used as design parameters for optimization of the metasurface y - phi mask shown in fig . for each set of the design parameters , transmission coefficients ty and tx were computed for the uniform array shown in supplementary fig . 7a using the rigorous coupled wave analysis ( rcwa ) technique with a freely available software package . these transmission coefficients were computed at 8 wavelengths : 650 nm to 720 nm , in steps of 10 nm . wavelength dependence of the refractive index and extinction coefficient of amorphous silicon ( a - si ) was considered in these simulations . the a - si refractive index and extinction coefficient values used in the simulations were measured using a variable angle spectroscopic ellipsometer , and are shown in supplementary fig . , the -to - y conversion efficiency ( ) was obtained at each wavelength using the simulated transmission coefficients ty and tx . a weighted average of the conversion efficiency values was used for comparison of different metasurfaces with different design parameters . the conversion efficiency values were weighted by the relative power density emitted from the fluorescent dye at each wavelength . the emission spectrum of dcdhf - a-6 was approximated by a gaussian function centered at 686 nm and with 60 nm full width at half maximum bandwidth . using this approach , optimum values of 365 nm , and 325 nm 7c shows the average conversion efficiency values for an array with the optimum lattice constant and post height as a function of the elliptical post diameters . diameters of dx = 180 nm , and dy = 110 nm ( indicated by a black dot in supplementary fig . 7c ) were selected to reduce the sensitivity to fabrication induced errors in the diameters of the elliptical posts , and to achieve an average conversion efficiency of 88% . the simulated conversion efficiency of the optimized metasurface mask as a function of wavelength is presented in supplementary fig . 7d . a metasurface y - phi mask with a diameter of 4.2 mm was fabricated on a 500-m - thick fused silica substrate using standard nanofabrication techniques . first , a 365-nm - thick layer of hydrogenated a - si was deposited on the substrate using a 5% mixture of silane in argon at 200 c through the plasma enhanced chemical vapor deposition technique . then , a ~300-nm - thick positive electron beam resist ( zep 520-a , zeon chemicals ) was spin - coated on the a - si layer and was baked at 180 c for 3 min . to avoid static charging effects during electron beam lithography , a ~60-nm - thick layer of a water - soluble charge dissipative conductive polymer ( aquasave , mitsubishi rayon ) was spin - coated on the resist and baked at 70 c for 5 min . after writing the nanopost pattern on the resist using electron beam lithography , the charge dissipative polymer was dissolved in water , and the resist was developed by immersing the sample in a solvent ( zed - n50 , zeon chemicals ) for 3 min followed by 30 sec rinse in methyl isobutyl ketone . the pattern was subsequently transferred through a lift - off process to a 70-nm - thick layer of aluminum oxide which was evaporated on the resist after its development . the patterned aluminum oxide layer was used as a hard mask for inductively coupled plasma reactive ion etching of the a - si layer in a mixture of sf6 and c4f8 gases . finally , the aluminum oxide mask was removed in a 1:1 mixture of ammonium hydroxide and hydrogen peroxide at 80 c. scanning electron microscope images of the top and tilted views of the final device are shown in fig . . additional characterization of the y - phi mask is described in the supplementary information and supplementary fig . dcdhf - a-6 was dissolved in 1% poly(methyl methacrylate ) in toluene to a final concentration of ~2 nm . the solution was spin - coated on ozone - etched glass coverslips , then samples were mounted on an inverted olympus ix71 microscope and pumped with a 561-nm laser ( crystalaser ) in epi - illumination mode ( peak intensity ~125 - 400 w / cm ) after reflection from a 561 dichroic beam splitter ( semrock ) . fluorescence was collected through an oil immersion objective ( olympus uplan - sapo 100/1.40 ) and passed through a 675/50 band - pass emission filter ( chroma technology ) . the linear polarizer ( newport 20lp - vis - b ) was placed just after the y - phi - mask . a broadband /2 wave plate ( b. halle 500 - 900 nm achr . l ) was placed just before the mask to act as a phase compensating element ( pce in fig . 3 ) in order to partially offset the phase incurred from reflection off the glass prism within the olympus ix71 plus the birefringence of the dichroic ( see supplementary information ) . images were recorded on an emccd camera ( andor ixon+ ) operating with em gain either 100 or 300 , at 3.33-hz frame rate . each field - of - view ( fov ) was imaged with the y - phi mask and linear polarizer in place while scanning the objective in z using a piezo ( mad city labs c - focus ) from 300 nm to + 300 nm ( only the center 400 nm was analyzed ) in 100-nm steps , with 10 camera acquisitions / step . the linear polarizer and y - phi mask were then removed and a z scan was taken of the fov in clear aperture . finally , a set of defocused ( ~1 m toward the air ) images was recorded ( supplementary fig . a small amount of asymmetry was noted in the intensity of the lobes of the y - phi mask , which may be a result of small residual aberrations ( e. g. coma ) in the imaging system ( supplementary fig . future applications may require further correction of these residual aberrations . in order to visualize single dl5-mg complexes , we prepared recombinant dl5 protein using a pet21a expression vector in rosetta gami ii de3 cells ( novagen ) . this protein stock was diluted in pbs ( ph 7.4 ) and supplemented with mg - ester such that the final concentration of dl5 and mg in the solution were 10 nm and 100pm , respectively . 50 l of this solution was incubated on etched glass coverslips ( fisherfinest , # 1.5 , 2222 mm ) that were coated with 0.01% ( w / v ) poly - l - lysine . after a 15 minute incubation , the solution was washed twice using pbs and dried for 2 hours at room temperature . imaging experiments with the dl5 samples were conducted the same manner as for dcdhf - a-6 , with minor differences . the spectrum of the labeled protein required a 647-nm laser pump ( coherent obis ) , which required a different dichroic beamsplitter ( semrock di01-r405/488/561/635 ) . this dichroic induced a different relative phase lag between x and y polarizations , so to compensate a second long - pass dichroic ( semrock ff650-di01 ) was added to the emission path in addition to the /2 wave plate from the dcdhf - a-6 measurement . the fluorescence was passed through a 650 long - pass filter ( omega optical ) and a 700/75 band - pass filter ( chroma technology ) . we noted a larger lobe asymmetry in these experiments than in the dcdhf - a-6 experiments , which may have resulted from a combination of the facts that the mask was not optimized for the dl5 emission spectrum , and that the different optical elements produced stronger aberrations . because the bound mg are inherently less bright and stable than dcdhf - a-6 , we binned image acquisitions into 3.9-s effective exposures to obtain sufficient signal - to - noise with either the standard or y - phi imaging configurations . simulated distributions of light at the bfp were produced by propagating dipole emission fields using the appropriate green 's tensor . the mathematical formalism , including symmetry considerations for the current application , is provided in the supplementary information . we assumed a numerical aperture of 1.4 and emission wavelength of 686 nm , consistent with the peak emission of dcdhf - a-6 . 3 assume the indices of refraction of both the sample and imaging medium are matched to that of immersion oil ( n = 1.518 ) . 5 considers an emitter placed in water 1 m above a water - glass interface ( e. g. as in a cell imaging measurement ) , while supplementary fig . 6 corresponds to the molecule embedded at the bottom of a 30-nm layer of pmma , as in our first demonstration . to simulate the action of the y - phi mask we effectively multiplied the field computed at the bfp by the spatially - varying jones matrix j : j=[cossinsincos ] simulated images were produced by taking the appropriately scaled fourier transform of the field at the bfp ( details in supplementary information ) , with 100x magnification . high - resolution images ( 20-nm sampling ) were first produced then binned into pixels of side length 160 nm to match the conditions of our imaging setup . for simulated data we realized 10 noisy images for each data point . 3 . mean signal photons were scaled as necessary for polarization , relative detection efficiency ( e.g. ~0.7 , 0.8 , and 0.9 for d = 30 , 45 , and 60 , resp . ) , and relative pumping efficiency sind ( assuming a collimated , circularly polarized source ) . crlb data is presented in supplementary figs . 3 , 5 , and 6 . to compute the crlb of r and d we assumed poisson noise and constructed the fisher information ( fi ) matrix as outlined in previous work . the square roots of the diagonal elements of the fi matrix give the crlb for each parameter . to compute r we constructed a 22 fi matrix with parameters ( x , y ) ; to compute d the fi matrix was 33 with parameters ( x , y , d ) . this distinction was made because we noted that r actually worsens when d is included because off - diagonal elements appear in the fi matrix , representing a propensity to confuse the estimated parameters . we chose to report the crlb of r in this way since typical position estimators of the standard psf do n't fit d . post height , lattice constant , and ellipse diameters were used as design parameters for optimization of the metasurface y - phi mask shown in fig . for each set of the design parameters , transmission coefficients ty and tx were computed for the uniform array shown in supplementary fig . 7a using the rigorous coupled wave analysis ( rcwa ) technique with a freely available software package . these transmission coefficients were computed at 8 wavelengths : 650 nm to 720 nm , in steps of 10 nm . wavelength dependence of the refractive index and extinction coefficient of amorphous silicon ( a - si ) was considered in these simulations . the a - si refractive index and extinction coefficient values used in the simulations were measured using a variable angle spectroscopic ellipsometer , and are shown in supplementary fig . , the -to - y conversion efficiency ( ) was obtained at each wavelength using the simulated transmission coefficients ty and tx . a weighted average of the conversion efficiency values was used for comparison of different metasurfaces with different design parameters . the conversion efficiency values were weighted by the relative power density emitted from the fluorescent dye at each wavelength . the emission spectrum of dcdhf - a-6 was approximated by a gaussian function centered at 686 nm and with 60 nm full width at half maximum bandwidth . using this approach , optimum values of 365 nm , and 325 nm 7c shows the average conversion efficiency values for an array with the optimum lattice constant and post height as a function of the elliptical post diameters . diameters of dx = 180 nm , and dy = 110 nm ( indicated by a black dot in supplementary fig . 7c ) were selected to reduce the sensitivity to fabrication induced errors in the diameters of the elliptical posts , and to achieve an average conversion efficiency of 88% . the simulated conversion efficiency of the optimized metasurface mask as a function of wavelength is presented in supplementary fig . a metasurface y - phi mask with a diameter of 4.2 mm was fabricated on a 500-m - thick fused silica substrate using standard nanofabrication techniques . first , a 365-nm - thick layer of hydrogenated a - si was deposited on the substrate using a 5% mixture of silane in argon at 200 c through the plasma enhanced chemical vapor deposition technique . then , a ~300-nm - thick positive electron beam resist ( zep 520-a , zeon chemicals ) was spin - coated on the a - si layer and was baked at 180 c for 3 min . to avoid static charging effects during electron beam lithography , a ~60-nm - thick layer of a water - soluble charge dissipative conductive polymer ( aquasave , mitsubishi rayon ) was spin - coated on the resist and baked at 70 c for 5 min . after writing the nanopost pattern on the resist using electron beam lithography , the charge dissipative polymer was dissolved in water , and the resist was developed by immersing the sample in a solvent ( zed - n50 , zeon chemicals ) for 3 min followed by 30 sec rinse in methyl isobutyl ketone . the pattern was subsequently transferred through a lift - off process to a 70-nm - thick layer of aluminum oxide which was evaporated on the resist after its development . the patterned aluminum oxide layer was used as a hard mask for inductively coupled plasma reactive ion etching of the a - si layer in a mixture of sf6 and c4f8 gases . finally , the aluminum oxide mask was removed in a 1:1 mixture of ammonium hydroxide and hydrogen peroxide at 80 c. scanning electron microscope images of the top and tilted views of the final device are shown in fig . . additional characterization of the y - phi mask is described in the supplementary information and supplementary fig . dcdhf - a-6 was dissolved in 1% poly(methyl methacrylate ) in toluene to a final concentration of ~2 nm . the solution was spin - coated on ozone - etched glass coverslips , then samples were mounted on an inverted olympus ix71 microscope and pumped with a 561-nm laser ( crystalaser ) in epi - illumination mode ( peak intensity ~125 - 400 w / cm ) after reflection from a 561 dichroic beam splitter ( semrock ) . fluorescence was collected through an oil immersion objective ( olympus uplan - sapo 100/1.40 ) and passed through a 675/50 band - pass emission filter ( chroma technology ) . the 4f system of fig . 3 consisted of two achromatic doublet lenses ( edmund optics , 50 mm dia . 150 mm fl , vis - nir coated ) . the linear polarizer ( newport 20lp - vis - b ) was placed just after the y - phi - mask . a broadband /2 wave plate ( b. halle 500 - 900 nm achr . l ) was placed just before the mask to act as a phase compensating element ( pce in fig . 3 ) in order to partially offset the phase incurred from reflection off the glass prism within the olympus ix71 plus the birefringence of the dichroic ( see supplementary information ) . images were recorded on an emccd camera ( andor ixon+ ) operating with em gain either 100 or 300 , at 3.33-hz frame rate . each field - of - view ( fov ) was imaged with the y - phi mask and linear polarizer in place while scanning the objective in z using a piezo ( mad city labs c - focus ) from 300 nm to + 300 nm ( only the center 400 nm was analyzed ) in 100-nm steps , with 10 camera acquisitions / step . the linear polarizer and y - phi mask were then removed and a z scan was taken of the fov in clear aperture . finally , a set of defocused ( ~1 m toward the air ) images was recorded ( supplementary fig . a small amount of asymmetry was noted in the intensity of the lobes of the y - phi mask , which may be a result of small residual aberrations ( e. g. coma ) in the imaging system ( supplementary fig . future applications may require further correction of these residual aberrations . in order to visualize single dl5-mg complexes , we prepared recombinant dl5 protein using a pet21a expression vector in rosetta gami ii de3 cells ( novagen ) . this protein stock was diluted in pbs ( ph 7.4 ) and supplemented with mg - ester such that the final concentration of dl5 and mg in the solution were 10 nm and 100pm , respectively . 50 l of this solution was incubated on etched glass coverslips ( fisherfinest , # 1.5 , 2222 mm ) that were coated with 0.01% ( w / v ) poly - l - lysine . after a 15 minute incubation , the solution was washed twice using pbs and dried for 2 hours at room temperature . imaging experiments with the dl5 samples were conducted the same manner as for dcdhf - a-6 , with minor differences . the spectrum of the labeled protein required a 647-nm laser pump ( coherent obis ) , which required a different dichroic beamsplitter ( semrock di01-r405/488/561/635 ) . this dichroic induced a different relative phase lag between x and y polarizations , so to compensate a second long - pass dichroic ( semrock ff650-di01 ) was added to the emission path in addition to the /2 wave plate from the dcdhf - a-6 measurement . the fluorescence was passed through a 650 long - pass filter ( omega optical ) and a 700/75 band - pass filter ( chroma technology ) . we noted a larger lobe asymmetry in these experiments than in the dcdhf - a-6 experiments , which may have resulted from a combination of the facts that the mask was not optimized for the dl5 emission spectrum , and that the different optical elements produced stronger aberrations . because the bound mg are inherently less bright and stable than dcdhf - a-6 , we binned image acquisitions into 3.9-s effective exposures to obtain sufficient signal - to - noise with either the standard or y - phi imaging configurations . | nanoscale localization of single molecules is a crucial function in several advanced microscopy techniques , including single - molecule tracking and wide - field super - resolution imaging 1 . to date , a central consideration of such techniques is how to optimize the precision of molecular localization . however , as these methods continue to push toward the nanometre size scale , an increasingly important concern is the localization accuracy . in particular , single fluorescent molecules emit with an anisotropic radiation pattern of an oscillating electric dipole , which can cause significant localization biases using common estimators 2 - 5 . here
we present the theory and experimental demonstration of a solution to this problem based on azimuthal filtering in the fourier plane of the microscope .
we do so using a high efficiency dielectric metasurface polarization / phase device composed of nanoposts with sub - wavelength spacing 6 .
the method is demonstrated both on fluorophores embedded in a polymer matrix , and in dl5 protein complexes that bind malachite green 7 , 8 . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
functional glycomics is an emerging field of science , aiming to create a cell - by - cell catalogue of glycosyltransferase expression and detected glycan structures in relation to health and diseases ( 13 ) . however , modern analytical methods such as mass spectrometry and nmr have afforded the ability to elucidate most structural details at the concentration levels required for glycomics ( 4,5 ) . several national and international initiatives aiming to decipher the biological function of carbohydrates have emerged for the recent years ( 68 ) . in a similar fashion to the finished human genome project which determined the sequences of the chemical base pairs that make up a human dna most of these glycomics projects intend to make their data freely accessible under an open access philosophy . unfortunately , the exchange of data between different glyco - related databases is seriously hampered by the dearth of generally accepted digital exchange formats and standardized structural and biological descriptions ( 9 ) . similar to the genomics and proteomics field , a description of glycan structures would be an appropriate way to establish an efficient connection of glyco - related information resources . however , glycan sequences can not be described by a simple linear one - letter code as each pair of monosaccharides can be linked in several ways and branched structures can be formed . the glycosciences.de portal ( 7 ) demonstrates that data originating from various resources can be efficiently integrated using a linear notation for unique description of carbohydrate sequences ( linucs ) ( 10 ) . the extended alphanumeric iupac description and glycosidic linking information are applied to build up a hierarchy of the various branches starting from the reducing end of the oligosaccharide chain , which is then converted into a linear representation . however the commercially available glycosuitedb ( 11 ) uses the so - called condensed form of the iupac description to create a linear representation , where four rules are applied to obtain a unique linear code . the glycan database of the us consortium for functional glycomics ( 6 ) uses the so - called linear code ( 12 ) , using a one or two character - based representation of saccharide units and linkages . the ordering of glycan branches is established using a special lookup table where the hierarchy of monosaccharide structures is defined . the kegg carbohydrate matcher ( kcam ) ( 8,13 ) uses a connection table based graph representation to encode carbohydrate structures , where monosaccharides are represented by nodes and glycosidic bonds as edges . glycosciences.de , glycosuite , cfg glycan database and kegg - glycan concentrate on glycan structures found in mammalian species . in contrast , the mission of the russian bacterial carbohydrate structure database ( bcsdb ) [ ( 14 ) , for urls see appendix ] is to provide all published glycan structures found in bacteria . since the monosaccharide namespace as well as the type of linkages found in bacterial polysaccharides differ considerably from those found in mammals , bcsdb uses an internal representation of glycans , which diverges from those used to describe structures found in mammals . looking at various existing carbohydrate databases accessible through the internet , it is obvious that diverse ways to encode and store complex carbohydrates are in use . however , users who would like to access all publicly available glyco - related data spread over many databases have not only to cope with varying graphical and non - graphical interfaces to input glycan structures , but also must be aware that the definition of building blocks and topologies may be different . each database has developed its own set of rules to solve some problematic encoding situations such as treatment of monovalent substituents , phosphates , sulphates , repeat units , unknown linkages and other uncertain structural features of glycan structures . it is , of course , an attractive vision [ expressed during the joint meeting of the japanese and american consortia for glycomics ( 15 ) ] to have a single user interface which will provide access to all relevant world - wide distributed resources without any technical and administrative barrier . a prerequisite for an efficient exchange of data is the agreement to a generally accepted exchange format as well as a common application programming interface . consequently , several proposals for an xml - based description of glycan structures have already been published ( 16,17 ) . to avoid any further confusion about xml descriptions of glycans , the seven larger initiatives in this field [ cfg , bcsdb , glycosciences.de , eurocarbdb , kegg , hgpi and ccrc ( for abbreviations see appendix ) ] agreed to further develop the xml description for the encoding of glycan structures on the basis of the already existing glycan data exchange ( glyde ) ( 17 ) . the progress discussion is open to all interested scientists and takes place at the forum pages of the eurocarbdb project . concerning the technical realization of the online connection between existing databases , it seems that the simple object access protocol ( soap ) is now the broadly accepted procedure for automated communication between web - applications . being designed to communicate via the internet , it is well suited to be also used for the exchange of glycan - related data between distributed computers . taken together , it seems like the field has matured to the point where it is feasible to establish an online connection of distributed databases , at least between the larger of the established projects . the bacterial carbohydrate structure database ( bcsdb ) [ ( 14 ) , for urls see appendix ] is a database containing data on natural carbohydrates with known structure . in addition to the structure and bibliography , each record in the bcsdb contains the abstract of the publication , data on the carbohydrate source , methods of structure elucidation , information on the availability of spectral data and assignment of nmr spectra when available , data on conformation , biological activity , chemical and enzymatic synthesis , biosynthesis , genetics and other related data . the search criteria can be fragment(s ) of the structure ; fragment(s ) of the nmr spectrum ; and indexed tags , including microorganism , bibliography and keywords . currently , the bcsdb contains 8200 records on bacterial carbohydrates , including the corresponding part of carbbank ( 18 ) ( 3500 records on structures reported before 1995 ) . data from both literature and carbbank have been carefully checked for consistency before the upload , and corrected when necessary . the bcsdb interface includes the web - based user part , web - based administrator part and programming gateways for the automated data interchange . the bcsdb is available on the internet for free usage and validated user data submission . the glycosciences.de portal ( 7 ) is an attempt to link glycan - related data originating from various resources through a unique structural description . the linucs ( linear notation for unique description of carbohydrate sequences ) ( 10 ) notation is used to uniquely encode fully characterized glycans . currently , the glycosciences portal provides access to 24 000 different entries with nearly 14 000 different carbohydrate moieties . these structures are sourced from a number of sources , including the former carbbank and sugabase - project ( 19 ) , automatic extraction from the protein data base ( pdb ) ( 20 ) , and the curation of new entries altogether . the structure - oriented approach to the database allows the data related to a single glycan , but originating from various sources ( e.g. experimental nmr spectra , theoretically calculated fragment ions for mass spectra interpretation or experimental or simulated 3d structures ) to be easily linked and accessed using a single database query . according to the varying needs of specific research questions , the glycosciences portal provides several structure - oriented options to recall glycan - related data . the retrieval of glycans matching an exact structure is the most traditional way to access a database . the motif search enables to retrieve all entries , which possess substructures having names such as lewis , blood group h antigen or gm3 . all glycan - related scientific data of the glycosciences.de portal are freely accessible via the internet following the open access philosophy : free availability and unrestricted use. the soap - based web - services are available on the websites of the two projects and are documented in the form of wsdl ( for urls see appendix ) descriptions that provide the possibility of platform - independent formalization of server - side features . wsdl files can be easily integrated into the existing code by using features from various soap libraries which allow the transparent work with the soap interface under perl , php , java , etc . glycan data exchange ( glyde ) version 1.2 ( 17 ) was chosen as the structure exchange format . it supports almost all known peculiarities of carbohydrate structures , such as uncertainities in configuration and ring sizes , various combinations of repeating and non - repeating parts , non - carbohydrate linkers , cyclic structures , etc .. glyde uses a tree - based approach to structure description . within this approach the tree root is the reducing and or the rightmost residue in the repeating unit , while all the substituents are the children of the residue they are attached to . configurations , ring - size and other related information is stored as attributes of the residue . two approaches are used : the raw data ( as array of strings corresponding to authors , title terms , journal name , etc . ) or pubmed xml . a well - known identifier for an organism is a taxid provided by ncbi taxonomy database . however , the ranking of taxid is limited to species ; thus , no possibility to cross - search for particular strains / serogroups is provided . as this detailed ranking is significant mainly for bacteria , the capability to perform deep species searching taxids are stored in the glycosciences.de database together with structures , while bcsdb generates taxids based on genus and species name , making use of an ncbi web service . example 1 , using the bibliographic search of glycosciences.de , shows all references found in both resources for author brade in year 2002 . example 2 depicts a substructure search containing a specified disaccharide fragment [ -d - neup5nac-(2 - 3)--d - galp ] in glycosciences.de . the data associated with two entries containing the disaccharide fragment are shown in example 2b . example 3 demonstrates a substructure search in bcsdb using glycosciences.de to input the trisaccharide fragment -d - galp-(1 - 3)--d - manp-(1 - 4)--l - rhap . substructure search for a - d - neup5nac-(2 - 3)-b - d - galp in glycosciences.de using the bcsdb input wizard . result querying glycosciences for all structures containing a specified disaccharide fragment -d - neup5ac-(23)-d - galp . querying bcsdb for all structures containing the specified trisaccharide fragment -d - galp-(1 - 3)--d - manp-(1 - 4)--l - rhap . the glycosciences.de substructure input spreadsheet is used . the data associated with bcsdb entry 10147 are additionally shown . the capability of web services to make distributed scientific data accessible is clearly demonstrated . to our knowledge , the implemented mutual online access between bcsdb and glycosciences.de is the first reported attempt of a structure - based interconnection of two glyco - related databases . for users the advantages are obvious : they can use and have to learn only one interface , always have access to the latest data from both services , and the results of both searches are presented in a consistent way . for the database design and its functionality the establishment of a connection helped to find shortcomings and inconsistencies in both underlying data concepts and structural representations . for the maintenance of the databases , duplication of work can be easily avoided . it can be expected that more frequent use of both services will improve the quality of data . this will hopefully lead to a better worldwide acceptance of both services within the community of glycoscientists . since the exchange of data is accomplished through standard , well - documented xml - based descriptions and soap protocols ; other interested providers of glyco - related databases may easily be linked so that a larger network could grow . it can be envisaged that online connection of thematically related scientific data collections will have a bright future , and not only in the area of glycosciences . one of the main bottlenecks is currently that broadly accepted standard xml exchange formats are often not yet available . it will definitively be a time - consuming task to come to agreements about such standard descriptions within the various communities . with glyde 1.2 an xml - based encoding scheme of glycan structures exists , which is sufficiently flexible to link the vast majority of structures contained in bcsdb and glycosciences.de . however , glyde 1.2 has some shortcomings regarding uncertainties in terminal residues and other fuzzy encodings , which will become more important for glycomics projects . the current focus of discussion is to base a more flexible encoding on the concept of a connection table approach , instead of a tree - like structure as used in glyde 1.2 . recently ( september 2006 , nih meeting frontier in glycomics ) , the seven larger projects already mentioned above have agreed to support glyde - ct as the main database format for the exchange of glycan structures . a less favourable situation would be that several exchange format exit and parsers must be available for each database . | functional glycomics , the scientific attempt to identify and assign functions to all glycan molecules synthesized by an organism , is an emerging field of science . in recent years ,
several databases have been started , all aiming to support deciphering the biological function of carbohydrates .
however , diverse encoding and storage schemes are in use amongst these databases , significantly hampering the interchange of data . the mutual online access between the bacterial carbohydrate structure database ( bcsdb ) and the glycosciences.de portal , as a first reported attempt of a structure - based direct interconnection of two glyco - related databases is described . in this approach
, users have to learn only one interface , will always have access to the latest data of both services , and will have the results of both searches presented in a consistent way .
the establishment of this connection helped to find shortcomings and inconsistencies in the database design and functionality related to underlying data concepts and structural representations .
for the maintenance of the databases , duplication of work can be easily avoided , and will hopefully lead to a better worldwide acceptance of both services within the community of glycoscienists .
bcsdb is available at and the glycosciences.de portal at |
You are an expert at summarizing long articles. Proceed to summarize the following text:
diaphragmatic dysfunction likely plays an important role in sepsis - related respiratory failure , a condition which occurs in a large proportion of patients admitted to the icu for severe sepsis . sepsis - induced diaphragmatic dysfunction is a pleiomorphic phenomenon manifested by decreased force - generating capacity of the muscle , an increased susceptibility to fatigue , and the presence of muscle fiber injury [ 27 ] . inflammatory mechanisms have been strongly implicated , with oxygen free radicals [ 810 ] , nitric oxide or its derivatives [ 4 , 5 ] , and tnf- [ 11 , 12 ] being amongst the major mediators identified thus far . chemokines are low - molecular - weight cytokines that play a key role in orchestrating leukocyte trafficking to infected or diseased tissues . the chemokines are subdivided into four main families ( cc , cxc , cx3c , and c ) based upon their structure . increased expression levels of several chemokine ligands and their cognate receptors , particularly of the cc family , have been found in muscle biopsies obtained from animal models and human patients suffering from muscular dystrophy or inflammatory myopathies [ 1416 ] . in this regard , we have recently reported a marked upregulation of ccr1 and two of its major ligands , rantes ( ccl5 ) and mip-1 ( ccl3 ) , in the diaphragms of muscular dystrophy ( mdx ) mice and demonstrated a relationship with the degree of muscle infiltration by mononuclear inflammatory cells . in addition to their classical role as leukocyte chemoattractants , the chemokines influence a diverse repertoire of normal and pathophysiologic processes , including myoblast behavior in skeletal muscle repair [ 1719 ] and tissue responses to oxidative stress . rantes and mip-1 can also act together with ifn- to synergistically upregulate tnf- , the most prototypical cytokine implicated in sepsis - induced diaphragmatic dysfunction . furthermore , it has recently been demonstrated that the ccr1-rantes axis is a key modulator of the host immune response to bacterial peritonitis as well as exogenously administered lps . in the present study , our primary objective was to determine whether rantes and mip-1 , as well as their major receptors , are upregulated within the diaphragm after the induction of sepsis in vivo . to examine this question , we employed two different models of sepsis in vivo , an acute model of systemic lps administration and a more clinically realistic model of sustained sepsis caused by pseudomonas lung infection . we further dissected the cellular mechanisms underlying chemokine upregulation in the septic diaphragm , by examining the responses of primary diaphragm muscle cells to lps stimulation in vitro . more specifically , we sought to ascertain whether these responses could be influenced by inhibiting either free radicals or the nf-b transcription factor pathway , since both have been implicated in sepsis - associated respiratory muscle dysfunction . male c57bl/6 mice ( charles river , quebec , canada ) aged 810 weeks ( 2326 g ) were studied . anesthesia was achieved with a mixture of ketamine ( fort dodge animal health , fort dodge , ia ) and xylazine ( bayer , shawnee mission , ks ) . two experimental models of infection were used : acute endotoxemia and pseudomonas aeruginosa lung infection . to produce endotoxemia , animals received an intraperitoneal injection of lps ( 25 mg / kg of serotype o55:b5 ; sigma chemical co. , st louis , mo ) diluted in saline . in previous studies , such a dose has been shown to produce a significant impairment of diaphragmatic force generation . a sham - treated control group underwent the same procedure with an equivalent volume of saline alone , and all mice were euthanized at either 6 or 24 hours after injection . the model of chronic pulmonary infection with p. aeruginosa was performed as originally reported by starke et al . the bacteria were entrapped within agar beads ( 100150 m ) , and the number of viable bacteria was measured by homogenizing the beads and then plating 10-fold serial dilutions on trypticase soy agar plates . sterile agar beads were produced in the same manner but with omission of bacteria and were confirmed to be free of colony - forming units ( cfus ) . the trachea of anesthetized mice was intubated to deliver either bacteria - containing or sterile agar beads to mouse lungs , and the mice were killed at either 2 or 7 days later . two different doses of inoculating bacteria ( 2 105 and 1 106 cfu ) were studied at 2 days after infection , whereas only the lower inoculating dose was used for the 7-day time point due to unacceptable signs of ill health at the higher dose in the 7-day group . the higher dose has been shown to produce a significant impairment of diaphragm force generation 2 days after infection , whereas the lower dose has been shown to produce similar effects 7 days after infection . primary diaphragm and tibialis anterior ( ta ) muscle cell cultures were established as previously described in detail , using single living muscle fibers to isolate adult myoblasts . briefly , excised diaphragm muscle strips were digested ( 0.2% collagenase ) , followed by trituration to liberate individual fibers . the fibers were transferred into matrigel ( becton dickinson , franklin lakes , nj)-coated ( 1 mg / ml in dmem ) 6-well plates . cultures were maintained in dmem with 10% horse serum and 0.5% chick embryo extract ( mp biomedical , aurora , oh ) for 4 days , during which myoblasts attached to the substratum . diaphragmatic myoblasts were then expanded in growth medium ( 20% fetal bovine serum , 10% horse serum , 1% chick embryo extract in dmem ) until attaining approximately 75% confluence . at this point , the cultures were placed in differentiation medium ( 2% fetal bovine serum , 10% horse serum , 0.5% chick embryo extract in dmem ) in order to induce myoblast fusion into differentiated myotubes . all experiments were performed on the fifth day in differentiation medium . for lps stimulation , the cells were washed with dmem and then stimulated during 4 hours with e. coli lps ( 1 g / ml ) . to study the involvement of nf-b , myotubes were treated one hour before lps exposure with pyrrolidine dithiocarbamate ( pdtc ; at 100 m ) ( fisher scientific , nepean , on ) , a compound which inhibits the ubiquitin e3 ligase function required for nf-b activation [ 26 , 27 ] . to study the potential role of free radicals , myotubes were treated with catalase ( at 2000 u / ml ) or n - acetylcysteine ( nac ; at 10 mm ) ( fisher scientific , nepean , on ) , initiated one hour before lps exposure . because the transfection efficiency of primary muscle cell cultures is very low , a murine myoblast cell line ( c2c12 ) was employed for transfection experiments . c2c12 cells ( 5 105 ) were seeded onto 60 mm plates , incubated overnight in growth medium ( dmem-10% fbs ) , and transfected the following day at approximately 60% confluence . to achieve transfection , lipofectamine 2000tm ( invitrogen , carlbad , ca ) and 8 g of plasmid dna were combined according to the manufacturer 's protocol . at 48 hours posttransfection , the cells were trypsinized , replated in 6-well plates and allowed to attach overnight . the next day the medium was switched to dmem-2% hs to induce differentiation , and lps stimulation experiments were performed on differentiated myotubes 5 days later , as described earlier for primary cells . cells transfected with pegfp - c1 ( bd bioscience , clontech , san diego , ca ) , expressing green fluorescent protein ( gfp ) , served as transfection controls . to evaluate transcriptional activity from the rantes promoter , muscle cells were transfected with a firefly luciferase expression plasmid under the control of the rantes promoter ( gift from moriuchi et al . ) . the cells were exposed to lps for 4 hours , then lysed and assayed for luciferase activity . to assess the involvement of nf-b in chemokine gene regulation , dominant negative mutant forms of the ib kinases ( gift from bhakar et al . ) , ikk , and ikk were employed to inhibit nf-b activation . the mrna expression levels of ccr1 and ccr5 as well as their two major ligands , rantes and mip-1 , were assessed in rna extracted from tissues or muscle cell cultures using trizol ( invitrogen , carlsbad , ca ) . the labelled riboprobes were hybridized with total rna overnight ( 20 micrograms from muscle tissues and 5 micrograms from muscle cell cultures ) and then processed using the manufacturer 's protocol ( bd bioscience , pharmingen , san diego , ca ) . protected mrnas were separated using a 5% acrylamide gel ( 19:1 acrylamide : bisacrylamide ) . gels were transferred to filter paper and dried under vacuum using a gel drier . dried gels were exposed to scientific x - ray imaging film ( kodak , rochester , ny ) , and the bands were quantified using an image analysis system ( fluorchem 8000 , alpha innotech corp , san leandro , ca ) . all signals were normalized to l32 as a loading control and expressed in absolute arbitrary units . for both in vivo and in vitro studies , 6 independent replicate analyses were performed for each experimental condition and time point , unless otherwise noted . for the same experimental condition , diaphragm and ta were compared using the wilcoxon sign rank test . for the same type of muscle , lps and saline groups were compared using the mann - whitney rank sum test . comparisons between three or more groups were performed using the kruskall - wallis test . when significant , post hoc multiple comparisons were archived using the student - newman - keuls method . the analysis was performed using sigmastat software ( spss , chicago , il ) , and statistical significance was set at p < .05 . to ascertain the effects of endotoxemia on ccr1 and ccr5 expression in the diaphragm and ta muscles , total rna was extracted at 6 or 24 hours after lps administration and analyzed by ribonuclease protection assays . a representative autoradiograph showing ccr1 expression levels at 24 hours after lps delivery , as well as group mean data ( expressed in arbitrary units ) for both time points , are provided in figure 1 . it should be noted that for these experiments , all of the rna samples evaluated for a specific time point ( n = 24 ) were loaded onto the same gel in order to ensure that the x - ray exposure conditions were identical . this allowed for valid quantitative comparisons at a given time point but not necessarily between different time points , and thus only the former was subjected to statistical analysis . as can be seen , ccr1 and ccr5 were both expressed at a higher constitutive level in the diaphragm than in the ta limb muscle under control ( saline ) conditions . however , only ccr1 showed upregulation in the diaphragm during sepsis , and this was observed at the 24-hour time point . interestingly , both ccr1 and ccr5 were significantly upregulated in the ta muscle at 6 hours after lps administration . we next sought to evaluate whether the observed upregulation of ccrs in the septic diaphragm and ta is associated with altered expression of their cognate ligands , rantes and mip-1 , at the same time points ( 6 and 24 hours ) after lps delivery in vivo . figure 2 shows that the overall patterns of rantes and mip-1 gene expression responses were similar . hence for both chemokines , increased mrna expression was induced by lps injection in the diaphragm at 6 hours , and levels remained elevated over control values at 24 hours . in addition , under control conditions , the baseline expression levels of both rantes and mip-1 were significantly higher in the diaphragm than in the ta limb muscle . similarly , the absolute magnitude of mrna expression for both chemokines after lps administration was generally greater in the diaphragm than in the limb muscle . to determine if similar patterns of chemokine upregulation within the diaphragm are observed in another septic situation relevant to the icu setting , namely , gram - negative bacterial pneumonia , a live infection with pseudomonas aeruginosa was introduced into the lungs . two different inoculating doses were studied at day 2 postinfection , whereas only the lower inoculating dose was used for day 7 due to excessive mortality at this time point with the higher dose . figure 3 shows that both rantes and mip-1 mrna expression were upregulated at day 2 postinfection with the higher inoculating dose ( 10 cfu ) only . in contrast , the lower inoculating dose ( 10 cfu ) also triggered an increased expression of the two chemokines at day 7 postinfection , and this was particularly striking in the case of rantes . interestingly , we did not observe any upregulation of either mip-1 and rantes within limb muscles under the same conditions ( data not shown ) , and the levels of chemokine upregulation in the diaphragm at both time points after pseudomonas infection were greater ( relative to control ) than following acute lps administration . having shown that both rantes and mip-1 are upregulated in the diaphragm by two different sepsis models in vivo , we next wished to establish whether stimulation by lps has direct effects on the expression of these chemokines by diaphragm muscle cells in vitro . ribonuclease protection assays were performed to quantify rantes and mip-1 expression levels in primary diaphragm muscle cell cultures ( differentiated myotubes ) at 4 hours poststimulation with lps . in the absence of lps stimulation , rantes expression by diaphragmatic myotubes was observed , but only at a very low level , whereas mip-1 was not detectable . however , exposure of the cultured diaphragmatic myotubes to lps markedly upregulated rantes ( see figure 4(a ) ) but had no measurable effect on mip-1 expression . to further explore the mechanism of rantes upregulation by muscle cells after lps stimulation , we transfected c2c12 cells ( skeletal muscle cell line ) with a plasmid containing the rantes promoter linked to a luciferase reporter gene , and the transfected cells were then once again stimulated with lps at the myotube stage . as shown in figure 4(b ) , there was a significant increase in luciferase expression in the lps - stimulated group in comparison to nonstimulated control cultures transfected with the same plasmid ( transfection efficiency of approximately 50% in both groups ) . these data suggest that lps - induced upregulation of rantes expression in muscle cells involves an increased level of transcription from the rantes promoter . because the rantes promoter is a target for the transcription factor nf-b , we also examined the effects of an nf-b inhibitor ( pdtc ) on rantes gene expression in primary cultured diaphragmatic myotubes stimulated with lps . these findings suggest that nf-b plays an important role in lps - induced rantes expression by diaphragmatic myotubes . on the other hand , nac and catalase , two potent antioxidant compounds , had no measurable effects on rantes expression ( figure 5(b ) ) , suggesting that the free radical generation which is known to occur in septic diaphragm muscle [ 3 , 9 ] is not a major factor in lps - induced rantes upregulation . finally , the role of nf-b in the lps - induced rantes gene upregulation within differentiated myotubes was further studied using a strategy of dominant negative inhibition . two different dominant negative constructs were transfected into c2c12 cells in order to inhibit ikk and ikk , which are involved in the alternate and classical nf-b pathways , respectively . figure 6 illustrates and confirms the role of the classical nf-b pathway in lps - induced rantes upregulation by muscle cells . hence when c2c12 muscle cells were transfected with the dominant negative ikk construct prior to lps stimulation at the myotube stage , there was a significant inhibition of subsequent lps - induced rantes expression , whereas ikk inhibition had no significant effect . chemokines are responsible for modulating multiple aspects of the host inflammatory response against infection , such as leukocyte adhesion and migration , inflammatory cell activation , and the balance between type i and type ii cytokine production [ 21 , 33 ] . serum levels of both rantes and mip-1 are elevated in septic patients , and modulating the biological activity of these chemokines has been shown to alter the clinical course of sepsis in animal models [ 22 , 35 ] . two major receptors targeted by rantes and mip-1 in vivo are ccr1 and ccr5 , which are both expressed by a broad spectrum of leukocytes . the authors and sell et al . have recently demonstrated both expression and function of these receptors in isolated skeletal muscle cells . to our knowledge , this is the first study to demonstrate an upregulation of cc class chemokine receptors and their ligands within septic skeletal muscles . the major findings of our study can be summarized as follows : ( 1 ) diaphragmatic ccr1 gene expression is upregulated in response to acute endotoxemia ; ( 2 ) both acute endotoxemia and pseudomonas lung infection lead to increased expression of the ccr1 ligands , rantes and mip-1 , and this occurs to a significantly greater extent within the diaphragm as compared to limb muscle ; ( 3 ) lps - induced upregulation of rantes expression by diaphragmatic muscle cells is transcriptionally regulated and dependent upon the nf-b transcription factor pathway , whereas oxygen free radicals do not appear to be involved in this process . in the present investigation , both of these models have previously been shown to be associated with sepsis - induced upregulation of a number of proinflammatory cytokines ( see table 1 ) , along with diaphragmatic dysfunction . the acute administration of lps has the advantage of being a well - established and easily quantifiable septic stimulus , whereas the pseudomonas lung infection model may be considered closer to most clinical situations , since it encompasses additional virulence factors within the bacteria besides endotoxin . interestingly , in both models , the sepsis - induced chemokine upregulation was more marked in the diaphragm than in limb muscles . this pattern of preferential diaphragmatic involvement mirrors the muscle functional impairment and injury observed in several previous studies [ 4 , 5 , 7 ] . moreover , the fact that the diaphragm showed greater constitutive as well as induced levels of chemokine receptor / ligand upregulation is consistent with an emerging concept that the diaphragm may be in certain respects immunologically primed to respond in a more vigorous fashion to inflammatory stimuli , perhaps to aid in its role as a physical barrier to the passage of infectious agents between the peritoneal and pleural cavities . we have recently reported that rantes and mip-1 are also upregulated to a greater extent in the inflammatory milieu of the muscular dystrophic diaphragm than in limb muscles from the same animals . in the present study , rantes also demonstrated more sustained upregulation than mip-1 in the chronic inflammatory situation associated with pseudomonas lung infection . this is consistent with previous data showing an important role for rantes in sustaining chronic inflammation [ 39 , 40 ] . stimulation of cultured primary diaphragmatic ( as well as c2c12 ) muscle cells with lps triggered an upregulation of rantes expression . interestingly , we did not detect an upregulation of mip-1 within muscle cells following exposure to lps in vitro , suggesting that sepsis - induced mip-1 expression by the diaphragm in vivo may originate from nonmuscle cell types within the whole muscle . we have previously reported that primary diaphragmatic myotubes also demonstrate significant upregulation of ccr1 but not ccr5 during proinflammatory cytokine stimulation in vitro , which is in keeping with the greater upregulation of ccr1 during endotoxemia shown in the present study . these observations reinforce the idea that skeletal muscle fibers have the ability to behave as immune cells and thus actively participate in the response to various inflammatory stimuli . for example , in cultured human muscle cells , proinflammatory cytokines induce the expression of class i and class ii major histocompatibility molecules . indeed , skeletal muscle cells have the ability to express a large repertoire of immunologically relevant molecules [ 38 , 4143 ] . to begin to dissect the molecular events underlying rantes upregulation within septic diaphragmatic muscle cells , we first determined that the rantes promoter is responsive to lps in skeletal muscle cells , thus pointing to a transcriptional mechanism . we then focused on examining the role of nf-b , a transcription factor that has been shown to activate a large number of proinflammatory genes in various cell types , including rantes . in addition , inhibition of the ikk-dependent nf-b has recently been shown to ameliorate muscle pathology in the setting of muscular dystrophy and after experimentally induced muscle injury . in our study , two distinct methods were used to evaluate the involvement of nf-b : pharmacological inhibition with pdtc and a dominant negative transfection approach . this combination of complementary methods allowed us to inhibit two critical protein complexes which are required for the phosphorylation and degradation of ib : the ikk and e3 ubiquitin ligase complexes , respectively . the sequential actions of these two complexes permit the liberation and subsequent translocation of nf-b into the nucleus in order to activate proinflammatory gene transcription . in keeping with our hypothesis , both pharmacological and dominant negative molecular inhibitions of the ikk-dependent classical nf-b pathway were effective in suppressing lps - induced upregulation of rantes . on the other hand , inhibition of the ikk-dependent alternative nf-b pathway , and blockade of free radicals with the antioxidants nac or catalase , did not have any effect on lps - induced rantes gene expression under the same experimental conditions , strongly suggesting that these pathways were not involved . what are the precise functional roles played by cc - class chemokine upregulation within the septic diaphragm muscle ? the results of our study do not allow this question to be definitively addressed , since this would require a model in which muscle tissue - specific knockout of the receptor - ligand interaction could be achieved in vivo . however , there are a number of plausible scenarios . the most intuitively obvious role for cc chemokines in this setting would be to stimulate the recruitment of inflammatory cells , as has been proposed in several other diseases involving skeletal muscle [ 1416 ] . for example , in the mdx mouse , a murine model of duchenne muscular dystrophy , the diaphragm exhibits a pronounced infiltration by macrophages and lymphocyctes , which parallels a marked overexpression of rantes and mip-1 . this is consistent with the inflammatory cell types known to be attracted by these chemokines . in animal models of sepsis , infiltration of the diaphragm by inflammatory cells has been observed in some but not all studies . hence in models of acute endotoxemia and bacterial peritonitis induced by cecal ligation and perforation , macrophage infiltration has been reported in the diaphragm [ 4 , 5 ] . the presence of inflammatory cells in the diaphragm was associated with contractile impairment and muscle fiber injury , both of which were partially prevented by inhibition of nitric oxide synthase [ 4 , 5 ] . along these same lines , supinski et al . reported neutrophilic infiltration of the diaphragm after systemic delivery of lps , along with an improvement in diaphragmatic contractility when the superoxide - generating nadph complex was inhibited . however , while we demonstrate in this study that cc chemokine upregulation occurs in the diaphragm during pseudomonas lung infection , we have previously reported that inflammatory cells are not recruited to the diaphragm in this model , suggesting the existence of alternative functions for chemokines in septic skeletal muscle . in this regard , another recently recognized function of cc chemokines is in the regulation of cytokine production . it has been found that both nk cells and cd8 + t cells use rantes and mip-1 to help drive the th1 cytokine response . in human monocytes , rantes is able to directly induce the expression of several proinflammatory cytokines / chemokines ( e.g. , il-1 , mcp-1 ) and molecules involved in extracellular matrix digestion ( e.g. , mmp-9 ) as well as its own receptor , ccr1 . in the cecal ligation and perforation model of sepsis , it was recently shown that administration of rantes significantly increases lethality , and this is associated with an increased production of multiple proinflammatory cytokines , which probably underlies the reduction in survival . taken together , these findings suggest that cc chemokines could act in an autocrine or paracrine fashion to upregulate the expression of other proinflammatory mediators within the diaphragm during sepsis , and thereby contribute to contractile dysfunction . there is also evidence for an important contribution of cc chemokines to muscle regeneration and recovery . rantes is reported to act as a chemotactic factor for myoblasts , and gene expression profiling studies have shown an enhanced expression of cc - class chemokines as well as their receptors in injured muscles [ 48 , 49 ] . other studies have shown that functional recovery from muscle injury is impaired in mice rendered deficient in mcp-1 or its receptor , ccr2 [ 17 , 18 ] . our group has recently shown that several cc chemokines , including mip-1 , are able to stimulate myoblast proliferation . collectively , these data suggest that at least under certain conditions , chemokines may also play a beneficial role in the repair of diaphragmatic muscle fiber injury triggered by sepsis [ 5 , 6 , 24 ] . the ultimate impact of these cc chemokines upon septic diaphragm function is likely dependent upon achieving the appropriate magnitude as well as spatial and temporal equilibrium of chemokine expression . | sepsis - induced diaphragmatic inflammation has been associated with
respiratory failure , but the role of chemokines in this process has
not been evaluated .
here we sought to study the local expression and
molecular regulation of the chemokines , regulated upon activation
normal t cell expressed and secreted ( rantes ) and macrophage
inflammatory protein ( mip)-1 , in the murine diaphragm during sepsis .
constitutive
expression levels of rantes and mip-1 , as well as their receptors , ccr1 and ccr5 , were
significantly higher in diaphragm than limb muscle .
sepsis was induced
by acute lipopolysaccharide ( lps ) delivery or subacutely by
intratracheal administration of live pseudomonas aeruginosa bacteria .
both sepsis models triggered a marked upregulation of rantes and mip-1 in the diaphragm . in vitro ,
stimulation of diaphragmatic
muscle cells with lps also led to rantes upregulation .
inhibition of
the nf - kb pathway using pharmacologic or dominant negative genetic
approaches blocked the lps - induced rantes upregulation , while free
radical scavengers had no effect .
we conclude that sepsis leads to
greatly increased expression of rantes , mip-1 and their cognate receptors in the diaphragm .
manipulation
of the nf - kb pathway and other regulators of chemokine expression in
the diaphragm could represent a novel method for mitigating the
skeletal muscle inflammatory response associated with sepsis - induced
diaphragmatic dysfunction . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the following mice strains were used : c57bl / j ins2 , which display elevated glucose at 46 weeks of age ; b6 ( c57bl/6 ) ( a gift from dr . s. smith , georgia health sciences university ) and db / db ( bks cg - dock7 m ) , which display elevated glucose at 48 weeks of age ; and blks ( c57blks / j ) ( diabetes and obesity discovery institute , georgia health sciences university ) . blood glucose was measured after a tail clip using accu - chek aviva ( roche , worldwide ) . fibroblasts were obtained through trypsin digestion of mouse ears for 1 h and cultured in dulbecco s modified eagle s medium ( dmem ) ( invitrogen , carlsbad , ca ) from rage - deficient ( rage , n = 3 ) ( 17 ) , c57bl / j ins2 ( n = 4 ) , and b6 ( n = 4 ) mice . all protocols for animal use and euthanasia were reviewed and approved by the animal use committee at georgia health sciences university and were in accordance with national institutes of health guidelines . solei were dissected from mice , secured to 35-mm culture dishes by a drop of new - skin ( medtech , irvington , ny ) liquid bandage , and kept ( < 1 h ) in ice - cold tyrode solution : 128 mmol / l nacl , 4.7 mmol / l kcl , 1.36 mmol / l cacl2 , 20 mmol / l nahco3 , 0.36 mmol / l nah2po4 , 1 mmol / l mgcl2 , and 10 mmol / l glucose , ph 7.4 ( 18 ) . laser analysis was carried out in 37c tyrode solution containing 1.36 mmol / l cacl2 , or tyrode without added cacl2 . fm 143 dye ( invitrogen ) , a membrane impermeant dye , was added ( 2.5 mol / l ) before laser injury ( 19 ) . intact plasma membranes of healthy myocytes , highlighted by staining of fm dye , were targeted for laser injury using a two - photon laser scanning confocal microscope ( lsm 510 ; zeiss , thornwood , ny ) coupled with a 10-w argon / ti - sapphire laser ( spectra - physics lasers ) operated at 100% power , 100 iterations , with a 5 35 pixels bleach area . fluorescence , associated with the injured domain of the myocyte , was measured in a 120-m diameter circular window centered on the myocyte injury site using zeiss lsm 510 software . fluorescence measured before the injury was subtracted . after laser injury , myocyte contractions were often observed , moving fibers out of focus , and manual refocusing was used . cultured cells were wounded in phosphate - buffered saline ( pbs ) containing 137.9 mmol / l nacl , 2.7 mmol / l kcl , 15.2 mmol / l na2hpo4 , 1.5 mmol / l kh2po4 , 1 mmol / l mgcl2 ( with or without 1.2 mmol / l ca ) ( sigma , st . louis , mo ) , and 2.5 mol / l fm 143 or fm 464 ( invitrogen ) for injury . the disruptions were made using 100% power and one laser iteration with a 15 15 pixels bleach area . ins2 mice ( n = 9 ) with blood glucose levels > 600 mg / dl ( accu - chek hi ) and b6 mice ( n = 9 ) with blood glucose levels of 151.11 7.95 mg / dl were run on a treadmill set at a 15-degree decline as previously described ( 11 ) ( omnipacer treadmill lc4/m - mga / at ; accuscan instruments , columbus , oh ) . these mice were subjected to a moderate downhill run of 10 m / min for 1 h. control mice remained in their cages ( n = 4/group ) . after completion of the downhill run , all mice were injected with evan s blue dye ( ebd ) ( 10 mg ebd/1 ml pbs , 100 l/10 g body wt ; sigma ) ( 20 ) , and 24 h later , the quadriceps and gastrocnemius were excised , weighed , and frozen in o.c.t . ebd stains albumin present within tissue , clearly highlighting the exterior of muscle fibers , while also staining albumin present within fibers that are no longer intact ( 16,20 ) . cross sections were cut at a 10-m thickness and analyzed microscopically ( 10 magnification ) for ebd - positive fibers . fluorescently labeled cells were scored per cross - section taken at four random muscle locations and averaged per muscle . c2c12 , bs - c-1 , and hela cells were cultured in dmem , passaged twice weekly , and supplemented with glucose ( glucose at 5.5 mmol / l simulates a fasting blood glucose of 99 mg / dl , 7.5 mmol / l glucose represents fed blood glucose level of 135 mg / dl , and 30 mmol / l glucose elevated blood glucose level of 540 mg / dl ) or mannitol ( 21 ) and , where noted , treated with 1 mmol / l aminoguanidine ( amg ) ( sigma ) . the 96-well plates of cells were subjected to scraping using a spring - loaded 96-well transfer device ( 22 ) . this device was used in a circular motion to scrape cells off their substratum , therefore creating plasma membrane disruptions ( 23 ) . survival in this population was assessed using a live / dead viability / cytotoxicity kit according to the manufacturer s instructions ( molecular probes , carlsbad , ca ) . the live / dead fluorescence ratio , calcein acetoxymethyl ester ( excitation / emission 495 nm/515 nm ) to ethidium homodimer-1 ( excitation / emission 495 nm/635 nm ) ratio , was determined after 1 h of incubation using a fluorescent plate reader ( flx800 ; biotek , winooski , vt ) . alternatively , after replating cells from the multiwell scrape assay , fresh dmem was added to each well . a dmem rinse was used to remove dead nonadherent cells from the plate after 4 h at 37c . the mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay was then performed using the methods previously described ( 24 ) . the cells surviving the scrape injury were compared with uninjured control populations to calculate percent survival . a rage construct was produced by subcloning the full - length human rage gene into the enhanced green fluorescent protein ( pegfp)-n1 vector to generate pegfr - n1-rage ( egfp is fused to the cooh terminus of rage ) . bs - c-1 cells were transfected with 1 g pefgp - n1-rage using lipofectamine ( invitrogen ) . cells were treated for 20 h with 5 mmol / l of age - modified bsa ( age - bsa ) ( sigma ) 4 h after transfection . microscope images of laser injury were taken using a 40 ir - acroplan lens , 0.8 numerical aperture ; a zeiss axioplan 2 microscope ; and lsm 510 software for image analysis . microscope images of ebd were taken using a 10 plan - neofluar lens , 0.3 numerical aperture ; a zeiss axioplan 2 microscope ; and a zeiss axiocam high - resolution camera . comparisons were made with prism 5.0 software using repeated - measures anova and tukey post - analysis tests for significance . a student t test was used to determine the difference between muscle diameters ( two - tailed ) , mouse weights ( two - tailed ) , and decreased muscle weights ( one - tailed ) . for all analysis , p < 0.05 was considered to be significant . in text and graphs , the following mice strains were used : c57bl / j ins2 , which display elevated glucose at 46 weeks of age ; b6 ( c57bl/6 ) ( a gift from dr . s. smith , georgia health sciences university ) and db / db ( bks cg - dock7 m ) , which display elevated glucose at 48 weeks of age ; and blks ( c57blks / j ) ( diabetes and obesity discovery institute , georgia health sciences university ) . blood glucose was measured after a tail clip using accu - chek aviva ( roche , worldwide ) . fibroblasts were obtained through trypsin digestion of mouse ears for 1 h and cultured in dulbecco s modified eagle s medium ( dmem ) ( invitrogen , carlsbad , ca ) from rage - deficient ( rage , n = 3 ) ( 17 ) , c57bl / j ins2 ( n = 4 ) , and b6 ( n = 4 ) mice . all protocols for animal use and euthanasia were reviewed and approved by the animal use committee at georgia health sciences university and were in accordance with national institutes of health guidelines . solei were dissected from mice , secured to 35-mm culture dishes by a drop of new - skin ( medtech , irvington , ny ) liquid bandage , and kept ( < 1 h ) in ice - cold tyrode solution : 128 mmol / l nacl , 4.7 mmol / l kcl , 1.36 mmol / l cacl2 , 20 mmol / l nahco3 , 0.36 mmol / l nah2po4 , 1 mmol / l mgcl2 , and 10 mmol / l glucose , ph 7.4 ( 18 ) . laser analysis was carried out in 37c tyrode solution containing 1.36 mmol / l cacl2 , or tyrode without added cacl2 . fm 143 dye ( invitrogen ) , a membrane impermeant dye , was added ( 2.5 mol / l ) before laser injury ( 19 ) . intact plasma membranes of healthy myocytes , highlighted by staining of fm dye , were targeted for laser injury using a two - photon laser scanning confocal microscope ( lsm 510 ; zeiss , thornwood , ny ) coupled with a 10-w argon / ti - sapphire laser ( spectra - physics lasers ) operated at 100% power , 100 iterations , with a 5 35 pixels bleach area . fluorescence , associated with the injured domain of the myocyte , was measured in a 120-m diameter circular window centered on the myocyte injury site using zeiss lsm 510 software . fluorescence measured before the injury was subtracted . after laser injury , myocyte contractions were often observed , moving fibers out of focus , and manual refocusing was used . cultured cells were wounded in phosphate - buffered saline ( pbs ) containing 137.9 mmol / l nacl , 2.7 mmol / l kcl , 15.2 mmol / l na2hpo4 , 1.5 mmol / l kh2po4 , 1 mmol / l mgcl2 ( with or without 1.2 mmol / l ca ) ( sigma , st . louis , mo ) , and 2.5 mol / l fm 143 or fm 464 ( invitrogen ) for injury . the disruptions were made using 100% power and one laser iteration with a 15 15 pixels bleach area . ins2 mice ( n = 9 ) with blood glucose levels > 600 mg / dl ( accu - chek hi ) and b6 mice ( n = 9 ) with blood glucose levels of 151.11 7.95 mg / dl were run on a treadmill set at a 15-degree decline as previously described ( 11 ) ( omnipacer treadmill lc4/m - mga / at ; accuscan instruments , columbus , oh ) . these mice were subjected to a moderate downhill run of 10 m / min for 1 h. control mice remained in their cages ( n = 4/group ) . after completion of the downhill run , all mice were injected with evan s blue dye ( ebd ) ( 10 mg ebd/1 ml pbs , 100 l/10 g body wt ; sigma ) ( 20 ) , and 24 h later , the quadriceps and gastrocnemius were excised , weighed , and frozen in o.c.t . ebd stains albumin present within tissue , clearly highlighting the exterior of muscle fibers , while also staining albumin present within fibers that are no longer intact ( 16,20 ) . cross sections were cut at a 10-m thickness and analyzed microscopically ( 10 magnification ) for ebd - positive fibers . fluorescently labeled cells were scored per cross - section taken at four random muscle locations and averaged per muscle . c2c12 , bs - c-1 , and hela cells were cultured in dmem , passaged twice weekly , and supplemented with glucose ( glucose at 5.5 mmol / l simulates a fasting blood glucose of 99 mg / dl , 7.5 mmol / l glucose represents fed blood glucose level of 135 mg / dl , and 30 mmol / l glucose elevated blood glucose level of 540 mg / dl ) or mannitol ( 21 ) and , where noted , treated with 1 mmol / l aminoguanidine ( amg ) ( sigma ) . the 96-well plates of cells were subjected to scraping using a spring - loaded 96-well transfer device ( 22 ) . this device was used in a circular motion to scrape cells off their substratum , therefore creating plasma membrane disruptions ( 23 ) . survival in this population was assessed using a live / dead viability / cytotoxicity kit according to the manufacturer s instructions ( molecular probes , carlsbad , ca ) . the live / dead fluorescence ratio , calcein acetoxymethyl ester ( excitation / emission 495 nm/515 nm ) to ethidium homodimer-1 ( excitation / emission 495 nm/635 nm ) ratio , was determined after 1 h of incubation using a fluorescent plate reader ( flx800 ; biotek , winooski , vt ) . alternatively , after replating cells from the multiwell scrape assay , fresh dmem was added to each well . a dmem rinse was used to remove dead nonadherent cells from the plate after 4 h at 37c . the mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) assay was then performed using the methods previously described ( 24 ) . the cells surviving the scrape injury were compared with uninjured control populations to calculate percent survival . a rage construct was produced by subcloning the full - length human rage gene into the enhanced green fluorescent protein ( pegfp)-n1 vector to generate pegfr - n1-rage ( egfp is fused to the cooh terminus of rage ) . bs - c-1 cells were transfected with 1 g pefgp - n1-rage using lipofectamine ( invitrogen ) . cells were treated for 20 h with 5 mmol / l of age - modified bsa ( age - bsa ) ( sigma ) 4 h after transfection . microscope images of laser injury were taken using a 40 ir - acroplan lens , 0.8 numerical aperture ; a zeiss axioplan 2 microscope ; and lsm 510 software for image analysis . microscope images of ebd were taken using a 10 plan - neofluar lens , 0.3 numerical aperture ; a zeiss axioplan 2 microscope ; and a zeiss axiocam high - resolution camera . comparisons were made with prism 5.0 software using repeated - measures anova and tukey post - analysis tests for significance . a student t test was used to determine the difference between muscle diameters ( two - tailed ) , mouse weights ( two - tailed ) , and decreased muscle weights ( one - tailed ) . for all analysis , p < to directly assess repair in the diabetic skeletal muscle environment , we used a novel in situ assay . the intact solei of mice were placed in physiological saline containing a fluorescent dye , fm 143 , which strongly stains the plasma membrane boundary only of intact cells . after laser disruption of the plasma membrane , fm 143 diffuses into the cytosol through the disruption site and begins to fluorescently stain the internal membrane . in the presence of calcium ( ca ) , membrane repair is activated , slowing dye entry into the cytosol and consequent staining of internal membranes ( supplementary movie 1 ) . a hot spot of fluorescence staining , the result of initial dye entry , marks the injury throughout the experiment . in the absence of ca , membrane repair fails and the internal cellular compartments begin to saturate with dye , a process that continues throughout the 445-s time course ( supplementary movie 2 ) . because of the contractions of the injured myocyte , refocusing is frequently necessary , so that measurement is centered on the initial disruption site . however , with this maneuver , fluorescence within the cell can be monitored and quantitated over time surrounding the disruption site . two diabetic mouse models , c57bl / j ins2 ( ins2 ) with type 1 diabetes and bks cg - dock7 m ( db / db ) with type 2 diabetes , were assessed using this in situ laser assay . in muscle from c57bl/6 ( b6 ) and c57blks / j ( blks ) control mice , laser injured in the presence of ca , a hot spot of fm 143 appeared at the membrane disruption site , but widespread staining of internal membrane was not observed , indicating repair had occurred ( fig . 1a , control b6 + ca , 445 s ; and supplementary movie 3 ) . however , when ca was absent , control fibers displayed continuous cytosolic filling with fm 143 , characteristic of failed membrane repair ( fig . 1a , control b6 ca ) . strikingly , in both diabetic models , dye entered continuously into diabetic fibers , even when ca was present , indicating repair failed to occur ( fig . 1a , type 1 ins2 + ca ; and supplementary movies 4 and 5 ) . 1b : all fibers initially displayed similar dye uptake kinetics in the presence and absence of ca , indicating the size of the disruption made was equivalent . however , at time points thereafter ( 375 s for the ins2 and 175 s for the db / db ) , significantly more dye uptake , compared with wild - type controls , in the presence of ca , was recorded in solei from both diabetic models . in fact , measured dye uptake in diabetic myocytes was indistinguishable , whether ca was present or absent . thus , using a highly sensitive in situ assay , we show that ca - dependent muscle membrane repair is deficient in both type 1 and type 2 diabetes mouse models . a : a myocyte within the soleus muscle of a wild - type ( control b6 , top two rows ) and type 1 diabetic ( type 1 ins2 , bottom two rows ) mouse was imaged before ( 0 s ) and after membrane irradiation with an infrared laser ( arrow indicates injury site ) in the presence of ca or its absence . repair ( control b6 + ca ) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury . failed repair ( control b6 ca , type 1 ins2 ca , and type 1 ins2 + ca ) results in a sustained filling of the entire fiber with dye . b : fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice . the red circle indicates the time point where the first significant difference of p < 0.05 is observed ( compared with all conditions ) . * p < 0.01 ; myofibers injured , n = 25 for b6 + ca , n = 25 for b6 -ca , n = 35 for ins2 + ca , n = 30 for ins ca , n = 47 for blks + c , n = 45 for blks ca , n = 50 for db / db + ca , and n = 49 for db / db ca ( a high - quality digital representation of this figure is available in the online issue . ) images obtained while performing the in situ laser assay suggested that there might be a size difference between normal and diabetic fibers . decreased fiber diameter was measured in both the soleus and quadriceps of diabetic mice , and ins2 mice also displayed significantly decreased body weight and muscle mass aged 67.5 months ( supplementary fig . 1 ) . these morphological changes confirm previous findings ( 25,26 ) that diabetes , as expected , negatively affected the skeletal muscle in these mouse models . plasma membrane disruptions are induced in vivo in muscle undergoing eccentric contractions , such as those produced by downhill running ( 11 ) . to determine if membrane repair fails at a greater rate in diabetic mice during eccentric contraction - induced injury , we monitored myocyte permeability to ebd . ebd is a fluorescent dye that enters only into cells lacking an intact plasma membrane barrier and is effective at identifying injured muscle fibers without the presence of a counter - stain ( 16,20 ) . we injected this dye into mice after running , so that myofibers that suffered a disruption and failed to repair would be labeled . as expected , in both normal and diabetic mice , running induced an increase in the number of ebd - positive fibers . however , running induced a significantly higher number of ebd - positive fibers in the diabetic mice ( fig . a : paired transmission and fluorescence micrographs showing ebd - labeled myofibers ( arrows ) in the gastrocnemius muscle from diabetic ( ins2 ) and control ( ctl ) ( b6 ) mice that run downhill for 60 min and then are injected with the ebd tracer . b : the number of ebd myofibers counted from such micrographs in the gastrocnemius and quadriceps muscles of diabetic ins2 and control b6 mice . data are presented as the mean sem . * p < 0.05 ; n = 5 mice for the run groups and 4 mice for the not run groups ( a high - quality digital representation of this figure is available in the online issue . ) thus , both our in situ and in vivo measurements strongly support the hypothesis that membrane repair fails in diabetes . the diabetic environment in vivo is a complicated one , in which elevated blood glucose is just one pathophysiological element . to test whether glucose elevation alone is sufficient to induce a repair defect , we used an in vitro model mimicking the blood glucose levels found in our diabetic mouse models ( supplementary table 1 ) . c2c12 cells , a skeletal muscle myocyte culture model , were assessed for membrane repair after growth for 18 weeks in medium containing normal glucose ( 7.5 mmol / l ) , elevated glucose ( 30 mmol / l ) , or mannitol ( 30 mmol / l ) . after 1 week of elevated glucose exposure , there was no qualitative change in repair ( for example , dye entry pattern ) ( fig . 3a , 1 week + ca all conditions ; and supplementary movie 6 ) . however , after 8 weeks of high glucose exposure , dye entry after laser injury was clearly elevated , relative to the normal glucose and mannitol controls ( fig . 3a , 8 weeks n gluc + ca and man + ca ) , indicating an inability to repair ( fig . thus , prolonged exposure to high glucose is required for inducing a repair defect , a finding that is not compatible with other short - term responses , such as induction of insulin resistance . a : c2c12 myoblasts were cultured in medium with 7.5 mmol / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol / l mannitol ( man ) and then subject to laser analysis of repair ( arrows indicate injury site ) at 1 or 8 weeks of exposure . the interval ( seconds ) postinjury is indicated . only cells grown in 30 mmol / l glucose for 8 weeks ( h gluc + ca ) b : dye uptake , fluorescence , measured within cells over time after injury . after 1 week of exposure to high glucose ( left panel ) , all cells displayed calcium - dependent repair with no significant difference in dye uptake between groups . by contrast , cells cultured for 8 weeks in high glucose ( right panel ) showed a significant increase in dye uptake compared with normal glucose and mannitol controls . * p < 0.05 ; 1 week , n = 15 for n gluc + ca , n = 14 for h gluc + ca , n = 13 for man + ca , and n = 17 for n gluc ca ; 8 weeks , n = 26 for n gluc + ca , n = 30 for h gluc + ca , n = 28 for man + ca , and n = 15 for n gluc ca . scale bar , 50 m and 40 magnification . ( a high - quality digital representation of this figure is available in the online issue . ) to confirm that this glucose - induced repair defect was not limited to laser - generated disruptions , adherent c2c12 cells were scraped to mechanically induce membrane disruptions ( 23 ) . consistent with the laser assay , there was no detectable difference in survival between treatment groups after 2 weeks of high glucose exposure ( fig . 4 , 2 weeks ) however , after 8 weeks of elevated glucose , cells displayed a significant decrease in survival when compared with those grown in normal glucose or mannitol ( fig . 4 , 8 weeks h gluc ) . thus , using an independent method for inducing membrane disruptions and for monitoring repair failure , we confirmed that high glucose leads to the development of a membrane repair defect . / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol / l mannitol ( man ) and then scraped from 96-well plates . no significant differences were demonstrated after 2 weeks of treatment . however , 8 weeks of high glucose treatment decreased survival as determined by both assays . * p < 0.01 ; n = 24 wells for each condition . to determine if other cell types develop a membrane repair defect after high glucose exposure , we applied the above analysis to bs - c-1 ( monkey kidney epithelial ) and hela ( human cervical ) cells . as was the case for c2c12 myoblasts , bs - c-1 cells developed a repair deficiency only after 8 weeks of high glucose treatment ( fig . bs - c-1 and hela survival of the scraping injury was also significantly decreased after 8 weeks of high glucose exposure , compared with cells grown in normal glucose or mannitol ( supplementary fig . this glucose - induced membrane repair defect remained even after cells were returned to a low glucose medium for over a week ( fig . additionally , fibroblasts obtained from diabetic ( ins2 ) mice also displayed a membrane repair defect after culturing in normal glucose medium for 1 week ( supplementary fig . 2b , ins2 ) . an epithelium - derived cell line , bs - c-1 , also develops repair failure in high glucose , and this defect is not readily reversible . a : bs - c-1 cells were cultured for 8 weeks in 7.5 mmol / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol / l mannitol ( man ) . laser analysis revealed that cells cultured in high glucose displayed a significant increase in dye uptake when compared with normal glucose and mannitol . b : bs - c-1 cells were cultured for 10 weeks in h gluc and then switched to l gluc ( 5.5 mmol / l glucose ) for 10 days ( h gluc / l gluc ) , or cells were cultured for 11 weeks in l gluc or h gluc only . laser analysis determined that the high glucose membrane repair defect remained 1 week after switching to low glucose ( h gluc / l gluc ) . 0.01 ; 2 weeks , n = 18 for n gluc + ca , n = 14 for h gluc + ca , n = 12 for man + ca , and n = 13 for h gluc ca ; 10 weeks , n = 14 for l gluc , n = 16 for h gluc , and n = 15 for h gluc / l gluc . thus , our in vitro analysis revealed that the membrane repair defect is glucose induced , not exclusive to skeletal muscle cells , and does not appear to be readily reversible . one well - established effect of elevated blood glucose is the production of ages , thought to contribute to the development of chronic diabetes complications ( 27,28 ) . numerous studies ( 29,30 ) have detected age generation in vitro as soon as 48 h after initiating high glucose incubation . to test if this age production is required for the development of the membrane repair defect , we simultaneously exposed bs - c-1 cells to elevated glucose and amg , a potent inhibitor of age formation and accumulation ( 31 ) . as expected , after 8 weeks of high glucose treatment in the absence of amg , membrane repair was defective ( fig . however , this high glucose repair defect was completely eliminated by cotreatment with amg ( fig . age / rage interaction is responsible for the repair defect . a : bs - c-1 cells were cultured in 30 mmol / l glucose supplemented with or without 1 mmol / l amg ( h gluc amg or h gluc ) , or 30 mmol / l mannitol ( man ) for 8 weeks . cells exposed to high glucose alone showed significantly increased ( * ) dye uptake when compared with mannitol controls . b : rage - deficient fibroblasts ( rage ) were cultured for 12 weeks in 30 mmol / l glucose ( h gluc ) , 5.5 mmol / l glucose ( l gluc ) , or 30 mmol / l mannitol ( man ) . rage cells exposed to high glucose and injured in the presence of calcium ( h gluc + ca ) did not exhibit a significant increase in dye uptake compared with controls ( l gluc + ca and man + ca ) . c : bs - c-1 cells were transfected with a rage - egfp expression vector ( pegfp - n1-rage ) . cells displaying egfp fluorescence ( fl rage ) , and as controls , nonfluorescent cells in the same culture ( nf ) , were selected for laser analysis of repair . one culture was exposed to 5 mmol / l age - bsa , and the other was not . laser analysis showed membrane repair of fluorescent cells ( fl rage ) to be indistinguishable from nonfluorescent controls ( nf ) in cultures not exposed to age - bsa . however , fluorescent cells treated with age ( fl rage age ) showed a significant ( * * ) elevation in dye uptake when compared with cells not treated with age , comparable to the dye uptake seen in cells wounded without calcium ( ca ) . d : bs - c-1 cells were treated or not with 1 or 5 mmol / l age - bsa for 3 days before laser injury . dye uptake was found to be significantly ( * ) greater for cells treated with age - bsa than cells left untreated and resembled the lack of repair for cells injured in the absence of ca ( ca ) . bs - c-1 : n = 14 cells for man , n = 12 for h gluc , and n = 12 for h gluc amg . fibroblasts : n = 23 cells for l gluc + ca , n = 23 for h gluc + ca , n = 24 for man + ca , and n = 19 for l gluc ca . transfected : n = 12 cells for nf , n = 9 for nf age , n = 10 for fl rage , n = 10 for fl rage age , and n = 15 for ca . n = 21 cells for + ca , n = 18 for ca , n = 22 for 1 mmol / l age , and n = 17 for 5 mmol / l age . age potently binds to a cell surface rage ( 27,28 ) , which in turn stimulates increased expression of this receptor and is hypothesized to contribute to the pathophysiology of diabetes ( 28,32 ) . to test if rage is implicated in the development of the membrane repair defect , we cultured rage - deficient cells ( rage ) in high glucose . after 12 weeks of elevated glucose exposure , rage cells did not display the drastic increase in dye uptake , as previously determined in other cells treated to high glucose ( fig . to further test the involvement of rage in promoting repair failure , we transfected bs - c-1 cells with an expression vector ( pegfp - n1-rage , where egfp is fused with the cooh terminus of rage ) to force rage expression . cells that were successfully transfected ( as indicated by egfp - derived fluorescence ) were selected for laser injury . dye entry kinetics into these rage - expressing cells were indistinguishable from nonfluorescent control cells in the same culture ( fig . however , when the mixed culture of egfp - positive and -negative cells were incubated with age - bsa , fluorescent ( egfp - positive ) cells displayed significantly enhanced dye entry relative to nonfluorescent cells and also relative to fluorescent cells not exposed to age - bsa ( fig . thus , rage expression alone is not sufficient for inducing membrane repair deficiency in cultured cells , but rage binding of age is clearly implicated in defective membrane repair . we noticed in these experiments a slight but not statistically significant increase in dye entry into the nonfluorescent cells exposed to age - bsa for 24 h ( fig . therefore , we investigated further whether age exposure by itself could negatively influence membrane repair . when we increased the exposure level of cell to age - bsa , we found that dye entry was significantly elevated ( fig . thus , age formation , and not some other metabolic change induced by high glucose exposure , is responsible for repair failure , and rage binding of age is identified as one mechanism by which age exerts its deleterious effect . to directly assess repair in the diabetic skeletal muscle environment , we used a novel in situ assay . the intact solei of mice were placed in physiological saline containing a fluorescent dye , fm 143 , which strongly stains the plasma membrane boundary only of intact cells . after laser disruption of the plasma membrane , fm 143 diffuses into the cytosol through the disruption site and begins to fluorescently stain the internal membrane . in the presence of calcium ( ca ) , membrane repair is activated , slowing dye entry into the cytosol and consequent staining of internal membranes ( supplementary movie 1 ) . a hot spot of fluorescence staining , the result of initial dye entry , marks the injury throughout the experiment . in the absence of ca , membrane repair fails and the internal cellular compartments begin to saturate with dye , a process that continues throughout the 445-s time course ( supplementary movie 2 ) . because of the contractions of the injured myocyte , refocusing is frequently necessary , so that measurement is centered on the initial disruption site . however , with this maneuver , fluorescence within the cell can be monitored and quantitated over time surrounding the disruption site . two diabetic mouse models , c57bl / j ins2 ( ins2 ) with type 1 diabetes and bks cg - dock7 m ( db / db ) with type 2 diabetes , were assessed using this in situ laser assay . in muscle from c57bl/6 ( b6 ) and c57blks / j ( blks ) control mice , laser injured in the presence of ca , a hot spot of fm 143 appeared at the membrane disruption site , but widespread staining of internal membrane was not observed , indicating repair had occurred ( fig . 1a , control b6 + ca , 445 s ; and supplementary movie 3 ) . however , when ca was absent , control fibers displayed continuous cytosolic filling with fm 143 , characteristic of failed membrane repair ( fig . 1a , control b6 ca ) . strikingly , in both diabetic models , dye entered continuously into diabetic fibers , even when ca was present , indicating repair failed to occur ( fig . 1a , type 1 ins2 + ca ; and supplementary movies 4 and 5 ) . 1b : all fibers initially displayed similar dye uptake kinetics in the presence and absence of ca , indicating the size of the disruption made was equivalent . however , at time points thereafter ( 375 s for the ins2 and 175 s for the db / db ) , significantly more dye uptake , compared with wild - type controls , in the presence of ca , was recorded in solei from both diabetic models . in fact , measured dye uptake in diabetic myocytes was indistinguishable , whether ca was present or absent . thus , using a highly sensitive in situ assay , we show that ca - dependent muscle membrane repair is deficient in both type 1 and type 2 diabetes mouse models . a : a myocyte within the soleus muscle of a wild - type ( control b6 , top two rows ) and type 1 diabetic ( type 1 ins2 , bottom two rows ) mouse was imaged before ( 0 s ) and after membrane irradiation with an infrared laser ( arrow indicates injury site ) in the presence of ca or its absence . repair ( control b6 + ca ) impedes further dye uptake and confines the resultant fluorescence to a hot spot at the site of injury . failed repair ( control b6 ca , type 1 ins2 ca , and type 1 ins2 + ca ) results in a sustained filling of the entire fiber with dye . b : fluorescence signal was monitored over time to determine uptake of dye by myocytes from diabetic and control mice . the red circle indicates the time point where the first significant difference of p < 0.05 is observed ( compared with all conditions ) . * p < 0.01 ; myofibers injured , n = 25 for b6 + ca , n = 25 for b6 -ca , n = 35 for ins2 + ca , n = 30 for ins ca , n = 47 for blks + c , n = 45 for blks ca , n = 50 for db / db + ca , and n = 49 for db / db ca ( a high - quality digital representation of this figure is available in the online issue . ) images obtained while performing the in situ laser assay suggested that there might be a size difference between normal and diabetic fibers . decreased fiber diameter was measured in both the soleus and quadriceps of diabetic mice , and ins2 mice also displayed significantly decreased body weight and muscle mass aged 67.5 months ( supplementary fig . 1 ) . these morphological changes confirm previous findings ( 25,26 ) that diabetes , as expected , negatively affected the skeletal muscle in these mouse models . plasma membrane disruptions are induced in vivo in muscle undergoing eccentric contractions , such as those produced by downhill running ( 11 ) . to determine if membrane repair fails at a greater rate in diabetic mice during eccentric contraction - induced injury , we monitored myocyte permeability to ebd . ebd is a fluorescent dye that enters only into cells lacking an intact plasma membrane barrier and is effective at identifying injured muscle fibers without the presence of a counter - stain ( 16,20 ) . we injected this dye into mice after running , so that myofibers that suffered a disruption and failed to repair would be labeled . as expected , in both normal and diabetic mice , running induced an increase in the number of ebd - positive fibers . however , running induced a significantly higher number of ebd - positive fibers in the diabetic mice ( fig . a : paired transmission and fluorescence micrographs showing ebd - labeled myofibers ( arrows ) in the gastrocnemius muscle from diabetic ( ins2 ) and control ( ctl ) ( b6 ) mice that run downhill for 60 min and then are injected with the ebd tracer . b : the number of ebd myofibers counted from such micrographs in the gastrocnemius and quadriceps muscles of diabetic ins2 and control b6 mice . data are presented as the mean sem . * p < 0.05 ; n = 5 mice for the run groups and 4 mice for the not run groups ( a high - quality digital representation of this figure is available in the online issue . ) thus , both our in situ and in vivo measurements strongly support the hypothesis that membrane repair fails in diabetes . the diabetic environment in vivo is a complicated one , in which elevated blood glucose is just one pathophysiological element . to test whether glucose elevation alone is sufficient to induce a repair defect , we used an in vitro model mimicking the blood glucose levels found in our diabetic mouse models ( supplementary table 1 ) . c2c12 cells , a skeletal muscle myocyte culture model , were assessed for membrane repair after growth for 18 weeks in medium containing normal glucose ( 7.5 mmol / l ) , elevated glucose ( 30 mmol / l ) , or mannitol ( 30 mmol / l ) . after 1 week of elevated glucose exposure , there was no qualitative change in repair ( for example , dye entry pattern ) ( fig . 3a , 1 week + ca all conditions ; and supplementary movie 6 ) . however , after 8 weeks of high glucose exposure , dye entry after laser injury was clearly elevated , relative to the normal glucose and mannitol controls ( fig . 3a , 8 weeks n gluc + ca and man + ca ) , indicating an inability to repair ( fig . thus , prolonged exposure to high glucose is required for inducing a repair defect , a finding that is not compatible with other short - term responses , such as induction of insulin resistance . a : c2c12 myoblasts were cultured in medium with 7.5 mmol / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol / l mannitol ( man ) and then subject to laser analysis of repair ( arrows indicate injury site ) at 1 or 8 weeks of exposure . the interval ( seconds ) postinjury is indicated . only cells grown in 30 mmol / l glucose for 8 weeks ( h gluc + ca ) were observed to fill with dye after laser injury in the presence of calcium . b : dye uptake , fluorescence , measured within cells over time after injury . after 1 week of exposure to high glucose ( left panel ) , all cells displayed calcium - dependent repair with no significant difference in dye uptake between groups . by contrast , cells cultured for 8 weeks in high glucose ( right panel ) showed a significant increase in dye uptake compared with normal glucose and mannitol controls . p < 0.05 ; 1 week , n = 15 for n gluc + ca , n = 14 for h gluc + ca , n = 13 for man + ca , and n = 17 for n gluc ca ; 8 weeks , n = 26 for n gluc + ca , n = 30 for h gluc + ca , n = 28 for man + ca , and n = 15 for n gluc ca . scale bar , 50 m and 40 magnification . ( a high - quality digital representation of this figure is available in the online issue . ) to confirm that this glucose - induced repair defect was not limited to laser - generated disruptions , adherent c2c12 cells were scraped to mechanically induce membrane disruptions ( 23 ) . consistent with the laser assay , there was no detectable difference in survival between treatment groups after 2 weeks of high glucose exposure ( fig . 4 , 2 weeks ) . however , after 8 weeks of elevated glucose , cells displayed a significant decrease in survival when compared with those grown in normal glucose or mannitol ( fig . 4 , 8 weeks h gluc ) . thus , using an independent method for inducing membrane disruptions and for monitoring repair failure , we confirmed that high glucose leads to the development of a membrane repair defect . c2c12 myoblasts were cultured for 2 or 8 weeks in 7.5 mmol / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol / l mannitol ( man ) and then scraped from 96-well plates . however , 8 weeks of high glucose treatment decreased survival as determined by both assays . data are presented as the mean sem . * p < 0.01 ; n = 24 wells for each condition . to determine if other cell types develop a membrane repair defect after high glucose exposure , we applied the above analysis to bs - c-1 ( monkey kidney epithelial ) and hela ( human cervical ) cells . as was the case for c2c12 myoblasts , bs - c-1 cells developed a repair deficiency only after 8 weeks of high glucose treatment ( fig . bs - c-1 and hela survival of the scraping injury was also significantly decreased after 8 weeks of high glucose exposure , compared with cells grown in normal glucose or mannitol ( supplementary fig . this glucose - induced membrane repair defect remained even after cells were returned to a low glucose medium for over a week ( fig . additionally , fibroblasts obtained from diabetic ( ins2 ) mice also displayed a membrane repair defect after culturing in normal glucose medium for 1 week ( supplementary fig . 2b , ins2 ) . an epithelium - derived cell line , bs - c-1 , also develops repair failure in high glucose , and this defect is not readily reversible . / l glucose ( n gluc ) , 30 mmol / l glucose ( h gluc ) , or iso - osmolar control 30 mmol laser analysis revealed that cells cultured in high glucose displayed a significant increase in dye uptake when compared with normal glucose and mannitol . b : bs - c-1 cells were cultured for 10 weeks in h gluc and then switched to l gluc ( 5.5 mmol / l glucose ) for 10 days ( h gluc / l gluc ) , or cells were cultured for 11 weeks in l gluc or h gluc only . laser analysis determined that the high glucose membrane repair defect remained 1 week after switching to low glucose ( h gluc / l gluc ) . * p < 0.01 ; 2 weeks , n = 18 for n gluc + ca , n = 14 for h gluc + ca , n = 12 for man + ca , and n = 13 for h gluc ca ; 10 weeks , n = 14 for l gluc , n = 16 for h gluc , and n = 15 for h gluc / l gluc . thus , our in vitro analysis revealed that the membrane repair defect is glucose induced , not exclusive to skeletal muscle cells , and does not appear to be readily reversible . one well - established effect of elevated blood glucose is the production of ages , thought to contribute to the development of chronic diabetes complications ( 27,28 ) . numerous studies ( 29,30 ) have detected age generation in vitro as soon as 48 h after initiating high glucose incubation . to test if this age production is required for the development of the membrane repair defect , we simultaneously exposed bs - c-1 cells to elevated glucose and amg , a potent inhibitor of age formation and accumulation ( 31 ) . as expected , after 8 weeks of high glucose treatment in the absence of amg , membrane repair was defective ( fig . however , this high glucose repair defect was completely eliminated by cotreatment with amg ( fig . a : bs - c-1 cells were cultured in 30 mmol / l glucose supplemented with or without 1 mmol / l amg ( h gluc amg or h gluc ) , or 30 mmol / l mannitol ( man ) for 8 weeks . cells exposed to high glucose alone showed significantly increased ( * ) dye uptake when compared with mannitol controls . b : rage - deficient fibroblasts ( rage ) were cultured for 12 weeks in 30 mmol / l glucose ( h gluc ) , 5.5 mmol / l glucose ( l gluc ) , or 30 mmol / l mannitol ( man ) . rage cells exposed to high glucose and injured in the presence of calcium ( h gluc + ca ) did not exhibit a significant increase in dye uptake compared with controls ( l gluc + ca and man + ca ) . c : bs - c-1 cells were transfected with a rage - egfp expression vector ( pegfp - n1-rage ) . cells displaying egfp fluorescence ( fl rage ) , and as controls , nonfluorescent cells in the same culture ( nf ) , were selected for laser analysis of repair . one culture was exposed to 5 mmol / l age - bsa , and the other was not . laser analysis showed membrane repair of fluorescent cells ( fl rage ) to be indistinguishable from nonfluorescent controls ( nf ) in cultures not exposed to age - bsa . however , fluorescent cells treated with age ( fl rage age ) showed a significant ( * * ) elevation in dye uptake when compared with cells not treated with age , comparable to the dye uptake seen in cells wounded without calcium ( ca ) . d : bs - c-1 cells were treated or not with 1 or 5 mmol / l age - bsa for 3 days before laser injury . dye uptake was found to be significantly ( * ) greater for cells treated with age - bsa than cells left untreated and resembled the lack of repair for cells injured in the absence of ca ( ca ) . bs - c-1 : n = 14 cells for man , n = 12 for h gluc , and n = 12 for h gluc amg . fibroblasts : n = 23 cells for l gluc + ca , n = 23 for h gluc + ca , n = 24 for man + ca , and n = 19 for l gluc ca . transfected : n = 12 cells for nf , n = 9 for nf age , n = 10 for fl rage , n = 10 for fl rage age , and n = 15 for ca . n = 21 cells for + ca , n = 18 for ca , n = 22 for 1 mmol / l age , and n = 17 for 5 mmol / l age . age potently binds to a cell surface rage ( 27,28 ) , which in turn stimulates increased expression of this receptor and is hypothesized to contribute to the pathophysiology of diabetes ( 28,32 ) . to test if rage is implicated in the development of the membrane repair defect , we cultured rage - deficient cells ( rage ) in high glucose . after 12 weeks of elevated glucose exposure , rage cells did not display the drastic increase in dye uptake , as previously determined in other cells treated to high glucose ( fig . this result indicates that rage is required for the glucose - induced repair defect . to further test the involvement of rage in promoting repair failure , we transfected bs - c-1 cells with an expression vector ( pegfp - n1-rage , where egfp is fused with the cooh terminus of rage ) to force rage expression . cells that were successfully transfected ( as indicated by egfp - derived fluorescence ) were selected for laser injury . dye entry kinetics into these rage - expressing cells were indistinguishable from nonfluorescent control cells in the same culture ( fig . however , when the mixed culture of egfp - positive and -negative cells were incubated with age - bsa , fluorescent ( egfp - positive ) cells displayed significantly enhanced dye entry relative to nonfluorescent cells and also relative to fluorescent cells not exposed to age - bsa ( fig . thus , rage expression alone is not sufficient for inducing membrane repair deficiency in cultured cells , but rage binding of age is clearly implicated in defective membrane repair . we noticed in these experiments a slight but not statistically significant increase in dye entry into the nonfluorescent cells exposed to age - bsa for 24 h ( fig . therefore , we investigated further whether age exposure by itself could negatively influence membrane repair . when we increased the exposure level of cell to age - bsa , we found that dye entry was significantly elevated ( fig . thus , age formation , and not some other metabolic change induced by high glucose exposure , is responsible for repair failure , and rage binding of age is identified as one mechanism by which age exerts its deleterious effect . the current study identifies a novel adverse effect of diabetes on skeletal muscle : we provide direct evidence that plasma membrane repair by myocytes is compromised in in vivo , in situ , and in vitro models . diabetic myopathy , generally speaking , has been viewed as a consequence of underlying neuropathy ( 5,33 ) . however , to our knowledge , there is no definitive evidence to suggest a causal relationship between diabetic neuropathy and myopathy . one study found that muscle contractions induced in diabetic rats by sciatic nerve stimulation resulted in focal ( limited domains of abnormal myofiber structure at the light microscope level ) muscle myocyte damage and elevated creatinine kinase levels ( 10 ) . this result suggested that the diabetic condition could directly affect the contracting skeletal muscle myocyte . what element of the diabetic environment might be detrimental and what exactly fails when the diabetic myocyte contracts remain open questions . using in vitro , in situ , and in vivo models , we demonstrate that elevated glucose results in defective plasma membrane repair and that , in diabetic mice , skeletal muscle myocytes are repair defective . in this study , using a novel in situ laser assay , we show that myofibers of both type 1 and type 2 diabetic mouse models display an inability to repair membrane disruptions . neuropathy can not be implicated in this repair defect , since our analysis of isolated muscle is inherently independent of the nervous system functioning . moreover , we use a vital dye ( ebd ) to show that repair of disruptions , induced by downhill running , is impaired in diabetic mice , indicating that the defect is not one unique to a laser injury , but one that arises under physiological conditions as well . plasma membrane repair is a complex and dynamic cell survival response activated by influx of extracellular ca through the disruption . this ca entry rapidly elicits homotypic vesicle - vesicle fusion in the cytoplasm surrounding the site of disruption ( 12,13,34 ) . vesicle thus forms from cytoplasmic membranes , such as endosomes ( 35 ) , lysosomes ( 36 ) , enlargesomes ( 37 ) , and yolk granules ( 38 ) . exocytotic annealing of this enlarging patch vesicle , also triggered by ca , to plasma membrane surrounding the injury site , restores membrane continuity ( 14,39 ) . not unexpectedly , these include known universal components of membrane fusion , such as the soluble n - ethylmaleimide sensitive factor attachment protein receptor family members ( 12,40 ) and synaptotagmin ( 41 ) . also demonstrated to be required for successful repair are dysferlin ( 16 ) , annexins ( 42 ) , actin and myosin ( 43 ) , calpain ( 44 ) , and mitsugumin 53 ( 45 ) . for the present work , it is important to also consider the heterogeneous array of membrane glycoproteins that decorate cell surfaces . multivalent plant lectins that bind these surface carbohydrates can cross - link these membrane glycoproteins . such lectin cross - linking potently inhibits membrane repair , and it is the exocytotic step of repair that is blocked by lectins ( 46 ) . this result suggested that age / rage interactions , which are elevated in diabetes , might be acting in an analogous fashion as an inhibitor of repair . in other words , rage binding and cross - linking of age ( 46 ) might be responsible for the repair defect in diabetic mice . we report here the numerous tests of the possibility that age / rage interactions are responsible for the repair defect in diabetes . our in vitro model system shows that development of the membrane repair defect can be driven by a prolonged ( at least 8 weeks ) exposure to high glucose . high glucose levels promote nonenzymatic cell surface hyperglycation of glycoproteins ( 47,48 ) ( e.g. , the production of age ) . we show that the accumulation of ages is responsible for the membrane repair defect seen in cell culture : the glucose - induced defect was not detectable when age generation was prevented by the addition of amg to cell culture medium . moreover , we show that age - bsa can , by virtue of its addition alone to culture medium , produces a repair defect in the absence of high glucose exposure . finally , we show that cells expressing egfp - rage , in the absence of high glucose exposure , develop a repair defect , but only if age - bsa is added to the culture medium , and that rage - deficient cells do not develop a repair defect when exposed to elevated glucose . taken together , these results strongly suggest that age and age / rage binding is involved in the development of the repair defect in diabetes . whether inhibition by age / rage occurs by a mechanism analogous to that mediated by lectin cross - linking ( see above ) remains a question for further research . normally produced at a relatively low rate under physiological conditions , reactive oxygen species are drastically elevated in diabetes ( 28 ) . one explanation for this is that age / rage binding , elevated in diabetes , stimulates production of reactive oxygen species ( 49 ) . another is that intracellular glucose can by itself increase production of superoxide radicals ( 1 ) . we have recently found that reactive oxygen species strikingly inhibit repair in skeletal muscle and that prior antioxidant treatment can prevent this ( a.c.h . thus , generation of reactive oxygen species in diabetes , whether by an age / rage - dependent or -independent mechanism , is a possible downstream mediator of membrane repair failure . additionally , the high glucose environment induces a variety of alterations in the cell , some of which ( such as hyperlipidemia , with consequent changes in membrane lipid composition ) might influence the membrane fusion events of repair . in summary , in vitro and in vivo assays show that the diabetic environment induces in the skeletal muscle myocyte a plasma membrane repair defect . although it is conceivable that this repair defect could negatively affect muscle health in diabetes , via muscle cell death and resulting atrophy , this conclusion must await further work directly demonstrating the extent , if any , of the connection : the pathogenesis of diabetes is , after all , thought to be complex and multifactorial . however , we have identified for the first time the skeletal muscle myocyte as a cell directly affected in this disease and the molecular players involved in this cellular pathology ( age / rage ) . because , as we show here , elevated glucose also induces a repair defect in cell types other than the myocyte , it is possible that defective membrane repair may contribute to other diabetes complications as well . | objectiveskeletal muscle myopathy is a common diabetes complication .
one possible cause of myopathy is myocyte failure to repair contraction - generated plasma membrane injuries . here
, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes.research design and methodsmyocytes in intact muscle from type 1 ( ins2akita+/ ) and type 2 ( db / db ) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency .
membrane repair defects were also assessed in diabetic mice after downhill running , which induces myocyte plasma membrane disruption injuries in vivo .
a cell culture model was used to investigate the role of advanced glycation end products ( ages ) and the receptor for age ( rage ) in development of this repair defect.resultsdiabetic myocytes displayed significantly more dye influx after laser injury than controls , indicating a repair deficiency .
downhill running also resulted in a higher level of repair failure in diabetic mice .
this repair defect was mimicked in cultured cells by prolonged exposure to high glucose .
inhibition of the formation of age eliminated this glucose - induced repair defect .
however , a repair defect could be induced , in the absence of high glucose , by enhancing age binding to rage , or simply by increasing cell exposure to age.conclusionsbecause one consequence of repair failure is rapid cell death ( via necrosis ) , our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes . |
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root canal preparation is considered satisfactory when adequate disinfection is obtained . in addition to disinfection , it is important to maintain the continuous tapered shape development of the root canal with the largest diameter in the cervical third . curved canals pose additional difficulties to endodontic treatment success , such as the maintenance of the original canal shape and the position of the apical foremen . such difficulties may be attributed to limitations of the endodontic instruments , which may be unable to adapt to the anatomical variations of the root canal system13,27 . the introduction of nickel - titanium ( ni - ti ) rotary instruments has allowed obtaining less canal transportation and more centered preparations6,7 . the ni - ti alloy is superelastic , that is , by curving the instrument it misshapes and comes back to its original position when the load is removed . in curved canals , ni - ti instruments adapt easily to canal anatomy at the same time that they return almost all the force needed bend them to the canal walls3,19 . the root canal system needs to be sufficiently enlarged to allow for removal of debris and microorganisms . it has been shown that apical third preparation with large - sized instruments allows that the irrigating chemical solution reaches this region and has a more effective action . several authors15,21,22 have demonstrated that when apical preparation is done with size 40 and 45 files there is a significantly greater reduction of the bacterial content from infected canals compared to the use of smaller size instruments . however , the incidence of canal transportation may become more evident when instrument size increases4,8 . wear and canal morphology after instrumentation has been widely investigated in the root canals of extracted teeth and simulated canals prepared in resin blocks9,11,14
griffiths , et al.9 analyzed superimposed images of simulated canals before and after instrumentation with quantec sc nickel - titanium rotary instruments and observed that the instruments removed more material from the outer aspect of the curve . the prevalence and severity of wear increased proportionally to the increase of the size of the instruments used to prepare the apical stop . miranzi , et al.14 compared the alterations occurred in simulated canals with 30 of curvature after preparation with stainless steel and nickel - titanium instruments . the authors evaluated the canal wear in three levels : 2 , 4 and 11 mm from the simulated canal apical termination by the superimposition of digital images using computer software . at 2 and 4 mm , there was greater wear towards the outside of the curve when stainless steel instruments were used . the stainless steel instruments also promoted greater wear at 11 mm towards the inner side of the curve . the authors concluded that both types of instruments formed the same areas of wear : towards the outer aspect of the curve in the apical third and to inside the curve in the third middle from the simulated canal . javaheri and javaheri11 observed the occurrence of canal transportation and changes in the curvature of mesiobuccal canals from extracted molars after instrumentation with the rotary systems protaper universal , hero 642 and race . apical preparation was performed up to a 30 instrument in the three systems . using a radiographic platform , the authors radiographed the tooth with a stainless steel file inserted in the canal , before and after preparation , and superimposed the two radiographs in the computer . the authors suggested that the protaper universal system may be implemented in canals with curvatures of 25 to 35 in combination with other systems with greater flexibility and less taper , such as race . since the apical third of curved canals is the most challenging area for instrumentation , undesirable morphological alterations in this region are common . this study aimed to evaluate the wear promoted by the protaper universal system in the apical third of simulated curved root canals and to determine whether the use of the complete set of instruments for apical stop preparation create inappropriate canal shape because of the increased stiffness of instruments with larger diamaters . twenty - four protaper universal system ( dentsply / maillefer , ballaigues , switzerland ) instrument sets , each set containing s1 , s2 , f1 , f2 , f3 , f4 and f5 instruments , were used . all instruments applied in this study were new and were autoclaved ( cristfoli , so paulo , sp , brazil ) at 1 atm and 127c , for 20 min before use . twenty - four simulated canals were prepared in clear epoxy resin blocks ( odontofix comrcio e indstria de materiais odontolgicos , ribeiro preto , sp , brazil ) . half of the canals were prepared with 20 curvature and 12 with 40 curvature , all of them presented 5 mm radius and 17 mm length ( 8 mm corresponded to upper straight portion and 9 mm corresponded to the final curved portion ) . the simulated canals were fixed in a flask with the canal curvature always turned to the operator 's right side . canals were prepared with the protaper instruments coupled to an electric engine ( endo pro torque ; vk driller electrical equipments ltda . , so paulo , sp , brazil ) at 300 rpm speed and 1 n / cm torque . instrumentation started with s1 instrument followed by s2 instrument with a paitn brush movements against the canal walls . subsequently , f1 , f2 , f3 , f4 and f5 instruments were used in the same way until reaching working length ( wl ) . the wl of the simulated canals was set at 16 mm , corresponding to a distance of 1 mm from the apical foramen . instrument penetration was limited by the placement of a rubber stopper prior to the start of treatment and was checked during the course of the experiment . the canal patency was permanently maintained with a size 10 stainless steel k - file ( dentsply / maillefer ) , irrigation with a sodium lauryl sulfate - based detergent ( tergipol ; biodinmica qumica e farmacutica ltda . , ibipor , pr , brazil ) , aspiration and inundation at each instrument change . the irrigating solution was flushed with a plastic syringe with an adaptor for a 27 g long anesthesia needle . the simulated canals were positioned in a wooden photographic platform that maintained the same distance between the digital camera and the simulated canal . the canals were photographed before instrumentation and after use of the f3 , f4 and f5 instruments . the images were stored in personal computer and transferred to adobe photoshop cs2 software version 9.0 ( adobe systems inc . , san jose , ca , usa ) . the image was converted into millimeters by associating the simulated canal original size with the image size in the computer screen . image pixels were not reduced from the image , preserving the clearness . in figure 1 , the simulated canal image may be observed before the preparation , where gray tone inversion was applied for better visualization of canal anatomy . each instrumented canal image ( surgical canal ) therefore , layers were created , the image was colored , and its opaque appearance was reduced . figure 2 presents a photograph of an instrumented canal superimposed on the photograph of a non - instrumented canal . later , the ruler tool was applied to locate exactly the areas in which wear would be measured in mm towards the outside of the root curvature . these locations were named a and b , which means , respective distances at 1 and 5 mm from the wl ( figure 3 ) . levels 1 and 5 were chosen for representing the apical third extremities . data referring to the amount of wear in the simulated canals ( in mm ) were analyzed by anova and tukey 's multiple - comparison test at significance level of 5% . there was no statistically significant interaction ( p>0.05 ) among the three factors ( canal curvature , instrument size and curve location ) . in two - by - two interactions , only the interaction between canal curvature and curve location was significant ( p<0.05 ) , which means that , independently on the instrument size , in location a , canals at 20 presented mean wear smaller than canals at 40 , while in point b this difference was not significant ( table 2 ) . in table 2 , the canals with 20 curvature showed no statistically significant difference ( p>0.05 ) in the wear mean in relation to the location . however , in canals with 40 curvature it may be observed that in point a , the mean wear was significantly greater ( p<0.05 ) than in point b. as the instrument size was significant in the interactions among the factors , its main effect was evaluated , that is , wear ( in mm ) . in this way , independently on the curve location and canal curvature , the instruments sizes f4 and f5 presented the greatest wear means , without statistically significant difference from each other ( p>0.05 ) . both instrument sizes produced significantly more wear than instrument f3 ( p<0.05 ) ( table 3 ) . standard deviation means followed by different uppercase letters in columns and different lowercase letters in rows differ significantly ( p<0.05 ; anova and tukey 's test ) . means followed by different letters differ significantly ( p<0.05 ; anova and tukey 's test ) . protaper instruments have multiple tapers within the same shaft . shaping files instruments ( sx , s1 and s2 ) have progressive taper ( increase from the tip towards the handle ) , which adds flexibility to the middle and apical portions of the files . the sx , s1 and s2 instruments have tip diameters of 0.19 mm , 0.17 mm and 0.20 mm respectively . on the other hand , the decreasing taper of the finishing instruments(f1 , f2 and f3 with tip diameters of 0.20 mm , 0.25 mm and 0.30 mm respectively ) affords a greater mechanical resistance and rigidity to the instruments3,15,16,28 . in 2006 , the manufacturer made some changes in the design of the instruments s2 , f1 , f2 and f3 . in addition , new instruments were introduced ( f4 and f5 ) and the system was renamed as protaper universal . the changes were complemented with the release of instruments f4 and f5 with tip diameter of 0.40 mm and 0.50 mm , respectively . the overall aim of this study was to analyze the wear promoted in the apical third after endodontic preparation with the protaperuniversal system , in simulated root canals under controlled laboratory conditions , in addition to determining whether the new instruments ( f4 and f5 ) create inappropriate canal shapes . simulated canals were chosen in order to standardize the groups , in accordance with previous studies14,25,29 . although simulated canals in acrylic blocks provide highly controlled in vitro conditions , such as standardized characteristics and resin hardness similar to that of root dentin , canal cross - sections differ from that of canals in extracted teeth16,26 . in the present study , these curvatures , with mild and moderate degrees , were standardized as reported in previous studies2,9,25 . the apical stop was prepared up to instrument f5 in both canal types in order to produce greater wear , which is desired in teeth with pulp necrosis . other authors also used instruments with larger diameter to prepare the apical stop : peters and barbakow17 used the profile system .04 until number 40 and al - sudani and al - shahrani1 used instruments with large taper ( 30/.06 ) in canals with curvature from 15 to 40. wear was measured in mm from the anatomical canal wall up to the surgical canal walls . in 72 images that were superimposed on the non - instrumented canal images wear was observed ( in points a and b ) only outside the curve . in the inner side of the curvature , there was a superpositioning of the anatomical and surgery canal walls . as no wear was observed in the inner side of the curvature , it is assumed that there was a canal transportation . during endodontic preparation of curved canals , the amount of wear must be similar in all walls ( buccal , lingual , mesial and distal ) in order to avoid the occurrence canal transportation . finally , it should be kept in mind that the methodology used in the present study does not provide a threedimensional image of the canals , as obtained in studies that used computed tomography for wear analysis10,18 . the present analysis was limited to the proximal aspect , which is considered a reliable , low - cost , interesting and easy technique . adobe photoshop computer software allows to superpose images and affords different visualizations , reducing the opaque appearance of images23 . from the obtained results , there was significant interaction between the curve location and the degree of canal curvature , which means , independently on the instrument size ( f3 , f4 or f5 ) , in point a , canals of 20 presented wear mean smaller than canals of 40 , but in point b this difference was not significant ( table 2 ) . such fact has been demonstrated in a previous12 study , where m4 system was used : canals with larger curvature degree showed greater apical wear . a possible explanation for not observing difference in the wear amount in point b , between the two canal types , was the fact that the curvature started at 8 mm from the canal entrance , becoming gradually curved from 7 mm , towards the apex . this may left the canals with similar curvature degree in point b ( 5 mm ) . the same wear values in point b were observed , after use of the instruments f3 and f5 : 0.57 mm , in the canals of 20and 40 and 0.33 mm in the canals of 20and 40. in canals with 20 curvature , there was no significant difference in the wear mean between points a and b , however in canals of 40 , the wear mean in a was larger than in b , what may be characterized as deviation . in agreement with that , in more curved canals , there is greater wear , and for this reason , caution must be taken during instrumentation to avoid having the instrument stopped during apical preparation5,25 . canal transportation is determined by the flexibility of the preparation instruments , the movement of the instruments in the canal , as well as the length of time the instrument is in contact with the canal wall during preparation20,24 . as according to loizides et al.13 , it is of great clinical importance when working with protaper finishing files not leaving the instruments preparing the root canal for longer than 1 second when reaching the required length . this means that finishing files should be immediately removed out of the canal once the working length is achieved . in this way , shaping aberrations in the apical part of the canals can be easily minimized . in both points a and b and regardless of the curvature degree , instruments f4 and f5 produced significantly greater wear than that produced by instrument f3 , but did not differ from each other ( table 3 ) . this occurs since , the larger metallic mass instrument quantity , the greater the resistance offered to file adaptation into the canal , because of its smaller flexibility2,3,19 . from the applied methodology and obtained results , it may be concluded that wear in points a and b occurred only in the outer side of the root curvature , producing canal transportation . in point a , canals with 20 curvature presented less wear than those with 40 curvature . in point b , this difference was not significant . in canals with 20 curvature , there was no significant difference in the mean wear between points a and b. in canals of 40 , the mean wear in point a was significantly greater than in point b. independently on the location and the canal curvature , instruments f4 and f5 , presented the greatest wear , significantly greater than the wear promoted by instrument f3 . all instruments produced canal transportation , but the f4 and f5 instruments produced more than the other instruments , and should thus be used with care in curved canals . | objective : this study evaluated the wear in the apical third of simulate canals after preparation with protaper universal rotary system.material and methods:24 sets of instruments were used in 24 simulated canals in transparent epoxy resin blocks with degree of curvature of either 20or 40. the canals were photographed preoperatively and after preparation of the apical stop with protaper f3 , f4 and f5 instruments .
the initial and final images were exported to adobe photoshop software and superimposed to detect the root canal wall differences ( in mm ) between them , in two points located 1 ( a ) and 5 ( b ) mm from the point where the working length was established .
data were subjected to analysis of variance to verify the existence of interaction among the factors : canal curvature , instrument size and curve location .
significant level was set at 5%.results : regardless of the location and the canal curvature , f4 and f5 instruments produced the greatest wear ( p<0.05).conclusions : there was a deviation from the original pathway towards the outside of the root curvature in both analyzed points .
all instruments produced canal transportation , but the f4 and f5 instruments produced more than the other instruments , and should thus be used with care in curved canals . |
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the human keratin 14 promoter ( k14 ) was used to either conditionally knockout yap or drive yap transgenic over - expression in mouse embryonic ectoderm - derived epithelial tissues . yap conditional knockout ( cko ) embryos were generated through crossing yap mice with k14-cre mice , in c57bl/6 background . yap over - expression ( oe ) embryos were generated through breeding col - teto - yap mice with k14-rtta mice , , , , in fvb / nj background , in which the constitutively active form of human yap1 protein with the ser127ala mutation was over - expressed upon doxycycline ( dox ) administration . 2 mg / ml of dox in drinking water was given starting from embryonic days 9.5 ( e9.5 ) to e14.5 . all mouse studies were performed in compliance with the protocols approved by the harvard university institutional animal care and use committee . three yap cko embryos at e14.5 , along with the three corresponding three litter controls , were used for tooth germ collection of the yap cko group . similarly , three yap over - expression and three corresponding litter control embryos at e14.5 were used for the yap oe group . the tooth germs were sequentially broken down by 18 , 21 and 25 gauge needles in trizol ( life technologies ) . then total rna was extracted and purified using rnaeasy mini kit ( qiagen ) with on - column dnase ( qiagen ) digestion , and evaluated by the agilent 2100 bioanalyzer ( agilent technologies , ca ) . 1 ) , indicating high quality of the rna samples . to minimize the individual difference from embryos , each rna - seq sample was pooled from the three biologically different e14.5 tooth germs with the same amount of total rna . rna - sequencing was performed on an illumina hiseq 2000 by the biopolymers facility at harvard medical school . paired end 50-nt sequencing strategy was used for all the rna - seq samples to minimize sequencing reading errors . mus
musculus genome ( mm9 ) and dnanexus software were used to extract sequencing reads of genes from the rna - seq raw data . rna - seq raw sequencing reads and aligned reads are available through the gene expression omnibus at the accession number of gse65524 . after rna - seq reads were extracted and aligned with the mm9 genome , the relative read ratio of each gene was calculated and further analysis was conducted . we compared the transcriptomic profilings between the yap cko and wild type tooth germs , as well as those between the yap oe and wild type mouse tooth germs . in comparison with the corresponding controls at the 1.5-fold change cutoff , there were 968 down - regulated genes and 979 up - regulated genes in yap cko tooth germs , while there were down - regulated 1289 genes and up - regulated 774 genes in yap over - expression tooth germs ( fig . further analysis revealed many differentially expressed genes between yap cko and yap oe tooth germs . interestingly , we found that some genes in the hox , wnt and laminin families were differentially expressed in the two kinds of the tooth germs . the transcription levels of several hox gene family members were decreased in yap cko tooth germs while their expression levels were increased in yap oe tooth germs ( fig . 3 ) , indicating that these genes could be potential downstream targets of yap in vivo . the regulation of the hoxa1 and hoxc13 gene expression by yap was further validated and functionally analyzed in both mouse and human epithelial cells . similar to the expression changes of the hox genes , several wnt and laminin family members also showed concurrent changes with yap transcripts ( fig . 3 ) , although the members of these two gene families do not have a linear genomic arrangement pattern . the members of the wnt family showing concurrent changes include wnt10b , wnt16 , wnt3 , wnt3a , wnt4 , wnt6 , wnt7a and wnt9b . the transcript levels of the latter six wnt genes were further validated by qpcr in both tooth germs and skin samples . the members of the laminin gene family with concurrent changes consist of lama3 , lama5 , lamb3 and lamc2 . the human keratin 14 promoter ( k14 ) was used to either conditionally knockout yap or drive yap transgenic over - expression in mouse embryonic ectoderm - derived epithelial tissues . yap conditional knockout ( cko ) embryos were generated through crossing yap mice with k14-cre mice , in c57bl/6 background . yap over - expression ( oe ) embryos were generated through breeding col - teto - yap mice with k14-rtta mice , , , , in fvb / nj background , in which the constitutively active form of human yap1 protein with the ser127ala mutation was over - expressed upon doxycycline ( dox ) administration . 2 mg / ml of dox in drinking water was given starting from embryonic days 9.5 ( e9.5 ) to e14.5 . all mouse studies were performed in compliance with the protocols approved by the harvard university institutional animal care and use committee . three yap cko embryos at e14.5 , along with the three corresponding three litter controls , were used for tooth germ collection of the yap cko group . similarly , three yap over - expression and three corresponding litter control embryos at e14.5 were used for the yap oe group . the tooth germs were sequentially broken down by 18 , 21 and 25 gauge needles in trizol ( life technologies ) . then total rna was extracted and purified using rnaeasy mini kit ( qiagen ) with on - column dnase ( qiagen ) digestion , and evaluated by the agilent 2100 bioanalyzer ( agilent technologies , ca ) . 1 ) , indicating high quality of the rna samples . to minimize the individual difference from embryos , each rna - seq sample was pooled from the three biologically different e14.5 tooth germs with the same amount of total rna . rna - sequencing was performed on an illumina hiseq 2000 by the biopolymers facility at harvard medical school . paired end 50-nt sequencing strategy was used for all the rna - seq samples to minimize sequencing reading errors . mus
musculus genome ( mm9 ) and dnanexus software were used to extract sequencing reads of genes from the rna - seq raw data . rna - seq raw sequencing reads and aligned reads are available through the gene expression omnibus at the accession number of gse65524 . after rna - seq reads were extracted and aligned with the mm9 genome , the relative read ratio of each gene was calculated and further analysis was conducted . we compared the transcriptomic profilings between the yap cko and wild type tooth germs , as well as those between the yap oe and wild type mouse tooth germs . in comparison with the corresponding controls at the 1.5-fold change cutoff , there were 968 down - regulated genes and 979 up - regulated genes in yap cko tooth germs , while there were down - regulated 1289 genes and up - regulated 774 genes in yap over - expression tooth germs ( fig . further analysis revealed many differentially expressed genes between yap cko and yap oe tooth germs . interestingly , we found that some genes in the hox , wnt and laminin families were differentially expressed in the two kinds of the tooth germs . the transcription levels of several hox gene family members were decreased in yap cko tooth germs while their expression levels were increased in yap oe tooth germs ( fig . 3 ) , indicating that these genes could be potential downstream targets of yap in vivo . the regulation of the hoxa1 and hoxc13 gene expression by yap was further validated and functionally analyzed in both mouse and human epithelial cells . similar to the expression changes of the hox genes , several wnt and laminin family members also showed concurrent changes with yap transcripts ( fig . 3 ) , although the members of these two gene families do not have a linear genomic arrangement pattern . the members of the wnt family showing concurrent changes include wnt10b , wnt16 , wnt3 , wnt3a , wnt4 , wnt6 , wnt7a and wnt9b . the transcript levels of the latter six wnt genes were further validated by qpcr in both tooth germs and skin samples . the members of the laminin gene family with concurrent changes consist of lama3 , lama5 , lamb3 and lamc2 . in this study , we used rna - seq analysis to compare the transcriptomic profilings of yap cko and yap over - expression mouse tooth germs . we found that some genes in the hox , wnt and laminin families exhibiting concurrent changes with yap transcripts and may be potentially targets of yap . the regulation of hoxa1 and hoxc13 genes by yap was further validated and functionally analyzed in different epithelial cells of mouse tooth germs , skin samples and human keratinocytes . the transcriptomic profiling comparison revealed that hoxa1 , hoxa2 , hoxa3 , hoxa5 , hoxb9 , hoxc13 , hoxc4 , hoxc8 and hoxd1 have differential expression in yap cko and yap oe tooth germs . however , our qpcr results showed that only hoxa1 and hoxc13 had significant concurrent changes in their transcripts . this discrepancy could be due to low abundance of the hox genes in mouse tooth tissues , which may cause high relative ratios . there are also some other gene families showing concurrent transcript changes with yap transcripts , such as the laminin , rho gtpas activating protein and ras - related protein gene family members . further investigation is required to understand the regulation of their expression by yap and the relationships among these gene families . | to identify the downstream target genes of yap , we used rna - seq technology to compare the transcriptomic profilings of yap conditional knockout ( yap cko ) and yap over - expression mouse tooth germs .
our results showed that some hox , wnt and laminin family genes had concurrent changes with yap transcripts , indicating that the expression of these genes may be regulated by yap . here , we provide the detailed experimental procedure for the transcriptomic profiling results ( ncbi geo accession number gse65524 ) .
the associated study on the regulation of hoxa1 and hoxc13 genes by yap was published in molecular cellular biology in 2015 [ liu et al . , 2015 ] . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
optimal fetal development and infant outcome depend on availability of specific nutrients during the preconceptual and gestational periods , including the omega-3 long chain polyunsaturated fatty acids ( lcpufas ) , docosahexaenoic acid ( dha ) , and eicosapentaenoic acid ( epa ) [ 1 , 2 ] . in response to maternal omega-3 lcpufa intake during pregnancy , infants have improved performance on cognitive and developmental tests [ 35 ] , accelerated maturation of the visual and autonomic nervous systems [ 68 ] , and leaner body composition [ 9 , 10 ] . the relationship between dietary omega-3 lcpufa intake and maternal mental health conditions ( depressive disorders during and after pregnancy ) has been examined . there is evidence that omega-3 lcpufa intake may benefit women with preexisting depressive illnesses [ 1114 ] . these findings are complemented by observational studies which point to an association between low dietary omega-3 lcpufa intake , especially dha , and increased risk of depressive disorders during and following pregnancy [ 15 , 16 ] . omega-3 lcpufas prolong pregnancy duration , reducing the risk of birth before 34 gestational weeks by 31% in normal and 61% in high - risk pregnancies [ 17 , 18 ] . increasing pregnancy duration has implications for decreasing incidence of preterm birth and intrauterine growth retardation . these measurable and documented benefits of omega-3 lcpufa underscore the recommendations of the 20152020 dietary guidelines for americans that pregnant women and women of childbearing ages consume 812 oz . , or two to three 4 oz . servings , of seafood per week , as cold water marine fish are the major dietary source of omega-3 lcpufas ( table 1 ) . in general , fish are regarded as good dietary sources of omega-3 lcpufas ; however , fatty acid content depends on variety , geographical location , method of farming / harvesting , and other factors . these recommendations translate to approximately 250 mg omega-3 lcpufas per day and are in line with the recommendation of 200 mg dha per day set forth by an international panel of experts in an earlier consensus statement . the food and drug administration and environmental protection agency further specify that servings should be from a variety of fish that have low levels of methylmercury . nearly all fish contain trace amounts of methylmercury ; however , larger fish with longer lifespans have greater accumulations of the neurotoxin . as methylmercury crosses the placenta , it accumulates in the fetus at higher concentrations than those in the mother [ 25 , 26 ] . fetal exposure to excess amounts of methylmercury in utero , when the brain is especially vulnerable to environmental insults , can negatively affect brain and nervous system development . tilefish from the gulf of mexico , shark , swordfish , and king mackerel contain high levels of methylmercury [ 21 , 23 ] ; thus , pregnant women and women of childbearing ages are advised to avoid these fish . during pregnancy , the fetus relies on maternal intake and placental transfer of nutrients to meet developmental demands . although prenatal vitamins and other vitamins / supplements are marketed to pregnant women , they may not contain omega-3 lcpufas or women may not consume them at all or with any regularity . thus , low fish intake during pregnancy could result in low fetal accumulation of dha and epa . previous estimates of dietary omega-3 lcpufa intake point to low consumption by pregnant women and women of childbearing ages . in a small sample ( n = 21 ) of pregnant women in baton rouge , louisiana , usa , data from our laboratory indicate the average dietary intake of dha is 72 mg per day , which translates to 95% of pregnant women not meeting the recommendation of 200 mg dha per day . when supplement intake was taken into consideration , 62% of pregnant women still failed to meet the recommended dha intake . in an earlier study , we reported that nonpregnant women of childbearing ages ( n = 183 ; average age : 20 years ; age range : 1828 years ) consumed an average of 66 mg dha per day , which included both dietary and supplemental sources of dha . given the role of omega-3 lcpufas in infant development , pregnancy outcome , and maternal health , it is important to assess if pregnant women are adhering to the dietary recommendation to include seafood in their diets and , if not , what foods they are choosing to consume instead . therefore , the aim of the study was to investigate the dietary habits of pregnant women in baton rouge , louisiana , usa . geographically , baton rouge is located directly on the mississippi river and approximately 157 miles north of the gulf of mexico . as louisiana is a coastal state and fish are an intricate part of the regional culture and cuisine , we hypothesized that pregnant women in the greater baton rouge area would meet the recommended seafood intake for pregnant women and women of childbearing ages . for this observational study , we approached women at an event held for expecting women and their partners at a hospital in baton rouge , louisiana . women were approached and invited to complete a survey about their dietary habits during pregnancy and respond to a demographic questionnaire ; the survey and questionnaire were provided as separate documents . all pregnant women who visited the research booth at the event were invited to participate ; the only inclusion criterion was current pregnancy and there was no selection bias . our efforts resulted in 221 completed surveys and questionnaires ; the responses from each were separated at the time of completion . compensation for study completion was entry into a raffle for free baby books and other materials for expecting families . women provided their first name and telephone number on a separate piece of paper ; this paper was not attached to the survey or questionnaire . when a name was drawn in the raffle , the woman was contacted by a call or text and returned to the booth to pick up her raffle prize . the survey included a statement that completion of the survey constituted consent to participate and participation were voluntary . all procedures involving human subjects were approved by the louisiana state university agcenter and woman 's hospital institutional review boards . contact and demographic information were not attached to the survey and , as such , responses were anonymous . the women were allowed to complete their survey and the questionnaire and provide their contact information on an individual clipboard standing away from the table at which the researchers were stationed . the survey was designed to be completed by participants in approximately 5 minutes with minimal input or direction from the researchers . women were instructed to complete the survey in accordance with their usual dietary habits during pregnancy . the survey has not been previously validated and was developed as a tool to provide preliminary , descriptive data that provides a direction and foundation upon which to build for future research . the survey contained four sections ( protein sources ) , labeled and ordered as follows : beef , chicken , fish , and pork . each section included a list of foods commonly consumed for that respective protein source ; these foods were subjectively chosen by the researchers . beef included , in order , steak , hamburger , stew meat , brisket , and roast . chicken included , in order , wings , breast , and legs . fish included , in order , canned tuna , tuna steak , tilapia , salmon , cod , catfish , swordfish , trout , bass , flounder , and herring . pork included , in order , chop , each question had six predefined responses to assess how frequently the women were consuming each : never , once / week , 2 - 2+/week , once / month , 2 - 3/month , and 4 - 4+/month . the majority of women checked only one box per food ; however , if multiple or none of the boxes were checked , that data point was entered as missing . as the primary focus of our study was fish consumption by pregnant women , we constructed our survey to include a variety of fish , including those that are poor and good sources of omega-3 lcpufas and those that are indigenous and nonnative to the area ( canned tuna , tuna steak , tilapia , salmon , cod , catfish , swordfish , trout , bass , flounder , and herring ) . the demographic questionnaire , included as a separate document , included questions about participant age , ethnicity , education level , and if she was a first - time mother . rather , we asked how often the women consumed each food and assumed portion sizes for each . in speculating whether pregnant women are meeting the omega-3 lcpufa recommendations by dietary fish intake , we assumed each serving to be 4 oz . this assumption was based on a table in the 20152020 dietary guidelines for americans . although the guidelines specify that pregnant women should consume 8 to 12 oz . of seafood per week from a variety of sources , a serving size is not defined . however , the food and drug administration and environmental protection agency specify that the recommended 812 oz . translates to two to three servings of fish per week . demographic data ( n = 221 ) for our survey population are provided in table 2 . the majority of the women in our population were caucasian ( 71% ) , 2630 years old ( 37% ) , and had completed some college ( 24% ) , a 4-year college degree ( 29% ) , or a graduate degree ( 29% ) . african american was the second most common ethnicity ( 20% ) and 2025 years of age was the second most common age range ( 29% ) . women responded to the frequency with which they ate stew meat least often ( i.e. , did not answer the question ; 88% response rate ) and chicken breast most often ( 96% response rate ) . twenty - two percent of women reported consuming fish , when consumption of all individual varieties was averaged ( figure 1 ) . tilapia , canned tuna , and salmon were consumed by 47 , 40 , and 35% of women , respectively . swordfish , herring , and flounder were consumed by less than 3% of women . the most common consumption frequency for catfish , tilapia , canned tuna , and salmon was once per month , followed by once per week . consumption rate and frequency for each fish variety are presented in figures 2 and 3 . consumption rate for beef , chicken , and pork , when all foods were averaged within protein source , was 71 , 74 , and 65% , respectively . hamburger , chicken breast , and pork chops were the most consumed foods for each protein source , with 90 , 92 , and 63% of women reporting that they consume each , respectively . brisket , chicken legs , and pork roast were the least consumed foods for each protein source , with 52 , 65 , and 58% of women reporting that they consume each , respectively . women most commonly reported intake of the most consumed beef ( hamburger ) and chicken foods ( chicken breast ) at a frequency of once or at least twice per week . a consumption frequency of once per month was the most common response for the least popular beef , chicken , and pork foods ( brisket , chicken legs , and pork roast , resp . ) . the most popular pork food ( pork chops ) was most often consumed at a frequency of once per month . consumption rate and frequency of consumption for the most and least consumed foods , grouped by protein source , are presented in figures 4 and 5 , respectively . the two most commonly consumed fish varieties by our population ( catfish and tilapia ) have significantly lower concentrations of omega-3 lcpufas than the varieties which were rarely consumed ( table 1 ) . of particular interest is the finding that canned tuna , which is widely available , is inexpensive , has a long shelf life , and is amenable to easy preparation , was not consumed by more women or more frequently . salmon , a similar variety to canned tuna in terms of preparation and favorable omega-3 lcpufa content , was also consumed at a low frequency . to meet the recommendation of an average intake of 250 mg omega-3 lcpufa per day , one would have to consume 12 oz . of farmed catfish or 9.7 this equates to approximately 21 servings of farmed catfish or 17 servings of tilapia per week , assuming a 4 oz . serving size . as the women in our population reported consuming catfish , tilapia , and canned tuna ( the three most consumed varieties ) each at a frequency of once per month , they are likely to not be achieving recommended intakes of omega-3 lcpufas . dietary incorporation of canned white tuna and/or salmon at a frequency of twice per week would satisfy recommended levels of omega-3 lcpufa intake , exclusive of intake of other varieties ( table 1 ) . the food and drug administration and environmental protection agency advise pregnant women and women of childbearing ages to avoid consumption of tilefish from the gulf of mexico , shark , swordfish , and king mackerel due to their high methylmercury content . in our population , three women ( 1.4% ) reported consumption of swordfish at a frequency of once per month and one ( 0.5% ) indicated she consumed swordfish at least four times per month . these data may point to a need to further emphasize the recommendation that pregnant women and women of childbearing ages should avoid fish known to contain high levels of methylmercury . in the current study , consumption rate of beef , chicken , and pork was at least threefold higher than that of fish . the most consumed fish varieties were consumed at a frequency that was similar to or less than that of the least consumed beef , chicken , and pork foods . clearly , when choosing a protein source , pregnant women are opting to consume beef , chicken , and/or pork in favor of fish . our data is in line with that of a previous investigation in which it was reported that 89% of pregnant women in massachusetts , usa , consume less than 3 fish meals per month and the average canned tuna consumption is 2 servings per month . similarly , pregnant women in ontario , canada , were reported to consume 0.7 fish meals per week , which equates to less than 3 fish meals per month . these findings are also similar to those for pregnant women in southwestern quebec , canada , where the women consumed 3.6 fish meals per month . there is a stark difference in dietary seafood habits of pregnant women between the north american countries of the united states and canada and that of other regions . a large , observational study found that 88% of pregnant women in the united kingdom consumed at least 8 seafood meals per month . pregnant norwegian women , on average , consumed approximately 45 seafood meals per month and 77% of pregnant women living on the faroe islands consumed at least 12 seafood meals per month . a comparison of two studies assessing pregnant women in denmark and netherlands revealed that 22% of the danish population consumed at least 560 g of fish per month ( equivalent to 4.9 servings ) versus 56% of the dutch population . these results are in agreement with those in a different danish population , where the average fish consumption for pregnant women was 3.9 meals per month . pregnant swedish women were reported to consume 6.7 fish / shellfish meals per month , with less than 1% of women reporting they never consumed fish at all . in spanish populations , 86% of pregnant women reported consuming at least 12 seafood meals per months and 61% of pregnant women ate canned tuna at a minimal frequency of 4 times per month . findings from a taiwanese study indicate 99% of pregnant women in taipei consumed fish during pregnancy at an average rate of 11 meals per month . although those north american populations outlined above [ 3234 ] , along with the population in the current study , all live within 160 miles of the coast or a major body of water , it is apparent that pregnant women in these locations eat less seafood than their european and east asian counterparts . the international difference in fish and seafood consumption is likely fueled by the typical western diet that is characteristic of north america , as supported by our current observation that pregnant women in baton rouge , louisiana , usa , have a strong preference for hamburger or chicken breast . given the wide availability of seafood in coastal regions , one may expect pregnant women living in inland locations in north america to have even lower seafood intakes than those reported in the current and previous studies . it is important to note , as well , that our population is well educated , with 90% having completed some college . thus , even pregnant women who are educated are not consuming recommended amounts of fish . the international disparity in seafood intake reflects the findings of a 2010 study , which qualitatively determined knowledge and behavior of pregnant women ( n = 22 ) in northeastern usa with regard to fish consumption . the researchers found that while a fair amount of pregnant women ( 46% ) was aware that fish contained a potential toxic contaminant ( methylmercury ) , less knew that fish contained dha ( 36% ) or a function of dha during pregnancy ( 23% ) . furthermore , none of the women ( 0% ) had been advised to consume fish during pregnancy . two studies [ 32 , 34 ] from 2003 and 2004 found that women from northeastern usa and canada more often maintained or reduced fish intake after becoming pregnant rather than increasing it . the authors speculated that this effect was a result of national mercury advisories in the early 2000s which recommended pregnant women limit consumption of certain fish . these findings contrast those of the aforementioned study in taiwan , conducted in 2006 , where the percentage of women who consumed fish increased from 95 to 99% upon becoming pregnant . thus , our data may suggest that , in 2015 , pregnant women in north america may remain uncomfortable incorporating fish as a dietary protein source and opt for chicken , beef , or pork foods instead . it is important to note that , for each study outlined above , dietary data were collected from and reported in a variety of ways . for comparison with our results , we converted the data to servings per month by assuming a serving was 4 oz . , if the data were reported as g consumed per unit of time . we note that dietary data were collected by various methods ( food frequency questionnaires , 24-hour dietary recalls ) but assumed each method to be equal . future studies should assess whether pregnant women and women of childbearing ages have knowledge of the dietary recommendations for seafood consumption . these efforts should aim to elucidate if ( 1 ) there are specific groups of pregnant women who are less likely to meet dietary fish recommendations and ( 2 ) why these women fail to meet those recommendations . replication of the current survey across different geographic areas would also provide insight into the effect of coastal versus inland location on fish intake and dietary protein preferences . our survey was conducted in a convenience sample and since the survey and demographic questionnaires were not connected , we are unable to examine potential group differences or correlations between demographic parameters and responses . although this assumption does not affect our observations of dietary habits , it does affect the precision of our calculations in regard to whether pregnant women are meeting omega-3 lcpufa recommendations or not . certain cooking styles are related to differences in the fatty acid content of the resulting product . the characteristics of our study population differ from those published by the united states census bureau for baton rouge , louisiana . the population of baton rouge are african americans ( 55% ) or caucasians ( 39% ) who have completed high school ( 26% ) or some college ( 23% ) . the overall population of louisiana is more similar to that of our study population ; 63% caucasian or 32% african americans who have completed high school ( 26% ) , some college ( 23% ) , or a 4-year college degree ( 19% ) . it is important to note that educational attainment data from the united states census bureau data reflects that of the population aged 25 years and older , without specificity to gender . additionally , the ethnic breakdowns provided by the united states census bureau data reflect that of the entire population in that region without regard to age , gender , or pregnancy status . these data reveal that pregnant women in baton rouge , louisiana , usa , are not meeting dietary recommendations for seafood consumption and , therefore , likely do not consume adequate amounts of omega-3 lcpufas for optimal maternal health , fetal development , and infant outcome . these data also reveal the protein sources and specific foods that pregnant women are consuming in lieu of fish . the apparent deficit in omega-3 lcpufa intake has major implications during and after pregnancy and should be addressed with intensified efforts to provide nutrition and lifestyle education to pregnant women and women of childbearing ages . although our data indicate pregnant women , in general , do not meet dietary seafood recommendations , future research will help us better understand the habits of pregnant women , directing us in our development of targeted education efforts which emphasize the importance of consumption of fish low in methylmercury during pregnancy . | background .
the 20152020 dietary guidelines for americans recommend that pregnant women and women of childbearing ages consume 812 oz . of seafood per week .
fish are the major dietary source of omega-3 long chain polyunsaturated fatty acids , which have benefits for the mother and fetus . methods . in this observational study
, we investigated dietary habits of pregnant women in baton rouge , louisiana , usa , to determine if they achieve recommended seafood intake . a print survey , which included commonly consumed foods from protein sources ( beef , chicken , pork , and fish ) , was completed by pregnant women at a single - day hospital convention for expecting families in october 2015 .
women ( n = 221 ) chose from six predefined responses to answer how frequently they were consuming each food . results .
chicken was consumed most frequently ( 75% of women ) , followed by beef ( 71% ) , pork ( 65% ) , and fish ( 22% ) , respectively .
consumption frequency for the most consumed fish ( catfish , once per month ) was similar to or lower than that of the least consumed beef , chicken , and pork foods .
consumption frequency for the most consumed chicken and beef foods was at least once per week .
conclusion .
our data indicate that pregnant women in louisiana often consume protein sources other than fish and likely fail to meet dietary seafood recommendations . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
atrial septal defect ( asd ) is a form of congenital heart defect that enables blood flow between two compartments of the heart called the left and right atria . normally , the right and left atria are separated by a septum called the interatrial septum . if this septum is defective or absent , then oxygen - rich blood can flow directly from the left side of the heart to mix with the oxygen - poor blood in the right side of the heart , or vice versa.1 asd is one of the most common congenital cardiac defects and accounts for approximately 610% of all congenital cardiac defects.2 young children with asd are usually asymptomatic and could wait for elective surgical or catheter - based closure for 3 years or more.34 shunts from asd are usually detected in childhood or young adulthood . the defect is generally well tolerated in infants and young children , but symptoms of mild fatigue , poor growth or dyspnoea on exertion may be present.5 therefore , early closure could be beneficial for such infants . if the asd is small , it may not require any intervention , or it may close on its own . asd , and therefore the shunt , tends to increase with age , and repair usually takes place when asd is diagnosed.5 in asymptomatic patients with a large left - to - right shunt , surgical closure should be performed between 2 and 5 years of age.6 asd is not without morbidity . about 40% of such patients may present with cryptogenic stroke at adulthood ; paradoxical embolism has also been reported.7 surgical repair of asd is a well - established procedure and is very safe , with a negligible mortality rate.8 transcatheter occlusion of an asd was first described by king and colleagues in 1976.9 although satisfactory results have been reported with transcatheter occlusion of these defects with a number of devices , only one of them has received food and drug administration ( fda ) approval : the amplatzer atrial septal occluder ( aga medical corporation ; golden valley , minn10 ) . this study is carried out to determine the pattern and clinical profile of children with asd . it is also aimed at determining the different types of interventions done for these patients and their complications . the study was conducted at a paediatric cardiac centre , innova heart hospital , hyderabad , india , established in 2007 . the hospital records showed that from april 2007 to june 2011 , a total of 69 patients underwent surgical closure of asd . chinese duct occluder manufactured by shanghai shape memory alloy material co. , ltd,11 which consists of metal mesh woven by nitinol wires with shape memory function , supper elasticity and polyethylene filled mesh . the occluder device has specifications of 4/6 , 6/8 , 8/10 , 10/12 , 12/14 , 14/16 and 16/18 mm ( diameter of waist pulmonary arterial end / diameter of aortic end ) , with lengths between 7 and 8 mm . the delivery system consists of loading sheath , delivery sheath and control wire , on the top of which there are screw threads . the diameter of outer sheath is 6 - 10 f. other duct occluders also included 4 . the ado12 received fda approval with the specific indication for nonsurgical closure of patent ductus arteriosus . this device is a self - expandable device made from a nitinol wire mesh and polyester fabric . as the occluder is implanted , it expands outward , and the wires push against the rims of the asd . the operation was performed after puncture and systemic heparinisation ( 100 iu / kg ) . intra - operative , intravenous administration of antibiotics was started and continued for 24 hours to prevent infection . for infants , the operation was performed under general anaesthesia , whereas for older children , it was done under courteous sedation . record showed that 69 patients presented with asd and underwent interventional closure of the defect during the study period . forty ( 58% ) patients were females while 29 ( 42% ) patients were males , giving a male to female ratio of 1:1.4 . their age ranged from 3.2 to 19 years , with a median age of 9.0 years . their weight ranged from 7.5 to 39.0 kg , whereas median weight was 31.5 kg . five patients ( 7.2% ) were young children aged between 3 and 5 years [ table 1 ] . forty - four ( 63.8% ) of these children presented with symptoms of heart failure , whereas 47 ( 68.1% ) of the cases repaired were large - sized asd . the majority had large - sized asd , whereas small asd accounted for 1.4% of cases . clinical profiles of asd and procedures used for repair the device used for the procedure was further shown to have a success rate of 88.4% , as shown in table 2 . the most common interventional closure procedures done were searcare heart and amplatzer technique with a highest success rate obtained in 2010 [ table 3 ] . this is in keeping with other studies.1314 the reason for this preponderance could be genetic . , a common genetic variation near a gene called msx1 is strongly associated with the risk of asd , and females with asd may have more of this variation near msx1 than their male counterpart.15 we noted that majority of our patients with asd presented with symptoms of heart failure . majority of young children with asd are usually asymptomatic and could wait for elective surgical or catheter - based closure for 3 years or more . however , some infants with asd who suffer from congestive heart failure , frequent respiratory infection , failure to thrive and progressive moderate - to - severe pulmonary hypertension do so because of late presentation.16 a good number of our patients had a large - sized asd , which explains why we have so many symptomatic patients in our study . it is the degree of shunting that predisposes to increased pulmonary flow and subsequent cardiac decompensation . the aso is a self - expanding double - disc device with a central connection waist to stent the asd . preliminary human experience regarding its safety and efficiency in closing asd has been very encouraging.171819 the aso is one of the most frequently used devices to close asd and has been proven to be highly effective and safe in the short term . previous reports have confirmed that transcatheter closure of asd with the aso achieved comparable efficacy and safety to that of surgical closure . it has some additional advantages , which include less complication rate , requirement of a shorter hospital stay and avoidance of a permanent scar.20 this is true because we had little or no complications in our patients and their hospital stay was short . the procedure is done in the hybrid theatre or cardiac catheterisation laboratory and requires a team that includes an interventional radiologist or interventional cardiologist . the capital outlay may be more expensive than that in open surgical programme , thereby making the unit cost of interventional treatment higher on the average . asd is a common congenital heart disease with a high success rate for those who underwent surgery . the use of aso percutaneous closure devices has become the procedure of choice for closure of asd in suitable patients . | background : atrial septal defect ( asd ) is a congenital heart defect that leads to shunting of blood between left and right atria
. it may be asymptomatic and sometimes may present with heart failure .
surgical repair is definitive , but currently non - surgical procedure is used to close the defect.materials and methods : it is a retrospective study of patients who underwent transcatheter closure of asd at innova heart hospital , hyderabad , india .
echocardiography was repeated at intervals of 24 hours , then at 1 , 3 and 6 months after the procedure to assess complications .
the morphological characteristics of the asd , including its diameter , location , shape and the width of surrounding septal margins , were also evaluated.results:from april 2007 to june 2011 , 69 consecutive children ( 29 males , 40 females ) with a median age of 9.0 years ( range = 3.219 years ) registered with diagnosis of asd .
the median weight was 31.5 kg ( range = 7.539.0 kg ) .
five patients ( 7.2% ) were young children aged 35 years .
forty - four ( 63.8% ) of these children presented with symptoms of heart failure , whereas 47 ( 68.1% ) of the cases repaired with device were large - sized asd .
the most common interventional procedures done were searcare heart and amplatzer technique with a highest success rate obtained in 2010.conclusions:asd is a common congenital heart disease with a high success rate for those who undergo intervention . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
pancreatic injuries are not common after blunt and penetrating trauma , but can be challenging to diagnose and manage . twenty - three year old man , injured during a fall from a motorcycle two days earlier , was admitted to department of surgery , university clinical centre tuzla because of suspicion of pancreatic trauma . immediately after hospitalization , patient underwent laboratory and radiological tests that revealed the existence of pancreatic trauma , so we opted for urgent surgical treatment . surgery and early postoperative course were normal and the patient was discharged on the ninth postoperative day . proper diagnosis and well - selected surgical treatment significantly increases the chances for recovery of these patients . pancreatic injuries are uncommon and can result from direct penetration into the organ or through the transmission of blunt force energy to the retroperitoneum . some studies report an incidence of 0.2% cases for blunt trauma up to 1.1% cases for penetrating trauma ( 1 ) . despite of low frequency of these injuries they are associated with significant morbidity and mortality , resulting in mortality rates of 23.4% and 30.2% for blunt and penetrating trauma , respectively ( 2 ) . the most commonly used system is the one devised by the american association for the surgery of trauma ( table 1 ) which is based on anatomic location of injury and status of pancreatic duct ( 3 ) . the diagnosis of pancreatic injuries is made by clinical examination , laboratory tests and imaging techniques such as ultrasound of the abdomen , contrast - enhanced ct scan , recap , or mcpc . the basic approach in the treatment of pancreatic injury is surgical and the choice of surgical procedure is govern by the location and the grade of injury . * proximal pancreatic injuries are injuries to the patient 's right of the superior mesenteric vein twenty - three year old man was hospitalized in general hospital tesanj due to injuries he sustained in motorcycle accident . on admission findings on ct scan of the brain , cervical spine and abdomen ( without contrast enhancement ) did nt suggest existence of any major injury . since the conducted diagnostic evaluation did nt verify the existence of traumatic changes , the patient was treated conservatively . however , two days after admission , his clinical status started to deteriorate . in order to evaluate the patient s status , control contrast - enhanced abdominal ct scan was made which suggested suspicion of a possible pancreatic injury . after that , patient was referred to university clinical center tuzla for further diagnostic and therapeutic evaluation . on admission to our hospital , physical examination revealed abdominal tenderness with signs of diffuse peritonitis . during preoperative evaluation laboratory tests and contrast - enhanced ct scan of the abdomen laboratory tests showed the elevation of leukocytes ( 17.42 , reference values 4.40 to 11.60 ) , c - reactive protein ( 180.5 , reference values 0.0 to 3.3 ) and mild elevation of serum amylase ( 618 u / l , reference values 23 to 135 ) . contrast - enhanced ct scan of the abdomen showed the presence of larger amounts of free fluid in the abdomen , and voluminous , inhomogeneous pancreas , transected at the body . ct scan also recorded laceration of the spleen , as well as diffuse imbibitions of adipose tissue and edema in the gastrocolic ligament ( contusion ) . given the clinical findings and the results of diagnostic evaluation , we decided for urgent surgical treatment . during surgery we found larger amount of free serohemoragic fluid inside the abdominal cavity , as well as incomplete transection of pancreas with disruption of the main duct to the patient s left of the superior mesenteric vein . according to aast organ injury scale it was grade iii injury of the pancreas . these intraoperative findings were consistent with ct scan we did during preoperative evaluation . with regard to intraoperative findings , we decided for distal pancreatectomy and splenectomy with external drainage ( figure 1 , 2 ) . en bloc resected distal pancreas and spleen remaining pancreas after distal pancreatectomy after surgery , the patient was admitted to the intensive care unit . the second postoperative day , the patient was haemodynamically and respiratory stable , moved from intensive care unit to the department of hepatobilliary and pancreatic surgery . on the eight postoperative day , the drains were removed and the patient was , on the next day , discharged home for outpatient follow - up . pancreatic injuries are relatively uncommon but , nevertheless , represent serious problem because of high morbidity and mortality rates that accompany them , with morbidity rates ranging between 45% and 60% , and mortality rates ranging between 23.4% and 30.2% ( 2,4 ) . the reason for these high morbidity and mortality rates can be attributed to delays in diagnosis and , consecutively , treatment . pancreatic enzymes are well known for their caustic nature , so every delay in management of pancreatic injuries results in massive systemic inflammation with subsequent poor outcomes . establishing the presence of pancreatic injury is a considerable challenge , as no single imaging modality has been found to highly effective . therefore , for early and timely diagnosis of pancreatic injury , a high index of suspicion must be maintained . patients with a history of acceleration / deceleration injury , forceful anterior compression of the abdomen and lower thoracic and upper lumbar vertebra fractures are at increased risk for pancreatic injury . a seatbelt sign across the upper and middle abdomen should suggest the possibility of serious intraabdominal injury ( 5 ) . in our case , the patient sustained pancreatic injury as a result of an impact of handlebars on the upper part of the abdomen . sometimes , abdominal pain and peritoneal signs take days to develop , which can be observed in our case , where more than 24 hours passed before abdominal pain and peritoneal signs developed . patients with elevations in white blood cell count , serum amylase , or lipase require further investigation . recent studies report that elevated serum amylase and lipase levels , obtained 6 hours after trauma , can be used as reliable indicator of pancreatic injury , although non diagnostic within 6 hours or less after trauma ( 6 ) . if the abdominal examination initially yields equivocal or unreliable results in a haemodynamically stable patient with hyperamylasemia , a contrast enhanced abdominal ct scan should be done . although it can indicate pancreatic injury , ct scan is only moderately sensitive and can underestimate or miss pancreatic injury . the sensitivity for detecting parenchymal or ductal injury ranges between 60% and 68% ( 7,8 ) . abdominal ct scan is unreliable in diagnosing pancreatic duct injury and should not be used to guide therapy ( 9 ) . for those patients endoscopic retrograde cholangiopancreatography may be helpful , as it is the best modality for imaging the pancreatic duct , and can be used as therapeutic procedure to stent lacerated ducts in selected patients ( 10,11 ) . minor pancreatic injuries ( grade i ) in haemodynamically stable patients can be treated conservatively ( 12 ) . however , the mainstream of therapy for pancreatic injuries is surgical . minor pancreatic contusions , hematomas , and capsular lacerations ( grade i ) , as well as lacerations of the pancreatic parenchyma without major duct disruption or tissue loss ( grade ii ) , are treated with hemostasis and adequate external drainage . distal pancreatic injuries with disruption of the main pancreatic duct ( grade iii ) are best treated with distal pancreatectomy . injuries to the pancreatic head , grade iv and v are the most difficult to manage . in rare instances when the proximal duct is injured in the absence of concomitant duodenal / ampullary injury , extended distal ( subtotal ) pancreatectomy or roux - en - y pancreaticojejunostomy ( tail - sparing ) may be performed . massive destruction of the pancreatic head , or combined pancreatic and duodenal injuries are best treated with pancreaticoduodenectomy ( 13,14 ) . in our case , physical examination , laboratory tests and contrast - enhanced ct scan of the abdomen , suggested pancreatic injury with duct disruption , so we decided for urgent surgical treatment which resulted in full recovery of the patient . pancreatic injuries are not frequent , but they have an important place in the abdominal trauma considering the high level of morbidity and mortality that is associates with these injuries . the diagnosis of pancreatic injuries is not simple and requires a high index of suspicion . delay of a timely diagnosis significantly reduces the chances of adequate treatment , thereby increasing the morbidity and mortality . the main stream of therapy for pancreatic injuries is surgical , and is determined by the grade and location of injury . since the majority of the pancreatic injuries are milder grade , most of the surgical interventions end with an exploration and external drainage . however , in cases in which pancreatic laceration with main duct injury is documented , some of the surgical resection procedures are indicated , such as distal pancreatectomy ( with or without splenectomy ) or cephalic duodenopancreatectomy ( trauma whipple ) . | introduction : pancreatic injuries are not common after blunt and penetrating trauma , but can be challenging to diagnose and manage.case report : twenty - three year old man , injured during a fall from a motorcycle two days earlier , was admitted to department of surgery , university clinical centre tuzla because of suspicion of pancreatic trauma . immediately after hospitalization , patient underwent laboratory and radiological tests that revealed the existence of pancreatic trauma , so we opted for urgent surgical treatment .
surgery and early postoperative course were normal and the patient was discharged on the ninth postoperative day.conclusion:proper diagnosis and well - selected surgical treatment significantly increases the chances for recovery of these patients . |
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meckel 's diverticulum ( md ) is the most common congenital gastrointestinal anomaly with incidence of 2% in general population and reported complication rate of 4 - 40% . the most common complications are bleeding , intestinal obstruction and diverticulitis . in 60% of cases md are lined with heterotopic mucosa , in the following manner : gastric mucosa , 62% ; of pancreatic , 6% ; both gastric and pancreatic , 5% , jejunal , 2% ; brunner 's glands , 2% ; and gastric and duodenal , 2% . based upon these histological facts , 99 m technetium - pertechnetate is the most commonly performed functional imaging for localization of md lined with heterotopic ( mucin secreting cells ) gastric mucosa . higher sensitivity ( 85 - 90% ) in the pediatric population than in adult ( 60% ) patients is reported . it is most commonly visualized in the right lower quadrant and umbilical regions and less commonly in other regions . wandering or mobile md during scintigraphy is a rare presentation and may pose a diagnostic challenge for nuclear physicians . we are presenting a case of wandering md during scintigraphy , which would help our readers to be cognizant of this atypical presentation in their clinical practice . a 2-year - old male child admitted to the emergency room with 2 days history of 3 episodes of painless per rectal fresh bleeding . on examination , child was irritable , grossly anemic , afebrile with mild abdomen distention , but audible gut sounds . his hemoglobin was 4.9 g / dl ( normal 11 - 14 g / dl ) while other biochemical and bleeding parameters were within the normal limits . ultrasound abdomen was unremarkable and child had blood transfusions with and started on oral ranitidine ( 2 mg / kg 12 hourly ) . on the 3 admission day , md scintigraphy was performed with 3 mci of 99 m technetium - pertechnetate injected intravenously with 3 h fasting . dynamic study for 60 min with a frame rate of 20s / frame was acquired in anterior projection under a dual head digital gamma camera ( ecam , siemens , germany ) fitted with low energy high resolution collimators . early images showed the appearance of focus of abnormal tracer deposition in hypogastric region concomitant with the appearance of gastric activity in left hypochondrium and better delineation in subsequent frames same as native gastric activity . sequential images revealed gross shifting of abnormal focus to left iliac region , hypogastrium , right lumbar , umbilical and finally in epigastric region [ figure 1 ] . as this abnormal focus remained compact during its tortuous path with no sign of change in its morphology during its whole path , an intraluminal source of radiotracer like bleed ( which keeps on changing its shape in lumen of small or large bowel ) was ruled out and provisional diagnosis of a wandering md with heterotopic gastric mucosa was made . the child underwent an exploratory laparotomy and md was resected and histopathology revealed md with heterotopic gastric and pancreatic mucosa . 99 m technetium - pertechnetate meckel 's diverticulum scintigraphy : sequential images showing appearance of abnormal focus ( arrow ) having temporal correlation with gastric activity and wandering in various quadrants maintaining its compactness md is the most common congenital intestinal anomaly with equal frequency in both genders , but higher incidence of complication in male gender . presence of adequate heterotopic gastric mucosa ( mucin secreting cells ) is mandatory for scintigraphic localization of md with good sensitivity in pediatric group . the characteristic diagnostic criteria are temporal correlation with gastric activity and presence of abnormal focus in umbilical or right lower quadrant . in this case , although there was a good temporal correlation , but abnormal focus was grossly mobile or wandering in various abdominal quadrants . this could easily be confused with ureteric activity or secreted gastric activity entering into duodenum . however furthermore , compactness of mobile focal uptake would be changing with time as it passes through ureter or small bowel lumen . to the best of our knowledge , primary reason for this feature is mobile and long mesentery and this was the perioperative finding in this child as well . although diagnosis of md having heterotopic gastric mucosa on scintigraphy is easy and straightforward but a dynamic or wandering md may pose a diagnostic challenge . however , temporal correlation with gastric activity and compactness of the focus during wandering period is two sentinel criteria favoring the diagnosis of a md lined with heterotopic gastric mucosa . we conclude that wandering md is a rare scintigraphic presentation , which may pose a diagnostic challenge , but the presence of temporal correlation with gastric activity and compactness of focus during wandering period could make this riddle easy . | meckel 's diverticulum ( md ) is the most common gastrointestinal anomaly with heterotopic mucosa in 60% of cases .
99 m technetium - pertechnetate scintigraphy is a sensitive tool for detection of md containing heterotopic gastric mucosa .
it is usually located in the right lower quadrant or umbilical region and less frequently in other quadrants .
a mobile or wandering md is a rare clinical presentation and may pose a diagnostic dilemma .
however , presence of temporal correlation with gastric activity and compactness of focus during wandering period could make this riddle easy . |
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the authors report a unique case of malignant mesothelioma with distant metastasis to the brain . pleural mesothelioma was thought to spread directly to local structures with distant metastasis found only on autopsy . the authors present this case to demonstrate that mesothelioma can have distant metastasis and that surgery can improve both survival and quality of life . we report a case of a 62-year - old gentleman who presented with shortness of breath and right - sided chest pain over the period of 12 months . a chest x - ray showed a right pleural plaque that subsequently caused a right pleural effusion ( fig . 1 ) . he declined chemotherapy or radiotherapy , although he was having regular follow - up by the oncologist . he confirmed being exposed to asbestos , having worked for 20 years in aluminum and margarine factories . eighteen months after his initial presentation , he experienced progressive left - sided weakness and left - sided homonymous hemianopia . an mri scan of his brain showed extra - axial right occipitoparietal enhancing mass ( 43 43 47 mm ) with surrounding vasogenic oedema . it was isointense to grey matter on t2 and abutting the falx cerebri with midline shift and effacement of the right lateral ventricle ( fig . 2 ) . the lesion was approached through a right occipital craniotomy and entirely removed in a piecemeal fashion . at surgery , it appeared as a greyish - white firm vascular mass with a clear cleavage plane with the surrounding brain . the patient had good recovery and was discharged home . on 6-week follow - up , the patient continued to improve with significant improvement in his quality of life . figure 2:mri image of brain with gadolinium contrast showing enhancing mass in the right occipital lobe . mri image of brain with gadolinium contrast showing enhancing mass in the right occipital lobe . section from the intraoperative specimen showed a large cell - epithelioid malignancy composed of markedly pleomorphic epithelial cells with large hyperchromatic nuclei . immunoperioxidase stains demonstrated in fact that the cells expressed vimentin , calretinine , ae1/ae3 and cam 5.2 as in the previously biopsied pleural lesion . histologically mesotheliomas may be purely epithelioid ( 50% ) or of mixed histology ( 30% ) . more than 80% of tumors arise from the pleura ; however , primary mesothelioma of the peritoneum , pericardium and tunica vaginalis have also been reported . the risk of developing mesothelioma after asbestos exposure is 8% , depending on the duration and intensity of exposure . malignant pleural mesothelioma usually presents between the fifth and seventh decade of life and can be difficult to diagnose due to the vagueness of the presenting symptoms including dyspnea and nonpleuritic chest pain . the median survival is only 9months from diagnosis with death usually due to thoracic disease . this rapid progression may have contributed to the belief that mesothelioma rarely has distant metastasis . reviewed 171 cases of malignant mesothelioma at autopsy and discovered that over 54% of patients had distant metastases . the sites most commonly affected were the liver , adrenal glands and kidneys ( 56 , 31 and 30% , respectively ) . wronski and burt also reviewed the post - mortem findings and they concluded that the incidence of distant metastasis from pleural mesothelioma is around 50% and the incidence of cerebral metastasis around 510% . although there are numerous reported cases of cerebral metastasis from malignant pleural mesothelioma , the majority of cases were post - mortem findings . furthermore , only eight patients had neurological manifestation of cerebral disease at the time of diagnosis [ 3 , 6 , 8 ] . the case we report is interesting for the presentation of the intracranial pathology 18months after the initial presentation , well beyond the reported median survival for this lesion . the good postoperative neurological recovery also emphasizes the role of surgery to extend survival in the presence of locally controlled primary disease . to the best of our knowledge , this case represents the third report of symptomatic cerebral metastases from malignant mesothelioma that was treated by surgical excision via craniotomy , where ante - mortem histological diagnosis was obtained . with an increasing incidence of mesothelioma , the treatments available are also expanding . in the past , single - modality therapy alone ( surgery , radiotherapy and chemotherapy ) has failed to improve survival significantly . furthermore , several new therapeutic techniques , such as photodynamic therapy , immunotherapy and intracavitary chemotherapy , are increasing survival and retarding disease progression . we present this case to demonstrate that metastatic mesothelioma can have distant metastasis to the brain . as a direct result of improvements in treatment , the survival of patients with malignant mesothelioma will continue to improve , and more patients are likely to present with metastatic disease . as we have demonstrated in this case , surgical excision of cerebral metastases can provide symptomatic relief , and surgical treatment may therefore be justified in enhancing the patient 's quality of life . | malignant mesothelioma is an uncommon , highly invasive tumor derived from the mesothelial cells of pleura or peritoneum characterized by poor outcome .
mesothelioma was thought to metastasize locally only via direct invasion and not have distant spread .
distant metastases were discovered mostly on post - mortem examination .
the authors present a case of 62-year - old man with pleural mesothelioma and brain metastasis . |
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psoriasis is an autoimmune , chronic , inflammatory skin disease affecting approximately 13% of the global population . psoriatic skin lesions are characterized by sharp - edged , erythematous , squamous plaques and are histologically characterized by epidermal changes , inflammatory cell infiltration , and increased angiogenesis . the pathogenesis is multifactorial , and genetic , environmental , and immunological factors have been implicated in its pathogenesis . narrow - band uvb ( nb - uvb ) phototherapy is an important tool for treating psoriasis , but its mechanism of action has not been completely explained . the clinical recovery in plaque - type psoriasis lesions after nb - uvb phototherapy is accompanied by a reduction in expression of interleukin 12 ( il-12 ) , il-18 , and il-23 . it has been shown that nb - uvb phototherapy suppressed the interferon ( ifn ) and t helper 17 ( th17 ) pathways in resolved psoriatic lesions . endocan is expressed by the vascular endothelium and its release is controlled by various cytokines and growth factors . experimental studies show that endocan plays a major role in cell adhesion and tumor progression . a relationship has been suggested between tumor prognosis , metastasis , angiogenesis , and endocan expression . elevated serum endocan levels have been reported in dermatological disorders characterized by systemic inflammation , such as behet s disease and psoriasis . nesfatin-1 is released in the spinal cord , hindbrain , brainstem , forebrain , and adipose tissue . nesfatin-1 has anorexigenic effects and it has a major regulatory role in food intake , energy homeostasis , water intake , and body temperature . in addition , nesfatin-1 has hypertensive effects . psoriasis is associated with an increased risk of systemic diseases , such as diabetes mellitus , metabolic syndrome , and cardiovascular disease . chronic inflammation is the pathogenetic process underlying the close relationship between psoriasis and metabolic diseases . a close relationship has been shown between nesfatin-1 and glucose , insulin metabolism , and insulin resistance . to the best of our knowledge , there is no study in the literature evaluating nesfatin-1 levels in patients with psoriasis . the neutrophil - to - lymphocyte ratio ( nlr ) is a marker of systemic inflammation , currently used in many diseases . there are studies evaluating nlr in dermatological disorders such as psoriasis and behet s disease . although there are reports evaluating underlying factors in the pathogenesis of psoriasis disease , as far as we know there is no report evaluating nlr , serum endocan , and nesfatin-1 levels all together . our study aimed to evaluate nlr , serum endocan , and nesfatin-1 levels in patients with psoriasis in comparison to a healthy control group . in addition , we also aimed to investigate the effect of nb - uvb phototherapy treatment on these parameters . the present study included 39 psoriasis vulgaris patients ( mean age 3416 years ) and 49 control subjects ( mean age 3811 years ) . the study protocol was carried out in accordance with the helsinki declaration as revised in 2013 . the study protocol was approved by the local ethics committee , and written informed consent was obtained from each subject . inclusion criteria were age older than 18 years and moderate - to - severe plaque psoriasis for at least 6 months . exclusion criteria were age younger than 18 years , pregnancy , lactation , infections , and a history of psoriasis treatment with systemic agents such as acitretin , methotrexate , cyclosporine , and biological agents within the last 4 weeks . subjects with chronic diseases such as endocrine disorders , cardiovascular disease , hepatic disorders , hematologic disease , chronic renal failure , hypertension , or cancer were also excluded from the study . skin biopsy was performed in all psoriasis patients . assessment of the disease severity was performed by using the psoriasis area and severity index ( pasi ) . the whole - body irradiation was performed in a phototherapy cabinet ( 7001 k cabinet , waldman , germany ) containing 21 uvb lamps ( philips tl-01/100 w ) radiating light at the 311-nm wavelength . the initial uvb dosage before the treatment was determined according to the fitzpatrick skin type ( between 20 and 40 mj / cm ) , and irradiation was performed 3 times a week and dosage increments were increased by 20% at each of the 2 sessions , depending on the erythema response . serum glucose , urea , creatinine , c reactive protein ( crp ) , total cholesterol , high - density lipoprotein ( hdl ) , and triglyceride were analyzed by use of a roche cobas integra 8000 ( germany ) otoanalyzer with roche cobas reagent kits ( germany ) . complete blood count was analyzed by using an abbott cell dyn ( abbott laboratories , il , usa ) analyser on the same day . the serum was obtained after centrifugation of the blood samples at 3500 rpm for 10 min and was kept frozen at 80c in aliquoted microcentrifuge tubes for endocan and nesfatin 1 analysis . the human endocan and nesfatin-1 levels were analyzed by enzyme - linked immunosorbent assay ( elisa ) using commercial kits ( aviscera bioscience , usa and biovendor laboratories , czech republic , respectively ) , according to the manufacturers instructions . intra - assay and inter - assay coefficient of variations ( cv ) of endocan assay were 68% and 1012% , respectively . upper limit of the standard was 25 ng / ml . intra - assay cvs of nesfatin-1 assay were 2.75% and 5.75% for the concentrations of 6.7 and 0.83 ng inter - assay cvs of nesfatin-1 assay were 5.43% and 6.29% for the concentrations of 3.81 and 6.89 ng sensitivity of the assay was 0.021 ng / ml and the upper limit of the standard was 4 ng / ml . measurements were carried out using an elisa plate reader ( biotek instruments inc , winooski , vt , usa ) and plate washer . the standard curve was drawn by plotting the mean absorbance ( y ) of standards against the known concentration ( x ) of standards in logarithmic scale , using the 4-parameter algorithm . results are presented as ng / ml . the statistical analyses were made using spss software version 16 . the distribution of continuous variables for normality was tested with a one - sample kolmogorov - smirnov test and data are presented as mean standard deviation ( sd ) or median and interquartile ranges , as appropriate . differences between individuals with and without psoriasis vulgaris in normally and nonnormally distributed variables were evaluated by the unpaired t - test and the mann the wilcoxon signed ranks test was used to compare the serum endocan and nesfatin-1 levels in patients with pre - and post - treatment status . the present study included 39 psoriasis vulgaris patients ( mean age 3416 years ) and 49 control subjects ( mean age 3811 years ) . the study protocol was carried out in accordance with the helsinki declaration as revised in 2013 . the study protocol was approved by the local ethics committee , and written informed consent was obtained from each subject . inclusion criteria were age older than 18 years and moderate - to - severe plaque psoriasis for at least 6 months . exclusion criteria were age younger than 18 years , pregnancy , lactation , infections , and a history of psoriasis treatment with systemic agents such as acitretin , methotrexate , cyclosporine , and biological agents within the last 4 weeks . subjects with chronic diseases such as endocrine disorders , cardiovascular disease , hepatic disorders , hematologic disease , chronic renal failure , hypertension , or cancer were also excluded from the study . skin biopsy was performed in all psoriasis patients . assessment of the disease severity was performed by using the psoriasis area and severity index ( pasi ) . the whole - body irradiation was performed in a phototherapy cabinet ( 7001 k cabinet , waldman , germany ) containing 21 uvb lamps ( philips tl-01/100 w ) radiating light at the 311-nm wavelength . the initial uvb dosage before the treatment was determined according to the fitzpatrick skin type ( between 20 and 40 mj / cm ) , and irradiation was performed 3 times a week and dosage increments were increased by 20% at each of the 2 sessions , depending on the erythema response . serum glucose , urea , creatinine , c reactive protein ( crp ) , total cholesterol , high - density lipoprotein ( hdl ) , and triglyceride were analyzed by use of a roche cobas integra 8000 ( germany ) otoanalyzer with roche cobas reagent kits ( germany ) . complete blood count was analyzed by using an abbott cell dyn ( abbott laboratories , il , usa ) analyser on the same day . the serum was obtained after centrifugation of the blood samples at 3500 rpm for 10 min and was kept frozen at 80c in aliquoted microcentrifuge tubes for endocan and nesfatin 1 analysis . the human endocan and nesfatin-1 levels were analyzed by enzyme - linked immunosorbent assay ( elisa ) using commercial kits ( aviscera bioscience , usa and biovendor laboratories , czech republic , respectively ) , according to the manufacturers instructions . intra - assay and inter - assay coefficient of variations ( cv ) of endocan assay were 68% and 1012% , respectively . upper limit of the standard was 25 ng / ml . intra - assay cvs of nesfatin-1 assay were 2.75% and 5.75% for the concentrations of 6.7 and 0.83 ng inter - assay cvs of nesfatin-1 assay were 5.43% and 6.29% for the concentrations of 3.81 and 6.89 ng sensitivity of the assay was 0.021 ng / ml and the upper limit of the standard was 4 ng / ml . measurements were carried out using an elisa plate reader ( biotek instruments inc , winooski , vt , usa ) and plate washer . the standard curve was drawn by plotting the mean absorbance ( y ) of standards against the known concentration ( x ) of standards in logarithmic scale , using the 4-parameter algorithm . the distribution of continuous variables for normality was tested with a one - sample kolmogorov - smirnov test and data are presented as mean standard deviation ( sd ) or median and interquartile ranges , as appropriate . differences between individuals with and without psoriasis vulgaris in normally and nonnormally distributed variables were evaluated by the unpaired t - test and the mann the wilcoxon signed ranks test was used to compare the serum endocan and nesfatin-1 levels in patients with pre - and post - treatment status . our study included 39 moderate - to - severe plaque - type psoriasis patients ( 17 males , 22 females ) and 49 healthy controls ( 24 males , 25 females ) . the mean ages of psoriasis patients and controls were 3416 and 3811 years , respectively . the demographic , clinical characteristics , and biochemical measurements of this study are summarized in table 1 . fasting blood glucose ( p=0.43 ) , creatinine ( p=0.08 ) , high - density lipoprotein ( hdl ) ( p=0.06 ) , and triglyceride ( p=0.74 ) levels were statistically similar between controls and psoriasis patients before nb - uvb phototherapy . white blood cell and neutrophile cell counts and nlr ( figure 1 ) were significantly higher in psoriasis patients before phototherapy ( p=0.001 , p<0.001 , and p<0.001 , respectively ) when compared to controls . however , lymphocyte , hemoglobin , platelet , and mean platelet volume levels were similar between the 2 groups ( table 1 ) ( p>0.05 ) . there was no significant difference between psoriasis patients and controls in terms of serum levels of endocan before nb - uvb phototherapy ( p>0.05 ) . in addition , no significant difference was detected between before - after nb - uvb phototherapy in psoriasis patients in serum levels of endocan ( p>0.05 ) ( table 2 ) . there was no significant difference between controls and psoriasis patients before phototherapy in serum levels of nesfatin-1 ( p>0.05 ) . of the patient group , the median serum nesfatin-1 levels increased after nb - uvb phototherapy , but was not significantly different from baseline ( p>0.05 ) ( table 3 ) . there was no significant difference in nlr before and after nb - uvb phototherapy in psoriasis patients ( p>0.05 ) . the biochemical measurements of psoriasis patients before vs. after nb - uvb phototherapy are summarized in table 1 . the correlation analyses in psoriasis patients showed that serum endocan levels were significantly correlated with pretreatment pasi score ( p : 0.006 , r : 0.483 ) . the main findings of our study were that there was no significant difference in serum endocan and nesfatin-1 levels between psoriasis patients and controls , but there was a significant difference in nlr . psoriasis patients had significantly higher nlr before nb - uvb phototherapy compared to the control group . it is remarkable that serum endocan levels were significantly correlated with disease activity before treatment . in addition , nb - uvb phototherapy did not affect nlr , serum endocan , or nesfatin-1 levels in psoriasis vulgaris patients . endocan is released by vascular endothelial cells , distal tubules of the kidneys , bronchi , and submucosal glands of the lungs . endocan expression is regulated by a series of growth factors and cytokines . in vitro endocan expression is induced by tumor necrosis factor - alpha ( tnf- ) and il-1 beta , while ifn - gamma has been shown to inhibit endocan expression induced by tnf- . endocan expression is strongly upregulated by vascular endothelial growth factor a ( vegf - a ) and vegf - c . a study reported that serum endocan levels were elevated in psoriasis patients compared with control subjects and a positive correlation was demonstrated between endocan levels and disease activity , as well as cardiovascular risk . similarly , serum endocan levels are increased in behet s disease patients compared to controls and endocan was reported to be a novel marker of disease activity . unlike these previous studies , the present study found no statistically significant difference in serum endocan levels between psoriasis patients and the control group . nesfatin-1 is a peptide released from the peripheral fat tissue , as well as from the central and peripheral nervous systems . it suppresses food intake and stimulates insulin release from pancreatic cells ; therefore , nesfatin-1 has been suggested to be a potential substance that could be used in the treatment of obesity and diabetes mellitus . nesfatin-1 has different effects on many organs and organ systems of the human body , such as the immune system , cardiovascular system , nervous system , and endocrine system . on the other hand , the effects of nesfatin-1 on dermatological diseases such as psoriasis have not yet been elucidated . there is no study in the literature that has evaluated nesfatin-1 levels in psoriasis patients . in the present study , there was no significant difference in serum nesfatin-1 levels between psoriasis patients and the control group , and we found that nb - uvb phototherapy did not affect serum nesfatin-1 levels . in recent years , nlr has been regarded as a marker of inflammation in cardiac and non - cardiac disorders [ 2628 ] and there have been many studies on nlr in inflammatory dermatological disorders . one study investigated nlr and platelet - to - lymphocyte ratio ( plr ) in patients with psoriasis or psoriatic arthritis , reporting a positive correlation between nlr , plr , and severity of disease in psoriasis patients . in addition , nlr and plr were higher in psoriatic arthritis compared to psoriasis patients . ataseven et al . reported that nlr levels were higher in psoriasis patients as compared to a control group ; however , there were no significant correlations between nlr and pasi . sen et al . reported nlr and high - sensitivity crp ( hscrp ) levels were higher in psoriasis patients than in a control group , and found a positive correlation between pasi levels and nlr and hscrp levels . in the present study , nlr levels were higher in psoriasis patients as compared to the control group , in agreement with previous reports . also , nb - uvb phototherapy did not affect nlr in patients with psoriasis . in this regard , the present study is the first to investigate the effects of nb - uvb phototherapy on nlr . this is the first study in the literature that evaluated nesfatin-1 levels in psoriasis patients , and it is also the first published study that evaluated the effects of nb - uvb phototherapy on nlr , serum endocan , and nesfatin-1 levels . the present study shows that nlr was higher in pretreatment psoriasis vulgaris patients when compared with controls , but serum endocan and nesfatin-1 levels were not significantly different . we found that serum endocan levels were significantly correlated with disease activity in pretreatment patients . in addition , nb - uvb phototherapy did not affect nlr , serum endocan , or nesfatin-1 levels . more comprehensive and larger - scale studies are needed to gain further insights into the role of endocan and nesfatin-1 in psoriasis pathogenesis and the effects of nb - uvb phototherapy on these molecules . | backgroundpsoriasis is an autoimmune , inflammatory , and chronic disease .
recent studies have evaluated serum endocan and nesfatin-1 levels in patients with inflammatory disorders .
the neutrophil - to - lymphocyte ratio ( nlr ) is an inflammatory marker currently used in many diseases .
the aim of the present study was to evaluate nlr , serum endocan , and nesfatin-1 levels in psoriasis vulgaris before and after narrow - band ultraviolet b ( nb - uvb ) phototherapy treatment and compared to healthy controls.material/methodsthis study was conducted on a total of 88 cases , 39 of which had psoriasis vulgaris and 49 were healthy volunteers .
thirty - nine psoriasis vulgaris patients underwent nb - uvb phototherapy treatment for 3 months .
nlr , serum endocan , and nesfatin-1 levels were measured in all psoriasis patients before and after nb - uvb phototherapy and in the control group.resultscompared with the control group , neutrophil count and nlr were significantly higher ( p<0.001 ) in psoriasis patients before nb - uvb phototherapy .
serum endocan levels were significantly correlated with disease activity before treatment .
there was no significant difference in nlr , serum endocan , and nesfatin-1 levels in psoriasis patients before and after nb - uvb phototherapy ( p>0.05).conclusionsthe current study shows that nlr was higher in psoriasis vulgaris patients when compared with the control group , whereas serum endocan and nesfatin-1 levels were not significantly different .
in addition , nb - uvb phototherapy did not affect nlr , serum endocan , or nesfatin-1 levels .
further larger - scale studies are required on this subject . |
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hematuria is a common problem in patients with polycystic kidney disease ( pckd ) , with 42% of patients experiencing at least one episode of gross hematuria . although self - limited , cumulative episodes of gross hematuria may have an unfavorable impact on long - term renal function . here we describe a case of recurrent severe hematuria in pckd treated successfully on six occasions with tranexamic acid ( ta ) , an antifibrinolytic agent that competitively inhibits the activation of plasminogen to plasmin . a 41-year - old caucasian woman was diagnosed at age 12 with autosomal dominant pckd . she presented to the emergency room 10 days after normal spontaneous vaginal delivery with a four - day history of gross hematuria and left flank pain . she was pale with hypoxemic o2 saturation 96% on 3 l nasal cannula , and she had fever ( 38.7 ) with normal bp and pulse rate ( bp 136/90 mmhg . she had high jvp ( 6 cm above the sternal angle ) , normal heart sounds , bilateral basal crackles on chest examination and markedly enlarged irregular nontender kidneys . mmol / l and creatinine 315 mol / l , egfr ( mdrd ) 15 ml / min/1.73 m. inr was 1.9 , ptt , and platelets were normal . unenhanced ct scan showed massively enlarged kidneys with multiple cysts ; there were several cysts bilaterally with high attenuation consistent with hemorrhage but no stones , masses , or tumours . mri showed a large hemorrhagic cyst within the left kidney but otherwise the same findings as the ct scan . patient underwent cystoscopy , and blood was seen coming from the left ureteric orifice , with no evidence of obstruction . the patient was treated for possible sepsis and urinary tract infection with intravenous antibiotics : cefazolin 1 gm every 12 hours , clindamycin 600 mg every 8 hours , and ciprofloxacin 400 mg daily . blood and urine cultures were negative , and antibiotics were stopped on the 2nd day of admission . the hematuria was managed conservatively with vitamin k 10 mg subcutaneously daily for 5 days , darbepoietin 80 mcg subcutaneously once weekly , plus 4 units of plasma to correct the coagulopathy . because of high surgical risk with poor cardiac status and the long - term impact of nephrectomy on kidney function , antifibrinolytic therapy was given with ta ( cyklokapron , pfizer canada inc . , qc ) 11 mg / kg ( 1000 mg ) orally three times a day for one day then once daily for 5 days . the hematuria stopped within 24 hours and did not recur after ta therapy was stopped . kidney function improved ; serum creatinine fell to 224 mol / l on discharge . she presented five days after discharge with dyspnea and bilateral lower limb edema and was thought to have a deep venous thrombosis . doppler ultrasound of the leg veins was normal but she again developed hematuria which persisted despite stopping the dalteparin . she was treated with ta 11 mg / kg orally twice a day for one day then once daily for 5 days . the patient presented 2 years later with a ten - day history of gross hematuria and left - sided flank pain . she had been given co - trimoxazole one ds tablet twice a day for five days for presumed urinary tract infection . g / l and 360 mol / l , respectively , a month before . coagulation profile was normal , urine grew normal flora , and co - trimoxazole was stopped . she was treated conservatively and received total of 4 units of packed red blood cells , and ddavp 20 mcg intravenously daily for 3 doses . ta 15 mg / kg was given 3 times a day for 3 doses then daily for 3 doses . six months later she presented with one - week history of gross hematuria . despite darbepoetin therapy she was transfused with 2 units packed red blood cells and given ta 10 mg / kg intravenously daily for 3 doses . the hematuria stopped within a day , and she was discharged with hemoglobin 91 g / l and renal function went back to baseline . twelve and sixteen months after her last admission , she presented with painless hematuria and was treated as an out patient with ta 11 mg / kg orally daily , and her hematuria resolved within 2436 hours after the first dose on both occasions . the patient progressed to end - stage renal disease and was started on hemodialysis four years after the initial presentation . spontaneous cyst bleeding is important to consider in this particular group of patients ; it can be prolonged by local activation of fibrinolysis by urokinase . the management of hematuria in pckd is usually conservative with bed rest , blood transfusion , correction of coagulopathies , use of ddavp , and erythropoietin stimulating protein . nonetheless , other modalities have been tried to avoid prolonged hospitalization and nephrectomy and to preserve kidney function . these include other antifibrinolytic agents such as aprotinin and epsilon aminocaproic acid ( eaca ) . we report rapid cessation of hematuria in pckd on four occasions with administration of ta , a potent fibrinolysis inhibitor . ta or trans-4-(aminomethyl ) cyclohexanecarboxylic acid is approximately 10 times more potent in binding to plasminogen than eaca and is better tolerated . it is a synthetic amino acid which reversibly blocks the lysine binding sites on plasminogen ; these are important for binding to fibrin and subsequent activation by plasminogen activators and fibrinolysis . in the setting of renal impairment ta it has also proved to be beneficial as adjunctive therapy in major upper gi bleeding including that occurring in dialysis patients . successful ta therapy of life - threatening hematuria in pckd has been reported previously in a single patient . ta was given at a dose of 20 mg / kg iv followed by 20 mg / kg orally every 2 days for 2 months . the nausea on this occasion may be due to the use of ta at a higher dose on the first day . in addition it could have been due to reduced renal function with transient uremia . in all four episodes kidney function either stabilized or improved after ta therapy . this is probably due to correction of a prerenal element since ta has no direct effect on kidney function in patients with chronic renal impairment . renal hematuria is considered a contraindication for ta treatment , because it may cause bladder and ureteral occlusion by clots . in addition there are reports of acute renal cortical necrosis after use of ta . in one case , a 37-year - old man with baseline serum creatinine of 80 mol / l received high dose ta to treat hemoptysis ( 3 gm iv daily for five days ) . l on the 6th day . in another case a 21-year - old man with hemophilia a who received ta for epistaxis at the same high dose ( 3 gm iv daily for 4 days ) , 3 days later his creatinine was 336 mol / l and after 6 days serum creatinine has risen to 716 mol / l . we believe that this complication may be due to the use of high dose ta over several days . however , in a recent review , tengborn questions the association of ta use and thrombosis given the lack of excess cases in ta studies involving cardiac surgery , total knee and hip arthroplasty , bleeding disorders of pregnancy , and menorrhagia . this risk is sometimes thought to be higher in hemophiliac patients with hematuria . however in our case the patient had no known history of bleeding disorder . ta accumulates in the presence of reduced kidney function as it is excreted by glomerular filtration . the usual dose is 10 mg / kg three or four a times a day . andersson et al . recommended dose reduction of ta in patients with renal impairment as follows : for serum creatinine of 120250 mol / l 10 mg / kg intravenously twice daily ; for serum creatinine of 250500 mol / l 10 mg / kg intravenously once daily ; for serum creatinine above 500 mol / l 10 mg / kg intravenously every 48 hr . similar doses have been successfully used in dialysis patients with upper gi bleeding . our patient received a range of ta doses from 15 mg / kg intravenously three times a day to 10 mg / kg once a day orally ; all were equally effective . we recommend the use of oral ta in the lowest possible effective dose for the shortest duration to reduce the risk of adverse effects . for our patient it is possible that ta therapy of less than three days duration might be just as effective but this has not been examined . early use of ta at home by patients with recurrent hematuria with pckd would be very cost effective if hospitalization could be prevented . ta can be used safely in some patients with chronic renal impairment and pckd to treat severe hematuria poorly responsive to conventional therapy . ta dose can be given orally or iv ; dose adjustment for renal impairment is important . | a 41-year - old woman with autosomal dominant polycystic kidney disease had chronic kidney disease class iv .
she presented 10 days postpartum with a 4-day history of severe hematuria , left flank pain , and anemia , hemoglobin 62
g / l .
ct scan showed massively enlarged kidneys with multiple cysts ; several cysts bilaterally had high attenuation consistent with hemorrhage .
hematuria persisted over several days despite intensive conservative measures that included vitamin k1 , 4 units of plasma , transfusion of 10 units of packed rbcs , darbopoeitin , and ddavp .
antifibrinolytic therapy was given with tranexamic acid 1000 mg p.o .
t.i.d for one day then od . the hematuria stopped within 24 hours and did not recur after tranexamic acid therapy ended .
over the next 4 years there were 3 hospitalizations each with severe gross hematuria requiring blood transfusion for acute anemia .
the hematuria responded well to further treatment with tranexamic acid .
tranexamic acid produces antifibrinolytic effects via complex interactions with plasminogen , displacing plasminogen from the fibrin surface .
chronic renal impairment is considered a relative contraindication to use of tranexamic acid due to reports of ureteric clots and acute renal failure from cortical necrosis .
we conclude that tranexamic acid can be used safely in some patients with ckd and polycystic kidney disease to treat severe hematuria . |
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implantable cardioverter defibrillators ( icds ) are currently an effective and accepted treatment for improving the outcomes of selected patients with ischemic and nonischemic cardiomyopathy with heart failure and severe left ventricular dysfunction.1 , 2 , 3 , 4 however , in the major randomized controlled trials determining guideline recommendations , women were markedly underrepresented.1 , 2 , 3 , 4
accordingly , the existence of sexrelated differences in outcomes among icd recipients is still controversial . while in north american registries,5 women seem to experience a lower incidence of appropriate therapies , these data were not confirmed in a large dutch singlecenter prospective cohort study6 and in the recent nationwide israeliicd registry.7
data on sexrelated survival differences are also contradictory . a large north american registry5 and the israeliicd registry7 have shown no differences in allcause death , while a 35% lower mortality was observed in women of the singlecenter dutch cohort.6
realworld data from european registries addressing these issues is still absent . in the present article , we aim to determine the proportion of female icd recipients , as well as differences in terms of characteristics at implant and outcomes ( therapies , overall and specific mortalities ) in women compared to men . we selected 5539 patients from the daipp study ( dfibrillateur automatique implantable prvention primaire ; nct01992458 ) for this analysis . to qualify for the study , patients had to be at least 18 years old at the time of icd implantation . overall , between 2002 and 2012 , all patients with ischemic cardiomyopathy or nonischemic cardiomyopathy , implanted with an icd ( biventricular , single chamber , or dual chamber ) in the setting of primary prevention in 12 reference french centers were considered and enrolled in the daipp followup program . primary prevention was defined when no prior history of sudden cardiac arrest and/or ventricular tachycardia / fibrillation was documented . ischemic cardiomyopathy was defined as presence of myocardial dysfunction in the context of previous myocardial infarction and/or history of coronary artery disease with or without revascularization ( angioplasty or bypass surgery ) . exclusion criteria included all patients having an icd implant for secondary prevention purposes or for primary prevention without structural heart disease ( including brugada , long qt syndrome , among others ) or structural heart disease other than ischemic or nonischemic cardiomyopathy ( hypertrophy cardiomyopathy , noncompaction cardiomyopathy , and arrhythmogenic right ventricular dysplasia ) . the study was funded by public sources , including the french institute of health and medical research ( inserm ) and the french society of cardiology , and was coordinated by clinique pasteur , toulouse and the paris cardiovascular research center , european georges pompidou hospital , paris , in france . the study complied with the declaration of helsinki , and the data file of the daipp study was declared to and authorized by the french data protection committee ( commission nationale informatique et libert , cnil ) . all variables at the time of the procedure were defined and categorized according to the literature or common practice . in addition to the new york heart association ( nyha ) functional class , we collected the etiology of the underlying heart disease ( ischemic cardiomyopathy or nonischemic cardiomyopathy ) . glomerular filtration rate was estimated with the formula of cockroft gault and categorized in 2 categories ( 60 and < 60 ml / min ) ; qrs duration was categorized as < 120 and 120 ms . atrial fibrillation ( af ) was defined as a history of af ( paroxysmal or persistent ) , documented on standard ecg or 24hour holter monitoring . comorbidities at the time of icd implantation were systematically collected from review of medical records : cancer , chronic obstructive pulmonary disease , chronic renal failure , chronic liver disease , history of transient ischemic neurological attack , and others ( including diabetes mellitus ) . the type of icd device implanted ( biventricular , single chamber or dual chamber no indication on manufacturers ) was recorded . data on device programming was not collected and was left to the discretion of individual investigators , according to each patient 's needs . programming rules were based on high detection windows , as suggested by local guidelines at the time.8 furthermore , french guidelines did not recommend special programming adjustments , namely , cutoffs for therapy zones , based on sex . information on medications at hospital discharge included blockers , amiodarone , ic class antiarrhythmic agents , sotalol , digoxin , calcium blockers , angiotensinconverting enzyme inhibitor or angiotensin ii receptor blocker , diuretics , antiplatelets , and vitamin k antagonists . followup information was obtained from appointments every 4 to 6 months for device evaluation , according to french guidelines.8 the different end points were occurrence of appropriate therapies , early complications , inappropriate shocks , as well as overall and specific mortalities . device interrogation printouts were checked by the local investigator for appropriate and inappropriate icd therapy . appropriate icd therapy was defined as an episode of ventricular tachycardia / ventricular fibrillation resulting in a single or multiple shocks or / and antitachycardia pacing for arrhythmia termination . the date of the first appropriate icd therapy was recorded , and the overall cumulative number of appropriate therapies was considered . adjudication as appropriate or inappropriate therapy was undertaken by the local electrophysiology ( ep ) investigator . early complications ( defined as those that appeared throughout the first 30 days after device implantation ) included leadrelated complications ( eg , failure of coronary sinus lead placement , rise of threshold , lead dislodgment with or without need of reintervention , phrenic nerve stimulation ) , bleeding ( ecchymosis , hematoma , other bleeds requiring transfusion , and procedurerelated anemia ) , sepsis , cardiac tamponade , pneumothorax , and death . vital status data were obtained from the hospital or the general practitioner , and were systematically controlled through the national institute of statistics economical studies ( insee ) . causes of death were obtained from the investigators and/or by the french center on medical causes of death ( cpidc inserm ) . inserm is an academic public institution focused on the analysis of circumstances and causes of death based on death certificate and medical records . causes of deaths were classified according to the international classification of diseases ( 10th revision ) . this information was reviewed by 2 investigators and causes of death were adjudicated after consideration of all the available information , and according to the following prespecified groups : cardiovascular ( including progressive heart failure death , stroke ) , noncardiovascular , icdunresponsive sudden cardiac death ( arrhythmic or not arrhythmic whenever the assessment was possible ) , icdrelated death , as well as unknown when the quality of the information could not allow the investigators to appropriately identify cause of death . overall , cause of death assessment was possible among 682 patients ( out of 826 deceased , 82.6% ) . preparation of this report was in accordance with the strengthening the reporting of observational studies in epidemiology ( strobe ) statement for reporting of observational studies.9
comparisons were performed between men and women . chisquare was used for comparison of nominal variables and student t test for continuous variables ; the levene 's test was used to check the homogeneity of variance ; when appropriate , nonparametric equivalent , mann when baseline differences were present , adjustment was performed using multivariate analysis with binary logistic regression . we compared the adjusted outcomes of appropriate shock and death in men and women by using cox proportional hazards regression analysis . hazard curves were traced for comparison of men and women after adjustment for baseline differences . data were filled into a predefined data introduction electronic sheet made available to all participant centers . after completion of followup , data from all centers were merged and analyzed at the paris cardiovascular research center ( inserm u970 , cardiovascular epidemiology unit ) using sas program v9.3 ( sas institute inc , cary , nc ) . all variables at the time of the procedure were defined and categorized according to the literature or common practice . in addition to the new york heart association ( nyha ) functional class , we collected the etiology of the underlying heart disease ( ischemic cardiomyopathy or nonischemic cardiomyopathy ) . glomerular filtration rate was estimated with the formula of cockroft gault and categorized in 2 categories ( 60 and < 60 ml / min ) ; qrs duration was categorized as < 120 and 120 ms . atrial fibrillation ( af ) was defined as a history of af ( paroxysmal or persistent ) , documented on standard ecg or 24hour holter monitoring . comorbidities at the time of icd implantation were systematically collected from review of medical records : cancer , chronic obstructive pulmonary disease , chronic renal failure , chronic liver disease , history of transient ischemic neurological attack , and others ( including diabetes mellitus ) . the type of icd device implanted ( biventricular , single chamber or dual chamber no indication on manufacturers ) was recorded . data on device programming was not collected and was left to the discretion of individual investigators , according to each patient 's needs . programming rules were based on high detection windows , as suggested by local guidelines at the time.8 furthermore , french guidelines did not recommend special programming adjustments , namely , cutoffs for therapy zones , based on sex . information on medications at hospital discharge included blockers , amiodarone , ic class antiarrhythmic agents , sotalol , digoxin , calcium blockers , angiotensinconverting enzyme inhibitor or angiotensin ii receptor blocker , diuretics , antiplatelets , and vitamin k antagonists . followup information was obtained from appointments every 4 to 6 months for device evaluation , according to french guidelines.8 the different end points were occurrence of appropriate therapies , early complications , inappropriate shocks , as well as overall and specific mortalities . device interrogation printouts were checked by the local investigator for appropriate and inappropriate icd therapy . appropriate icd therapy was defined as an episode of ventricular tachycardia / ventricular fibrillation resulting in a single or multiple shocks or / and antitachycardia pacing for arrhythmia termination . the date of the first appropriate icd therapy was recorded , and the overall cumulative number of appropriate therapies was considered . adjudication as appropriate or inappropriate therapy was undertaken by the local electrophysiology ( ep ) investigator . early complications ( defined as those that appeared throughout the first 30 days after device implantation ) included leadrelated complications ( eg , failure of coronary sinus lead placement , rise of threshold , lead dislodgment with or without need of reintervention , phrenic nerve stimulation ) , bleeding ( ecchymosis , hematoma , other bleeds requiring transfusion , and procedurerelated anemia ) , sepsis , cardiac tamponade , pneumothorax , and death . vital status data were obtained from the hospital or the general practitioner , and were systematically controlled through the national institute of statistics economical studies ( insee ) . causes of death were obtained from the investigators and/or by the french center on medical causes of death ( cpidc inserm ) . inserm is an academic public institution focused on the analysis of circumstances and causes of death based on death certificate and medical records . causes of deaths were classified according to the international classification of diseases ( 10th revision ) . this information was reviewed by 2 investigators and causes of death were adjudicated after consideration of all the available information , and according to the following prespecified groups : cardiovascular ( including progressive heart failure death , stroke ) , noncardiovascular , icdunresponsive sudden cardiac death ( arrhythmic or not arrhythmic whenever the assessment was possible ) , icdrelated death , as well as unknown when the quality of the information could not allow the investigators to appropriately identify cause of death . overall , cause of death assessment was possible among 682 patients ( out of 826 deceased , 82.6% ) . preparation of this report was in accordance with the strengthening the reporting of observational studies in epidemiology ( strobe ) statement for reporting of observational studies.9
comparisons were performed between men and women . chisquare was used for comparison of nominal variables and student t test for continuous variables ; the levene 's test was used to check the homogeneity of variance ; when appropriate , nonparametric equivalent , mann when baseline differences were present , adjustment was performed using multivariate analysis with binary logistic regression . we compared the adjusted outcomes of appropriate shock and death in men and women by using cox proportional hazards regression analysis . hazard curves were traced for comparison of men and women after adjustment for baseline differences . data were filled into a predefined data introduction electronic sheet made available to all participant centers . after completion of followup , data from all centers were merged and analyzed at the paris cardiovascular research center ( inserm u970 , cardiovascular epidemiology unit ) using sas program v9.3 ( sas institute inc , cary , nc ) . the mean age of the sample was 62.511.2 years . among the 5539 patients included in the study , most ( 60.2% ) had an ischemic cardiomyopathy and af was present in 24.0% . mean left ventricular ejection fraction and nyha class were 26.77.2% and 2.40.7% , respectively . cardiac resynchronization therapy with defibrillator ( crtd ) more information on baseline sample data and medical treatment at the time of implant can be found in table 1 . aa indicates antiarrhythmic agent ; acei , angiotensinconverting enzyme inhibitor ; arb , angiotensinii receptor blocker ; crtd , cardiac resynchronization therapy with defibrillator ; gfr , glomerular filtration rate ; icd , implantable cardioverter defibrillators ; lvef , left ventricular ejection fraction ; nyha , new york heart association . number of comorbidities among the following : cancer , chronic kidney disease , chronic lung disease , hepatic failure , diabetes mellitus , and previous stroke . women accounted for 15.1% ( n=837 ) of the whole study sample and had higher prevalence of patients in higher nyha classes ( nyha iii : 54.2% versus 47.8% , p=0.014 ) . men presented a higher prevalence of af , ischemic cardiomyopathy , and had a higher prevalence of narrow qrs complex width ( qrs < 120 ms ) . no significant differences were found regarding age , mean left ventricular ejection fraction , and number of comorbidities . on univariate analysis , women were more frequently implanted with crtd devices ( 61.0% versus 52.5% ; p<0.001 ) . however , after adjustment for baseline differences , this became no longer significant ( odds ratio=1.28 , 95% ci 0.981.66 ; p=0.07 ) . conversely , men received amiodarone , vitamin k antagonists , and antiplatelet agents more frequently and had a trend for higher use of calcium channel blockers . however , after adjustment for baseline differences , only spironolactone was used more frequently among women ( odds ratio=1.30 , 95% ci 1.061.59 ; p=0.014 ) , whereas all other drugs presented similar use among men and women ( table 1 ) . no significant differences were observed in the occurrence of early complications ( 12.8% versus 13.3% ; p=0.789 ; adjusted odds ratio=1.00 , 95% ci 0.751.32 ; p=0.992 ) ( figure 1 ) . except for a higher incidence of pneumothorax in women ( 0.6% versus 1.9% , p<0.001 ) , the remaining complication types were evenly distributed between sexes : bleeding ( 4.8% versus 3.2% ) , cardiac tamponade ( 0% in both groups ) , infection ( 1.0% versus 0.8% ) , leadrelated ( 3.4% versus 4.2% ) , and death ( 0.2% versus 0.1% ) ( all p = ns ) . overall view of outcomes in the male and female sex . during 16 786 patientyears of followup , corresponding to a median of 994 days ( 95% ci 512623 without differences between sexes , p=0.997 ) , appropriate therapies were documented in 1181 ( 22.3% ) patients , corresponding to an annual incidence of 8.2 per 100 patientyears ( 95% ci 7.88.7 ) ( table 2 ) . on univariate analysis , women had a significantly lower likelihood of receiving appropriate therapies ( 23.0% versus 17.4% ; p<0.001 ) . after adjustment for all baseline intersex differences , on multivariate coxregression female sex remained an independent predictor of lower incidence of appropriate therapies ( hazard ratio [ hr]=0.59 , 95% ci 0.450.76 ; p<0.001 ) ( figure 2 ) . even though the presence of crt was not significantly associated with appropriate therapies ( hr=0.978 , 95% ci 0.801.20 , p=0.830 ) , analysis of different device strata suggests a larger effect size and more pronounced reduction in the incidence of appropriate therapies in female crtd recipients versus men ( hr=0.49 , 95% ci 0.340.70 ; p<0.001 ) , than in their single and dualchamber icd counterparts ( hr=0.76 , 95% ci 0.521.11 ; p=0.159 ) ( table 2 ) . study outcomes in the global sample : analysis by sex and device interaction crtd indicates cardiac resynchronization therapy with defibrillator ; dddicd , dual chamber deviceimplantable cardioverter defibrillators ; hr , hazard ratio ; or , odds ratio ; vvi , single chamber device . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . adjusted event curves for first appropriate therapy . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of cardiac resynchronization therapy with defibrillator , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . regarding inappropriate shocks , on univariate analysis , no sexrelated benefit was observed ( 6.7% versus 6.7% ; odds ratio=1.00 , 95% ci 0.741.35 , p=0.997 ) . on binary logistic regression , after adjustment for baseline differences , the absence of sexspecific interaction was confirmed . death occurred in 15.2% ( 6.0 per 100 patientyears ; 95% ci 5.66.3 ) . despite the fact that women experienced a slightly lower annual mortality rate ( 5.0 per 100 patient years versus 4.2 per 100 patient years ) on univariate coxregression , after adjustments for all baseline differences , this was no longer observed : female sex was not an independent predictor of mortality ( hr=0.87 , 95% ci 0.661.15 ; p=0.324 ) . by contrast , af ( hr=1.41 ; 95% ci 1.131.77 , p=0.003 ) , nyha iii ( hr=2.25 ; 95% ci 1.812.79 , p<0.001 ) , left ventricular ejection fraction < 30% ( hr=1.50 ; 95% ci 1.211.86 , p<0.001 ) , ischemic heart disease ( hr=1.66 , 95% ci 1.342.00 , p<0.001 ) , and glomerular filtration rate 60 ml / min ( hr=2.31 , 95% ci 1.882.84 , p<0.001 ) were associated with increased mortality . on coxregression multivariate analysis , when restricting the analysis only to patients implanted with crtds , a significant survival benefit was seen in women implanted with a crtd ( hr=0.68 , 95% ci 0.470.97 ; p=0.034 ) ( table 2 ) . in their single and dualchamber icd counterparts , this was not observed ( hr=1.50 , 95% ci 0.962.34 ; p=0.072 ) . meier curves illustrating this interaction ( figure 3 ) show that male and female patients implanted with crtds diverge right from the beginning . on the other hand , for female and male patients implanted with single and dualchamber icds , curves are overlapping for the first 4 years , and suddenly diverge after that , at a time where only close to 100 female patients are being followed . these curves also show that male patients with crtds do much worse than males implanted with single and dual chamber icds . crtd indicates cardiac resynchronization therapy with defibrillator ; hr , hazard ratio ; icd , single or dualchamber implantable cardioverter defibrillator . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . the specific analysis of causes of death ( table 3 ) , after adjustment for baseline differences , revealed a significant interaction of sex with crt as regards nonarrhythmic cardiovascular mortality : women with single and dualchamber icd implants fared worse than men ( hr=2.49 , 95% ci 1.404.45 , p=0.002 ) , but those implanted with crtds were less likely to die of nonarrhythmic cardiovascular causes ( hr=0.50 , 95% ci 0.350.99 , p=0.048 ) . causes of death : analysis by sex and device interaction crtd indicates cardiac resynchronization therapy with defibrillator ; dddicd , dual chamber deviceimplantable cardioverter defibrillators ; hr , hazard ratio ; icd , implantable cardioverter defibrillators ; nyha , new york heart association ; vvi , single chamber device . adjusted for baseline differences in nyha class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . women accounted for 15.1% ( n=837 ) of the whole study sample and had higher prevalence of patients in higher nyha classes ( nyha iii : 54.2% versus 47.8% , p=0.014 ) . men presented a higher prevalence of af , ischemic cardiomyopathy , and had a higher prevalence of narrow qrs complex width ( qrs < 120 ms ) . no significant differences were found regarding age , mean left ventricular ejection fraction , and number of comorbidities . on univariate analysis , women were more frequently implanted with crtd devices ( 61.0% versus 52.5% ; p<0.001 ) . however , after adjustment for baseline differences , this became no longer significant ( odds ratio=1.28 , 95% ci 0.981.66 ; p=0.07 ) . conversely , men received amiodarone , vitamin k antagonists , and antiplatelet agents more frequently and had a trend for higher use of calcium channel blockers . however , after adjustment for baseline differences , only spironolactone was used more frequently among women ( odds ratio=1.30 , 95% ci 1.061.59 ; p=0.014 ) , whereas all other drugs presented similar use among men and women ( table 1 ) . no significant differences were observed in the occurrence of early complications ( 12.8% versus 13.3% ; p=0.789 ; adjusted odds ratio=1.00 , 95% ci 0.751.32 ; p=0.992 ) ( figure 1 ) . except for a higher incidence of pneumothorax in women ( 0.6% versus 1.9% , p<0.001 ) , the remaining complication types were evenly distributed between sexes : bleeding ( 4.8% versus 3.2% ) , cardiac tamponade ( 0% in both groups ) , infection ( 1.0% versus 0.8% ) , leadrelated ( 3.4% versus 4.2% ) , and death ( 0.2% versus 0.1% ) ( all p = ns ) . overall view of outcomes in the male and female sex . during 16 786 patientyears of followup , corresponding to a median of 994 days ( 95% ci 512623 without differences between sexes , p=0.997 ) , appropriate therapies were documented in 1181 ( 22.3% ) patients , corresponding to an annual incidence of 8.2 per 100 patientyears ( 95% ci 7.88.7 ) ( table 2 ) . on univariate analysis , women had a significantly lower likelihood of receiving appropriate therapies ( 23.0% versus 17.4% ; p<0.001 ) . after adjustment for all baseline intersex differences , on multivariate coxregression female sex remained an independent predictor of lower incidence of appropriate therapies ( hazard ratio [ hr]=0.59 , 95% ci 0.450.76 ; p<0.001 ) ( figure 2 ) . even though the presence of crt was not significantly associated with appropriate therapies ( hr=0.978 , 95% ci 0.801.20 , p=0.830 ) , analysis of different device strata suggests a larger effect size and more pronounced reduction in the incidence of appropriate therapies in female crtd recipients versus men ( hr=0.49 , 95% ci 0.340.70 ; p<0.001 ) , than in their single and dualchamber icd counterparts ( hr=0.76 , 95% ci 0.521.11 ; p=0.159 ) ( table 2 ) . study outcomes in the global sample : analysis by sex and device interaction crtd indicates cardiac resynchronization therapy with defibrillator ; dddicd , dual chamber deviceimplantable cardioverter defibrillators ; hr , hazard ratio ; or , odds ratio ; vvi , single chamber device . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . adjusted event curves for first appropriate therapy . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of cardiac resynchronization therapy with defibrillator , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . regarding inappropriate shocks , on univariate analysis , no sexrelated benefit was observed ( 6.7% versus 6.7% ; odds ratio=1.00 , 95% ci 0.741.35 , p=0.997 ) . on binary logistic regression , after adjustment for baseline differences , the absence of sexspecific interaction was confirmed . death occurred in 15.2% ( 6.0 per 100 patientyears ; 95% ci 5.66.3 ) . despite the fact that women experienced a slightly lower annual mortality rate ( 5.0 per 100 patient years versus 4.2 per 100 patient years ) on univariate coxregression , after adjustments for all baseline differences , this was no longer observed : female sex was not an independent predictor of mortality ( hr=0.87 , 95% ci 0.661.15 ; p=0.324 ) . by contrast , af ( hr=1.41 ; 95% ci 1.131.77 , p=0.003 ) , nyha iii ( hr=2.25 ; 95% ci 1.812.79 , p<0.001 ) , left ventricular ejection fraction < 30% ( hr=1.50 ; 95% ci 1.211.86 , p<0.001 ) , ischemic heart disease ( hr=1.66 , 95% ci 1.342.00 , p<0.001 ) , and glomerular filtration rate 60 ml / min ( hr=2.31 , 95% ci 1.882.84 , p<0.001 ) were associated with increased mortality . on coxregression multivariate analysis , when restricting the analysis only to patients implanted with crtds , a significant survival benefit was seen in women implanted with a crtd ( hr=0.68 , 95% ci 0.470.97 ; p=0.034 ) ( table 2 ) . in their single and dualchamber icd counterparts , this meier curves illustrating this interaction ( figure 3 ) show that male and female patients implanted with crtds diverge right from the beginning . on the other hand , for female and male patients implanted with single and dualchamber icds , curves are overlapping for the first 4 years , and suddenly diverge after that , at a time where only close to 100 female patients are being followed . these curves also show that male patients with crtds do much worse than males implanted with single and dual chamber icds . crtd indicates cardiac resynchronization therapy with defibrillator ; hr , hazard ratio ; icd , single or dualchamber implantable cardioverter defibrillator . adjusted for baseline differences in new york heart association class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . the specific analysis of causes of death ( table 3 ) , after adjustment for baseline differences , revealed a significant interaction of sex with crt as regards nonarrhythmic cardiovascular mortality : women with single and dualchamber icd implants fared worse than men ( hr=2.49 , 95% ci 1.404.45 , p=0.002 ) , but those implanted with crtds were less likely to die of nonarrhythmic cardiovascular causes ( hr=0.50 , 95% ci 0.350.99 , p=0.048 ) . causes of death : analysis by sex and device interaction crtd indicates cardiac resynchronization therapy with defibrillator ; dddicd , dual chamber deviceimplantable cardioverter defibrillators ; hr , hazard ratio ; icd , implantable cardioverter defibrillators ; nyha , new york heart association ; vvi , single chamber device . adjusted for baseline differences in nyha class , atrial fibrillation , ischemic cardiomyopathy , qrs width , use of crtd , treatment with blockers , amiodarone , spironolactone , calcium channel blockers , antiplatelet agents , and vitamin k antagonists . our multicentric realworld data show that similar to randomized controlled trials10 and other registries of icd recipients in daily clinical practice,11 , 12 , 13 women account for only a minority of our cohort . also , they present with a different clinical profile , with higher prevalence of nonischemic cardiomyopathy , more advanced heart failure , broader qrs complex width , and lower prevalence of af . women , overall , presented with lower incidence of appropriate icd therapies and similar mortality compared with men . however , our results suggest that differences in sexrelated outcomes in primary prevention icds may be observed among crt recipients . whereas female crtd recipients present with lower mortality and incidence of appropriate icd interventions than their male counterparts , outcomes of single and dualchamber icd recipients seem to be more comparable . our findings , suggesting a lower incidence of appropriate icd therapies among women , have also been reproduced in other studies : the prospective ontario registry5 and a singlecenter north american propensitymatched observational study.14 however , only the latter has shown that this effect was more pronounced among crtd recipients.14 this is similar to what we observed in our cohort , where the lower incidence of appropriate therapies among women was mostly driven by a significant difference in crtd recipients . we believe these differences may partially result from different clinical risk profiles or ethnic / regional factors in the different samples . therefore , identifying specific subgroups of primary prevention icd female recipients who can derive higher benefit from this therapy may be of interest . the underlying causes for the more favorable arrhythmic profile of women are not entirely clear but may be related to the lower propensity to sustained ventricular tachycardia in the nonischemic heart failure setting15 , 16 but also for ischemic cardiomyopathy associated sudden cardiac death.17 , 18 , 19 in our sample , despite having more women with nonischemic cardiomyopathy , the overall reduction in arrhythmic burden was still present even after adjustment for baseline differences . several mechanisms have been proposed for sex differences regarding the risk of ventricular arrhythmia : higher resting heart rate , different autonomic response to stress , degree of vagal activation , differences in cardiac repolarization , hormonal differences affecting arrhythmic vulnerability , genetic variants influencing qt interval length or adrenergic receptors , and even nutritional factors , adherence to a lowrisk lifestyle , and behavorial and psychological factors.20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 overall , the interplay between vulnerable substrate ( underlying structural and electric heart substrate ) , triggers , and autonomic tone may be slightly different.30
the reason for the sexrelated benefit among women implanted with crtd is still unclear . arhsad et al have proposed 2 hypothetical explanations32 : first , a likely possible greater risk of progression to overt heart failure among women , resulting in a greater preventive benefit of crtd therapy . second , women may have , on average , a 10 ms shorter qrs duration than men in subjects without heart disease.33 therefore , on a relative basis , for the same degree of qrs duration , more pronounced conduction disturbances and cardiac dyssynchrony may occur in women , explaining the higher response to cardiac resynchronization therapy . thus , a higher prevalence of response to crt and reverse remodeling , as observed in the maditcrt trial,32 can possibly explain the survival and arrhythmic benefit of our population . regarding inappropriate shocks , all existing data have found a similar risk in both men and women,5 , 9 , 34 as we have observed . furthermore , we have assessed the underlying reasons for inappropriate therapies and found no intersex differences ( namely , a similar incidence of supraventricular tachycardia despite the fact that af was more frequent among men ) . macfadden and colleagues reported a 50% significantly higher ( 5.4% versus 3.3% ) occurrence of any major or minor complications in women at 45day followup.5 recent data from the united states national cardiovascular data registry icd registry show that devicerelated complications are more common in women ( 7.2% versus 4.8% ; p<0.001).35 unlike the latter study , we observed no differences regarding 30day mortality and observed a much lower rate of cardiac tamponade . possible explanations for the observed lack of sexrelated differences and the overall higher complication rate in our sample may be the very inclusive definition of end points such as bleeding events and leadrelated complications , and having local investigatoradjudicated end points instead of using the centers for medicare and medicaid services inpatient claims . lastly , the use antiplatelet agents and oral anticoagulants in our sample was more common in males , which may account for the numerically , but not statistically significant , higher rate of bleeding observed in male patients . as regards mode of death , we have observed that the incidence of specific causes of death in women , namely , nonarrhythmic cardiovascular mortality , may depend on the type of implanted device . in single or dualchamber icd recipients , nonarrhythmic cardiovascular death occurred more commonly in women whereas in those implanted with a crtd it happened less frequently . these findings are contrary to previously published data showing no sexrelated differences in cause of death.6 , 36 we believe this may occur because these 2 studies have smaller samples , and therefore are not statistically powered to assess for interactions between sex , device type , and specific mortalities . in patients who are eligible for an icd , it is known that sudden cardiac death seems to occur less frequently in women.37 our data seem to suggest that after device implantation , the favorable arrhythmic profile may still exist because , notwithstanding the similar incidence of icd unresponsive sudden death , women present with fewer appropriate therapies , which can be considered , to a certain level , a sudden cardiac death surrogate . second , device programming may have been a source of some interindividual variability , but this factor has been minimized by proposed programming rules recommending no differences based on sex.8 last , no central adjudication for classification of appropriate and inappropriate therapies was used in this registry . second , device programming may have been a source of some interindividual variability , but this factor has been minimized by proposed programming rules recommending no differences based on sex.8 last , no central adjudication for classification of appropriate and inappropriate therapies was used in this registry . in our reallife registry , women seem to account for the minority of icd recipients and present with a different clinical profile . even though the incidence of early complications and inappropriate shocks was similar , lower allcause mortality and incidence of appropriate therapies was observed in female crtd recipients compared with men . among single and dualchamber icd recipients , the incidence of icd therapies was comparable but nonarrhythmic cardiovascular death was more common in women . the following investigators and institutions participated in the conception of the registry , and in the organization , collection , storage , and analysis of the data : coprincipal investigators : serge boveda , md , clinique pasteur , toulouse ; eloi marijon , md , phd , hpital europen georges pompidou , paris , france . coinvestigators in charge of the data collection and analysis at each medical center : vincent algalarrondo , md , phd , chu antoine bclre , clamart ; dominique babuty , md , phd , chu trousseau , tours ; pierre bordachar , md , phd , chu haut lvque , bordeaux ; abdeslam bouzeman , md , serge boveda , md , rui providncia , md , phd , clinique pasteur , toulouse ; pascal defaye , md , chu michallon , grenoble ; daniel gras , md , nouvelles cliniques nantaises , nantes ; jeanclaude deharo , md , phd , chu la timone , marseille ; didier klug , md , phd , chru lille , lille ; christophe leclercq , md , phd , chu pontchaillou , rennes ; eloi marijon , md , phd ; hpital europen georges pompidou , paris ; olivier piot , md , centre cardiologique du nord , saint denis ; nicolas sadoul , md , phd , chu brabois , nancy . data storage , quality control , and statistical analyses : frankie beganton , ms , marieccile perier , mph , cardiovascular epidemiology unit , paris cardiovascular research center ( inserm unit 970 ) , hpital europen georges pompidou , paris . steering committee : serge boveda , md , clinique pasteur , toulouse ; pascal defaye , md , chu michallon , grenoble ; christophe leclercq , md , phd , chu pontchaillou , rennes ; eloi marijon , md , phd ; hpital europen georges pompidou , paris ; nicolas sadoul , md , phd , chu brabois , nancy . this work was supported by the following independent institutions : the toulouse association for the study of rhythm disturbances ; the french institute of health and medical research ; and the french society of cardiology ( nct 01992458 ) . dr providencia has received training grant from boston scientific , and sorin group and a research grant from medtronic . dr babuty has received travel support and clinical study support from biotronik , boston scientific , medtronic , st . dr sadoul has received personal fees from biotronik , boston scientific , medtronic , sorin group , and st . dr boveda has received consulting fees from medtronic , boston scientific , and sorin group . all other authors have reported that they have no relationships relevant to the contents of this article to disclose . | backgroundthere are limited data describing sex specificities regarding implantable cardioverter defibrillators ( icds ) in the realworld european setting.methods and resultsusing a large multicenter cohort of consecutive patients referred for icd implantation for primary prevention ( 20022012 ) , in ischemic and nonischemic cardiomyopathy , we examined the sex differences in subjects ' characteristics and outcomes .
of 5539 patients , only 837 ( 15.1% ) were women and 53.8% received cardiac resynchronization therapy . compared to men , women presented a significantly higher proportion of nonischemic cardiomyopathy ( 60.2% versus 36.2% , p<0.001 ) , wider qrs complex width ( qrs > 120 ms : 74.6% versus 68.5% , p=0.003 ) , higher new york heart association functional class ( iii in 54.2% versus 47.8% , p=0.014 ) , and lower prevalence of atrial fibrillation ( 18.7% versus 24.9% , p<0.001 ) . during a 16 786 patientyears followup , overall , fewer appropriate therapies were observed in women ( hazard ratio=0.59 , 95% ci 0.450.76 ; p<0.001 ) . by contrast , no sexspecific interaction was observed for inappropriate shocks ( odds ratio =0.84 , 95% ci 0.501.39 , p=0.492 ) , early complications ( odds ratio=1.00 , 95% ci 0.751.32 , p=0.992 ) , and allcause mortality ( hazard ratio=0.87 95% ci 0.661.15 , p=0.324 ) .
analysis of sexby cardiac resynchronization therapy interaction shows than female cardiac resynchronization therapy recipients experienced fewer appropriate therapies than men ( hazard ratio=0.62 , 95% ci 0.500.77 ; p<0.001 ) and lower mortality ( hazard ratio=0.68 , 95% ci 0.470.97 ; p=0.034).conclusionsin our reallife registry , women account for the minority of icd recipients and presented with a different clinical profile . whereas female cardiac resynchronization therapy recipients had a lower incidence of appropriate icd therapies and allcause death than their male counterparts , the observed rates of inappropriate shocks and early complications in all icd recipients were comparable.clinical trial registrationurl : https://www.clinicaltrials.gov/. unique identifier : nct01992458
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a finalidade do trabalho foi avaliar as alteraes da microdureza e o desgaste provocado pelo jato de bicarbonato de sdio em esmalte bovino e o posterior efeito remineralizador da saliva artificial . utilizaram - se 15 espcimes de esmalte ( 4,0 mm 4,0 mm ) que constituram os grupos : sem tratamento ( mi ) ; tratamento com jato de bicarbonato de sdio ( mii e di ) ; tratamento com jato de bicarbonato de sdio e imerso em saliva artificial por uma hora ( miii e dii ) , 24 horas ( miv e diii ) e sete dias ( mv e div ) . foram realizados testes de microdureza com um microdurmetro nos grupos m e testes de desgaste com um rugosmetro nos grupos d. os dados foram avaliados pela anlise de varincia a um critrio e pelo teste de tukey . o valor das mdias da microdureza , em khn , nos grupos mi , mii , miii , miv e mv foram 359,80 ; 335,46 ; 369,20 ; 377,73 e 341,86 ; respectivamente , enquanto que os valores mdios , em m , do desgaste para o grupo di , dii , diii e div foram 0,564 ; 0,519 ; 0,441 e 0,428 , respectivamente . o jato de bicarbonato de sdio causou desgaste e diminuio da microdureza superficial ; a saliva promoveu o retorno da microdureza superficial condio inicial e reduziu o desgaste ; o efeito reparador da saliva sobre as alteraes na microdureza superficial j ocorreu com uma hora de tratamento , no havendo diferena estatisticamente significante do efeito obtido com 24 horas ; o melhor efeito reparador da saliva sobre o desgaste ocorreu com 24 horas de tratamento . on the development of dental caries , bacterial plaque , plays an essential role , and thus , both the chemical and mechanical plaque control methods have been enphasized in modern dentistry . one of the available professional and prophylactic methods consists in employing a sodium bicarbonate jet , which acts promoting a mechanical remotion of plaque and also a polishing from the dental surfaces ( professional prophylaxis ) . when a professional prophylaxis is carried out , a wear of the dental surface occurs . several studies4,12,13,16,19 have quantified the ocurrence of sound enamel wear , but there is no consence as to the results , although it is known that the wear is higher when the prophylaxis is performed on the previously demineralized enamel3,5,8 . besides , there is a lack of information about the consequences related to this procedure on the superficial microhardness of enamel . however , it is known that when there is a mineral loss on a tooth , a remineralization by the action of saliva occurs 1,10,17 . saliva contains in its composition the main components of dental structure , as calcium and phosphate having a protective action on enamel and dentine 11 . although it is undoubtfull the benefit for caries prevention resulting from the plaque control , it is known that : the mechanical plaque remotion causes a certain wear of the enamel surface which quantification is not completely clarified yet ; there is a lack of information on what happens to the enamel microhardness submitted to the prophylaxis ; the enamel alterations originated from prophylaxis can be minimized by the remineralizator power of saliva , but it is still questionable how much of repair occurs and in which time span it occurs . the present study intended to contribute to a better understanding of those aspects having as a purpose : to evaluate in vitro whether the application of the sodium bicarbonate jet on bovine enamel promotes wear and reduction of its surface microhardness and which is the effect of artificial saliva , in different periods of action , on the repairing of the possible occurred alterations . after the remotion of the roots from 30 incisors bovine , the crowns were imbedded in thermoactivated godiva in a cristal acrilic plate , which has been coupled to a precision cutting device ( isomet low speed saw ) . with the aid of two duble faced diamond discs xli 2205 , " high concentration " , and a stainless steel spreader ( 7 cm diameter , 4 mm wide , and central orifice 1.3 cm ) between the discs , 30 enamel specimens with 4 mm x 4 mm taken from the plane portion of the crown , making a double secction in cervico - incisal direction and other in the mesiodistal direction . then , the enamel blocks were fixed with sticking wax in the center of a cristal acrilic disc ( 30 mm x 8 mm ) , with the purpose of firstly perform the dentine planification . the set disc / tooth was adapted in a metalographic polishing device ( apl4 , arotec , cotia , sp ) . for the planification a carbide silicon sandpaper granulation 320 ( extec corp . ) following , the blocks were inverted and now fixed with the enamel placed on the upside face . the set was adapted to the polishing device and the enamel was polish with a carbide silicone sandpaper ( extec corp . ) granulation 600 , and further with a granulation 1200 sandpaper . in order to finish the polishing , a wet felt with a diamond suspention of 1m ( buehler ) , was used . the initial enamel surface microhardness was assessed using a microhardness measuring machine ( hmv-2000/shimadzu corporation , japan ) coupled to a microcomputer and a software specific for analysing images ( cams - win - new age industries / usa ) . a knoop indenter was used , with static load of 25 g , applied for 5 seconds . in each sample the blocks which microhardness was 10% over or under the average of all blocks , and those whose standard deviation was 10% above the value of its own mean were discarted . from the 30 specimens , the research was carried out with the same 15 bovine enamel specimens wich were submited to surface microhardness and enamel wear tests . for the study related to the surface microhardness ( m ) and enamel wear ( d ) five groups of specimens were constituted , according to the stage of treatment they were , such as : mi group no treatment ; mii and di groups treatment with sodium bicarbonate jet ; groups miii and dii treatment with sodium bicarbonate jet and immersion in artificial saliva for one hour ; miv and diii groups - treatment with sodium bicarbonate jet and immersion in artificial saliva for 24 hours ; mv and div groups - treatment with sodium bicarbonate jet and immersion in artificial saliva for seven days . for the treatment with sodium bicarbonate jet , half of the surface of each specimen was protected with red nail polishing . on the other half was applied the sodium bicarbonate jet ( dabi atlante industrias mdico odontolgicas ltda ) with a distance of 5 mm from the bovine enamel , during 10 seconds , with a 90 angulation , without interruption . after this procedure , the nail polishing film was removed and , in this right moment , the specimens constituted the groups mii and di . the enamel surface was evaluated with a rugosimeter hommel tester t 1000 ( hommelwerke , gmbh , alte tuttinger strebe 20 d-7730 vs schwenningen ) which was connected to a microcomputer . with the aid of a equipment software ( turbo datawin - nt version 1.34 , copwright 2001 ) , besides the rugosity data , the measurement was carried out in five different sites of each block and the mean was obtained . twenty ml of saliva were utilized which were individually stored in covered plastic vials , in incubator at 37 c , for different periods of time , constituting the groups miii and dii ( 1 hour ) , miv and diii ( 24 hours ) and mv and div ( 7days ) . the obtained data were submmited to a statistical procedure by the variance analysis at one criterium and by the tukey test . the mean value ( khn ) of superficial microhardness of bovine enamel , in the diferent experiment estages , can be seen in table 1 . same letters denote no statistically significant difference ( p>0.05 ) by tukey test there was a statistically significant reduction in the enamel superficial microhardness following the treatment with sodium bicarbonate jet ( mii ) , when compared to the initial superficial microhardness . data have shown that the permanency of the specimens in artificial saliva permited that the enamel surface microhardness , which was low after the prophylaxis ( mii ) , has returned to the initial condition , because the values , both from group miii , and group miv as well as group mv , did not present statistically significant differences related to the values of initial surface microhadness ( mi ) . considering the immersion period of time of the specimens in saliva , there was no statistically significant difference in enamel superficial microhardness in group miii ( 1 hour ) , compared to group miv ( 24 hours ) . table 2 contain the mean values ( m ) of the wear observed in bovine enamel , in the different phases of the experiment . same letters denote no statistically significant difference ( p>0.05 ) by tukey test the simulation of prophilaxis ( di ) caused a enamel wear of 0.564 m which was repaired by the treatment with saliva . within one hour immersion of the specimens in saliva ( dii ) the wear value had reduced to 0.519 m , but the difference compared to the wear obtained following the prophylaxis simulation ( di ) was not statistically significant . the treatment with saliva for 24 hours ( diii ) and by seven days ( div ) presented better results , because the wear values in these groups were lower than the value found in the specimens immediately after the prophilaxis simulation procedure ( di ) at a level statistically significant . this research was carried out with the same 15 bovine enamel specimens which were submitted to sucessive treatments . the employed methodology permitted this procedure . in the wear testing , the rugosimeter was used , which does not alter the tested surface allowing the further performance of other stages of the experiment . in the microhardness test , the knoop indenter was used , which neither produces distortions nor damages the enamel structure15 . these methods have made possible to evaluate sucessive alterations occurring in a same specimen . the enamel surface profile was evaluated by a rugosimeter . as the bovine enamel was polished , the surface outline not treated by the sodium bicarbonate jet was similar to a straight line and thus , the alteration of this line observed on the enamel expressed the result of the prophylaxis simulation . by the literature , it was possible to infer that , even though the enamel abrasion has not been considerable , it has been observed after prophylaxis procedures . although there is report of wear on the enamel of deciduous tooth with the sodium bicarbonate jet9 , it was not verified abrasion in sound enamel of permanent tooth with this procedure 3,6,13,16 . the wear caused by the sodium bicarbonate jet only occurred in human demineralized enamel3 . as the human enamel is less porous than the bovine enamel 2,14,20 , results of wear of both of them must not be compared without a careful understanding of this fact . in bovine enamel , it was evaluated the wear caused by the sodium bicarbonate . gerbo7 ( 1993 ) had observed no wear , whereas honrio8 ( 2003 ) and fraga5 ( 2005 ) , verified the presence of rugosity , which was higher on enamel previously demineralized than on the sound enamel . in the present study , it was found a mean wear of 0.564m , following the simulation of prophylaxy with sodium bicarbonate jet . this value is higher than the one reported by honrio8 ( 2003 ) , which was of 0.418m , and by fraga5 ( 2005 ) which verified a wear of 0.319 m . it is known that small alterations on the enamel surface can reflect in its physical properties , one of them is the microhardness . in this study the microhardness mean value of the initial bovine enamel surface , before any treatment , was 359.80 khn , very close to the value reported by newbrun ; timberlake ; pigman15 ( 1959 ) , and higher than the value reported by fraga5 ( 2005 ) which was of 300.47 khn and lower compared to honrio8 , wich was 394.0 khn . with the treatment of profylaxis simulation with sodium bicarbonate jet , the bovine enamel microhardness has reduced from 359.80 khn to 335.46 khn , being the difference statistically significant . there was , therefore a loss of enamel hardness . in a research carried out by fraga5 ( 2005 ) the application of the sodium bicarbonate jet has not altered the superficial microhardness of sound bovine enamel , but its mean value on the enamel presenting artificial carious lesion increased in a significant level , following the simulation of prophylaxis . it is difficult to explain how , working in similar conditions , there was a discordance of results as to the sound enamel surface microhardness observed in the present study as well as in fragas study5 . the lack of previous studies in the literature , assessing the effect of sodium bicarbonate on the enamel related to the microhardness , impossibilitates a comparison of the values found in this research . this makes difficult to afirm if the result obtained here was the one expected or not . once that , in this in vitro study , alterations on the bovine dental enamel were found by the action of sodium bicarbonate jet , it is important to consider that if those alterations can occur in the clinical practice , although minimally , it can be expected the return to the initial conditions , simply by the fact that the tooth is constantly bathed by saliva . in the present study , the specimens were treated by artificial saliva by periods of one hour , 24 hours , and 7 days . these periods of time were based on the study carried out by fraga5 ( 2005 ) due to the lack of informations in the literature about the right moment when the effects of saliva can be detected in the repairing of the alterations resulting from the prophylaxis in sound enamel . fraga5 ( 2005 ) could not detect any alterations in the microhardness value in sound teeth subjected to the prophylaxis simulation with sodium bicarbonate jet . thus it is natural that the further treatment with saliva has not been reflected in this value . only with the immersion period in saliva for 28 days fraga5 ( 2005 ) obtained an alteration in wear and microhardness of enamel . in the present research , in which there was a reduction in initial microhardness value following the treatment with the sodium bicarbonate jet , when the specimens were immersed in saliva , it could really be verified its effect in the restitution of the tissue integrity . the specimens immersion in saliva , for just one hour , was enough for the microhardness to reach a value that was not different , statistically significant , from the initial value . the same has occurred with the immersion of the blocks in saliva for 24 hours and for 7 days , permiting to afirm that the repairing of the alterations generated by simulation prophylaxis already occurs , right after the contact with the saliva . the treatment with saliva also had a repairing effect on the wear . with one hour of the specimens immersion in saliva , the wear value of 0.564m caused by the prophylaxis simulation was reduced to 0.519m , although the difference has not been statistically significant . however the treatment with saliva by 24 hours , had an expressive reduction of wear , which value was 0.441 with statistically significant difference from the value found after the application of the sodium bicarbonate jet . there was no higher value in the result of wear with the extended period of immersion of the specimens in saliva for 7 days . great discrepancy was found between these results and the fragas results5 which study the immersion period of 4 hours in saliva was not sufficient to alter the microhardness and the wear of hygid bovine enamel . only with 28 days period of the specimens immersion in saliva , fraga5 ( 2005 ) however , the too long interval between the two observations ( 4 hours and 28 days ) did not permit to know which was the period of time necessary to visualize an effect of treatment . the results of the current research are in agreement to the johansson10 ( 1965 ) . the repairing effect of saliva , in the present work on the alterations on superficial microhardness and wear enamel was revealed in , at most , 24 hours , and , with the aditional time of 7 days no benefit was noted . in the study of johansson10 ( 1965 ) using demineralized human teeth , the remineralizing process by saliva occurred rapidly within the first 24 hours and there was no increment of mineral deposition with the immersion in saliva by a period of up to 3 weeks . a possible explanation for this fact is that , with the passing time , must occur an ionic difusion blocking toward the inner enamel , due to the mineral deposition in the most external layer . this immediate remineralizing of enamel , when saliva or artificial remineralizing solutions with high calcium concentration are used , was verified in a literature review carried out by silverstone19 ( 1977 ) . also , gaard ; ten bosch18 ( 1994 ) observed in vivo that the remineralization in the presence of saliva is a relatively rapid process . although it is not known , in which extension , the obtained results can be transfered to the clinical reality , the indication that the remineralizing process occurs very fast , simply by the action of saliva , this fact gives the dentist reassurement to perform the professional prophylaxis how many times it is necessary . the application of sodium bicarbonate jet caused a wear and a reduction in the surface microhardness on bovine enamel . the repairing effect of saliva on the surface microhardness alterations has occured as fast as one hour of treatment , presenting no statistically significant difference from the effect obtained with 24 hours . the wear resulting from the application of sodium bicarbonate jet were repaired in , at most , 24 hours , following the immersion in artificial saliva . | purpose : the aim of the present study was to evaluate the alterations of surface microhardness and wear caused by the sodium bicarbonate jet on bovine enamel and the further remineralizing effect of artificial saliva.methods:fifteen enamel samples ( 4,0 mm 4,0 mm ) were used , which constituted the groups : no treatment ( mi ) ; treatment with sodium bicarbonate jet ( mii and di ) ; treatment with sodium bicarbonate jet and immersion in saliva for one hour ( miii and dii ) , 24 hours ( miv and diii ) and 7 days ( mv and div ) .
microhardness tests were carried out using a microdurometer in groups m and wear tests by a rugosimeter in groups d. the data were assessed by the one criterion variance analysis and tukey test.results:the mean value of microhardness , in khn , in groups mi , mii , miii , miv and mv were 359,80 ; 335,46 ; 369,20 ; 377,73 and 341,86 , respectively , whereas the mean values in m , of wear for group di , dii , diii and div were 0,564 ; 0,519 ; 0,441 and 0,428 , respectively.conclusions:the sodium bicarbonate jet caused a wear and a reduction in microhardness on the enamel surface ; saliva promoted the recovery of initial condition surface microhardness and reduced the wear ; the repairing effect of saliva on the surface microhardness alterations occurred within one hour of treatment , having no significant statistical difference from the effect obtained in 24 hours ; the best saliva repairing effect on the wear occurred with treatment of 24 hours . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the eukaryotic genome is under constant mutational stress through exposure to exogenous and endogenous agents that damage dna . these external and internal factors damage dna introducing a wide variety of genetic alterations including : deletions , translocations , and chromosome loss , which can result in cell death . one type of genetic alteration , dna double - stranded breaks ( dsbs ) , poses a serious threat to cell viability and genome stability if left unrepaired or repaired incorrectly . in order to repair these dsbs , cells have evolved the complex repair pathways : nonhomologous end - joining ( nhej ) and homologous recombination ( hr ) [ 1 , 2 ] . in mammalian cells , the majority of dsbs produced by ionizing radiation are repaired by nhej , whereas dsbs produced by replication fork collapses are repaired by hr [ 24 ] . the nhej pathway occurs mainly in the g0 and g1 phase of the cell cycle by joining dsb ends directly or after limited processing , whereas hr uses a sister homolog as a template for repair during late s and g2 phases . encompassing these repair pathways the ddr is responsible for sensing dna damage and activating signaling cascades necessary to initiate repair and to stall damaged cells until the damage is resolved [ 68 ] . regulation of repair proteins in nhej , hr , and ddr pathways is key to maintaining proper repair mechanisms and eliminating defects in these pathways that can cause genomic instability and promote tumorigenesis . regulating repair proteins through posttranslational modification , such as phosphorylation , provides molecular switches for regulating dsb repair . phosphorylation is the addition of a phosphate ( po4 ) group to a protein in order to induce a conformational change that initiates protein activation or deactivation . in dna repair proteins , phosphorylation has been found to occur on serine and threonine residues , but it can also occur on tyrosine , histidine , arginine , or lysine residues as well . phosphorylation of dna repair proteins generally results in activation of the proteins to facilitate dna repair . the mechanism of reversible phosphorylation in proteins is an important regulatory mechanism for dna repair pathways . the primary goal of nonhomologous end - joining ( nhej ) is to resolve dsbs generated by exogenous and endogenous agents that damage dna . key factors for facilitating proper repair through nhej include the ku70/80 heterodimer , the dna - dependent protein kinase catalytic subunit ( dna - pkcs ) , artemis , x - ray repair complementing defective repair in chinese hamster cells 4 ( xrcc4 ) , dna ligase iv , and xrcc4-like factor ( xlf ) . as shown in figure 1 , nhej proceeds through three key steps : recognition of the break , dna processing to remove nonligatable ends or other forms of damage at the termini , and finally ligation of the dna ends . structural studies of the ku heterodimer reveal that it exists as a ring structure that binds dna , interacts tightly with dna - pkcs forming the protein complex dna - dependent - protein kinase , dna - pk . first , the ku70/80 heterodimer recognizes and binds the dsb , recruiting dna - pkcs to the ends of the dsb and inducing inward translocation of ku . dna - pkcs protects the dna ends from exonuclease activities and stimulates juxtaposition of dna ends [ 13 , 14 ] . secondly , depending on the type and complexity of the dsb break , the dna ends are processed by different processing factors such as , artemis , polynucleotide kinase 3-phosphatase ( pnkp ) , dna polymerases , or the mrn complex ( mre11/rad50/nijmegen breakage syndrome 1 ( nbs1 ) ) . once bound to the broken ends , dna - pkcs become activated via autophosphorylation , dissociate from the dsbs , and phosphorylate other targets including replication protein a ( rpa ) , wrn , artemis , and sometimes h2ax [ 1618 ] . lastly , xlf stimulates the xrcc4/dna ligase iv complex to seal the dna ends [ 2 , 9 , 19 ] . researchers have recently implicated dna - pk as a central regulator of dna end access via phosphorylation . dna - pk is composed of dna - pkcs and the ku 70/80 subunits , which act as the catalytic and regulatory subunits , respectively . initially , the ku subunits recognize dsbs and recruit dna - pkcs , forming the dna - pk complexes on both ends of the dsb . assembly of the dna - pk complex induces a conformational change in dna - pkcs such that two globular domains interact creating channels that enclose the two dna ends and protect them from degradation . next , the ku heterodimer , which exists as a ring structure , translocates to a more interior position on the dna strand . finally , autophosphorylation of dna - pkcs must occur before repair of the dsb [ 20 , 21 ] . the catalytic subunit of dna - pk , dna - pkcs , is responsible for the kinase activity . dna - pkcs is a member of the phosphatidylinositol-3-kinase - like ( pikk ) family of serine / threonine protein kinases , which also includes ataxia telangiectasia - mutated ( atm ) and atm- and rad3-related ( atr ) . in response to dna damage , dna - pkcs phosphorylation occurs on t2609 which causes colocalization with ku 70/80 at the dsb site . in order to release dna termini , dna - pkcs undergo autophosphorylation on clusters of serine and threonine residues to mechanistically regulate dna end access during nhej . these dna - pkcs residues are phosphorylated by dna - pkcs itself and occasionally by atm or atr , which causes the release of dna - pkcs from the dna and allows nhej to proceed [ 24 , 25 ] . regulation of dna - pkcs autophosphorylation and subsequent removal from dna termini appears to be essential to the continuation of nhej . several studies have discovered two functionally relevant autophosphorylation clusters : abcde ( t2609 , s2612 , t2620 , s2624 , t2638 , and t2647 ) and pqr ( s2023 , s2029 , s2041 , s2051 , s2053 , and s2056 ) [ 13 , 18 ] . these sites have been studied by ablating the phosphorylation sites via alanine substitutions or by creating mutants that mimic the phosphorylated sites via aspartic acid substitutions . phosphorylated mutants with any one or two of the conserved sites mutated to aspartic acid on the abcde or pqr clusters are sufficient for nhej to proceed . however , mutations that ablate the phosphorylation of all sites of the pqr or abcde clusters impart a severe to modest radiosensitive phenotype , respectively . both cluster mutants are fully functional kinases and are similar to wild - type dna - pkcs in every aspect except end - processing . autophosphorylation of the abcde cluster promotes end - processing , whereas pqr cluster autophosphorylation inhibits end - processing . therefore , autophosphorylation of the abcde cluster is responsible for dna - pkcs dissociation from ku and dsbs , allowing downstream repair factors to access the damaged dna . conversely , pqr cluster autophosphorylation retains dna - pkcs at damaged dna ends and limits processing of the ends by downstream repair factors . thus , it appears that autophosphorylation within one cluster opposes the effects of autophosphorylation within the other cluster [ 13 , 21 , 2628 ] . additionally , dna - pkcs contains a phosphorylation site within the activation loop ( t site ) of the kinase ( t3950 ) . mimicking phosphorylation at the t site inactivates the kinase and promotes severe defects in both nhej and homologous recombination . this suggests that dephosphorylation of dna - pkcs at t3950 may be required for dna - pk activity and function . mutants with alanine substitutions at the abcde cluster , pqr cluster , and t site autophosphorylate at 40% of wild - type levels and undergo autophosphorylation - induced dissociation . recent research has indicated three more phosphorylation sites which appear to play a role in dna - pk function ( s3821 , s4026 , and t4102 ) ; however no clear biological function in nhej has been determined . in conclusion , dna - pkcs undergoes distinct phosphorylation events that determine functional consequences in nhej [ 13 , 29 , 31 ] . several studies have associated full kinase activation with the combined dna - pk complexes rather than a single dna - pk complex , suggesting that the dna - pkcs must phosphorylate only in trans . these findings suggest that paired ends of dna are required before phosphorylation of dna - pkcs can occur and , subsequently , before other repair enzymes can gain access to damaged dna ends . this mechanism allows for damaged dna ends to be protected by dna - pkcs until the two damaged dna ends are juxtaposed , and dna - pk complexes can coordinate with one another to repair the dsbs [ 13 , 14 ] . additionally , dna - pkcs can phosphorylate many other essential substrates in vitro , such as rpa , h2ax , ku70/80 , xrcc4 , xlf , artemis , dna ligase iv , wrn , and hnrnp - u . however , phosphorylation of many of these substrates does not appear to be functionally important for nhej to repair dsbs in vivo [ 34 , 35 , 38 ] . therefore , the most relevant substrate for dna - pkcs in nhej is dna - pkcs itself . hr repairs dsbs by using homologous sequences elsewhere in the genome to prime repair synthesis , especially dsbs produced by replication fork collapse . hr is considered 100% accurate for repairing dsbs if the repair template is perfectly homologous . homologous sequences in the genome can include sister chromatids , homologous chromosomes , or repeated regions on the same or different chromosomes . however , regulation of hr is key to maintaining genomic integrity . as shown in figure 2 , initiation of hr begins with extensive 5 to 3 end - processing by the mrn complex , exo1 , and other exonucleases . brca1 regulates mrn complex end - processing after phosphorylation and subsequent activation by atm and chk2 , a checkpoint kinase . next , the 3 single - stranded dna ( ssdna ) ends are bound by rpa , which is phosphorylated and actively displaced by rad52 to allow for rad51 binding . brca2-bound rad51 is phosphorylated by cyclin - dependent kinases ( cdks ) , releasing rad51 to bind the ssdna end , forming a nucleoprotein filament that searches for and invades a homologous sequence . once all intermediates are resolved , any ssdna gaps and nicks are closed , and the dsb is repaired . hr - mediated repair utilizes the phosphorylation of rpa , breast cancer type 1 susceptibility protein type 1 and 2 ( brca1 and brca2 ) as molecular switches for regulating dna repair . mammalian replication protein a ( rpa ) is a dna - binding protein that plays an essential role at replication centers as well as stalled replication forks in the hr repair system . , rpa acts to recruit and activate key checkpoint mediators . after dna damage , rpa is recruited to dna - damage - dependent nuclear foci and interacts with repair and recombination factors to resolve the dsb . pikks and cyclin - cdk2 complexes regulate rpa via phosphorylation on the extreme n - terminus of the rpa2 subunit . two of the rpa phosphorylation sites , s23 and s29 , are phosphorylated by cyclin - cdk kinases , and at least five of the other seven sites ( s4 , s8 , s11 , s12 , s13 , t21 , and s33 ) are phosphorylated in response to ionizing radiation , most likely by pikks [ 46 , 47 ] . to better understand the role of rpa as a molecular switch rpa2 mutants that mimic the hyperphosphorylated form are unable to associate with replication centers , but still localize to dna damage foci . therefore , following dna damage , rpa is hyperphosphorylated , limiting dna replication but aiding dna repair by marking sites of dna damage for recruitment of repair factors . the breast cancer susceptibility genes ( brca1 and brca2 ) and rad51 colocalize to sites of dna damage and have a role in both the detection and repair of dsbs . brca1 binds dna directly to activate the repair of dsbs and initiate hr . more specifically , brca1 regulates the length of ssdna generation by inhibiting mre11 activity in the mrn complex . after dna damage is inflicted , brca1 is phosphorylated on the following residues : s1387 by atm , s988 by chk2 , as well as s1423 and s1524 by other kinases [ 7 , 49 , 50 ] . brca2 directly interacts with rad51 through the brct domain on the c - terminus and regulates rad51 function and localization [ 5153 ] . the carboxy - terminal region of brca2 is phosphorylated at s3291 by cyclin - dependent kinases , which blocks c - terminal interactions between brca2 and rad51 . rad51 is then activated to play a major role in hr repair of dsbs by coating ssdna to form a nucleoprotein filament that invades and pairs with a homologous region in duplex dna . brca1 , brca2 , and rad51 are important molecular switches for dna repair that initiate hr . in addition to the nhej and hr pathways , cells possess the dna damage response ( ddr ) which functions as a cellular defense against the accumulation of genetic mutations associated with cancer progression . the ddr safeguards the genomic integrity of cells . after dna damage is inflicted , cells undergo the ddr leading to the activation of cell cycle checkpoints and apoptosis . initially , cells deploy groups of dna damage - sensor proteins to dsbs , which delay cell - cycle progression and activate the correct dna repair pathways . these ddr proteins relay signals via signal transducers to a set of downstream effectors , which affect cellular events such as repair , cell cycle checkpoint , telomere stability maintenance , transcription control , and apoptosis . many proteins in the nhej pathway , hr pathway , and ddr overlap and have critical roles in repair pathways as well as other biological pathways . regulating the phosphorylation of proteins necessary for dna repair is key to maintaining proper repair mechanisms . key factors in the ddr regulate repair via a series of phosphorylation events as seen in figure 3 . members of the pikk family ( atm , atr , and dna - pk ) are first to the dna damage site and phosphorylate histone h2ax . as previously mentioned , dna - pk is also a critical kinase in the nhej pathway . phosphorylated h2ax recruits and aids in the phosphorylation of mediator of damage checkpoint protein 1 ( mdc1 ) . once phosphorylated mdc1 then serves as a platform for further recruitment of ddr factors , including nbs1 and rnf8 . the pikks , atm and atr , play a crucial role in ddr by relaying and amplifying the dsb damage signal . in response to dsbs , both atm and atr phosphorylate a multitude of substrates , including p53 , and the checkpoint kinases , chek1 and chek2 . arresting the cell cycle allows for enzymes to repair the dna before dna synthesis or chromosome segregation initiates . recruitment of pikks to dsbs is required for pikk - dependent signaling and subsequent dna repair . atm and atr are activated and recruited to the site of dsbs via distinct interacting partners in a phosphodependent manner . the activation of the protein kinase atm initiates phosphorylation of dsb repair and cell cycle control proteins . however , efficient activation requires the mrn complex and the autophosphorylation of specific atm serine and threonine residues . atm is recruited by the mrn complex via an interaction between atm and the c - terminus of nbs1 , a component of the mrn complex . atm is constitutively present as an inactive dimer and dissociates into monomers once activated by autophosphorylation , acetylation ( k3016 ) , and recruitment to sites of dsbs [ 6063 ] . several studies have revealed five functionally relevant autophosphorylation sites ( s367 , s1893 , s1981 , t1885 , and s2996 ) [ 6062 ] . phosphorylation mutants at s367 , s1893 , and s1981 are each defective at atm signaling in vivo and fail to correct radiation - induced cell death . additionally , phosphorylation mutants at these sites were defective in correcting the s phase checkpoint defect , and phosphospecific antibodies against s367 and s2996 revealed phosphorylation of both sites was rapidly induced by radiation . therefore , phosphorylation of atm at these five sites is required for activation of atm and subsequent phosphorylation of ddr proteins involved in dna repair and cell cycle control . another pikk , the atr kinase , phosphorylates atr substrates in order to inhibit dna replication and promote dna repair . activation of atr in the ddr requires the recruitment of atr to rpa - coated ssdna breaks via an interacting partner , atr - interacting protein ( atrip ) . disruption of the atr - atrip complex prevents recruitment to the sites of the dsb and leads to defects in repair . also , atr activation requires co - localization of the atr - atrip complex with the rad9-rad1-hus1 complex , which creates a heterotrimeric ring - shaped structure that recognizes a dna end that is adjacent to a stretch of rpa - coated ssdna . the presence of rpa is required for the binding of the complexes and imparts specificity in loading for the dna strands giving a preference to the 5 primer end [ 59 , 64 ] . in response to dna damage , atm and atr phosphorylates ddr proteins which participate in repair of dsbs and cell cycle control . most notably , atm phosphorylates and activates chek2 on the sq / tq cluster domain containing t68 , whereas atr phosphorylates chek1 on s317 and s345 [ 58 , 65 , 66 ] . atm also phosphorylates many other substrates , including but not limited to mdm2 and mdmx , which influence the stabilization of p53 . likewise , atr is capable of phosphorylating brca1 , rad17 , mcm proteins , rpa , pcna , and a multitude of other substrates [ 43 , 67 ] . atm and atr both phosphorylate p53 , atm on s15 and atr on s15 and s37 . both pikks also phosphorylate h2ax , leading to chromatin modification and promoting global genomic nucleotide excision repair [ 67 , 6971 ] . a crucial regulator of the ddr is histone variant h2ax , a member of the h2a family of histones , which packages and organizes dna into chromatin . h2ax is at the center of cellular responses to dna dsbs . in response to dna damage , h2ax is phosphorylated on a conserved serine residue at the carboxyl terminus by pikk family , including atm , atr , and dna - pkcs . phosphorylation of h2ax recruits ddr proteins to regions of damaged dna , leading to delays in the cell cycle and/or dna repair [ 72 , 73 ] . the phosphorylated h2ax , -h2ax , is required for dna damage signal amplification and the accumulation of ddr factors in subnuclear foci called ionizing radiation - inducing foci ( irif ) . the signal amplification perpetuated by h2ax phosphorylation is not limited to the immediate substrate , but instead spreads to a large chromatin region surrounding the dsb . this major amplification begins with a few damaged base pairs and produces nearly 30 mbp span modification of chromatin . in addition , dna dsb repair is enhanced after the phosphorylation of h2ax nucleates the formation of a large complex of factors including mdc1 , rnf8 , brca1 , rap80 , 53bp1 . in order to negatively regulate this amplification process , -h2ax is dephosphorylated by multiple protein phosphatases including pp2a , pp4 , pp6 , and wip1 . this impressive amplification and regulation of this ddr regulator indicate that h2ax phosphorylation plays a major role in the ddr and the subsequent initiation of dsb repair processes . another ddr factor , mediator of damage checkpoint protein 1 ( mdc1 ) , is a large adaptor protein required for irif formation and binds -h2ax through the brct domains . more specifically , mdc1 recognizes a phosphorylation site on the c - terminus of -h2ax . six repeating residue clusters ( sdtdxd / e ) within mdc1 are phosphorylated by casein kinase 2 ( ck2 ) to generate recognition sites for nijmegen breakage syndrome 1 ( nbs1 ) and the rest of the mrn complex ( mre11 and rad50 ) [ 1 , 75 ] . additionally , e3 ubiquitin ligase , rnf8 , is recruited by phosphorylated mdc1 and recruits 53bp1 , brca1 , and rap80 to damage sites through ubiquitination of -h2ax . overall , mdc1 is a key upstream determinant of the ddr that relies on the phosphorylation of h2ax for recruitment as well as phosphorylation of itself to recruit other ddr factors to initiate dna repair and delay cell cycle progression . together , the reversible phosphorylation of dna repair factors provides a foundation for a more complete understanding of the role of molecular switches in dna repair . regulation of repair proteins through posttranslational modification , such as phosphorylation , provides cells with a mechanism for managing dna repair processes . nhej , hr , and ddr operate together to repair damaged dna and utilize phosphodependent binding to begin repair , recruit factors to the damage sites , and initiate signaling cascades . | repair of double - stranded breaks ( dsbs ) is vital to maintaining genomic stability . in mammalian cells ,
dsbs are resolved in one of the following complex repair pathways : nonhomologous end - joining ( nhej ) , homologous recombination ( hr ) , or the inclusive dna damage response ( ddr ) .
these repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate dna repair .
many of these molecular switches overlap and play key roles in multiple pathways .
for example , the nhej pathway and the ddr both utilize dna - pk phosphorylation , whereas the hr pathway mediates repair with phosphorylation of rpa2 , brca1 , and brca2 .
also , the ddr pathway utilizes the kinases atm and atr , as well as the phosphorylation of h2ax and mdc1 .
together , these molecular switches regulate repair of dsbs by aiding in dsb recognition , pathway initiation , recruitment of repair factors , and the maintenance of repair mechanisms . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
chronic subdural hematoma ( sdh ) is known to have a good prognosis after simple burr hole drainage , and thus , most neurosurgeons do not consider chronic sdh seriously . contralateral or bilateral development of an acute subdural hematoma immediately after removal of chronic sdh have been previously reported to be rare but devastating postoperative complications3,12 ) . however , massive intracerebral hemorrhage ( ich ) caused by coagulopathy after evacuation of a recurrent chronic sdh has not been previously reported . here , we report a rare case of fulminant ich after the evacuation of a chronic sdh and include a review of the literature . a 62-year - old man was admitted to our institute with a 3-week history of trivial head injury with complaints of mild headache and left side motor weakness . he was afebrile and results of blood tests , including erythrocyte sedimentation rate and c - reactive protein , were all within normal limits . he had taken low dose aspirin 100 mg per day for 4 years , but routine laboratory test results , which included platelet count , prothrombin time ( pt ) , activated partial thromboplastin time ( aptt ) and bleeding time were within normal limits . brain computed tomography ( ct ) scan depicted a hypodense lesion in the right frontotemporal ( f - t ) region , suggesting chronic sdh with midline shift ( fig . the hematoma was evacuated through one burr hole using a 5-l catheter under local anesthesia ; dark old blood was removed and no evidence of active bleeding was confirmed during the operation . after setting a simple closed system in the subdural space , the wound was closed layer by layer and the operation was completed in the usual manner . on the postoperative ct scan performed at the next day , we could find the resolution of hematoma without midline shifting ( fig . postoperatively , the headache was immediately relieved and the patient was able to ambulate independently without difficulty , after 5-l catheter removal . hematological investigation revealed prolongation of pt prolongation to 14.0 sec but other parameters including fibrinogen were all normal range after trephination surgery . however , 3 days after the operation , the patient 's level of consciousness gradually deteriorated to a drowsy state , and brain ct revealed recurrent sdh on rt . the hematoma was re - evacuated through one burr hole using a 5-l catheter under local anesthesia ; dark old blood was removed and no evidence of active bleeding was also confirmed . after setting a simple closed system in the subdural space , the wound was closed layer by layer and the operation was completed without any problem . after revision surgery , his mental status was recovered to alert and follow - up ct scan at following day after revision surgery revealed the resolution of hematoma without midline shifting ( fig . 4 ) . however , hematological investigation revealed prolongation of pt to 14.1 sec and a decline of fibrinogen to 81 mg / dl ( normal range > 150 mg / dl ) . as a result , cryoprecipitate , fresh frozen plasma , and platelet concentrates were transfused . however , 5 days after revision surgery , the patient 's level of consciousness deteriorated to semicomatose state . brain ct revealed inracerebral hemorrhage in right basal ganglia and with midline shifting ( fig . 5 ) . at this time , despite continuous replacement of coagulation factors , hematological investigation revealed disseminated intravascular coagulation ( dic ) ; pt prolongation ( 14.5 sec ) , decreased fibrinogen ( 27 mg / dl ) , elevated fibrin degradation product test ( 10.5 ml / ml ) and d - dimer ( 575 mg / dl ) . emergency decompressive surgery was planned , but his family refused the operation for profound neurological deficits and hematological laboratory results of dic . despite conservative treatment to lower intracranial pressure and normalize the hematological parameters , his condition deteriorated rapidly , culminating in multiple organ failure and cardiorespiratory arrest with dilated non - reactive pupils . evacuation of a chronic sdh by burr hole drainage using a 5 l - catheter is an effective and minimally invasive technique . although the prognosis of chronic sdh is relatively good , some unusual and devastating complications may occur , such as , subdural hematoma , infection , seizure , hydrocephalus , and failure of the brain to expand , however , postoperative ich is rare8,9,11 ) . intracranial hematoma after chronic sdh removal has been recently reported to be a rare but near devastating postoperative complication3,12 ) . however , most patients with this complication develop an ipsilateral acute subdural hematoma , and massive ich caused by coagulopathy after drainage of chronic sdh has not been previously reported . in our patient , laboratory tests for clotting profiles revealed aggravated dic at 8 days after trephination . dic , whether acute or chronic is usually associated with an underlying causative condition . some diseases and conditions can disrupt the body 's normal blood clotting system and lead to dic . they include sepsis , cancer and massive tissue injury ( surgery and trauma ) . in this case , mori and maeda10 ) reported the surgical results of 500 consecutive patients with chornic sdh treated by burr hole craniotomy with closed drainage system . in his study , three patients ( 0.6% ) died due to dic and he insisted coagulopathy was an ominous indicator of poor prognosis in patients with chronic sdh . the loss of equilibrium among the tightly regulated coagulation factors can lead either to hypercoagulable states with microthrombosis and ischemia or to hypocoagulable states with possible progression of hemorrhagic lesions after traumatic brain injury . kawakami et al.5 ) also reported marked reductions of factors ii , v , vii and x , and antithrombin iii , and an increase in fibrinopeptide a in hematomas compared with venous blood in 19 patients . these results strongly suggest that coagulation is excessively activated regulatory mechanisms for coagulation and fibrinolysis are depressed in chronic sdh . modesti et al.9 ) undertook a detailed clinical review of this entity , reporting a 5% incidence of intracerebral hematomas among 140 surgically treated patients with chronic extracerebral fluid collections . they noticed no initial abnormality in clotting profiles , including bleeding time , pt , aptt , and platelets , in their series , as was observed in our patient . the pathophysiologic mechanisms of intracranial hematomas occurring after evacuation of chronic extracerebral fluid collections are unclear . many pathological events may contribute to the development of an ich after the evacuation of a chronic subdural hematoma . for example , damage to the cerebral vasculature secondary to perioperative parenchymal shift , a sudden increase in blood flow combined with defective vascular autoregulation , and hemorrhage into a previously undetected contusion have been proposed to explain the occurrence of delayed intracranial hematomas or sdh1,2,7 ) . focal cerebral edema beneath the compressed surface of the brain due to impeded venous drainage , can reduce cerebral blood flow in the affected hemisphere . chronic dilatation of small arterial vessels and loss of carbon dioxide reactivity in the ischemic hemisphere could also contribute to the pathogenesis6 ) . as a matter of course , aggravated coagulopathy might exert a baneful influence on recurrence of chronic sdh and intracerebral hematoma . to avoid this catastrophic complication , gradual drainage under a closed system and the effort to prevent the coagulopathy are mandatory . some authors have proposed that twist drill holes with closed drainage provide the safest and most effective means in the presence of a chronic extracerebral fluid collection4 ) . this procedure is effective and provides complete decompression with gradual brain re - expansion , and can also ameliorate rapid dynamic intracranial changes . although delayed intracerebral hemorrhage after drainage of a chronic sdh is a rare complication , it can occur , as demonstrated by our case . it is not easy to predict the possibility of postoperative intracerebral hemorrhage , especially when initial coagulation parameters are normal , and thus , it is important to monitor the patients intensively after surgical evacuation of hematoma . although ich caused by coagulopathy following drainage of a recurrent chronic sdh is an unusual complication , the possibility of ich must be considered when neurologic status deteriorates . the effort to prevent coagulopathy and a slow drainage system appear to be mandatory to prevent this catastrophic complication . | burr hole drainage has been widely used to treat chronic subdural hematomas ( sdh ) , and most of them are easily treated by simple trephination and drainage . however , various complications , such as , hematoma recurrence , infection , seizure , cerebral edema , tension pneumocephalus and failure of the brain to expand due to cerebro - cranial disproportion may develop after chronic sdh drainage . among them ,
intracerebral hemorrhage after evacuation of a recurrent chronic sdh is very rare . here
, we report a fatal case of delayed intracerebral hemorrhage caused by coagulopathy following evacuation of a chronic sdh . possible pathogenic mechanisms of this unfavorable complication are discussed and a review of pertinent literature is included . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
recent successes in gene transfer ( gt ) for ocular applications have revived long - standing hopes for the field . in the 1990s , gt was hailed as a miracle technology and was misleadingly referred to as gene therapy in spite of its experimental nature . despite high hopes , serious setbacks damaged gt 's reputation and resulted in significant public scrutiny . in 2007 , however , phase i trials for leber congenital amaurosis ( lca ) , an infant - onset retinopathy , displayed short - term safety and efficacy . optimistic clinicians described these early results as a paradigm shift in our management of retinal dystrophies of all types , previously thought to be an untreatable group of human diseases . while adults derived some visual benefits in the lca trials ( improvements in visual acuity , visual field , pupillary reaction , and nystagmus ) , children showed the most dramatic responses in later trials indeed , after gt , an 8-year - old child displayed comparable light sensitivity levels to age - matched controls , indicating that early gt application may lead to greater visual improvements . the results therefore suggested a limited therapeutic window for visual gain , a finding that has fueled patient urgency to access gt , especially as lca trials are now entering phase iii ( clinicaltrials.gov nct00999609 ) . while concerns have recently been raised about the long - term efficacy of the intervention in the lca trials , further early - stage gt trials for retinopathies now include stargardt disease ( clinicaltrials.gov nct01367444 ) , retinitis pigmentosa ( clinicaltrials.gov nct01482195 ) , and usher syndrome ( clinicaltrials.gov nct01505062 ) . in october 2011 , a phase i choroideremia trial was initiated in the united kingdom focused on safety and dosage ( clinicaltrials.gov nct01461213 ) . choroideremia is a sex - linked retinopathy affecting ~1 of 50,000 males . in the absence of a treatment , choroideremia causes progressive vision loss from childhood to legal blindness by middle age . positive media coverage of ocular gt of early evidence from the lca trials combined with optimistic statements by researchers and clinicians have ignited patient hopes for a successful treatment for a range of retinopathies . high hopes based on limited data , however , present challenges for communicating about risks of harm and potential benefits of early - stage trials as well as time frames for clinical application of therapies . of greatest concern , communication deficits may compromise the integrity of informed consent for potential trial participants . participants may misunderstand the safety focus of early - stage trials , expecting therapeutic benefit . termed therapeutic misconception ' , patients commonly conflate the goals of research and those of clinical care . similarly , patients may overestimate therapeutic benefits or underestimate risks of harm associated with research , termed therapeutic misestimation ' . therapeutic misconception and misestimation are not only characteristic of patients , but also affect stakeholders involved directly and indirectly in the informed consent process , such as clinical investigators and research oversight bodies . despite the rapid advances in ocular gt , little research has been directed at exploring the values and priorities of key stakeholders such as patients , patient advocates , and clinicians . similar research gaps are evident in the translation of other novel biotechnologies from bench research to clinical trials . our study , therefore , addresses multistakeholder perspectives about ocular gt trials , focusing on choroideremia . it examines the commonalities and differences in views between stakeholder groups ( patients , clinicians , and patient advocates ) . we specifically address the following issues : ( i ) potential benefits and therapeutic hopes ; ( ii ) risks of harm ; ( iii ) urgency to access gt ; and ( iv ) time frames for clinical implementation . we conclude with recommendations for responsible communications for gt trials that counter the historical sensationalism associated with this field of biotechnology . we interviewed 20 north american choroideremia patients ( p ) ; 15 clinicians from north america and europe ( c ) ; and 6 representatives of north american and european patient advocacy organizations ( advocates / a ) . patients were affected males older than 18 years of age who may be eligible to participate in the upcoming choroideremia trials . five of the 15 clinicians , ophthalmologists and genetic counselors who specialized in ocular genetics , were directly involved in ocular gt trials . advocates were representatives from the boards of patient advocacy organizations involved in fundraising for choroideremia research and patient education . we recruited patients from the regional eye centre , royal alexandra hospital , edmonton , and via notices on patient advocacy organization websites . s.b . conducted 45 min to 1 h semistructured interviews with each participant between june 2011 and june 2012 . the interview guides were informed by research on communication in genetics and were reviewed for depth and breadth of coverage by experts in ocular genetics , risk communication , and bioethics . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . patient interviews focused on experiences living with choroideremia ; understanding of diagnosis ; awareness of ocular gt research ; sources of communications about gt ; and views about participation in chm trials . clinician interviews focused on diagnosis ; management strategies for visual impairment ; perspectives on patient hopes ; and communication strategies with other stakeholders . interviews with advocates focused on the supports and information they provided for members ; position on gt ; communication with members , including about gt ; advocacy role ; and external stakeholder communications strategies . the health panel of the ethics review board at the university of alberta approved this study . we analyzed verbatim transcripts of digitally recorded interviews using nvivo 9.1 data analysis software ( qsr international 2010 ) . our qualitative analysis of interview transcripts , using the constant comparison method ' , involved a rigorous and iterative process . coded each line of a subset of transcripts , and analyzed these codes for similarities and differences between transcripts . starting with this close examination of the transcripts , and through ongoing discussions with other investigators , s.b . developed a preliminary codebook representative of key perspectives and ideas . analyzed the remaining transcripts and reexamined the already analyzed transcripts as new codes became apparent . our coding method provided a rich analysis of the data as multiple codes could apply to different dimensions of the same text . as explained by illes et al.,17
the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . a third coder external to the project reviewed the final codebook and transcripts to provide an objective assessment . the external coder suggested collapsing closely related themes , adding nuanced subthemes to emphasize key ideas , and refining the definitions of several constructs . finally , s.b . revised transcript coding again in light of the final codebook . to ensure that we captured participant views accurately , s.b . , i.m . , and t.b . prepared reports explaining the main themes that emerged from the interviews . we sent these reports to participants , inviting them to ask additional questions or provide feedback . we also reviewed all selected quotes in the context of the original transcripts to ensure that they retained their meaning . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . choroideremia trials in north america have not yet started , limiting our patient interviews to potential research participants . finally , we interviewed only a small number of advocates , who had the most heterogeneous perspectives of all stakeholders . we interviewed 20 north american choroideremia patients ( p ) ; 15 clinicians from north america and europe ( c ) ; and 6 representatives of north american and european patient advocacy organizations ( advocates / a ) . patients were affected males older than 18 years of age who may be eligible to participate in the upcoming choroideremia trials . five of the 15 clinicians , ophthalmologists and genetic counselors who specialized in ocular genetics , were directly involved in ocular gt trials . advocates were representatives from the boards of patient advocacy organizations involved in fundraising for choroideremia research and patient education . we recruited patients from the regional eye centre , royal alexandra hospital , edmonton , and via notices on patient advocacy organization websites . the interview guides were informed by research on communication in genetics and were reviewed for depth and breadth of coverage by experts in ocular genetics , risk communication , and bioethics . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . patient interviews focused on experiences living with choroideremia ; understanding of diagnosis ; awareness of ocular gt research ; sources of communications about gt ; and views about participation in chm trials . clinician interviews focused on diagnosis ; management strategies for visual impairment ; perspectives on patient hopes ; and communication strategies with other stakeholders . interviews with advocates focused on the supports and information they provided for members ; position on gt ; communication with members , including about gt ; advocacy role ; and external stakeholder communications strategies . the health panel of the ethics review board at the university of alberta approved this study . we analyzed verbatim transcripts of digitally recorded interviews using nvivo 9.1 data analysis software ( qsr international 2010 ) . our qualitative analysis of interview transcripts , using the constant comparison method ' , involved a rigorous and iterative process . coded each line of a subset of transcripts , and analyzed these codes for similarities and differences between transcripts . starting with this close examination of the transcripts , and through ongoing discussions with other investigators , s.b . developed a preliminary codebook representative of key perspectives and ideas . analyzed the remaining transcripts and reexamined the already analyzed transcripts as new codes became apparent . our coding method provided a rich analysis of the data as multiple codes could apply to different dimensions of the same text . as explained by illes et al.,17
the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . a third coder external to the project reviewed the final codebook and transcripts to provide an objective assessment . the external coder suggested collapsing closely related themes , adding nuanced subthemes to emphasize key ideas , and refining the definitions of several constructs . finally , s.b . revised transcript coding again in light of the final codebook . to ensure that we captured participant views accurately , s.b . , i.m . , and t.b . prepared reports explaining the main themes that emerged from the interviews . we sent these reports to participants , inviting them to ask additional questions or provide feedback . we also reviewed all selected quotes in the context of the original transcripts to ensure that they retained their meaning . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . choroideremia trials in north america have not yet started , limiting our patient interviews to potential research participants . finally , we interviewed only a small number of advocates , who had the most heterogeneous perspectives of all stakeholders . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . clinician and advocate visual benefit perspectives , mediated by the outcomes of the lca trials , converged from slowing down vision loss , halting vision loss , to a partial reversal of lost vision . these stakeholders emphasized that gt could provide a treatment but not a cure because gt is unlikely to provide regenerative benefits to retinal cells that have already degenerated . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . some clinicians raised patient difficulties in conceptualizing the meaning of a treatment for choroideremia in light of their understanding of treatment for other diseases : people say therapy like : oh , we can treat the pneumonia , therefore my lungs are normal again . i 'm going to have gene therapy all my vision is going to be restored.c11 . some clinicians also believed that patients lack the tools to distinguish between the potential visual outcomes of choroideremia gt . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . much like other stakeholders , most patients hoped for a treatment rather than a cure . patients were focused on gaining access to gt rather than on risks of harm ( table 2 ) . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . a quarter of patients even expressed a no risk perspective : for me there 's no risks . most patients , however , acknowledged the risk of accelerated vision loss , but opinions diverged with respect to the personal relevance of this risk . others , especially those who described their visual field as significantly deteriorated , expressed a willingness to accept the risk of accelerated vision loss : if i lose my sight , it 's going anyway.p2 patients also described factors that attenuated their risk perspectives . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . i think there 's urgency , and [ gt ] would allow me to keep more of my vision the sooner i get it.p8 . others expressed frustration with the slow regulatory approval process : the treatment is not available fast enough i know everybody wants to do it in careful way ; but for me , i 'd rather take the chance and save my eyes.p20 many patients explained that they would do anything to access gt : incur financial burdens ; take time off of work ; or travel . some advocates echoed this perspective , revealing their personal stakes : i will go for the second mortgage on my home if that 's what it takes.a1 . i think anybody faced with the prospect of blindness [ would ] say that i would do anything to undertake clinical trial patients have said to me i 'd rather have a heart attack than lose my vision.c9 . uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . like , when will a certain thing be standard of care ? it will become the five to eight year time frame.a2 . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? so their main question is when are we going to see the fruit of all of the research that has been done over the years that 's going to and they 're blunt about it cure the disease ? pa3 . nevertheless , most clinicians did not communicate effectively about time frames , frustrating patients with vague or dismissive responses . left to interpret vague time frames , patients shared their estimates . however , some , who already had significantly deteriorated visual fields , understood that their therapeutic window had passed . for other patients , including affected advocates , the success of the lca trials fueled hopes that adults , including themselves , will benefit from gt : it 's going to be too late for us then in the blink of an eye [ following the lca trials] that whole mindset did a complete 180 . now there 's a chance for my sight to be saved by a genetic therapy.a1 . in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . while some clinicians quantified their predictions , others made general remarks about the progress in the field or refused to provide estimates altogether . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . we can say where we hope to go , but be very clear that that 's a wish , that is not reality right now.c3 . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . clinician and advocate visual benefit perspectives , mediated by the outcomes of the lca trials , converged from slowing down vision loss , halting vision loss , to a partial reversal of lost vision . these stakeholders emphasized that gt could provide a treatment but not a cure because gt is unlikely to provide regenerative benefits to retinal cells that have already degenerated . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . some clinicians raised patient difficulties in conceptualizing the meaning of a treatment for choroideremia in light of their understanding of treatment for other diseases : people say therapy like : oh , we can treat the pneumonia , therefore my lungs are normal again . i 'm going to have gene therapy all my vision is going to be restored.c11 . some clinicians also believed that patients lack the tools to distinguish between the potential visual outcomes of choroideremia gt . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . much like other stakeholders , most patients hoped for a treatment rather than a cure . patients were focused on gaining access to gt rather than on risks of harm ( table 2 ) . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is not their first concern.c10 . supporting this view , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . a quarter of patients even expressed a no risk perspective : for me there 's no risks . most patients , however , acknowledged the risk of accelerated vision loss , but opinions diverged with respect to the personal relevance of this risk . some patients were hesitant in risking their remaining functional vision . others , especially those who described their visual field as significantly deteriorated , expressed a willingness to accept the risk of accelerated vision loss : if i lose my sight , it 's going anyway.p2 patients also described factors that attenuated their risk perspectives . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . i think there 's urgency , and [ gt ] would allow me to keep more of my vision the sooner i get it.p8 . others expressed frustration with the slow regulatory approval process : the treatment is not available fast enough i know everybody wants to do it in careful way ; but for me , i 'd rather take the chance and save my eyes.p20 many patients explained that they would do anything to access gt : incur financial burdens ; take time off of work ; or travel . some advocates echoed this perspective , revealing their personal stakes : i will go for the second mortgage on my home if that 's what it takes.a1 . i think anybody faced with the prospect of blindness [ would ] say that i would do anything to undertake clinical trial patients have said to me uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? so their main question is when are we going to see the fruit of all of the research that has been done over the years that 's going to and they 're blunt about it cure the disease ? pa3 . nevertheless , most clinicians did not communicate effectively about time frames , frustrating patients with vague or dismissive responses . however , some , who already had significantly deteriorated visual fields , understood that their therapeutic window had passed . for other patients , including affected advocates , the success of the lca trials fueled hopes that adults , including themselves , will benefit from gt : it 's going to be too late for us then in the blink of an eye [ following the lca trials] that whole mindset did a complete 180 . now there 's a chance for my sight to be saved by a genetic therapy.a1 . in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . while some clinicians quantified their predictions , others made general remarks about the progress in the field or refused to provide estimates altogether . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . we can say where we hope to go , but be very clear that that 's a wish , that is not reality right now.c3 . the prospect of gt for choroideremia has fueled patient hopes for therapeutic benefit ; provided a sense of urgency for its clinical implementation ; and , in the interim , motivated patients to privilege considerations of potential benefits over risks of harm in anticipated clinical trial recruitment . most participants cited qualified hopes for potential benefits rather than confident expectations . in other words , they remained optimistic without being able to express specific likelihoods for benefits and acknowledged the possibility that gt may not generate visual benefits . similar to other studies , patient hope for direct benefits greatly surpassed aspirational and collateral benefits as a motivator for trial participation , a finding indicative of a therapeutic misconception . when stakeholders view cts as both science and a source of succor or an opportunity for patient care , problems arise because there is a less than 1% chance for clinical improvement in phase i gt trials . nevertheless , lazarus - like responses have occurred in phase i trials , generating debate on the appropriateness of discussing direct benefits . in oncology trials , critics raise concerns about the use of surrogate measures ( e.g. , tumor response , stable disease ) , rather than end point measures ( e.g. , increased survival , improved quality of life ) . surrogate measures may not be clinically meaningful and , at best , are suggestive of direct benefit . gt , as a novel and complex intervention , is at the forefront adapted trial design use , such as the use of secondary outcome measures . these may obscure the research - treatment distinction in early - phase trials , further confounding communications about the safety focus of phase i trials . secondary outcome measures in ocular phase i gt trials ( e.g. , microperimetry , fundus imaging ) confirm safety , but also measure potential efficacy . the lca trials employed surrogate end point measures with significant shortcomings : end point measures for long - term safety and efficacy are still under development , as are measures for clinically meaningful improved visual function and quality of life . although disputed by lead researchers of the lca trials , a recent analysis indicates photoreceptor degradation continues after gt despite short - term improvements in visual function , raising concerns about long - term efficacy of gt . other clinical trial design decisions , such as the inclusion of children in a phase i lca trial to accommodate the limited therapeutic window of patients , further blurs the research - treatment distinction . the implications of such adaptations to standard phase i clinical trial designs for the therapeutic hopes of patients and clinical investigators require further investigation . however , our results show that patients overestimated therapeutic benefit compared with other stakeholder groups . gt is not regenerative ; it can not revive degenerated photoreceptor cells and so can not cure adult patients . discrepancies may be due to heterogeneous interpretations of benefit from disease amelioration to cure . therapeutic misestimation engages not only overestimation of potential benefits , but also underestimation risks of harm . as such , patient attention was diverted from risks of harm and directed toward gaining access to trials . trust also mediated patient risk perspectives : patients revealed trust in science and trust in their physicians . trust allows patients to reinterpret uncertainties in phase i trials as opportunities for potential benefits rather than as risks of harm . despite prominent discussion of therapeutic benefits , gt has often been characterized as permanently 5 years away from clinical application . media portrayals of gt as an imminent cure , supported by positive quotations by leading researchers , reinforce this perspective . overly optimistic hopes about time frames may not only lead to disappointment but also undermine the stated trust in researchers and threaten funding support . while predicting the future of ocular gt is impossible , the frustration of patients and advocates about uncertain or delayed time frames necessitates improved communication strategies . research on communicating time frames , however , has focused primarily on communicating patient prognoses . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . to address therapeutic misestimation , patient communications should be clear about the full spectrum of theoretically feasible visual outcomes from gt , in general , and in light of patient - specific therapeutic windows . clinicians and advocates should be aware of the dominance of curative public discourse in the media and beyond and be prepared to counter such representations . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . as an exemplar , the lca trials began in 2007 , but are only now , in 2013 , beginning to enroll patients in phase iii studies ( clinicaltrials.gov nct00999609 ) . historical evidence suggests that it takes 1014 years to move from novel target to drug approval . novel biotechnologies , such as gt , given their unique risk profile and additional regulatory steps , may take even longer . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . a two - pronged approach to patient communications balances such hope with pragmatic discussion about strategies for living with progressive visual impairment . patient advocacy organizations , in particular , should carefully balance their communications to raise funds for promising research against more pragmatic information on disease management , peer support , and access to credible information sources . finally , patients demonstrated a more nuanced understanding of potential visual outcomes than suggested by clinicians . it is therefore important for other stakeholder groups to recognize patients as critically thinking experts , while acknowledging patient vulnerabilities . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . to address therapeutic misestimation , patient communications should be clear about the full spectrum of theoretically feasible visual outcomes from gt , in general , and in light of patient - specific therapeutic windows . clinicians and advocates should be aware of the dominance of curative public discourse in the media and beyond and be prepared to counter such representations . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . as an exemplar , the lca trials began in 2007 , but are only now , in 2013 , beginning to enroll patients in phase iii studies ( clinicaltrials.gov nct00999609 ) . historical evidence suggests that it takes 1014 years to move from novel target to drug approval . novel biotechnologies , such as gt , given their unique risk profile and additional regulatory steps , may take even longer . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . a two - pronged approach to patient communications balances such hope with pragmatic discussion about strategies for living with progressive visual impairment . patient advocacy organizations , in particular , should carefully balance their communications to raise funds for promising research against more pragmatic information on disease management , peer support , and access to credible information sources . finally , patients demonstrated a more nuanced understanding of potential visual outcomes than suggested by clinicians . it is therefore important for other stakeholder groups to recognize patients as critically thinking experts , while acknowledging patient vulnerabilities . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities . | purpose : ocular gene transfer clinical trials are raising patient hopes for the treatment of choroideremia a blinding degenerative retinopathy .
phase i choroideremia gene transfer trials necessitate communicating about the risks of harm and potential benefits with patients while avoiding the sensationalism that has historically undermined this field of translational medicine.methods:we conducted interviews between june 2011 and june 2012 with 6 choroideremia patient advocates , 20 patients , and 15 clinicians about their hopes for benefits , perceived risks of harm , and hopes for the time frame of clinical implementation of choroideremia gene transfer.results:despite the safety focus of phase i trials , participants hoped for direct visual benefits with evident discrepancies between stakeholder perspectives about the degree of visual benefit .
clinicians and patient advocates were concerned by limited patient attention to risks of harm .
interviews revealed confusion about the time frames for the clinical implementation of choroideremia gene transfer and patient urgency to access gene transfer within a limited therapeutic window.conclusion:differences in stakeholder perspectives about choroideremia gene transfer necessitate strategies that promote responsible communications about choroideremia gene transfer and aid in its translation .
strategies should counter historical sensationalism associated with gene transfer , promote informed consent , and honor patient hope while grounding communications in current clinical realities . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
prokaryotes and lower eukaryotes are
able to move towards favorable and away from unfavorable conditions including
chemicals and light , referred to as chemotaxis and phototaxis , respectively [ 1 , 2 ] . repellent phototaxis in the halophilic archaeon natronomonas pharaonis ( also known as natronobacterium pharaonis ) is initiated by light activation of
sensory rhodopsin ii ( psrii ) , a 7 -helical membrane receptor related to
bacteriorhodopsin . activation of psrii induces structural changes in its cognate
transducer protein , phtrii , a 2 -helical membrane protein with a long
cytoplasmic extension that transmits the signal further to induce subsequent
phosphorylation processes that control flagellar motion . the crystal structures of psrii alone [ 4 , 5 ] and in complex with the transmembrane domain of phtrii ( residues 1114 ,
electron density was observed for residues 2282 ) have been recently solved ,
both in the inactive dark state and in the light - activated conformation [ 6 , 7 ] . a peptide fragment corresponding to the juxtamembrane residues 83149
of the above protein was shown by nmr spectroscopy to be unstructured in
solution , but to undergo a structural transition upon interaction with psrii . epr spectroscopy of the n - terminal phtrii protein fragment ( residues
1157 ) also showed that the juxtamembrane domain has molten globule - like characteristics
. however , the structure of the long cytoplasmic domain of phtrii
( phtrii - cyt ) , which directly interacts with the proteins of the signaling
cascade , remains unknown . phtrii - cyt exhibits
sequence homology to the cytoplasmic domains of receptors in eubacteria
including the well - studied escherichia
coli serine ( tsr - cyt ) and aspartate ( tar - cyt ) receptors . the identity between phtrii - cyt and the corresponding sequence of
tsr - cyt is 24% . for comparison , these sequences belong to the methyl - accepting chemotaxis protein
( mcp ) family , which currently ( as of march 2008 ) has more than 5000 identified
members . the functional significance of the homology between phototaxis
transducer proteins and chemoreceptors has been proven through studies of
chimera . tsr - cyt has been
crystallized and its crystal structure corresponds to a helical coiled - coil dimer
. a comparison of the crystal structure of the methylated with the structure of the non - methylated signaling state revealed little changes in the structure , but
significant differences in b - factor values supporting a model , in which receptor activity is determined by the
flexibility of the cytoplasmic domain . furthermore , the helical tar - cyt fragment is easily denatured
thermally [ 16 , 17 ] and has the characteristics of a molten globule . nmr amide - exchange data revealed the presence of a small ( 10% ) core of
slowly exchanging residues , which presumably are involved in tertiary packing
contacts . the size of the core increased as a result of mutation or ph change [ 16 , 17 , 19 ] . we have previously shown that
phtrii - cyt assumes a shape consistent with the tsr - cyt helical coiled - coil
dimer structure , but cd spectra lacked evidence for a predominantly helical
conformation . here , we present an in - depth analysis of phtrii - cyt using cd ,
fourier - transform ir ( ftir ) , nmr spectroscopy , and statistical sequence
analysis . from these studies , a picture emerges in which phtrii - cyt has a high
propensity for helical coiled - coil structure , but maintains flexibility to such
an extent that cd is not able to reliably detect it . all sources of materials
including preparation of phtrii - cyt were as described previously , unless otherwise stated . buffers used were phosphate - buffered saline ,
0.9 mm cacl2 , 0.5 mm mgcl2 , 2.7 mm kcl , 138 mm nacl , 1.5 mm kh2po4 , 8 mm na2hpo4 ( pbs ) ; 10 mm napi ph 6.0 ( buffer a ) ; 10 mm tris - hcl ph 8.0 ( buffer b ) ; 10 mm
tris - hcl ph 8.0 in 99% d2o ( buffer c ) ; 10 mm napi ph 6.0 ,
10% d2o ( buffer d ) . cd spectra were acquired using a jasco 810 spectropolarimeter ( jasco inc . , easton ,
md , usa ) as described previously . experimental data exp were recorded in a 0.2-cm quartz cell at phtrii - cyt concentration of 0.1 mg / ml and
then converted to the mean residue ellipticity according to [ ]mrw = exp /(10*l*c*n ) where l is the path length
of the cell in cm , c is the protein concentration in m , and n is the number of
residues . [ ]mrw was used as the input data for the contin / ll secondary structure prediction
program from the cdpro software package [ 21 , 22 ] . the dataset including reference spectra of denaturated proteins
( sdp48 ) was chosen as the reference one . the error bars represent the standard deviation of values predicted by
contin / ll assuming 10% error in the determination of concentration . the values
obtained from deconvolution should be considered approximate because of inherent
difficulties in cd spectra deconvolution [ 24 , 25 ] and the known effects of dynamics on reliability of secondary structure
prediction from cd data . however , we use them here to allow quantitative comparison of
structure and dynamics of phtrii - cyt under different solvent and temperature
conditions . solutions for nmr
spectroscopic analysis contained 5 mg / ml uniformly n - labeled
phtrii - cyt in buffer d. h - n - hsqc , longitudinal ( t1 )
and transverse ( t2 ) relaxation data , and h - n
heteronuclear noe ( hetnoe ) data of phtrii - cyt in the absence and presence of
4 m salt were recorded at + 10c and + 20c
using a bruker 600 mhz spectrophotometer . standard bruker sequences were used to
record h- , h - n - hsqc , and het - noe spectra . the t1 , t2 spectra were recorded using
the pulse sequence schemes described previously . we used a 90 pulse of 50 microseconds for n for all the experiments and a 90 pulse of 14 microseconds and 9.6 microseconds for the h in 4 m salt and water samples ,
respectively . the r1(1/t1 ) and r2(1/t2 )
rates of the 4 m salt sample were measured using the relaxation delays of 40 ,
80 , 160 , 320 , 480 , 640 milliseconds and 30 , 64 , 96 , 128 , 192 , 240 milliseconds ,
respectively . further , the water relaxation rate values were obtained at
relaxation delays of 32 , 64 , 128 , 256 , 384 , 512 milliseconds for r1 and 32 , 64 , 128 , 192 , 256 , 320 milliseconds for the r2 . hetnoe experiments
were obtained by collecting the spectra with and without h
presaturation . a proton presaturation period of 3 seconds was applied before
acquiring each hn hsqc spectra . the data recorded
were processed using the nmrpipe , nmrview , and sparky software packages for
further analysis . the r1 and r2 rates were estimated by
fitting the peak intensities at different relaxation delays using the
two - parameter exponential decay function in the numerical recipes package [ 27 , 28 ] . the hetnoe values for each
peak were estimated by finding the ratios between the peak intensities of the h - n hsqc spectra with and without
proton presaturation . ftir absorbance spectra were acquired at room temperature using a bruker vector
22 ir - spectrometer equipped with a dtgs detector . for film preparation , 150 g of
phtrii - cyt in buffer b were dried under n2 gas flow and then sealed .
for measurements in solution , phtrii - cyt was concentrated to 10 mg / ml , and buffer
b was exchanged to buffer c to reduce background absorbance in the amide i
region . the spectrum of residual water vapor was measured with an empty chamber and
then subtracted manually from the protein spectra . sequence - based
secondary structure predictions were performed using all of the prediction
programs ( sopm , sopma , hnn , mlrc , dpm , dsc , gor i , gor iii , gor iv , phd ,
predator , and simpa96 ) available on the nps@web server . the predictions of coiled - coil regions were done by the coils
prediction server using the recommended window of 28 residues , mtk matrix ( derived from
the sequences of myosins , tropomyosins , and keratins ) . since coiled - coils are
often fibrous , solvent - exposed structures , all residues except for the internal a and d have a high probability of being hydrophilic . giving equal
weights to all positions therefore means being biased towards hydrophilic ,
charge - rich sequences . to avoid highly charged false positives a 2.5-fold
weighting of positions a sequence - based predictions of the dynamic properties
of individual proteins were obtained from the predictors of natural disordered
regions ( pondr ) prediction server . the analysis of the disorder propensities of the mcp signal family was
carried out using ronn disorder prediction software [ 32 , 33 ] . phtrii - cyt is predicted to be
a helical coiled - coil based on homology to the serine receptor ( figure 1(a ) ) ,
high coiled - coil structure propensity ( figure 1(b ) , top panel ) , and high helix
propensity with 63% to 97% -helix content ( figure 1(b ) ,
bottom panel ) , depending on the prediction algorithm used ( see section 2.5 ) . these predictions are not consistent with the cd spectra recorded in the
commonly used buffers , pbs , buffer a and buffer b , which were all
indistinguishable and essentially lacked negative ellipticity at 222 nm
characteristic for helix . the values obtained from deconvolution of the spectra were 77% unstructured ,
7% turn , 13% -sheet ,
and 3% -helix .
however , control experiments with helix - inducing solvent 2,2,2-trifluoroethanol
( tfe ) showed a gradual rise in negative ellipticity at 222 nm with increasing
amounts of tfe added ( figure 1(d ) ) , experimentally validating the high helix
propensity inferred from the phtrii - cyt sequence . figure 1(c ) shows ftir spectra
in the amide i region for phtrii - cyt in buffer c ( solid line ) and in dry film
( dashed line ) . the position of the maximum of the amide i band in buffer c at
1645 cm is characteristic of a disordered conformation . in dry
film , a shift to 1654 cm was observed , a value that is
characteristic of -helical secondary structure [ 25 , 34 ] . addition of glycerol to pbs
at concentrations of 60% ( v / v ) and
higher greatly stabilized helix content which based on spectral deconvolution
rose from 19% in 60% ( v / v ) glycerol
to 59% in 90% ( v / v ) glycerol . -sheet and turn contributed ,
respectively , 5% and 12% to the spectrum in 90% ( v / v ) glycerol ( figure 1(e ) ) . in sucrose , at 90% ( w / v ) saturation , -helix
content was 37% , where -sheet and turn contributed ,
respectively , 10% and 21% , to the spectrum ( data not shown ) . ammonium sulfate , a common
agent to precipitate , stabilize , or facilitate refolding of proteins , up to 30%
saturation , did not significantly alter the cd spectra of phtrii - cyt obtained
in pbs alone ( figure 1(f ) ) . between 30% and 60% , phtrii - cyt adopted approximately
5056% -helical conformation , while at above 60% saturation , helical content
was reduced and that of random coil increased . previous crosslinking and
analytical gel - filtration experiments showed that the structural transition was
accompanied by protein aggregation at high ammonium sulfate concentrations . since the native
intracellular environment for n.
pharaonis includes 4 m kcl , we also investigated the effect of monovalent
and bivalent cationic solutions on phtrii - cyt cd spectra . in solutions of up to
2 m of either licl , nacl , kcl , rbcl , or cscl in pbs and cacl2 and
mgcl2 in buffer b , however , at 4 m salt concentrations , helix conformation was
stabilized to different degrees depending on the nature of the salt added ( figure 2(a ) ) . a plot of hydrated ion size ( ion radii taken from ) versus ellipticity ( figure 2(d ) ) shows a minimum at the position of
nacl , indicating that nacl is best in stabilizing phtrii - cyt structure . we note that maximum helix induction by the
high salt additions required buffer d ( containing phosphate and other salts ) as
the base buffer , suggesting that there is additional complexity in the factors
contributing to maximum stability than can be explained by a single solvent
component . for example , studies in h.
marismortui indicated that although kcl was the predominant salt , there
were also considerable amounts of sodium and magnesium salts present . the negative ellipticity at 222 nm as a
function of temperature is shown in figures 2(b ) and 2(c ) . in pbs , cd spectra were
essentially independent of temperature , characteristic of random coil at all
temperatures probed ( 0c to + 99c ) . in the presence of 4 m salt ( figure 2(b ) ) ,
helicity was lost cooperatively with rising temperature with a single
transition point , observed at + 41.5c in 4 m licl , + 37.9c in 4 m nacl , + 36.3c
in 4 m kcl , and + 32.4c in 4 m rbcl . the melting temperature was thus linearly
dependent on ionic radius of the cations , with smaller ions stabilizing helix
stronger ( figure 2(e ) ) . denaturation in 90% ( v / v ) glycerol proceeded similar to that
observed in the presence of high salt concentrations , that is , cooperatively
with a transition point of + 38.3c . thermal denaturation of phtrii - cyt in pbs +
40% saturation ammonium sulfate was fitted by a sigmoidal curve with a
transition point significantly higher than those obtained under any other
condition , at + 66 3c . however , the shape of
the curve was not symmetric indicating the presence of multiple species with
different melting temperatures . in 98.5% ( v / v ) tfe , a transition point of + 36 4.5c was obtained , but seemed to follow a linear
dependency with temperature , suggesting a gradual melting with little
cooperativity . resonances
in one - dimensional h spectra of phtrii - cyt in buffer d ( data not
shown ) were very sharp and clustered around 8.3 ppm . these spectral features are characteristic of
disordered or globally dynamic proteins . h - n - hsqc spectra of uniformly n - labeled
phtrii - cyt at + 10c and + 20c in buffer d with and without 4 m kcl are
shown in figures 3(a ) , 3(b ) , respectively . spectra recorded at + 4c , + 10c ,
+ 25c , + 37c , and + 41c were qualitatively
similar in chemical shift dispersion but varied greatly with respect to
individual chemical shifts ( data not shown ) . a maximum of 254 of the expected
271 backbone nh resonances were resolved at + 4c in buffer d , while only 216
were resolved at + 41c in buffer d. only 27 peak positions were independent of
temperature for low salt and 44 peak positions for high salt samples . to quantify the
dynamic properties of htrii - cyt , we measured n relaxation parameters t1 , t2 , and hetnoe
at two different temperatures ( + 10c and + 20c ) in the presence and absence of
4 m kcl . values could only be obtained for 141148
of the 251258 peaks observed in the four respective hsqc spectra , while
106110 of the peaks decayed so rapidly that reliable relaxation parameters
could not be determined . this indicates that the backbone resonances of these
residues are most likely rigid or undergo conformational exchange on the microsecond - to - millisecond
time scale . for the 141148 peaks for which relaxation parameters were
determined in buffer d at + 10c and + 20c ,
the t1 , t2 and hetnoe values are characteristic of random
coil structure as , for example , observed in unfolded lysozyme in 8 m urea . in contrast , the observed t1 , t2 , and hetnoe values are far from those observed in a rigid helical
protein . there was very little deviation from the average in all relaxation
parameters at both temperatures suggesting that the subset of resonances for
which relaxation parameters could be measured experiences similarly restricted
motions , on the nanosecond - to - subnanosecond time scale . in the presence of 4 m
kcl , t2 average values were overall slightly increased as compared to those observed in
the absence of high salt , probably due to the difference in solvent viscosity . importantly , however , the standard deviation in t2 times was much
higher in the presence of salt , reflecting the appearance of resonances with
lower t2 times ( higher relaxation rates ) , indicating restriction in
conformational space similar to what has been observed in hydrophobic clusters
in unfolded
lysozyme . these results strongly
suggest that both in the presence and absence of salt , there is significant
restriction in the conformational space being explored by the ensemble of
structures . the nmr results provide detailed information on the number of
resonances whose dynamics are affected by changes in solvent conditions . for
141148 backbone amide groups out of 271 phtrii - cyt residues ( 5255% ) , the
presence of 4 m kcl diminishes the degree of flexibility significantly . in the
remaining 4548% of
resonances , there is stronger restriction in conformational space that is
present both in low and high salt conditions . these residues experience
significant changes in their environment when comparing low and high salt
conditions because the chemical shift values change and a broader range of
values is found under high salt conditions indicating more highly structured
states . the
experimental evidence presented above uniformly suggests that the phtrii - cyt
structure is highly dynamic . since the tar - cyt and tsr - cyt structures are also
believed to be dynamic ( see section 1 ) , we investigated the propensity for
disorder in all three proteins and in the entire mcp family using two methods : pondr
and ronn [ 32 , 33 ] ( figure 4 ) . y - axis values ( probability of disorder ) higher than 0.5
generally indicate disordered regions . in all three proteins pondr predicts
substantial disorder for the cytoplasmic domain , while overall the ronn
probabilities for disorder were more moderate ( figure 4(a ) ) . using ronn , we
investigated the propensity for disorder in the entire mcp family ( figure 4(d ) ) . a mean value of 0.5 with a low standard deviation of ~0.09 was obtained . tsr - cyt and phtrii - cyt closely follow the mean ronn disorder values in the mcp
family at the majority of sites , indicating that high flexibility but not
complete lack of structure is conserved in the mcp family . notably , some regions
at cytoplasmic domain alignment position 100110 , 190200 and at the extreme
c - terminus ( > 275 ) displayed least flexibility in the mcp family ( figure 4(d ) ) . these correspond to regions of lowest b - factors observed in the tsr - cyt crystal
structure . previous studies showed that
the shape of phtrii - cyt is an elongated rod with a radius of gyration of 54 , both as a monomer in low salt and as a dimer in 4 m kcl . this shape is consistent with the helical coiled - coil dimer of the
tsr - cyt crystal structure . however , this shape is not consistent with cd data lacking evidence
for helix altogether for the monomer , and indicating only 24% helix for the
dimer in 4 m kcl . here , we provide evidence that despite random coil spectroscopic
features , phtrii - cyt can exist as a highly flexible , loosely packed but folded
helical coiled - coil : ( i ) sequence - based structure predictions all strongly
support a helical coiled - coil structure for phtrii - cyt , and addition of tfe , a
well - known helix inducing agent , is able to generate cd spectra that are
characteristic for ~80% helix ; ( ii ) both , uncharged electrolytes and salts
induce negative ellipticity in cd spectra of phtrii - cyt , but the thermal
midpoint transitions occur at very low temperatures indicating thermal
instability ; ( iii ) the majority of signals in h , n - hsqc spectra
are highly temperature - sensitive , with the largest number of visible peaks
observed at low temperatures and best spectral dispersion in high salt at low
temperatures . because small angle neutron scattering indicates an identical
radius of gyration regardless of the differences in helix content in cd spectra and
spectral changes in the nmr spectra , we propose that stabilization of more
folded conformations by added stabilizing agents in the case of phtrii - cyt
means a reduction in dynamics leading to a more compact but overall similar
structure . the stabilization of folded
structures of proteins by additives has been extensively studied beginning with
the seminal work by hofmeister . careful experiments , thermodynamic analyses , and discussions over the
years ( [ 4245 ] and references within ) , including those for halophilic proteins [ 4649 ] , have led to the relatively recent unifying theory that both , hard
factors , referring to excluded volume effects and soft factors , referring to
interactions between additives , water , and proteins / nucleic acids together
contribute to the mechanisms underlying the effects of cosolvents on
protein / nucleic acid structural stability . while it is possible to empirically
estimate the overall contribution of hard versus soft effects , the current uncertainty of the nature of unfolded states will preclude
quantitative modeling in the near future . the very high density of acidic amino acids in phtrii - cyt typical of
halophilic proteins suggests that soft effects will be important in the case
of stabilization of the helical coiled - coil in phtrii - cyt . to illustrate this
point , we generated homology models of each monomer in the dimer from the tsr - cyt
crystal structure ( figure 5(a ) ) . fastcontact analysis of the model reveals that this dimeric structure is not stable because
of electrostatic repulsion between the acidic residues at the dimer interface
( figure 5(c ) ) , while each helix in the monomer is favorably packed through hydrophobic
interactions ( figure 5(b ) ) . as a result , docking of the two helical monomers
using cluspro software does not reproduce the homology model , and instead , only results in
physical interaction in an area in which hydrophobic interactions are possible
( figure 5(d ) ) . presumably , the high salt concentrations in vivo overcome the electrostatic repulsions . to test this
hypothesis , we conducted systematic comparison of monovalent and bivalent
cations in the presence of a constant anion ( chloride ) . we found that the
extent and stability of the ellipticity signal in cd spectra in the presence of
different cations follows exactly that predicted by collins ' law of matching
water affinities : the maximum stabilization is with the ion ( na ) that has a
water affinity closest to those of the acidic amino acid residues in
phtrii - cyt , and not the one with highest charge density ( ca ) , or
smallest ionic radius ( li ) . however , even in the presence of 4 m
nacl , phtrii - cyt is still a dynamic ensemble of folded coiled - coil structures ,
rather than a single stable conformation . the
dynamic properties of phtrii - cyt reported here for high salt concentrations
physiological for n. pharaonis are in
good agreement with those reported for tar - cyt under conditions physiological
for e. coli . furthermore , tar - cyt and tsr - cyt were found to be globally dynamic in
solution , similar to our observations with phtrii - cyt . finally , disorder
predictions indicate conservation of flexible properties in the entire mcp
family . this evolutionary conservation of flexibility suggests a role in
function . activation of chemotaxis receptors and phototaxis transducers occurs
through very subtle effects such as small helix movements during activation and
only dimers bind and activate chea [ 5456 ] . the compaction coupled to dimerization of a preformed ensemble of
coiled - coil helical - like structures proposed here could contribute to the
mechanism for activation . in support of this hypothesis , the strength of the
interaction between helices in the chemoreceptor cytoplasmic domain was found
to modulate kinase activity [ 54 , 55 , 57 ] . recently ,
it was estimated that as much as 50% of human proteins belong to the
intrinsically unstructured or natively unfolded class . these estimates are based on predictors of disorder such as pondr that
are trained in part on sequence data of proteins with cd , ftir , or nmr spectra
like the ones we measured for phtrii - cyt as sole evidence for disorder . the findings presented in this paper supporting that phtrii - cyt is not
a random coil suggest that the terms intrinsically unstructured and natively
unfolded would more appropriately be termed the notion of residual structure is analogous to that observed in
chemically denatured proteins , where it has been proposed that a true random
coil may never exist . the case of phtrii - cyt is particularly intriguing because its
nonspherical shape allows us to clearly distinguish between a true random coil
that may or may not retain residual structure and a near - native but highly
dynamic structural ensemble . this case also reminds us that local , that is ,
secondary structure in the classical sense and long - range tertiary structure
defining overall shape are not independent of each other . understanding the
amplitude of the motions observed in this and other systems would be an
important future goal towards defining this interrelationship in more detail . investigating how much motion is needed to lose the characteristic ellipticipty
in cd spectra would be a good starting point . it is an unanswered question
to what extent proteins experimentally observed to be dynamic in vitro are as dynamic in vivo [ 42 , 61 ] . evidence suggests that crowding inside the cell can be mimicked with
solutes in vitro . for example , flgm ,
an intrinsically disordered protein gains structure in the presence of high
concentrations of solutes in vitro
and inside living e. coli cells . low stability of intrinsically unstructured or highly dynamic protein
ensembles has been shown to be related to functions such as membrane insertion
or fusion events , protein interactions , and signal transduction . | chemo- and phototaxis systems in bacteria and archaea serve as models for more complex signal transduction mechanisms in higher eukaryotes .
previous studies of the cytoplasmic fragment of the phototaxis transducer ( phtrii - cyt ) from the halophilic archaeon natronomonas pharaonis showed that it takes the shape of a monomeric or dimeric rod under low or high salt conditions , respectively .
cd spectra revealed only approximately 24% helical structure , even in 4 m kcl , leaving it an open question how the rod - like shape is achieved . here , we conducted cd , ftir , and nmr spectroscopic studies under different conditions to address this question .
we provide evidence that phtrii - cyt is highly dynamic with strong helical propensity , which allows it to change from monomeric to dimeric helical coiled - coil states without undergoing dramatic shape changes .
a statistical analysis of predicted disorder for homologous sequences suggests that structural flexibility is evolutionarily conserved within the methyl - accepting chemotaxis protein family . |
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mice were maintained on a 12-h light - dark cycle with ad libitum access to tap water ( reverse osmosis purified ) and a chow diet containing 21% calories from fat , 23% from protein , and 55% from carbohydrates ( mouse diet 9f ; pharmaserv , framingham , ma ) . for the feeding studies , 6-week - old mice were submitted to low - fat diet ( rodent nih-31 m auto : 14% calories from fat , 25% from protein , and 61% from carbohydrates [ taconic ] ) or high - fat diet ( td.93075 : 55% calories from fat , 21% calories from protein , and 24% calories from carbohydrates [ harlan teklad , madison , wi ] ) for 18 weeks before they were killed . in addition to fat content , these diets differ in other components that could also influence the results of our analysis . for example , our chow diet contained fat from animal sources , whereas both low - fat and high - fat diets contained vegetable fat . these differences may have contributed to the fact that a few metabolic parameters displayed in table 1 , such as insulin levels , did not necessarily track with the percentage of fat in the diet . however , in this study and in others , these diets have been primarily used as independent paradigms to investigate the impact of the environment on weight gain of animal models , showing metabolic effects that closely resembled different characteristics of human obesity ( 8) . caloric restriction was initiated at 14 weeks of age with a 10% decrease in calories , increased to 25% restriction at 15 weeks , and to 40% restriction at 16 weeks , which was maintained until the age of 6 months when the mice were killed . protocols for animal use were reviewed and approved by the animal care committee of the joslin diabetes center and were in accordance with the national institutes of health guidelines . metabolic parameters in b6 vs. 129 mice at different ages and dietary conditions values are expressed as mean se of 8 animals per group . two to five affymetrix murine chips u74av.2 ( santa clara , ca ) were used per mouse strain , for each comparison of b6 versus 129 mice . quantitative rt - pcr was performed using a sybr green - based abi prism 7900 sequence detection system ( applied biosystems , foster city , ca ) . for details , refer to the supplementary methods in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0367/dc1 . a novel version of gene network enrichment analysis ( 16 ) was applied to each comparison of b6 versus 129 mice to identify gene networks ( subnetworks of genes with edges connecting physically interacting protein products ) with differential activation between these mice , and to test such networks for statistically significant , over - represented pathways and processes . briefly , the algorithm was composed of the following steps ( see supplementary methods for details ) . first , for each gene , a differential expression significance value was calculated in b6 versus 129 mice , converted to z scores , and used to annotate the corresponding proteins in a protein - protein interaction network curated from literature ( 17 ) ( fig . individual comparisons of b6 and 129 mice were performed in different tissue types ( adipose , skeletal muscle , and liver ) , ages ( 6 weeks and 6 months ) , and diets ( high fat , low fat , and standard chow ) . a : for each comparison , a differential expression significance value was determined for each gene , converted to a z score , and mapped onto a network of protein - protein interactions . b : a stochastic search algorithm ( 18 ) was then applied to search for subnetworks with high mean z score ( high significance ) . each application of the algorithm identifies one subnetwork ; hence , to detect a multitude of subnetworks , the algorithm was run many times . the consensus among these subnetworks represented approximations of the true , differentially active network in b6 vs. 129 mice . c : a membership in differentially active subnetwork ( man ) score was determined for each gene . man scores are p values and represent the probability of genes to belong to the true , differentially active network . this score was calculated as the random chance probability that the gene is detected in the observed number of subnetworks computed over permutations of the data . d : biological processes and pathways were tested for enrichment in genes with high man scores . statistically significant pathways and processes were predicted to be differentially active in b6 vs. 129 mice . e : for each significant process and pathway , a network visualization was generated to aid interpretation . subsequently , a computationally intensive , stochastic optimization algorithm ( 18 ) was employed to identify a subnetwork of genes with a high mean z score ( reflecting high significance ) . the hypothesis was that a consensus among the stochastically detected subnetworks represented a prediction of the true , differentially active gene network in b6 versus 129 mice . thus , each gene was associated with a p value based on the observed number of subnetworks in which it appeared , reflecting its chance probability of belonging to the true network ( fig . each biological process and pathway from the gene ontology and molecular signatures database were tested for over - representation of genes with low p values , using standard statistical methods ( fig . finally , for each statistically significant pathway or biological process , the algorithm produced a gene network consisting of pathway member genes and all additional genes with significant , adjusted p values ( false discovery rate 0.25 ) that shared an edge with a pathway member gene in the protein - protein interaction network ( fig . 1e ) , providing an intuitively appealing visualization aid that helped in interpreting the enrichment results . erythrocyte - free stromavascular cells were isolated from epididymal fat pads after collagenase digestion as previously described ( 19 ) . these cells were incubated with a mix of antibodies against different surface markers and analyzed using the lsrii flow cytometer ( bd biosciences , san jose , ca ) . rna pooled from tissues of two or three mice was used for microarray analysis . two to five affymetrix murine chips u74av.2 ( santa clara , ca ) quantitative rt - pcr was performed using a sybr green - based abi prism 7900 sequence detection system ( applied biosystems , foster city , ca ) . for details , refer to the supplementary methods in the online appendix available at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0367/dc1 . a novel version of gene network enrichment analysis ( 16 ) was applied to each comparison of b6 versus 129 mice to identify gene networks ( subnetworks of genes with edges connecting physically interacting protein products ) with differential activation between these mice , and to test such networks for statistically significant , over - represented pathways and processes . briefly , the algorithm was composed of the following steps ( see supplementary methods for details ) . first , for each gene , a differential expression significance value was calculated in b6 versus 129 mice , converted to z scores , and used to annotate the corresponding proteins in a protein - protein interaction network curated from literature ( 17 ) ( fig . individual comparisons of b6 and 129 mice were performed in different tissue types ( adipose , skeletal muscle , and liver ) , ages ( 6 weeks and 6 months ) , and diets ( high fat , low fat , and standard chow ) . a : for each comparison , a differential expression significance value was determined for each gene , converted to a z score , and mapped onto a network of protein - protein interactions . b : a stochastic search algorithm ( 18 ) was then applied to search for subnetworks with high mean z score ( high significance ) . each application of the algorithm identifies one subnetwork ; hence , to detect a multitude of subnetworks , the algorithm was run many times . the consensus among these subnetworks represented approximations of the true , differentially active network in b6 vs. 129 mice . c : a membership in differentially active subnetwork ( man ) score was determined for each gene . man scores are p values and represent the probability of genes to belong to the true , differentially active network . this score was calculated as the random chance probability that the gene is detected in the observed number of subnetworks computed over permutations of the data . d : biological processes and pathways were tested for enrichment in genes with high man scores . statistically significant pathways and processes were predicted to be differentially active in b6 vs. 129 mice . e : for each significant process and pathway , a network visualization was generated to aid interpretation . subsequently , a computationally intensive , stochastic optimization algorithm ( 18 ) was employed to identify a subnetwork of genes with a high mean z score ( reflecting high significance ) . the hypothesis was that a consensus among the stochastically detected subnetworks represented a prediction of the true , differentially active gene network in b6 versus 129 mice . thus , each gene was associated with a p value based on the observed number of subnetworks in which it appeared , reflecting its chance probability of belonging to the true network ( fig . each biological process and pathway from the gene ontology and molecular signatures database were tested for over - representation of genes with low p values , using standard statistical methods ( fig . finally , for each statistically significant pathway or biological process , the algorithm produced a gene network consisting of pathway member genes and all additional genes with significant , adjusted p values ( false discovery rate 0.25 ) that shared an edge with a pathway member gene in the protein - protein interaction network ( fig . 1e ) , providing an intuitively appealing visualization aid that helped in interpreting the enrichment results . erythrocyte - free stromavascular cells were isolated from epididymal fat pads after collagenase digestion as previously described ( 19 ) . these cells were incubated with a mix of antibodies against different surface markers and analyzed using the lsrii flow cytometer ( bd biosciences , san jose , ca ) . at 6 months of age , on a chow diet , b6 mice were overweight , hyperglycemic , and exhibited higher plasma leptin and insulin levels than 129 mice of the same age ( table 1 , supplementary fig . b6 mice showed clear signs of insulin resistance when compared with 129 mice , even on low - fat diet ( lfd ) , and this difference was further exacerbated when the mice were placed on a high - fat diet ( table 1 ) ( 8) . at 6 weeks of age , however , no phenotypic differences or serum markers indicative of diabetes and obesity could be detected between the two strains ( table 1 ) ( 8) . indeed , 6-week - old 129 mice have the same weight as b6 mice and actually have significantly more fat mass , as indicated by higher fat pad weight ( table 1 ) . by studying b6 versus 129 mice at different ages and on different diets , one may therefore elucidate the background inherited phenomena predisposing to obesity - accompanied insulin resistance . to identify gene networks that might provide early biomarkers of adult metabolic disease , we initially performed microarray analyses on rna from adipose tissue , liver , and skeletal muscle of b6 and 129 mice at 6 weeks and 6 months of age on a standard chow diet . we then applied gnea to detect networks that were differentially active in a predisease stage and to estimate how they changed over time in disease - prone versus diabetes - resistant strains ( fig . since disease susceptibility is an inherited phenomenon in these animals , we hypothesized that the predisposing hereditable biological processes would be those showing differential activity in young mice and maintained or further increased with age . among the three different tissues in which b6 versus 129 mice were compared , only adipose tissue exhibited significantly enriched biological processes ( q value < 0.33 ) at 6 weeks of age by gnea , with 12 enriched gene sets , and this increased to 25 enriched gene sets at 6 months of age . of these , only two sets ( immune system process and regulation of cytokine secretion ) were found enriched at both ages ( supplementary table 1 ) , and both of these related to inflammatory pathways . furthermore , the immune system process was the most significantly enriched gene set at 6 months of age ( q value = 0.008 ; supplementary table 1 ) . in contrast , in the liver , 37 gene sets ( q value < 0.33 ) were differentially regulated in b6 versus 129 at 6 months of age , but none was significantly different in the 6-week - old animals . in skeletal muscle , no significant difference was observed at either age ( data not shown ) . in addition to gnea , the adipose tissue dataset was analyzed using gene set enrichment analysis ( gsea ) ( 20 ) and the standard hypergeometric test ( 21 ) . on the other hand , gsea failed to identify any gene set that was differentially expressed at both ages ( supplementary table 3 ) . by comparison , the standard hypergeometric test ( 21 ) failed to identify either a clear immune signature at 6 weeks or any enriched gene sets at 6 months ( supplementary tables 2 and 3 ) . to determine how genes related to the immune response were differentially expressed in the fat tissue of young b6 and 129 mice and changed in expression as the mice aged , we identified the network associated with the immune system process . s2 ) and plotted the histogram of differential expression for its genes in b6 versus 129 mice at 6 weeks and 6 months ( fig . a few individual genes , such as apolipoprotein a ( apoa1 and apoa2 ) and mannose - binding lectin ( mbl2 ) were more strongly downregulated in b6 versus 129 mice at 6 months and less so at 6 weeks . nonetheless , much of the immune system process network was upregulated at 6 months compared with 6 weeks ( note the right - shift in the histogram for 6 months vs. 6 weeks in fig . 2 ) . lymphoid and myeloid markers , such as cd45 , cd3 , and chemokine ( c - c motif ) receptor 5 ( ccr5 ) , monocyte chemotactic protein-1 ( mcp1 ) , as well as proinflammatory genes such as chemokine ( c - c motif ) ligand 5 ( ccl5 ) and interleukin-6 ( il6 ) , were more highly expressed in b6 compared with 129 mice at 6 weeks , and this increased further at 6 months of age ( supplementary fig . 2 ) , many of the immune system process genes already showed twofold or greater differences in expression in b6 versus 129 mice at 6 weeks , an age when no significant metabolic differences were observed between strains . histogram of differential expression between b6 and 129 mice at 6 weeks and 6 months of age for genes in the immune system process network . the x - axis values are the upper bounds of the histogram bins and correspond to the log2 fold - change difference in b6 vs. 129 mice . few network genes are unchanged ( absolute log2 fold change < 1 ) in b6 vs. 129 mice at 6 months . the majority are strongly upregulated ( log2 fold change 1 ) , although a few are strongly downregulated ( log2 fold change 1 ) . a network view of the same biological process demonstrating the fold changes of individual genes is provided in supplementary fig . genes that have protein products that physically interact tend to correlate in expression ( 22 ) and are likely related by function or membership in the same signaling pathways . we consequently examined the subnetworks of genes that interact at the protein level with inflammatory markers identified in the strain comparisons ( fig . minor changes in the expression of its interacting genes were observed in the 6-week - old b6 versus 129 mice comparisons ( fig . however , by 6 months of age , more than 90% of the nodes in the network were differentially active between the two strains ( large green triangles in fig . hence , genes interacting with thy1 were dramatically more upregulated with age - associated weight gain in adipose tissue of b6 mice than in 129 mice , suggesting that b6 mice have more t - cells recruited to adipose tissue in response to increasing adiposity than 129 mice . the same pattern was observed in subnetworks generated for cd45 , a leukocyte marker ( fig . 3c ) ; and tumor necrosis factor ( tnf ) , a proinflammatory cytokine ( supplementary fig . together these data suggest that b6 mice show a proinflammatory tendency in fat at 6 weeks of age not observed in 129 mice , and that this differentially increases with weight gain promoted by aging , affecting the pool of inflammatory cells . gene networks associated with inflammatory markers in adipose tissue of b6 and 129 mice at 6 weeks ( left ) and 6 months ( right ) of age . gene networks were generated by mapping genes that were significantly overrepresented ( q value < 0.25 ) among the gene network enrichment analysis results at 6 months intersected with those at 6 weeks onto protein - protein interaction networks involving at least one interactor to each inflammatory marker : ( a ) thy1 , ( b ) cd45 , and ( c ) mcp1 . the cd45 network was further restricted only to genes showing a greater than twofold magnitude difference in b6 vs. 129 mice between 6 weeks and 6 months . this was done to aid visualization since the unrestricted network consisted of 416 interactions and 141 genes . colors range from bright red to green , corresponding to twofold less and twofold greater differences in expression between b6 and 129 at each age , respectively . nodes point upward if the fold - change difference between b6 and 129 is greater at 6 months than at 6 weeks , and downward otherwise . the node size corresponds to the magnitude of that fold - change difference between the two ages . networks around different biomarkers are not to scale with one another . to validate the observations obtained using network analysis , we measured markers of inflammatory cells and cytokine / chemokine genes in the adipose tissue of b6 and 129 mice using quantitative rt - pcr ( qpcr ) ( fig . , there were higher levels of macrophage / myeloid cell markers ( cd68 , cd11b , and cd18 ) , lymphocyte markers ( cd3 , cd72 , and cd80 ) , t - cell attractants / activators ccl5/rantes , stroma cell - derived factor-1 ( sdf1 ) , and interferon- ( ifn ) , among others , in b6 versus 129 mice ( fig . 4a and b ) . for most of these genes , the differences between strains were maintained or further exacerbated at 6 months of age . mcp1 , a monocyte attractant chemokine , despite being similarly expressed in young b6 and 129 mice , exhibited a 4.4-fold increase in b6 mice between 6 weeks and 6 months of age , whereas in 129 mice , there was a 78% reduction with age ( fig . 4b ) . for a few cytokines , namely tnf , il6 , and macrophage migration inhibitory factor ( mif ) , 4b ) . expression of inflammatory markers in adipose tissue of 6-week - old ( 6 weeks ) or 6-month - old ( 6 months ) b6 and 129 mice . a : leukocyte markers , including selective myeloid cell markers ( e.g. , macrophages and dendritic cells ) and lymphoid markers ( e.g. , lymphocytes ) . all values are normalized by tata - binding protein ( tbp ) and expressed as fold change of the 6-week - old b6 average value . results represent mean sem of 57 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 . # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. 6 weeks . we also validated by rt - pcr the expression of selected genes identified as differentially present in the differentially active subnetworks identified by gnea . two mediators of lipopolysaccharide ( lps ) signaling , i.e. , the lymphocyte antigen 86 ( ly86 ) and lps - binding protein ( lbp ) , were highly ranked by gnea and were substantially higher in b6 compared with 129 mice at both ages ( fig . 4c ) . to determine if the higher expression of inflammation markers in b6 versus 129 mice was restricted to the adipose tissue or present in other tissues , we assessed the expression of two macrophage markers ( cd68 and f4/80 ) , two t - cell markers ( cd3 and thy1 ) , and two chemokines ( mcp1 and sdf1 ) in the liver , skeletal muscle , and spleen of b6 and 129 mice at both ages using qpcr . the pattern of expression in liver was qualitatively similar to that in adipose tissue , but with smaller fold differences ( supplementary fig . , no differences were observed in the skeletal muscle or spleen in the expression for most of these genes ( supplementary fig . the two exceptions were mcp1 , which was increased in all tissues in the 6-month - old b6 mice when compared with 129 mice , and cd3 , which increased in both b6 and 129 mice with age in skeletal muscle only ( supplementary fig . s4 ) . to determine if there was a cellular shift in the adipose tissue contributing to our computational and qpcr results , we analyzed the inflammatory cell repertoire in the fat tissue of b6 versus 129 mice using flow cytometry ( fig . 5 ) . this revealed a significant twofold increase in the number of nonerythrocyte stromavascular fraction cells per gram of epididymal fat in b6 compared with 129 mice at both ages ( fig . although the number of leukocytes per gram of adipose tissue decreased in both strains with age as adipose mass increased , at both time points the number of leukocytes was about 2.5-fold higher in the stromavascular fraction from b6 fat than in 129 fat ( fig . , the number of macrophages per gram of fat decreased with age because of growing fat mass , but was higher in the b6 versus 129 mice at both ages ( fig . 5a ) . these data correlated with histologic evidence of crown - like structures , i.e. , collections of macrophages surrounding dead or dying adipocytes , in the adipose tissue of 6-month - old b6 mice ( supplementary fig . s1 ) . immune cell repertoire in adipose tissue of 6-week - old ( 6 weeks ) or 6-month - old ( 6 months ) b6 and 129 mice . cells in the stromavascular fraction were labeled with fluorescence - conjugated antibodies against different myeloid cell and lymphoid markers and counted using flow cytometry . nonerythrocytes stromavascular cells , pi-/ter-119- ; leukocytes , pi-/ter-119-/cd45 + ; macrophages , pi-/ter-119-/cd45+/cd11b+/f4/80+/cd11c- ; cd11c - myeloid cells , pi-/ter-119-/cd45+/cd11b+/f4/80+/cd11c+ ; t - cells , pi-/ter-119-/cd45+/cd3 + ; t - helper cells , pi-/ter-119-/cd45+/cd3+/cd4 + . a : represents number of cells per gram of fat tissue and ( b ) the total number of cells in the epididymal fat depot . all values are mean sem of 48 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 mice . # p < 0.05 ; # # p somewhat surprisingly , the density of cd11c - myeloid cells , which have been previously associated with insulin resistance states in mice ( 23 ) , was decreased in b6 versus 129 mice at 6 weeks and 6 months of age by 65 and 37% , respectively ( fig . the t - cell population was dramatically increased by 7.1-fold in b6 mice at 6 weeks of age ( fig . the density of t - cells increased by 25-fold in b6 mice with age - associated weight gain , but showed only a threefold increase in 129 mice ( fig . 5a ) . thus , at 6 months of age , b6 mice had > 55-fold more total t - cells and > 60-fold more cd4 t - helper cells in the adipose tissue compared with the 129 mice ( fig . b - cells were also present in the adipose tissue of these mice ; however , they were not considered in the analysis because of their modest numbers in comparison with other leukocytes ( < 1% ) . a similar pattern of lymphocyte / monocyte infiltration was observed in the 6-week - old mice when the total number of cells in the epididymal fat pads was calculated , i.e. , cells per gram total grams of fat ( fig . when expressed as total cells , however , it was apparent that the number of each of the myeloid and lymphoid cell population increased with age in both strains . importantly , the differences between strains were maintained in both younger and older mice ( fig . these results confirm the gene network and expression analysis and demonstrate that most of these differences are consistent with differences in the lymphoid / myeloid population in the adipose tissue of b6 versus 129 mice . to investigate how differences in the genetic background could impact the effects of diet - induced obesity on the inflammatory cell repertoire of the adipose tissue , cohorts of b6 and 129 mice were placed on a high - fat diet ( hfd ) for 18 weeks and compared with mice on lfd . on both lfd and hfd , the mrna expression of the macrophage marker cd68 was higher by twofold or greater in b6 compared with 129 mice ( fig . similarly , mcp1 expression was higher in b6 than in 129 on lfd , and this was maintained with hfd ( fig . the macrophage marker f4/80 and sdf1 both trended higher in b6 than in 129 mice on lfd , but neither reached statistical significance ( fig . interestingly , the hfd - induced increases in the expression of the t - cell markers cd3 and thy1 were completely abrogated in the 129 mice ( fig . caloric restriction , an intervention that drastically reduces fat mass , helps prevents age - associated diseases , and prolongs life span ( 24 ) , decreased the expression of cd3 and thy1 in the adipose tissue of b6 mice by 41 and 51% , respectively , without affecting the expression of macrophage markers ( supplementary fig . s5 ) . these results demonstrate a positive correlation between adiposity and the expression of t- cells in adipose tissue of b6 mice , a phenomenon that is significantly impaired in 129 mice . expression of inflammatory markers in adipose tissue of 6-month - old b6 and 129 mice fed with lfd or hfd . all values are normalized by tbp and expressed as fold change of the average value of the lfd b6 mice . results represent mean sem of 46 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 mice . b : the network view of the immune system process differences between b6 and 129 mouse strains on a lfd ( left ) and hfd ( right ) . the gene network was generated by mapping genes that were significantly overrepresented in b6 vs. 129 mice ( q value < 0.25 ) among the gnea results on hfd intersected with those on lfd onto protein - protein interaction networks involving genes annotated with the immune system process gene set . genes in red are more than twofold higher in 129 mice compared with b6 mice ; genes in green are more than twofold higher in b6 mice compared with 129 mice . genes are denoted by a downward triangle if the fold change of the hfd animals is less than those on the lfd , and by an upward triangle for the converse ; the size of the triangle denotes the magnitude of the difference . accordingly , analysis of b6 versus 129 mice under lfd and hfd using network analysis revealed a substantial number of genes in the immune system process gene set with higher expression in b6 compared with 129 mice , as evidenced by the large , bright green nodes in fig . the majority of these genes had greater differences between b6 and 129 mice under hfd than under lfd conditions , as shown by the high number of upward triangles in fig . in particular , t - cell marker cd3 and the subnetwork around t - helper marker cd4 were among the most upregulated genes with hfd in b6 versus 129 mice ( arrows in fig . conversely , anti - inflammatory cytokines , such as interleukin-4 ( il-4 ) and interleukin-10 ( il-10 ) were reduced after hfd in the b6 mice , but not in 129 mice ( arrows in fig . at 6 months of age , on a chow diet , b6 mice were overweight , hyperglycemic , and exhibited higher plasma leptin and insulin levels than 129 mice of the same age ( table 1 , supplementary fig . b6 mice showed clear signs of insulin resistance when compared with 129 mice , even on low - fat diet ( lfd ) , and this difference was further exacerbated when the mice were placed on a high - fat diet ( table 1 ) ( 8) . at 6 weeks of age , however , no phenotypic differences or serum markers indicative of diabetes and obesity could be detected between the two strains ( table 1 ) ( 8) . indeed , 6-week - old 129 mice have the same weight as b6 mice and actually have significantly more fat mass , as indicated by higher fat pad weight ( table 1 ) . by studying b6 versus 129 mice at different ages and on different diets , one may therefore elucidate the background inherited phenomena predisposing to obesity - accompanied insulin resistance . to identify gene networks that might provide early biomarkers of adult metabolic disease , we initially performed microarray analyses on rna from adipose tissue , liver , and skeletal muscle of b6 and 129 mice at 6 weeks and 6 months of age on a standard chow diet . we then applied gnea to detect networks that were differentially active in a predisease stage and to estimate how they changed over time in disease - prone versus diabetes - resistant strains ( fig . since disease susceptibility is an inherited phenomenon in these animals , we hypothesized that the predisposing hereditable biological processes would be those showing differential activity in young mice and maintained or further increased with age . among the three different tissues in which b6 versus 129 mice were compared , only adipose tissue exhibited significantly enriched biological processes ( q value < 0.33 ) at 6 weeks of age by gnea , with 12 enriched gene sets , and this increased to 25 enriched gene sets at 6 months of age . of these , only two sets ( immune system process and regulation of cytokine secretion ) were found enriched at both ages ( supplementary table 1 ) , and both of these related to inflammatory pathways . furthermore , the immune system process was the most significantly enriched gene set at 6 months of age ( q value = 0.008 ; supplementary table 1 ) . in contrast , in the liver , 37 gene sets ( q value < 0.33 ) were differentially regulated in b6 versus 129 at 6 months of age , but none was significantly different in the 6-week - old animals . in skeletal muscle , no significant difference was observed at either age ( data not shown ) . in addition to gnea , the adipose tissue dataset was analyzed using gene set enrichment analysis ( gsea ) ( 20 ) and the standard hypergeometric test ( 21 ) . on the other hand , gsea failed to identify any gene set that was differentially expressed at both ages ( supplementary table 3 ) . by comparison , the standard hypergeometric test ( 21 ) failed to identify either a clear immune signature at 6 weeks or any enriched gene sets at 6 months ( supplementary tables 2 and 3 ) . to determine how genes related to the immune response were differentially expressed in the fat tissue of young b6 and 129 mice and changed in expression as the mice aged , we identified the network associated with the immune system process gene set ( supplementary fig . s2 ) and plotted the histogram of differential expression for its genes in b6 versus 129 mice at 6 weeks and 6 months ( fig . a few individual genes , such as apolipoprotein a ( apoa1 and apoa2 ) and mannose - binding lectin ( mbl2 ) were more strongly downregulated in b6 versus 129 mice at 6 months and less so at 6 weeks . nonetheless , much of the immune system process network was upregulated at 6 months compared with 6 weeks ( note the right - shift in the histogram for 6 months vs. 6 weeks in fig . lymphoid and myeloid markers , such as cd45 , cd3 , and chemokine ( c - c motif ) receptor 5 ( ccr5 ) , monocyte chemotactic protein-1 ( mcp1 ) , as well as proinflammatory genes such as chemokine ( c - c motif ) ligand 5 ( ccl5 ) and interleukin-6 ( il6 ) , were more highly expressed in b6 compared with 129 mice at 6 weeks , and this increased further at 6 months of age ( supplementary fig . 2 ) , many of the immune system process genes already showed twofold or greater differences in expression in b6 versus 129 mice at 6 weeks , an age when no significant metabolic differences were observed between strains . histogram of differential expression between b6 and 129 mice at 6 weeks and 6 months of age for genes in the immune system process network . the x - axis values are the upper bounds of the histogram bins and correspond to the log2 fold - change difference in b6 vs. 129 mice . few network genes are unchanged ( absolute log2 fold change < 1 ) in b6 vs. 129 mice at 6 months . the majority are strongly upregulated ( log2 fold change 1 ) , although a few are strongly downregulated ( log2 fold change 1 ) . a network view of the same biological process demonstrating the fold changes of individual genes is provided in supplementary fig . genes that have protein products that physically interact tend to correlate in expression ( 22 ) and are likely related by function or membership in the same signaling pathways . we consequently examined the subnetworks of genes that interact at the protein level with inflammatory markers identified in the strain comparisons ( fig . minor changes in the expression of its interacting genes were observed in the 6-week - old b6 versus 129 mice comparisons ( fig . however , by 6 months of age , more than 90% of the nodes in the network were differentially active between the two strains ( large green triangles in fig . hence , genes interacting with thy1 were dramatically more upregulated with age - associated weight gain in adipose tissue of b6 mice than in 129 mice , suggesting that b6 mice have more t - cells recruited to adipose tissue in response to increasing adiposity than 129 mice . the same pattern was observed in subnetworks generated for cd45 , a leukocyte marker ( fig . 3c ) ; and tumor necrosis factor ( tnf ) , a proinflammatory cytokine ( supplementary fig . together these data suggest that b6 mice show a proinflammatory tendency in fat at 6 weeks of age not observed in 129 mice , and that this differentially increases with weight gain promoted by aging , affecting the pool of inflammatory cells . gene networks associated with inflammatory markers in adipose tissue of b6 and 129 mice at 6 weeks ( left ) and 6 months ( right ) of age . gene networks were generated by mapping genes that were significantly overrepresented ( q value < 0.25 ) among the gene network enrichment analysis results at 6 months intersected with those at 6 weeks onto protein - protein interaction networks involving at least one interactor to each inflammatory marker : ( a ) thy1 , ( b ) cd45 , and ( c ) mcp1 . the cd45 network was further restricted only to genes showing a greater than twofold magnitude difference in b6 vs. 129 mice between 6 weeks and 6 months . this was done to aid visualization since the unrestricted network consisted of 416 interactions and 141 genes . colors range from bright red to green , corresponding to twofold less and twofold greater differences in expression between b6 and 129 at each age , respectively . nodes point upward if the fold - change difference between b6 and 129 is greater at 6 months than at 6 weeks , and downward otherwise . the node size corresponds to the magnitude of that fold - change difference between the two ages . to validate the observations obtained using network analysis , we measured markers of inflammatory cells and cytokine / chemokine genes in the adipose tissue of b6 and 129 mice using quantitative rt - pcr ( qpcr ) ( fig . , there were higher levels of macrophage / myeloid cell markers ( cd68 , cd11b , and cd18 ) , lymphocyte markers ( cd3 , cd72 , and cd80 ) , t - cell attractants / activators ccl5/rantes , stroma cell - derived factor-1 ( sdf1 ) , and interferon- ( ifn ) , among others , in b6 versus 129 mice ( fig . mcp1 , a monocyte attractant chemokine , despite being similarly expressed in young b6 and 129 mice , exhibited a 4.4-fold increase in b6 mice between 6 weeks and 6 months of age , whereas in 129 mice , there was a 78% reduction with age ( fig . 4b ) . for a few cytokines , namely tnf , il6 , and macrophage migration inhibitory factor ( mif ) , 4b ) . expression of inflammatory markers in adipose tissue of 6-week - old ( 6 weeks ) or 6-month - old ( 6 months ) b6 and 129 mice . a : leukocyte markers , including selective myeloid cell markers ( e.g. , macrophages and dendritic cells ) and lymphoid markers ( e.g. , lymphocytes ) . all values are normalized by tata - binding protein ( tbp ) and expressed as fold change of the 6-week - old b6 average value . results represent mean sem of 57 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 . # p < 0.05 ; # # p < 0.01 ; # # # p < 0.001 vs. 6 weeks . we also validated by rt - pcr the expression of selected genes identified as differentially present in the differentially active subnetworks identified by gnea . two mediators of lipopolysaccharide ( lps ) signaling , i.e. , the lymphocyte antigen 86 ( ly86 ) and lps - binding protein ( lbp ) , were highly ranked by gnea and were substantially higher in b6 compared with 129 mice at both ages ( fig . 4c ) . to determine if the higher expression of inflammation markers in b6 versus 129 mice was restricted to the adipose tissue or present in other tissues , we assessed the expression of two macrophage markers ( cd68 and f4/80 ) , two t - cell markers ( cd3 and thy1 ) , and two chemokines ( mcp1 and sdf1 ) in the liver , skeletal muscle , and spleen of b6 and 129 mice at both ages using qpcr . the pattern of expression in liver was qualitatively similar to that in adipose tissue , but with smaller fold differences ( supplementary fig . in contrast , no differences were observed in the skeletal muscle or spleen in the expression for most of these genes ( supplementary fig . the two exceptions were mcp1 , which was increased in all tissues in the 6-month - old b6 mice when compared with 129 mice , and cd3 , which increased in both b6 and 129 mice with age in skeletal muscle only ( supplementary fig . to determine if there was a cellular shift in the adipose tissue contributing to our computational and qpcr results , we analyzed the inflammatory cell repertoire in the fat tissue of b6 versus 129 mice using flow cytometry ( fig . this revealed a significant twofold increase in the number of nonerythrocyte stromavascular fraction cells per gram of epididymal fat in b6 compared with 129 mice at both ages ( fig . although the number of leukocytes per gram of adipose tissue decreased in both strains with age as adipose mass increased , at both time points the number of leukocytes was about 2.5-fold higher in the stromavascular fraction from b6 fat than in 129 fat ( fig . , the number of macrophages per gram of fat decreased with age because of growing fat mass , but was higher in the b6 versus 129 mice at both ages ( fig . 5a ) . these data correlated with histologic evidence of crown - like structures , i.e. , collections of macrophages surrounding dead or dying adipocytes , in the adipose tissue of 6-month - old b6 mice ( supplementary fig . s1 ) . immune cell repertoire in adipose tissue of 6-week - old ( 6 weeks ) or 6-month - old ( 6 months ) b6 and 129 mice . cells in the stromavascular fraction were labeled with fluorescence - conjugated antibodies against different myeloid cell and lymphoid markers and counted using flow cytometry . nonerythrocytes stromavascular cells , pi-/ter-119- ; leukocytes , pi-/ter-119-/cd45 + ; macrophages , pi-/ter-119-/cd45+/cd11b+/f4/80+/cd11c- ; cd11c - myeloid cells , pi-/ter-119-/cd45+/cd11b+/f4/80+/cd11c+ ; t - cells , pi-/ter-119-/cd45+/cd3 + ; t - helper cells , pi-/ter-119-/cd45+/cd3+/cd4 + . a : represents number of cells per gram of fat tissue and ( b ) the total number of cells in the epididymal fat depot . all values are mean sem of 48 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 mice . # p < 0.05 ; # # p somewhat surprisingly , the density of cd11c - myeloid cells , which have been previously associated with insulin resistance states in mice ( 23 ) , was decreased in b6 versus 129 mice at 6 weeks and 6 months of age by 65 and 37% , respectively ( fig . the t - cell population was dramatically increased by 7.1-fold in b6 mice at 6 weeks of age ( fig . furthermore , the density of t - cells increased by 25-fold in b6 mice with age - associated weight gain , but showed only a threefold increase in 129 mice ( fig . 5a ) . thus , at 6 months of age , b6 mice had > 55-fold more total t - cells and > 60-fold more cd4 t - helper cells in the adipose tissue compared with the 129 mice ( fig . b - cells were also present in the adipose tissue of these mice ; however , they were not considered in the analysis because of their modest numbers in comparison with other leukocytes ( < 1% ) . a similar pattern of lymphocyte / monocyte infiltration was observed in the 6-week - old mice when the total number of cells in the epididymal fat pads was calculated , i.e. , cells per gram total grams of fat ( fig . when expressed as total cells , however , it was apparent that the number of each of the myeloid and lymphoid cell population increased with age in both strains . importantly , the differences between strains were maintained in both younger and older mice ( fig . these results confirm the gene network and expression analysis and demonstrate that most of these differences are consistent with differences in the lymphoid / myeloid population in the adipose tissue of b6 versus 129 mice . to investigate how differences in the genetic background could impact the effects of diet - induced obesity on the inflammatory cell repertoire of the adipose tissue , cohorts of b6 and 129 mice were placed on a high - fat diet ( hfd ) for 18 weeks and compared with mice on lfd . on both lfd and hfd , the mrna expression of the macrophage marker cd68 was higher by twofold or greater in b6 compared with 129 mice ( fig . 6a ) . similarly , mcp1 expression was higher in b6 than in 129 on lfd , and this was maintained with hfd ( fig . the macrophage marker f4/80 and sdf1 both trended higher in b6 than in 129 mice on lfd , but neither reached statistical significance ( fig . interestingly , the hfd - induced increases in the expression of the t - cell markers cd3 and thy1 were completely abrogated in the 129 mice ( fig . caloric restriction , an intervention that drastically reduces fat mass , helps prevents age - associated diseases , and prolongs life span ( 24 ) , decreased the expression of cd3 and thy1 in the adipose tissue of b6 mice by 41 and 51% , respectively , without affecting the expression of macrophage markers ( supplementary fig . these results demonstrate a positive correlation between adiposity and the expression of t- cells in adipose tissue of b6 mice , a phenomenon that is significantly impaired in 129 mice . expression of inflammatory markers in adipose tissue of 6-month - old b6 and 129 mice fed with lfd or hfd . all values are normalized by tbp and expressed as fold change of the average value of the lfd b6 mice . results represent mean sem of 46 animals . * p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 vs. 129 mice . b : the network view of the immune system process differences between b6 and 129 mouse strains on a lfd ( left ) and hfd ( right ) . the gene network was generated by mapping genes that were significantly overrepresented in b6 vs. 129 mice ( q value < 0.25 ) among the gnea results on hfd intersected with those on lfd onto protein - protein interaction networks involving genes annotated with the immune system process gene set . genes in red are more than twofold higher in 129 mice compared with b6 mice ; genes in green are more than twofold higher in b6 mice compared with 129 mice . genes are denoted by a downward triangle if the fold change of the hfd animals is less than those on the lfd , and by an upward triangle for the converse ; the size of the triangle denotes the magnitude of the difference . accordingly , analysis of b6 versus 129 mice under lfd and hfd using network analysis revealed a substantial number of genes in the immune system process gene set with higher expression in b6 compared with 129 mice , as evidenced by the large , bright green nodes in fig . the majority of these genes had greater differences between b6 and 129 mice under hfd than under lfd conditions , as shown by the high number of upward triangles in fig . in particular , t - cell marker cd3 and the subnetwork around t - helper marker cd4 were among the most upregulated genes with hfd in b6 versus 129 mice ( arrows in fig . conversely , anti - inflammatory cytokines , such as interleukin-4 ( il-4 ) and interleukin-10 ( il-10 ) were reduced after hfd in the b6 mice , but not in 129 mice ( arrows in fig . although a relationship between obesity and inflammation in fat has been previously observed , in most cases these inflammatory changes have been viewed as being secondary to obesity . our data , comparing diabetes - prone b6 mouse and diabetes - resistant 129 mouse at 6 weeks of age , show alterations in the inflammatory process in adipose tissue even before differences in metabolic parameters can be detected . thus , b6 mice exhibit increased expression of the t - cell chemokines sdf1 and ccl5/rantes and an increased number of t - cells in the fat tissue . these differences are associated with higher ifn and cd80 levels in the b6 mice both molecules are known to participate in t - cell function and activation ( 25 ) ( summarized in the model shown in fig . 7 ) . schematic model illustrating the potential causes and consequences related to the different repertoire of immune cells in adipose tissue of b6 and 129 mice . solid arrows , secretion ; dashed arrows , migration ; dotted arrows , migration / differentiation . recent work has shown that t - cells infiltrate into the visceral adipose tissue of obese animals and humans with type 2 diabetes , and this is followed by recruitment of macrophages and development of insulin resistance ( 2629 ) . based on these findings , a model for the role of t - cells in the pathogenesis of obesity and insulin resistance has been proposed in which increases in sdf1 and ccl5/rantes levels in adipose tissue occur in response to an obesogenic environment and promote infiltration with t lymphocytes ( 29 ) . ifn derived from these t - cells then promotes mcp1 secretion by preadipocytes ( and possibly other cell types ) , resulting in recruitment of macrophages that further contribute to insulin resistance by production of proinflammatory cytokines ( 29 ) . our study provides evidence to support a major impact of the genetic background of different mouse strains in the migration of t - cells to the adipose tissue , both in the basal state and in response to weight gain . in b6 mice , the number of t - cells in the adipose tissue correlates positively with the increase in adipose mass as a consequence of aging or hfd . by contrast , this response is practically absent in 129 mice despite a significant increase in adiposity in response to age or hfd . thus , similar to mice with ablation of t - cells ( 27,28 ) , 129 mice develop only mild insulin resistance in response to obesity . to what extent aging and the composition of the diet impact the infiltration of t - cells into adipose tissue , in addition to the effects of weight gain , remain to be determined . it is clear though that insulin resistance in response to increased adiposity differ substantially between mouse strains , and this phenomenon is correlated with the migration of t - cell to the adipose tissue . thus , inflammation in the adipose tissue does not always correlate with weight gain and is strongly dependent on the genetic background of the host . similar differences dependent on genetic background are likely to occur in humans and contribute to differences in obesity - induced diabetes risk in different ethnic groups or even different individuals , allowing for some of the fat - fit phenotype . among other implications , these observations clearly impact the choice of models for metabolic studies , in particular when knockout mice are used . knockout mice are often derived from the embryonic stem cells of 129 mice , and therefore , in many cases , studies are performed on mice on a pure 129 background or mixed 129/b6 background . in these cases , backcrossing to b6 mice for several generations usually potentiates the inflammatory and metabolic response of the model to environmental factors . however , it is not clear which of these backgrounds ( b6 , 129 , or mixed ) most closely mimic the human condition . other recent reports support our findings that metabolic disease traits can be associated with alterations of inflammatory gene expression networks in the adipose tissue and liver ( 30,31 ) . however , to our knowledge , no study has previously demonstrated a measurable difference in inflammation preceding any measurable phenotypic difference associated with metabolic diseases . the novelty of our study is that , by comparing young b6 and 129 animals using a sensitive computational approach , we can focus on factors that can potentially predispose to disease in a prospective manner and avoid findings that are mainly secondary to obesity or metabolic differences . our investigation also examined differences between b6 and 129 mice in multiple organs , allowing us to conclude that inflammation in adipose tissue , and to a lesser extent in liver , but not in skeletal muscle or spleen , is associated with the predisposition to insulin resistance . in addition , our computational analysis was able to set forth hypotheses that led to subsequent biologic validation experiments which provided further insight into the components of the immune system that may contribute to metabolic diseases ( i.e. , t - cell recruitment ) . in addition to inflammation , other phenomena have been shown to participate in the multifactorial pathophysiology of insulin resistance . alterations in insulin receptor levels and insulin signaling through irs - proteins ( 32 ) , induction of the unfolded protein response ( 33 ) and oxidative stress pathways ( 34 ) , and changes in lipid ( 35 ) and amino acid metabolism ( 36 ) can all promote insulin resistance and contribute to the final phenotype . many of these pathways act by producing post - translational modifications of signaling proteins , such as phosphorylation or alterations in compartmentalization , which would not be detected as changes at the gene expression level . in this regard it is worth noting that in addition to differences in inflammation , gnea analysis was able to identify other gene sets differentially expressed between the mouse strains in adipose tissue at 6 weeks of age ( supplementary table s2 ) , including networks related to signal transduction , protein secretion pathways , and glucose catabolism . many of these pathways can interact with inflammatory pathways , and this crosstalk could represent an entry point to the manifestation of metabolic diseases . thus , although changes in the immune response are definitely one of the factors that precedes and predicts the tendency of b6 mice to have greater insulin resistance than 129 , it is unlikely that inflammation is the only predisposing risk factor associated with diabetes between these two mouse strains . in summary , it has been proposed that type 2 diabetes and obesity are diseases associated with an immune system that can not cope appropriately with environmental threats ( 3739 ) . based on this hypothesis , anti - inflammatory drugs , such as salicylates ( 40,41 ) and interleukin-1 blockers ( 42 ) , have been used to improve glycemia in individuals with type 2 diabetes . in this study , we demonstrate that pre - existing differences in the inflammatory milieu in metabolically active tissues may represent an important component of the genetic background as a risk factor to metabolic diseases . thus , inflammation can not be viewed as only a mechanism that links susceptibility factors such as overfeeding , underactivity , aging , and stress to metabolic diseases it may also link these pathologies to heritability . our study indicates that inflammation is an important early variable in the metabolic response to environmental challenges and suggests several potential targets for intervention , including lbp , ly86 , sdf1 , ccl5/rantes , and mcp1 . this provides new strategies for reducing the epidemic of type 2 diabetes and metabolic diseases in spite of increasing obesity by attacking the variable genetic risk . | objectivetype 2 diabetes and obesity are increasingly affecting human populations around the world .
our goal was to identify early molecular signatures predicting genetic risk to these metabolic diseases using two strains of mice that differ greatly in disease susceptibility.research design and methodswe integrated metabolic characterization , gene expression , protein - protein interaction networks , rt - pcr , and flow cytometry analyses of adipose , skeletal muscle , and liver tissue of diabetes - prone c57bl/6ntac ( b6 ) mice and diabetes - resistant 129s6/svevtac ( 129 ) mice at 6 weeks and 6 months of age.resultsat 6 weeks of age , b6 mice were metabolically indistinguishable from 129 mice , however , adipose tissue showed a consistent gene expression signature that differentiated between the strains .
in particular , immune system gene networks and inflammatory biomarkers were upregulated in adipose tissue of b6 mice , despite a low normal fat mass .
this was accompanied by increased t - cell and macrophage infiltration .
the expression of the same networks and biomarkers , particularly those related to t - cells , further increased in adipose tissue of b6 mice , but only minimally in 129 mice , in response to weight gain promoted by age or high - fat diet , further exacerbating the differences between strains.conclusionsinsulin resistance in mice with differential susceptibility to diabetes and metabolic syndrome is preceded by differences in the inflammatory response of adipose tissue .
this phenomenon may serve as an early indicator of disease and contribute to disease susceptibility and progression . |
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the online version of this article ( doi:10.1007/s00392 - 011 - 0368 - 2 ) contains supplementary material , which is available to authorized users . recent randomized controlled trials have demonstrated the mortality benefit of an implantable cardioverter - defibrillator ( icd ) over optimal medical therapy in patients with left ventricular ( lv ) dysfunction [ 1 , 2 ] . the multicenter automatic defibrillator implantation trial ( madit ) ii showed 31% reduction in relative risk of mortality among patients with prior myocardial infarction and a lv ejection fraction ( lvef ) 30% . the sudden cardiac death in heart failure trial ( scd - heft ) showed 23% reduction in relative risk of mortality in patients with lvef 35% and new york heart association ( nyha ) class ii iii congestive heart failure due to ischemic or non - ischemic etiology . despite the significant reduction in mortality shown in madit ii and scd - heft , the absolute benefits were relatively small : 5.6% over 20 months and 7.2% over 5 years , respectively [ 1 , 2 ] . because of concerns not only for the potential side effects of icd [ 35 ] but also for the huge economic burden , further risk stratification to identify patients who are likely or unlikely to benefit from icd seems to be necessary . microvolt - level t - wave alternans ( twa ) in a body - surface electrocardiogram ( ecg ) was proposed as a non - invasive method to evaluate risk of sudden cardiac death ( scd ) or lethal ventricular tachyarrhythmia . although high negative predictive values of twa have been reported [ 611 ] , recent studies have reported conflicting data on the prognostic value of twa [ 1214 ] . furthermore , twa can not be assessed in patients with persistent atrial fibrillation ( af ) , ventricular pacing or exercise intolerance , who account for a sizable portion of the entire population of patients with lv dysfunction [ 1517 ] . to address these issues , we have conducted a multicenter , prospective study assessing the predictive value of twa for scd or lethal ventricular tachyarrhythmia in a broad population of japanese patients with lv dysfunction , including those ineligible for the twa test . the prospective evaluation of ventricular tachyarrhythmic events and sudden cardiac death in patients with left ventricular dysfunction ( prevent - scd ) study is a prospective multicenter registry of japanese patients with lv dysfunction , designed to evaluate the prognostic value of twa and other potential arrhythmia risk factors for ventricular tachyarrhythmia . . 1study design of the prevent - scd study study design of the prevent - scd study patients with lvef 40% without treatable cardiac ischemia , who had nyha class i iii heart failure , were eligible for the study . all patients had to be maintained on an optimal dose of angiotensin - converting enzyme inhibitors or angiotensin receptor blockers and beta - blockers before enrollment in the study unless these drugs were contraindicated or patients were intolerant . patients were excluded if they had a history of sustained ventricular fibrillation ( vf ) or ventricular tachycardia ( vt ) , had previous icd implantation , had nyha class iv heart failure , had a myocardial infarction ( mi ) within the past month , had undergone coronary revascularization within the past 3 months , had treatable cardiac ischemia , had scheduled coronary revascularization or cardiac surgery , or had any condition other than cardiac disease with expectation of survival less than 1 year . lv function was assessed by echocardiography using modified simpson s method . when echocardiography was considered inappropriate due to poor images , then magnetic resonance imaging , angiography or radionuclide scanning ischemic cardiomyopathies were defined as lv dysfunction associated with a documented history of mi or severe narrowing of at least one of the three major coronary arteries . absence of treatable cardiac ischemia was confirmed by a stress test and/or coronary angiography . between june 2004 and february 2007 , a total of 453 patients were enrolled from 38 centers in japan ( appendix 1 ) . patients were scheduled to undergo twa and other risk stratification tests , including 12-lead ecg and holter ecg as mandatory tests and signal - averaged ecg and electrophysiological ( ep ) study as optional tests . therapeutic strategies based on the results of the risk stratification tests were determined within 2 months of enrollment . the choice of therapeutic strategy , such as icd implantation , was left to the discretion of the attending physicians . the relevant review boards or ethics committees in all participating centers approved the research protocol . written informed consent patients underwent the twa test while taking their regular medication , including beta - blockers . careful skin preparation was performed and high - resolution electrodes were used to minimize noise . ecg leads were placed at the standard 12-lead positions and in an orthogonal x , y and z configuration . the result of the twa test was automatically interpreted by the alternans report classifier within the ch2000 or heartwave system , reviewed by ep cardiologists at each individual center , and classified into positive , negative or indeterminate according to previously described criteria . because patients with indeterminate and positive twa tests have been reported to have a similar arrhythmic or mortality risk , they were prospectively combined for analysis as non - negative twa tests . in this study , therefore , comparisons were made among patients with negative twa , patients with non - negative ( positive or indeterminate ) twa , and those ineligible for the twa test . resting 12-lead ecg was evaluated for basic cardiac rhythm , heart rate , qrs duration , and qrs morphology . holter ecg was evaluated for number of premature ventricular contractions ( pvcs ) , as well as the presence or absence of non - sustained vt ( nsvt ) . other potential arrhythmia risk predictors included age , sex , etiology of lv dysfunction , nyha class , diabetes , hypertension , hyperlipidemia , af , ventricular pacing , blood pressure , lvef , lv end - diastolic dimension ( lvedd ) , mitral regurgitation , renal failure , anemia , brain natriuretic peptide ( bnp ) level and medication at the time of enrollment . all patients were followed in each participating center at least yearly after enrollment , either by hospital visits or telephone contact with the patients . when clinical events were suspected by telephone interview with the patients , they were confirmed by contact with the referring physicians . all icd patients were followed at least every 6 months by hospital visits , and all icd data were obtained by interrogation of the device . the prespecified primary end point of the study was a severe ventricular tachyarrhythmic event ( svte ) , defined as a composite of scd , sustained rapid - vt ( 188/min ) or vf , and appropriate icd therapy for rapid - vt ( 188/min ) or vf . the secondary end points were all - cause death , cardiac death , and a ventricular tachyarrhythmic event ( vte ) defined as a composite of svte , sustained vt ( < 188/min ) and appropriate icd therapy for vt ( < 188/min ) . scd was defined as unexpected natural death occurring instantaneously or within 24 h of the onset of acute symptoms or signs , of which the cause was proved to be cardiac at necropsy or assumed to be cardiac because of clinical circumstances . all of the primary and secondary events were adjudicated by a clinical events committee ( appendix 2 ) . statistical significance between the groups was assessed by student s t test for parametrically distributed continuous variables , the wilcoxon rank sum test for non - parametrically distributed continuous variables , and pearson s test for categorical variables . the significance of differences in event - free survival between groups was assessed with a log - rank test . two dummy variables were created to reflect three levels of twa status in the cohort : a negative test , a non - negative ( positive or indeterminate ) test , and no test ( ineligible for the twa test ) . we first selected variables with p values < 0.1 in the univariable cox models and then included them simultaneously in the multivariable models . the independent predictors of svte were identified by the forward and backward stepwise methods with inclusion and exclusion p values < 0.05 . relative risks are expressed as hazard ratios ( hrs ) with 95% confidence intervals ( cis ) . all tests were 2-sided , and a p value < 0.05 was considered statistically significant . the prospective evaluation of ventricular tachyarrhythmic events and sudden cardiac death in patients with left ventricular dysfunction ( prevent - scd ) study is a prospective multicenter registry of japanese patients with lv dysfunction , designed to evaluate the prognostic value of twa and other potential arrhythmia risk factors for ventricular tachyarrhythmia . . 1study design of the prevent - scd study study design of the prevent - scd study patients with lvef 40% without treatable cardiac ischemia , who had nyha class i iii heart failure , were eligible for the study . all patients had to be maintained on an optimal dose of angiotensin - converting enzyme inhibitors or angiotensin receptor blockers and beta - blockers before enrollment in the study unless these drugs were contraindicated or patients were intolerant . patients were excluded if they had a history of sustained ventricular fibrillation ( vf ) or ventricular tachycardia ( vt ) , had previous icd implantation , had nyha class iv heart failure , had a myocardial infarction ( mi ) within the past month , had undergone coronary revascularization within the past 3 months , had treatable cardiac ischemia , had scheduled coronary revascularization or cardiac surgery , or had any condition other than cardiac disease with expectation of survival less than 1 year . lv function was assessed by echocardiography using modified simpson s method . when echocardiography was considered inappropriate due to poor images , then magnetic resonance imaging , angiography or radionuclide scanning ischemic cardiomyopathies were defined as lv dysfunction associated with a documented history of mi or severe narrowing of at least one of the three major coronary arteries . absence of treatable cardiac ischemia was confirmed by a stress test and/or coronary angiography . between june 2004 and february 2007 , a total of 453 patients were enrolled from 38 centers in japan ( appendix 1 ) . patients were scheduled to undergo twa and other risk stratification tests , including 12-lead ecg and holter ecg as mandatory tests and signal - averaged ecg and electrophysiological ( ep ) study as optional tests . therapeutic strategies based on the results of the risk stratification tests were determined within 2 months of enrollment . the choice of therapeutic strategy , such as icd implantation , was left to the discretion of the attending physicians . the relevant review boards or ethics committees in all participating centers approved the research protocol . written informed consent patients underwent the twa test while taking their regular medication , including beta - blockers . careful skin preparation was performed and high - resolution electrodes were used to minimize noise . ecg leads were placed at the standard 12-lead positions and in an orthogonal x , y and z configuration . the result of the twa test was automatically interpreted by the alternans report classifier within the ch2000 or heartwave system , reviewed by ep cardiologists at each individual center , and classified into positive , negative or indeterminate according to previously described criteria . because patients with indeterminate and positive twa tests have been reported to have a similar arrhythmic or mortality risk , they were prospectively combined for analysis as non - negative twa tests . in this study , therefore , comparisons were made among patients with negative twa , patients with non - negative ( positive or indeterminate ) twa , and those ineligible for the twa test . resting 12-lead ecg was evaluated for basic cardiac rhythm , heart rate , qrs duration , and qrs morphology . holter ecg was evaluated for number of premature ventricular contractions ( pvcs ) , as well as the presence or absence of non - sustained vt ( nsvt ) . other potential arrhythmia risk predictors included age , sex , etiology of lv dysfunction , nyha class , diabetes , hypertension , hyperlipidemia , af , ventricular pacing , blood pressure , lvef , lv end - diastolic dimension ( lvedd ) , mitral regurgitation , renal failure , anemia , brain natriuretic peptide ( bnp ) level and medication at the time of enrollment . all patients were followed in each participating center at least yearly after enrollment , either by hospital visits or telephone contact with the patients . when clinical events were suspected by telephone interview with the patients , they were confirmed by contact with the referring physicians . all icd patients were followed at least every 6 months by hospital visits , and all icd data were obtained by interrogation of the device . the prespecified primary end point of the study was a severe ventricular tachyarrhythmic event ( svte ) , defined as a composite of scd , sustained rapid - vt ( 188/min ) or vf , and appropriate icd therapy for rapid - vt ( 188/min ) or vf . the secondary end points were all - cause death , cardiac death , and a ventricular tachyarrhythmic event ( vte ) defined as a composite of svte , sustained vt ( < 188/min ) and appropriate icd therapy for vt ( < 188/min ) . scd was defined as unexpected natural death occurring instantaneously or within 24 h of the onset of acute symptoms or signs , of which the cause was proved to be cardiac at necropsy or assumed to be cardiac because of clinical circumstances . all of the primary and secondary events were adjudicated by a clinical events committee ( appendix 2 ) . all continuous variables are expressed as mean sd or median . statistical significance between the groups was assessed by student s t test for parametrically distributed continuous variables , the wilcoxon rank sum test for non - parametrically distributed continuous variables , and pearson s test for categorical variables . the significance of differences in event - free survival between groups was assessed with a log - rank test . two dummy variables were created to reflect three levels of twa status in the cohort : a negative test , a non - negative ( positive or indeterminate ) test , and no test ( ineligible for the twa test ) . we first selected variables with p values < 0.1 in the univariable cox models and then included them simultaneously in the multivariable models . the independent predictors of svte were identified by the forward and backward stepwise methods with inclusion and exclusion p values < 0.05 . relative risks are expressed as hazard ratios ( hrs ) with 95% confidence intervals ( cis ) . all tests were 2-sided , and a p value < 0.05 was considered statistically significant . the clinical characteristics of the 453 patients enrolled in the prevent - scd study are listed in table 1 . the average age was 65.1 12.6 years , and 367 ( 81% ) were male . the etiology was ischemic in 189 ( 42% ) and non - ischemic in 264 ( 58% ) . the mean lvef was 29.2 7.4% . among the 189 ischemic patients , 170 ( 90% ) had had prior coronary revascularization.table 1patient characteristicsbaseline characteristics age65.1 12.6 male367 ( 81% ) nyha - iii128 ( 28% ) ischemic189 ( 42% ) non - ischemic264 ( 58%)risk factors and co - morbidities prior mi180 ( 40% ) cabg / pci170 ( 38% ) stroke or tia41 ( 9% ) diabetes125 ( 28% ) hypertension207 ( 46% ) hyperlipidemia144 ( 32% ) af120 ( 26% ) persistent af65 ( 14% ) pacemaker56 ( 12% ) systolic bp ( mmhg)119 22 systolic bp < 100 mmhg74 ( 16% ) diastolic bp ( mmhg)69 12 hemoglobin < 12 g / dl108 ( 24% ) egfr < 40 ml / min/1.73 m90 ( 20% ) bnp ( pg / ml)211 bnp 200 pg / ml228 ( 52%)echocardiography lvef ( % ) 29.2 7.4 lvef 30%228 ( 50% ) lvedd ( mm)62.3 8.9 lvedd 70 mm90 ( 20% ) mr grade 3 or 458 ( 13%)medications aceis or arbs375 ( 83% ) beta - blockers331 ( 73% ) diuretics350 ( 77% ) spironolactone236 ( 52% ) digitalis139 ( 31% ) amiodarone81 ( 18% ) aspirin229 ( 51% ) statin131 ( 29%)risk stratification tests twa ( n = 280 ) negative82 ( 29% ) positive138 ( 49% ) indeterminate60 ( 21%)resting 12-lead electrocardiogram ( n = 453 ) heart rate ( /min)71 qrs ( ms)113 qrs 120 ms199 ( 44%)holter monitoring ( n = 433 ) pvc > 10/h231 ( 53% ) nsvt157 ( 36%)antiarrhythmic therapies icd111 ( 25% ) icd with crt46 ( 10%)aceis angiotensin - converting enzyme inhibitors , af atrial fibrillation , arbs angiotensin receptor blockers , bnp brain natriuretic peptide , bp blood pressure , cabg coronary artery bypass grafting , crt cardiac resynchronization therapy , egfr estimated glomerular filtration rate , icd implantable cardioverter - defibrillator , lvedd left ventricular end - diastolic dimension , lvef left ventricular ejection fraction , mi myocardial infarction , mr mitral regurgitation , nsvt non - sustained ventricular tachycardia , nyha new york heart association , pci percutaneous coronary intervention , pvc premature ventricular contraction , tia transient ischemic attack , twa t - wave alternansmeasured by modified simpson s method . for 33 patients with poor echo images , magnetic resonance imaging was used in 15 patients , angiography in 15 patients , and radionuclide scanning in 3 patients patient characteristics aceis angiotensin - converting enzyme inhibitors , af atrial fibrillation , arbs angiotensin receptor blockers , bnp brain natriuretic peptide , bp blood pressure , cabg coronary artery bypass grafting , crt cardiac resynchronization therapy , egfr estimated glomerular filtration rate , icd implantable cardioverter - defibrillator , lvedd left ventricular end - diastolic dimension , lvef left ventricular ejection fraction , mi myocardial infarction , mr mitral regurgitation , nsvt non - sustained ventricular tachycardia , nyha new york heart association , pci percutaneous coronary intervention , pvc premature ventricular contraction , tia transient ischemic attack , twa t - wave alternans measured by modified simpson s method . for 33 patients with poor echo images , magnetic resonance imaging was used in 15 patients , angiography in 15 patients , and radionuclide scanning in 3 patients twa could be evaluated in 280 patients ( 62% ) , and the results were negative in 82 ( 29% ) , positive in 138 ( 49% ) , and indeterminate in 60 ( 21% ) . the twa test was performed by an exercise test in 251 patients ( 90% ) and by atrial pacing in 29 ( 10% ) . the reason for ineligibility for the twa test was presence of af in 65 patients ( 38% ) , presence of ventricular pacing in 34 ( 20% ) , presence of both af and ventricular pacing in 12 ( 7% ) , exercise intolerance in 50 ( 29% ) , and other causes in 12 ( 7% ) . the reason for the indeterminate twa results was an inadequate heart rate increase in 29 patients ( 48% ) , frequent ectopies in 13 ( 22% ) , excessive noise in 10 ( 17% ) , and other causes in 8 ( 13% ) . resting icd was implanted in 111 patients ( 25% ) , 46 of which were in combination with cardiac resynchronization therapy . twa - non - negative patients had a higher prevalence of frequent pvcs than twa - negative patients . those ineligible for the twa test were older , were associated with more severe heart failure symptoms , had higher bnp levels , had a wider qrs duration , and had a higher prevalence of af or digitalis use as compared with twa - negative patients . an icd was used more often in twa non - negative patients than in twa - negative patients.table 2comparison of patients according to twa statustwa - negative ( n = 82)twa - non - negative ( n = 198)ineligible for twa ( n = 173)age60.5 15.463.8 12.368.7 10.2ischemic41 ( 50%)94 ( 57%)54 ( 31%)nyha - iii13 ( 16%)34 ( 17%)81 ( 47%)af6 ( 7%)21 ( 11%)93 ( 54%)systolic bp ( mmhg)120 23120 20117 23hemoglobin ( g / dl)13.6 1.813.4 3.213.3 2.2bnp ( pg / ml)155174286lvef ( % ) 29.8 7.229.2 7.228.8 7.6lvedd ( mm)61.2 8.063.2 8.961.7 9.2mr grade 3 or 48 ( 10%)23 ( 12%)27 ( 16%)digitalis21 ( 26%)41 ( 21%)77 ( 45%)qrs ( ms)103109127pvc > 10/h31 ( 39%)105 ( 54%)*95 ( 60%)nsvt25 ( 31%)66 ( 34%)66 ( 42%)icd12 ( 15%)60 ( 30%)*39 ( 23%)abbreviations as in table 1 * p < 0.05 ; p < 0.005 in comparison with twa - negative group comparison of patients according to twa status abbreviations as in table 1 * p < 0.05 ; p < 0.005 in comparison with twa - negative group during the median follow - up of 36 months ( interquartile range 2741 months ) , there were 47 first svtes , 70 first vtes , and 68 deaths including 44 cardiac deaths ( table 3 ) . figure 2 shows the event - free curves for the primary end point of svte according to twa status . the event - free rate for svte in twa - negative patients was high , 100% at 1 year , 98.6% at 2 years , and 97.0% at 3 years . both twa - non - negative patients and those ineligible for the twa test had significantly worse 3-year event - free rates for svte as compared with twa - negative patients ( 89.5% with p = 0.037 and 84.4% with p = 0.003 , respectively ) . when twa - non - negative patients were separated into twa - indeterminate and twa - positive patients , the corresponding 3-year event - free rate for svte was 92.5% ( p = 0.22 ) and 88.1% ( p = 0.02 ) , respectively . among patients ineligible for the twa test , those with af , ventricular pacing or exercise intolerance had similar 3-year event - free rates for svte ( 81.2 , 85.7 , and 86.5% , respectively ; p = 0.72).table 3primary and secondary end points during follow - upfirst svte47 ( 10.4% ) scd15 sustained rapid - vt / vf ( 188/min)8 icd therapy for rapid - vt / vf ( 188/min)24first vte70 ( 15.5% ) svte31 sustained vt ( < 188/min)3 icd therapy for vt ( < 188/min)36death68 ( 15.0% ) cardiac death44 ( 9.7% ) scd17 ( 3.8%)scd sudden cardiac death , svte severe ventricular tachyarrhythmic event , vf ventricular fibrillation , vt ventricular tachycardia , vte ventricular tachyarrhythmic eventout of 17 scds , 15 were first svteout of 47 svtes , 31 were first vtefig . 2event - free curves for the primary end point of svte according to twa status primary and secondary end points during follow - up scd sudden cardiac death , svte severe ventricular tachyarrhythmic event , vf ventricular fibrillation , vt ventricular tachycardia , vte ventricular tachyarrhythmic event out of 17 scds , 15 were first svte out of 47 svtes , 31 were first vte event - free curves for the primary end point of svte according to twa status univariable and multivariable predictors of the primary end point are shown in table 4 . multivariable analysis identified both non - negative twa ( hr 4.43 ; 95% ci 1.0219.2 ; p = 0.047 ) and ineligibility for the twa test ( hr 6.89 ; 95% ci 1.5929.9 ; p = 0.010 ) to be independent predictors of svte . when non - negative twa was separated into indeterminate and positive twa , the corresponding adjusted hr for indeterminate and positive twa was 2.67 ( 95% ci 0.4814.7 ; p = 0.26 ) and 5.36 ( 95% ci 1.2223.7 ; p = 0.027 ) , respectively . there was no significant interaction between twa status and other clinical variables , including age , etiology of lv dysfunction , lvef and qrs duration ( table 5).table 4univariable and multivariable predictors of svtevariablesunivariable analysismultivariable analysisunadjusted hr ( 95% ci)p valueadjusted hr ( 95% ci)p valuetwa ineligible6.69 ( 1.5928.2)0.0106.89 ( 1.5929.9)0.010 non - negative4.15 ( 0.9717.8)0.0564.43 ( 1.0219.2)0.047mr grade 3 or 44.69 ( 2.568.61)<0.0013.61 ( 1.807.27)<0.001age < 65 years1.70 ( 0.953.02)0.0722.48 ( 1.324.68)0.005systolic bp < 100 mmhg2.91 ( 1.585.38)0.0012.43 ( 1.254.73)0.009pvc > 10/h3.01 ( 1.525.95)0.0022.35 ( 1.164.76)0.017nsvt2.56 ( 1.414.64)0.002lvedd 70 mm2.22 ( 1.214.06)0.010bnp 200 pg / ml2.13 ( 1.143.96)0.017qrs 120 ms1.91 ( 1.073.40)0.029af1.87 ( 1.043.37)0.037digitalis1.78 ( 1.003.16)0.050lvef 35%2.12 ( 0.904.96)0.060hemoglobin < 12 g / dl1.75 ( 0.953.23)0.087ci confidence interval , hr hazard ratio . other abbreviations as in table 1table 5risk of svte according to twa status in patient subgroupssubgroupsevents / n ( % ) hazard ratio ( 95% ci)interaction p*age0.09/0.20 < 65 years twa - negative1/46 ( 2.2% ) twa - non - negative13/91 ( 14.3%)7.69 ( 1.0158.8 ) ineligible for twa12/58 ( 20.7%)11.21 ( 1.4686.2 ) 65 years twa - negative1/36 ( 2.8% ) twa - non - negative6/107 ( 5.6%)1.92 ( 0.2316.0 ) ineligible for twa14/115 ( 12.2%)4.44 ( 0.5833.8)etiology of lv dysfunction0.19/0.10 ischemic twa - negative2/41 ( 4.9% ) twa - non - negative8/94 ( 8.5%)1.87 ( 0.408.79 ) ineligible for twa6/54 ( 11.1%)2.38 ( 0.4811.8 ) non - ischemic twa - negative0/41 ( 0% ) twa - non - negative11/104 ( 10.6%) ineligible for twa20/119 ( 16.8%)lvef0.19/0.05 35% twa - negative1/63 ( 1.6% ) twa - non - negative17/160 ( 10.6%)7.06 ( 0.9453.09 ) ineligible for twa23/130 ( 17.7%)12.52 ( 1.6992.74 ) > 35% twa - negative1/19 ( 5.3% ) twa - non - negative2/38 ( 5.3%)1.06 ( 0.1412.51 ) ineligible for twa3/43 ( 7.0%)1.30 ( 0.1011.71)qrs duration0.46/0.05 120 ms twa - negative0/24 ( 0% ) twa - non - negative7/72 ( 9.7%) ineligible for twa20/103 ( 19.6%) < 120 ms twa - negative2/58 ( 3.4% ) twa - non - negative12/126 ( 9.5%)2.91 ( 0.5112.6 ) ineligible for twa6/70 ( 8.6%)2.51 ( 0.6513.0 ) * p for interaction between twa and each variable based on difference in probabilities of events ; twa - negative versus twa - non - negative ( left ) and twa - negative versus ineligible for twa ( right)indeterminate because of no events in twa - negative group univariable and multivariable predictors of svte ci confidence interval , hr hazard ratio . other abbreviations as in table 1 risk of svte according to twa status in patient subgroups * p for interaction between twa and each variable based on difference in probabilities of events ; twa - negative versus twa - non - negative ( left ) and twa - negative versus ineligible for twa ( right ) indeterminate because of no events in twa - negative group event - free curves for the secondary end points are shown in fig . 3 . those ineligible for the twa test had significantly worse event - free rates for all secondary end points as compared with twa - negative patients with a hr of 3.15 for vte ( 95% ci 1.337.47 ) , 3.57 for all - cause death ( 95% ci 1.409.11 ) , and 12.5 for cardiac death ( 95% ci 1.6991.9 ) . twa - non - negative patients had significantly worse cardiac survival than twa - negative patients ( hr 7.71 ; 95% ci 1.0457.7).fig . event - free curves for vte ( a ) , all - cause death ( b ) and cardiac death ( c ) event - free curves for the secondary end points according to twa status . event - free curves for vte ( a ) , all - cause death ( b ) and cardiac death ( c ) twa negative patients had higher 3-year event - free rate for a composite of all - cause death and svte ( 91.9% ) as compared with twa - non - negative patients ( 81.7% , p = 0.03 ) and those ineligible for the twa test ( 74.5% , p = 0.0004 ) . the 3-year event - free rate for cardiac death or svte was 97.0 , 86.2 ( p = 0.008 ) , and 78.9% ( p the clinical characteristics of the 453 patients enrolled in the prevent - scd study are listed in table 1 . the average age was 65.1 12.6 years , and 367 ( 81% ) were male . the etiology was ischemic in 189 ( 42% ) and non - ischemic in 264 ( 58% ) . the mean lvef was 29.2 7.4% . among the 189 ischemic patients , 170 ( 90% ) had had prior coronary revascularization.table 1patient characteristicsbaseline characteristics age65.1 12.6 male367 ( 81% ) nyha - iii128 ( 28% ) ischemic189 ( 42% ) non - ischemic264 ( 58%)risk factors and co - morbidities prior mi180 ( 40% ) cabg / pci170 ( 38% ) stroke or tia41 ( 9% ) diabetes125 ( 28% ) hypertension207 ( 46% ) hyperlipidemia144 ( 32% ) af120 ( 26% ) persistent af65 ( 14% ) pacemaker56 ( 12% ) systolic bp ( mmhg)119 22 systolic bp < 100 mmhg74 ( 16% ) diastolic bp ( mmhg)69 12 hemoglobin < 12 g / dl108 ( 24% ) egfr < 40 ml / min/1.73 m90 ( 20% ) bnp ( pg / ml)211 bnp 200 pg / ml228 ( 52%)echocardiography lvef ( % ) 29.2 7.4 lvef 30%228 ( 50% ) lvedd ( mm)62.3 8.9 lvedd 70 mm90 ( 20% ) mr grade 3 or 458 ( 13%)medications aceis or arbs375 ( 83% ) beta - blockers331 ( 73% ) diuretics350 ( 77% ) spironolactone236 ( 52% ) digitalis139 ( 31% ) amiodarone81 ( 18% ) aspirin229 ( 51% ) statin131 ( 29%)risk stratification tests twa ( n = 280 ) negative82 ( 29% ) positive138 ( 49% ) indeterminate60 ( 21%)resting 12-lead electrocardiogram ( n = 453 ) heart rate ( /min)71 qrs ( ms)113 qrs 120 ms199 ( 44%)holter monitoring ( n = 433 ) pvc > 10/h231 ( 53% ) nsvt157 ( 36%)antiarrhythmic therapies icd111 ( 25% ) icd with crt46 ( 10%)aceis angiotensin - converting enzyme inhibitors , af atrial fibrillation , arbs angiotensin receptor blockers , bnp brain natriuretic peptide , bp blood pressure , cabg coronary artery bypass grafting , crt cardiac resynchronization therapy , egfr estimated glomerular filtration rate , icd implantable cardioverter - defibrillator , lvedd left ventricular end - diastolic dimension , lvef left ventricular ejection fraction , mi myocardial infarction , mr mitral regurgitation , nsvt non - sustained ventricular tachycardia , nyha new york heart association , pci percutaneous coronary intervention , pvc premature ventricular contraction , tia transient ischemic attack , twa t - wave alternansmeasured by modified simpson s method . for 33 patients with poor echo images , magnetic resonance imaging was used in 15 patients , angiography in 15 patients , and radionuclide scanning in 3 patients patient characteristics aceis angiotensin - converting enzyme inhibitors , af atrial fibrillation , arbs angiotensin receptor blockers , bnp brain natriuretic peptide , bp blood pressure , cabg coronary artery bypass grafting , crt cardiac resynchronization therapy , egfr estimated glomerular filtration rate , icd implantable cardioverter - defibrillator , lvedd left ventricular end - diastolic dimension , lvef left ventricular ejection fraction , mi myocardial infarction , mr mitral regurgitation , nsvt non - sustained ventricular tachycardia , nyha new york heart association , pci percutaneous coronary intervention , pvc premature ventricular contraction , tia transient ischemic attack , twa t - wave alternans measured by modified simpson s method . for 33 patients with poor echo images , magnetic resonance imaging was used in 15 patients , angiography in 15 patients , and radionuclide scanning in 3 patients twa could be evaluated in 280 patients ( 62% ) , and the results were negative in 82 ( 29% ) , positive in 138 ( 49% ) , and indeterminate in 60 ( 21% ) . the twa test was performed by an exercise test in 251 patients ( 90% ) and by atrial pacing in 29 ( 10% ) . the reason for ineligibility for the twa test was presence of af in 65 patients ( 38% ) , presence of ventricular pacing in 34 ( 20% ) , presence of both af and ventricular pacing in 12 ( 7% ) , exercise intolerance in 50 ( 29% ) , and other causes in 12 ( 7% ) . the reason for the indeterminate twa results was an inadequate heart rate increase in 29 patients ( 48% ) , frequent ectopies in 13 ( 22% ) , excessive noise in 10 ( 17% ) , and other causes in 8 ( 13% ) . resting icd was implanted in 111 patients ( 25% ) , 46 of which were in combination with cardiac resynchronization therapy . twa - non - negative patients had a higher prevalence of frequent pvcs than twa - negative patients . those ineligible for the twa test were older , were associated with more severe heart failure symptoms , had higher bnp levels , had a wider qrs duration , and had a higher prevalence of af or digitalis use as compared with twa - negative patients . an icd was used more often in twa non - negative patients than in twa - negative patients.table 2comparison of patients according to twa statustwa - negative ( n = 82)twa - non - negative ( n = 198)ineligible for twa ( n = 173)age60.5 15.463.8 12.368.7 10.2ischemic41 ( 50%)94 ( 57%)54 ( 31%)nyha - iii13 ( 16%)34 ( 17%)81 ( 47%)af6 ( 7%)21 ( 11%)93 ( 54%)systolic bp ( mmhg)120 23120 20117 23hemoglobin ( g / dl)13.6 1.813.4 3.213.3 2.2bnp ( pg / ml)155174286lvef ( % ) 29.8 7.229.2 7.228.8 7.6lvedd ( mm)61.2 8.063.2 8.961.7 9.2mr grade 3 or 48 ( 10%)23 ( 12%)27 ( 16%)digitalis21 ( 26%)41 ( 21%)77 ( 45%)qrs ( ms)103109127pvc > 10/h31 ( 39%)105 ( 54%)*95 ( 60%)nsvt25 ( 31%)66 ( 34%)66 ( 42%)icd12 ( 15%)60 ( 30%)*39 ( 23%)abbreviations as in table 1 * p < 0.05 ; p < 0.005 in comparison with twa - negative group comparison of patients according to twa status abbreviations as in table 1 * p < during the median follow - up of 36 months ( interquartile range 2741 months ) , there were 47 first svtes , 70 first vtes , and 68 deaths including 44 cardiac deaths ( table 3 ) . figure 2 shows the event - free curves for the primary end point of svte according to twa status . the event - free rate for svte in twa - negative patients was high , 100% at 1 year , 98.6% at 2 years , and 97.0% at 3 years . both twa - non - negative patients and those ineligible for the twa test had significantly worse 3-year event - free rates for svte as compared with twa - negative patients ( 89.5% with p = 0.037 and 84.4% with p = 0.003 , respectively ) . when twa - non - negative patients were separated into twa - indeterminate and twa - positive patients , the corresponding 3-year event - free rate for svte was 92.5% ( p = 0.22 ) and 88.1% ( p = 0.02 ) , respectively . among patients ineligible for the twa test , those with af , ventricular pacing or exercise intolerance had similar 3-year event - free rates for svte ( 81.2 , 85.7 , and 86.5% , respectively ; p = 0.72).table 3primary and secondary end points during follow - upfirst svte47 ( 10.4% ) scd15 sustained rapid - vt / vf ( 188/min)8 icd therapy for rapid - vt / vf ( 188/min)24first vte70 ( 15.5% ) svte31 sustained vt ( < 188/min)3 icd therapy for vt ( < 188/min)36death68 ( 15.0% ) cardiac death44 ( 9.7% ) scd17 ( 3.8%)scd sudden cardiac death , svte severe ventricular tachyarrhythmic event , vf ventricular fibrillation , vt ventricular tachycardia , vte ventricular tachyarrhythmic eventout of 17 scds , 15 were first svteout of 47 svtes , 31 were first vtefig . 2event - free curves for the primary end point of svte according to twa status primary and secondary end points during follow - up scd sudden cardiac death , svte severe ventricular tachyarrhythmic event , vf ventricular fibrillation , vt ventricular tachycardia , vte ventricular tachyarrhythmic event out of 17 scds , 15 were first svte out of 47 svtes , 31 were first vte event - free curves for the primary end point of svte according to twa status univariable and multivariable predictors of the primary end point are shown in table 4 . multivariable analysis identified both non - negative twa ( hr 4.43 ; 95% ci 1.0219.2 ; p = 0.047 ) and ineligibility for the twa test ( hr 6.89 ; 95% ci 1.5929.9 ; p = 0.010 ) to be independent predictors of svte . when non - negative twa was separated into indeterminate and positive twa , the corresponding adjusted hr for indeterminate and positive twa was 2.67 ( 95% ci 0.4814.7 ; p = 0.26 ) and 5.36 ( 95% ci 1.2223.7 ; p = 0.027 ) , respectively . there was no significant interaction between twa status and other clinical variables , including age , etiology of lv dysfunction , lvef and qrs duration ( table 5).table 4univariable and multivariable predictors of svtevariablesunivariable analysismultivariable analysisunadjusted hr ( 95% ci)p valueadjusted hr ( 95% ci)p valuetwa ineligible6.69 ( 1.5928.2)0.0106.89 ( 1.5929.9)0.010 non - negative4.15 ( 0.9717.8)0.0564.43 ( 1.0219.2)0.047mr grade 3 or 44.69 ( 2.568.61)<0.0013.61 ( 1.807.27)<0.001age < 65 years1.70 ( 0.953.02)0.0722.48 ( 1.324.68)0.005systolic bp < 100 mmhg2.91 ( 1.585.38)0.0012.43 ( 1.254.73)0.009pvc > 10/h3.01 ( 1.525.95)0.0022.35 ( 1.164.76)0.017nsvt2.56 ( 1.414.64)0.002lvedd 70 mm2.22 ( 1.214.06)0.010bnp 200 pg / ml2.13 ( 1.143.96)0.017qrs 120 ms1.91 ( 1.073.40)0.029af1.87 ( 1.043.37)0.037digitalis1.78 ( 1.003.16)0.050lvef 35%2.12 ( 0.904.96)0.060hemoglobin < 12 g / dl1.75 ( 0.953.23)0.087ci confidence interval , hr hazard ratio . other abbreviations as in table 1table 5risk of svte according to twa status in patient subgroupssubgroupsevents / n ( % ) hazard ratio ( 95% ci)interaction p*age0.09/0.20 < 65 years twa - negative1/46 ( 2.2% ) twa - non - negative13/91 ( 14.3%)7.69 ( 1.0158.8 ) ineligible for twa12/58 ( 20.7%)11.21 ( 1.4686.2 ) 65 years twa - negative1/36 ( 2.8% ) twa - non - negative6/107 ( 5.6%)1.92 ( 0.2316.0 ) ineligible for twa14/115 ( 12.2%)4.44 ( 0.5833.8)etiology of lv dysfunction0.19/0.10 ischemic twa - negative2/41 ( 4.9% ) twa - non - negative8/94 ( 8.5%)1.87 ( 0.408.79 ) ineligible for twa6/54 ( 11.1%)2.38 ( 0.4811.8 ) non - ischemic twa - negative0/41 ( 0% ) twa - non - negative11/104 ( 10.6%) ineligible for twa20/119 ( 16.8%)lvef0.19/0.05 35% twa - negative1/63 ( 1.6% ) twa - non - negative17/160 ( 10.6%)7.06 ( 0.9453.09 ) ineligible for twa23/130 ( 17.7%)12.52 ( 1.6992.74 ) > 35% twa - negative1/19 ( 5.3% ) twa - non - negative2/38 ( 5.3%)1.06 ( 0.1412.51 ) ineligible for twa3/43 ( 7.0%)1.30 ( 0.1011.71)qrs duration0.46/0.05 120 ms twa - negative0/24 ( 0% ) twa - non - negative7/72 ( 9.7%) ineligible for twa20/103 ( 19.6%) < 120 ms twa - negative2/58 ( 3.4% ) twa - non - negative12/126 ( 9.5%)2.91 ( 0.5112.6 ) ineligible for twa6/70 ( 8.6%)2.51 ( 0.6513.0 ) * p for interaction between twa and each variable based on difference in probabilities of events ; twa - negative versus twa - non - negative ( left ) and twa - negative versus ineligible for twa ( right)indeterminate because of no events in twa - negative group univariable and multivariable predictors of svte ci confidence interval , hr hazard ratio . other abbreviations as in table 1 risk of svte according to twa status in patient subgroups * p for interaction between twa and each variable based on difference in probabilities of events ; twa - negative versus twa - non - negative ( left ) and twa - negative versus ineligible for twa ( right ) indeterminate because of no events in twa - negative group event - free curves for the secondary end points are shown in fig . those ineligible for the twa test had significantly worse event - free rates for all secondary end points as compared with twa - negative patients with a hr of 3.15 for vte ( 95% ci 1.337.47 ) , 3.57 for all - cause death ( 95% ci 1.409.11 ) , and 12.5 for cardiac death ( 95% ci 1.6991.9 ) . twa - non - negative patients had significantly worse cardiac survival than twa - negative patients ( hr 7.71 ; 95% ci 1.0457.7).fig . event - free curves for vte ( a ) , all - cause death ( b ) and cardiac death ( c ) event - free curves for the secondary end points according to twa status . event - free curves for vte ( a ) , all - cause death ( b ) and cardiac death ( c ) twa negative patients had higher 3-year event - free rate for a composite of all - cause death and svte ( 91.9% ) as compared with twa - non - negative patients ( 81.7% , p = 0.03 ) and those ineligible for the twa test ( 74.5% , p = 0.0004 ) . the 3-year event - free rate for cardiac death or svte was 97.0 , 86.2 ( p = 0.008 ) , and 78.9% ( p < 0.0001 ) , respectively . the present study focused on the predictive value of twa for lethal ventricular tachyarrhythmia in a broad population of patients with lv dysfunction . the event - free rate for svte in twa - negative patients was excellent , 100% at 1 year , 98.6% at 2 years , and 97.0% at 3 years , which may suggest that twa - negative patients can be safely excluded as icd candidates . when those ineligible for the twa test were taken into account , however , the twa - negative patients accounted for only 18% of the entire study population . thus , despite the strikingly high negative predictive value of twa for svte , the safety assurance afforded by a negative result of the twa test was applicable to only a limited proportion of patients with lv dysfunction . the other important finding of this study was the poor long - term prognosis of patients ineligible for the twa test . ineligibility for the twa test was associated with highest risk not only for the primary end point of svte but for all of the secondary end points , including vte , all - cause death and cardiac death . exclusion of those patients might be associated with considerable selection bias toward patients with good exercise capacity and those without af or ventricular pacing . although the prevalence of negative twa among eligible patients with lv dysfunction has been reported to be 2237% [ 814 ] , a value coincident with the 29% observed in the present study , the proportion of twa - negative patients among all patients with lv dysfunction has not previously been established . in addition , the long - term arrhythmia and mortality risk of patients ineligible for the twa test has been uncertain . to the best of our knowledge , our study is the first multicenter study evaluating the prognostic utility of twa in a real - world population of lv dysfunction patients , including those ineligible for the twa test . twa showed high negative predictive ability for lethal ventricular tachyarrhythmia and cardiac mortality , but the proportion of twa - negative patients was less than one - fifth of the entire study population . to date , seven multicenter studies assessing the predictive value of twa for lethal ventricular tachyarrhythmia in patients with lv dysfunction have been reported ( supplementary table ) [ 814 ] . four of those studies reported the significant prognostic utility of twa with high negative predictive values for arrhythmic events [ 811 ] , as shown in our study . however , the other three more recent studies have reported conflicting data on the predictive value of twa [ 1214 ] . the microvolt t wave alternans testing for risk stratification of post - myocardial infarction patients ( master ) trial and the scd - heft substudy showed no difference in arrhythmic event rates between twa - negative and twa - non - negative patients [ 12 , 13 ] . furthermore , in the alternans before cardioverter defibrillator ( abcd ) trial , twa showed limited predictive ability for lethal ventricular tachyarrhythmia . the difference in the primary event rates between twa - negative and twa - non - negative patients was significant up to 9 months , but no longer after 12 months . first , among the studies there were substantial differences in twa protocol , which might have influenced the predictive accuracy of the twa test . in four studies including master , scd - heft and abcd , beta - blockers were withheld for 24 h before the twa test in order to reduce indeterminate twa results due to an inadequate heart rate increase during exercise . however , klingenheben et al . reported that metoprolol and sotalol decreased twa amplitude by 35 and 38% , respectively . also , in a study by rashba et al . , intravenous beta - blocker of esmolol reduced the number of positive twa tests by 50% . these data suggest that beta - blockers should be continued at the time of the twa test . in a recent meta - analysis of twa studies by chan et al . , non - negative twa was associated with a fivefold increased risk for ventricular tachyarrhythmia as compared with negative twa in studies where beta - blockers were continued during the twa test , while only a much weaker association was observed in studies where beta - blockers were withheld prior to the twa test . second , it may be that previous studies did not exclude patients with treatable cardiac ischemia , which could affect the prognostic value of the twa test . patients with heavy ischemic burden are considered to have higher risk for a future coronary event , which is often associated with lethal ventricular tachyarrhythmia in patients with lv dysfunction . in the present study , coronary artery disease was well treated among ischemic patients , none of whom had treatable cardiac ischemia . third , the prevalence of icd use was much higher in master and abcd , 100 and 87% , respectively , as compared with 25% in the present study . the use of appropriate icd therapy for ventricular tachyarrhythmia as a surrogate end point for scd may overestimate arrhythmic events , by counting potential self - terminating tachycardia as events . thus , a higher prevalence of icd use may lead to a higher possibility of overestimating arrhythmic events . the low prevalence of icd use in the present study was largely due to the fact that reimbursement officially covers icds only for secondary prevention of scd in japan . because patient enrollment of this study started in june 2004 , when the results of madit ii and scd - heft had been already published or announced , therapeutic strategies ( i.e. , whether to implant icd or not ) were left to the discretion of the attending physician . as a result , icd patients were much sicker than non - icd patients in terms of lower systolic blood pressure , higher bnp level , lower lvef , larger lvedd , wider qrs duration , lower prevalence of negative - twa , and higher prevalence of moderate to severe mitral regurgitation , pvc > 10/h , or nsvt ( data not shown ) . the twa test was initially developed as risk stratification test for lethal ventricular tachyarrhythmia mainly in ischemic patients [ 68 ] , but recent studies indicate that predictive values of twa might be higher in non - ischemic patients than in ischemic patients [ 9 , 13 ] . consistently , in the present study , the negative predictive value of twa for svte was 100% in non - ischemic patients , as compared with 95.1% in ischemic patients , although the difference was not statistically significant ( table 5 ) . because follow - up duration of previous studies reporting high predictive values of twa ranged from 17 to 20 months [ 611 ] , it has been uncertain whether the prognostic utility of twa remains robust beyond 2 years . in the present study , the median of follow - up was 36 months , which was the longest among the multicenter twa studies . the 3-year event - free rates for svte and cardiac death in twa - negative patients were extremely high , 97.0 and 98.6% , respectively . our data suggest that the predictive value of twa remains robust over 3 years . first , the number of patients enrolled in this study was not large enough , considering the broad spectrum of its patient population . second , the number of patients screened and excluded in this study was not recorded . considering the relatively slow rate of patient enrolment third , inclusion of appropriate icd therapy in the composite primary end point may overestimate arrhythmic events , as discussed above . however , the prevalence of icd use in the present study was relatively low as compared with other recent studies . fourth , the rate of self - terminating ventricular tachycardia in patients without an icd could not be determined . all intra - cardiac ecgs of icd therapies were , however , reviewed by the clinical events committee . finally , the results of the twa test were determined at each participating center without a core laboratory so that the data would more closely represent general clinical practice . conversely , there may be some heterogeneity in the interpretation of the twa test results . the present study demonstrated high negative predictive ability of twa for lethal ventricular tachyarrhythmia in a real - world japanese population of lv dysfunction patients during the median follow - up of 3 years . however , the twa - negative patients accounted for only 18% of the entire population , including those ineligible for the twa test . thus , despite the strikingly high negative predictive value of twa for lethal ventricular arrhythmia , the safety assurance given by a negative result of the twa test was applicable to only a limited proportion of patients with lv dysfunction . those ineligible for the twa test were associated with highest risk for lethal ventricular tachyarrhythmia , as well as highest overall and cardiac mortality . our study warrants further investigation with a larger patient population and a longer follow - up period . the following centers and investigators participated in the prevent - scd study ( listed in alphabetical order according to center ) : ehime prefectural central hospital : yukio kazatani , shinichi hiramatsu , tsuyoshi matsunaka , sumiko sato ; fujita health university hospital : hitoshi hishida , eiichi watanabe ; funabashi municipal medical center : shun ozawa , masayuki inagaki ; himeji cardiovascular center : teishi kajiya , kohei yamashiro , katsunori okajima ; hirosaki university school of medicine & hospital : ken okumura , atsushi iwasa , masaomi kimura , hyogo prefectural amagasaki hospital : yoshiki takatsu , ryoji taniguchi ; international medical center of japan : mitsuo kashida , osamu okazaki ; kagoshima university medical and dental hospital : chuwa tei , shuichi hamasaki , kobe university hospital : mitsuhiro yokoyama , akihiro yoshida ; kokura memorial hospital : masakiyo nobuyoshi , kenji ando , hitoshi okabayashi , jyota nakano ; kurashiki central hospital : kazuaki mitsudo satoki fujii ; kurume university hospital : teruhisa yoshida , yasutsugu nagamoto , eiichi takii ; kyorin university hospial : hideaki yoshino , takanori ikeda ; kyoto university graduate school of medicine : takeshi kimura , satoshi shizuta , yoshisumi haruna , takahiro doi , yukiko nishio , neiko ozasa , kei nishiyama , toru kita , takeshi morimoto ; kyoto - katsura hospital : shigeru nakamura , yoshihisa enjoji ; mitsubishi kyoto hospital : shinji miki , yutaka kono ; nagoya university hospital : toyoaki murohara , yasuya inden ; national defense medical college hospital : fumitaka ohsuzu , koh arakawa ; niigata university medical & dental hospital : yoshifusa aizawa , hiroshi furushima ; nippon medical school hospital : takao kato , yoshinori kobayashi ; oita university hospital : tetsunori saikawa , naohiko takahashi ; okayama university hospital : tohru ohe , satoshi nagase ; ome municipal general hospital : kenichiro otomo ; sakurabashi watanabe hospital : hiroshi ito , koichi inoue ; shiga university of medical science hospital : minoru horie , takashi yamamoto , masanori fujii , makoto ito ; showa university fujigaoka hospital : youichi takeyama , yukei higashi ; st . marianna university school of medicine hospital : fumihiko miyake , ryouji kishi , kiyoshi nakazawa ; st . marianna university school of medicine , yokohama city seibu hospital : haruki musha , osamu miyatsu ; teikyo university hospital : takaaki isshiki , taiji furukawa ; teikyo university school of medicine university hospital , mizonokuchi : tomoyuki kunishima ; the hospital of hyogo college of medicine : mitsumasa ohyanagi , hiroki shimizu ; toho university ohashi medical center : kaoru sugi , takao sakata ; tokyo medical and dental university hospital faculty of medicine : mitsuaki isobe , kenzo hirao ; tokyo metropolitan hiroo hospital : harumizu sakurada , tsuyoshi sakai , hidetaka okazaki , akiko tatsumoto ; toyama university hospital : hiroshi inoue , tomoki kameyama ; tsuchiya general hospital : yasuhiko hayashi , yuji muraoka ; yamaguchi university hospital : masunori matsuzaki , akihiko shimizu ; yokohama city university medical center : kazuo kimura , toshiaki ebina . asuka saeki , hiromi yoshida , yuko yamamoto , kumiko kitagawa , misato yamauchi , naoko okamoto , yumika fujino , izumi miki , yuki sato , yoko fujiki , rei fujita , miya hanazawa , miho hayashikawa , chikako hibi , takami hiraoka , sachiko maeda , saeko minematsu , ayu motofusa , satoko nishida , hitomi sasae , yayoi sugimoto , emi takinami , saori tezuka , ayumi yamamoto , mai yoshimoto . | backgroundthe predictive value of t - wave alternans ( twa ) for lethal ventricular tachyarrhythmia in patients with left ventricular ( lv ) dysfunction is controversial .
also , long - term arrhythmia risk of patients ineligible for the twa test is unknown.methodsthis was a multicenter , prospective observational study of patients with lv ejection fraction 40% due to ischemic or non - ischemic cardiomyopathies , designed to evaluate the prognostic value of twa for lethal ventricular tachyarrhythmia .
the primary end point was a composite of sudden cardiac death , sustained rapid ventricular tachycardia ( vt ) or ventricular fibrillation ( vf ) , and appropriate defibrillator therapy for rapid vt or vf.resultsamong 453 patients enrolled in the study , 280 ( 62% ) were eligible for the twa test .
twa was negative in 82 patients ( 29% ) , who accounted for 18% of the total population .
the median of follow - up was 36 months .
the 3-year event - free rate for the primary end point was significantly higher in twa - negative patients ( 97.0% ) than in twa non - negative patients ( 89.5% , p = 0.037 ) and those ineligible for the twa test ( 84.4% , p = 0.003 ) .
multivariable analysis identified both non - negative twa [ hazard ratio ( hr ) 4.43 ; 95% confidence interval ( ci ) 1.0219.2 ; p = 0.047 ) and ineligibility for the twa test ( hr 6.89 ; 95% ci 1.5929.9 ; p = 0.010 ) to be independent predictors of the primary end point.conclusionstwa showed high negative predictive ability for lethal ventricular tachyarrhythmia in patients with lv dysfunction , although the twa - negative patients accounted for only 18% of the entire population . those ineligible for the twa test had the highest risk for lethal ventricular tachyarrhythmia.electronic supplementary materialthe online version of this article ( doi:10.1007/s00392 - 011 - 0368 - 2 ) contains supplementary material , which is available to authorized users . |
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osteoporosis is the condition in which a low bone mass and altered microarchitecture of the bone leads to increased risk of fracture . primary osteoporosis refers to osteoporotic conditions which are not related to other chronic illnesses and is usually associated with aging and decreased gonadal function , such as decreased level of estrogen , whereas secondary osteoporosis is the type of osteoporosis caused by other health problems . disuse is one of the many reasons inducing bone loss and resulting in secondary osteoporosis . disuse osteoporosis has been shown to be a regional phenomenon in the areas with tremendous decrease in weight bearing like lower limbs . bones of lower limbs are subjected to mechanical stimulations during daily life provided by static gravity - related weight - bearing , ground reaction forces , and dynamic loading generated by muscle contractions during locomotion . milliken et al . have investigated the effect of 1-year supervised weight training exercise on bone mineral density ( bmd ) in postmenopausal women . the result showed higher bmds of trochanter and femoral neck in women with weight training exercise than in those lacking exercise . chan et al . have studied the effect of tai - chi excercise on bone quality in postmenopausal women . subjects in the exercise group performed 5 sessions of 45 minutes tai - chi per week . after 1 year of tai - chi exercise , a greater percentage loss in bone density was observed in the control group when compared to the exercise group , suggesting that performing tai - chi exercise could decline bone loss in postmenopausal women . besides , feskanich et al . have studied prospectively a cohort on the relationship between walking , leisure time activity , and the risk of hip fracture , showing that physical activity was inversely associated with the risk of hip fracture and that the effect was dose dependent . although there are many effective treatments available for primary osteoporosis , there is a lack of effective treatments for disuse osteoporosis . this is because of the fact that the aetiology , pathophysiology , and resultant pathology of disuse osteoporosis differ from those of primary osteoporosis . the objective of this paper is to examine the unique pathology and underlying pathophysiology of disuse osteoporosis . disuse bone loss in general is a reduction of bone mass in relation to bone volume , while the ratio of bone mineral to collagen remains unchanged . the loss of trabecular bone is more rapid and dramatic , while the cortical loss continues for a longer period . however , bones of lower limbs are subjected to three categories of mechanical loadings during daily life , namely , static gravity - related weight bearing , ground reaction forces , and dynamic loading generated by muscle contractions during locomotion . different health problem associates with absence or decrease in one or more of these mechanical stimulations and will result in bone loss differently in anatomical location , quantity , velocity , and through different mechanisms . long - term bed rest results in the absence of ground force reaction and reduction of muscle contractions . . have carried out a 35 days bed rest trial and assessed bone density 2 weeks after the bed rest . they reported reduction of bone mass in the cancellous bone - rich areas , 1% at distal femur , 3% at patella , and 2% at distal tibia while no changes in distal radius . the same group has observed that bone mass in distal radius remained unchanged after 56 days and 90 days bed rest , while bone mass in distal tibia declined 3.6% and 6% correspondingly [ 8 , 9 ] . the decreases of cortical bone thickness and density were below 2% after as long as 90 days bed rest . these results suggest that long - term bed rest does not affect balance of bone metabolism very much . disuse osteoporosis includes the reduction of bone mass after spinal cord injury ( sci ) and other brain neurologic conditions as well . sci leads to substantial reduction in ground force reaction and muscle contraction in the lower limbs resulting in dramatic reduction in bone mass . . demonstrated more than 20% bone loss at distal femur 3 months after injury in posttraumatic paraplegic and quadriplegic sci patients . the researchers reported also that the rate of bone loss below pelvis was rapid and linear in the acute stages . in another cross - sectional study with larger sample size , kiratli et al . found reduction of bone mineral density by 27% , 25% , and 43% in femoral neck , mid - shaft , and distal femur , respectively , compared with the controls . beside of sci , lower limb amputation , acquired brain injury , and other neurologic conditions can also lead to disabilities which , in turn , result in disuse osteoporosis . a recent cross - sectional study by smith et al . showed that 42.4% and 23.5% of disabled patients after neurologic traumas , such as sci or other conditions for at least 3 months , had developed osteopenia and osteoporosis , respectively . the researchers suggested further that ambulatory status and duration of disability were independent predictors of bone mineral densities at femoral neck and total proximal femur . exposure to microgravity would lead to reduced weight - bearing and ground reaction forces that result in reduction in bone mass . several studies on effects of microgravity on skeleton focused on the impacts on skeletons of astronauts after spaceflights . collet et al . analyzed the bmd and biochemical parameters of 2 astronauts who stayed 1 and 6 months , respectively , in space . a slight decrease in trabecular bone mass in distal tibia metaphysis was observed at the end of the first month of spaceflight , whereas remarkable bone losses in both trabecular and cortical bones was observed after 6 months of spaceflight . after 6 month of recovery , the trabecular bone mass was still significantly lower than normal , whereas no difference could be seen in cortical bone . another study on 11 astronauts by vico et al . showed greater bone losses occurred in cancellous bone compared to cortical bone . the mean decrease in cancellous bmd of the 11 astronauts was 5.4% after 6 months of spaceflight but the range of reduction varied from 0.4% to 23.4% . the astronaut who spent the longest time in space did not have the greatest bone loss . in comparison of the three causes of disuse osteoporosis , that is , long - term bed rest , paralysis , and microgravity , all of them involve the reduction of ground reaction forces and weight - bearing activities . however , patients with long - term bed rest or paralysis are further subjected to reduced or even absence of muscular contraction . this situation is different in the case of astronauts whose muscular contractions are not restricted , and this may be a possible reason account for the great variations of bone loss in previous findings . furthermore , muscular contraction can be the most important force out of the 3 categories of mechanical loading for keeping bone mass . disuse osteoporosis can be resulted from failure for the bone to achieve the optimal peak bone mass and strength , if disuse occurred during the period of bone mass accumulation . on the other hand , disuse osteoporosis can be the result of an accelerated rate of bone resorption and slower bone formation in adults . ralston indicated that peak bone mass and strength could be determined by genetic factors which affect the level of bmd , biochemical markers of bone turnover , and mechanical properties of bone . results of association studies have suggested that polymorphisms of components in the gene - signaling pathway of genes such as col1a1 , esr1 , and lrp5 were associated with bone mass level and fracture risk . the impact of genetic factors diminishes with age because of the increasing impact of environmental and nutritional factors . skeletal growth and repair occur through bone - remodeling which is a tightly regulated process . the normal bone mass is maintained during remodeling , based on the balance between bone formation and bone resorption . the bone - remodeling cycle begins with the resorption phase during which osteoclasts are recruited to the remodeling site on the bone surface , and these osteoclasts will then excavate the bone surface in the subsequent 2 to 4 weeks . after the resorption phase , the osteoclasts move away from the site , and osteoblast precursors move to the site and differentiate to become osteoblasts . during the subsequent 2 to 4 months of formation phase , mature osteoblasts deposit an organ matrix , which will then be mineralized . however , because of certain events such as hormonal changes at menopause , the balance between bone formation and resorption is disturbed , and resorption occurs at a higher rate than that of formation leading to osteoporosis . osteoclasts are multinucleated cells derived from monocyte / macrophage lineage ( figure 1 ) and are the only type of cells capable of resorbing bone . the rate of bone resorption is determined by the number and activity of osteoclasts . during bone resorption , osteoclasts adhere to the bone matrix forming a deeply folded membrane and secrete protons and hydrolytic enzymes to the lacuna . the lacuna is then demineralized by the acidic environment due to proton secretion , leading to the exposure of organic components of the bone , such as collagen , to the hydrolytic enzymes , resulting in degradation of the organic components . however , increased rate of bone resorption may result in the depletion of bone mass and to disruption of skeletal microarchitecture leading to skeletal fragility . receptor activator of nuclear factor kappab ligand ( rankl ) is a cytokine that belongs to the tnf family ; it is essential for osteoclast formation and function . rankl is found on the surface of osteoblasts and the interaction between rankl and its receptors rank on osteoclast precursors triggers the maturation of osteoclasts , thus inducing bone resorption . the rankl - rank interaction is prevented by the natural rankl inhibitor , osteoprotegerin ( opg ) . opg is also a tnf family member that binds to rankl and , hence , inhibits the binding of rankl to rank . therefore , the activity of osteoclasts is partially dependent on the balance between rankl and opg ( figure 2 ) . in addition to rankl and opg , there are many other cytokines such as il-1 , tnf- and prostaglandin e2 that have been identified as regulators for osteoclastic activity [ 23 , 24 ] . . showed that estrogen was able to negatively regulate the formation and function of osteoclast by reducing the lifespan of the cells by promoting apoptosis . however , excessive osteoclastic activity without the complementary actions by osteoblasts will result in skeletal fragility . based on previous studies , it is likely that bone resorption is influenced by complex interactions between different factors with osteoclasts , and also among the different factors . in which the rate of bone resorption is increased , there could be no apparent bone loss if the rate of bone formation is matched , since the bone removed will be replaced by new bone formation . osteoporosis is , thus , an imbalance condition where the rate of bone resorption is higher than the one of bone formation , resulting in continuous loss of bone and deterioration of skeletal microarchitecture . hence , it is important to determine what factors will affect the rate of bone formation as well as the coupling process between bone resorption and formation . osteoblasts are mononucleate cells which are responsible for bone formation . during ossification , or bone forming , osteoblasts produce an unmineralized , organic portion of the bone matrix known as osteoid . the osteoids produced will then mineralize , with minerals such as calcium and zinc , to form new bone tissue . osteoblasts arise from mesenchymal progenitor cells , which possess the master gene of osteoblast differentiation , runx2 ( previously known as cbfa1 , aml3 , or pebp2a1 ) . in 1997 , komori and coworkers demonstrated that intramembranous and endochondral ossification were completely blocked in mice with cbfa1 gene mutated , where cbfa1 was a transcription factor belonging to the runt - domain gene family . the same year , otto et al . showed that homozygous cbfa1-deficient mice died of respiratory failure shortly after birth . in that study , they found that there was neither osteoblast nor bone in the skeletons of the homozygous cbfa1-deficient mice . also , heterozygous mutants exhibited specific skeletal abnormalities that were characteristic of a human heritable skeletal disorder , cleidocranial dysplasia . these findings suggested that the cbfa1 ( runx2 ) gene is essential for osteoblast differentiation and bone formation . the differentiation of osteoblasts from mesenchymal progenitor cells is influenced by growth factors such as fibroblasts growth factor ( fgf ) , transforming growth factor beta ( tgf- ) , and bone morphogenetic proteins ( bmps ) ( figure 3 ) . . pointed out that the number of osteoblasts tended to decrease as people reach old age . this leads to the decreased rate of bone formation and results in osteoporosis . according to a review by rosen , the insulin - like growth factor ( igf ) system is linked to the process of skeletal acquisition , and igf-1 is essential for normal bone formation . previous study by sakata et al . showed that skeletal unloading led to resistant to the anabolic actions of igf - i on bone , and this was associated with the reduction in integrin expression . this result suggests that igf - i may be involved in the reaction mechanism of disuse osteoporosis . beside of igf - i , the levels of various factors and hormones such as bmps and pth have been demonstrated to be changed in response to skeletal unloading . however , the exact mechanisms of how these factors respond to changes in mechanical loading are still unclear . sclerostin is the product of the sost gene that has been found to bind to lrp5/6 receptors . a recent study by robling showed that mechanical stimulation decreased sclerostin expression , whereas significant increase in sost expression in tibias was observed in hindlimb unloaded animals . thus , the level of sclerostin , and hence bone formation , appears to be affected by mechanical stimulation . a recent study by lin et al . further suggested that the responses of bone to mechanical unloading were mediated via sclerostin , probably by antagonizing wnt/-catenin signaling . in the study , wild - type and sost knockout mice were unloaded for 2 weeks . decreased wnt/-catenin signaling in association with increased expression of sost was observed in wild - type mice upon unloading . however , in the absence of sclerostin in sost / mice , decrease in wnt/-catenin signaling and inhibition of osteoblast activity upon unloading were diminished . the results showed that bone masses of sost / mice in both loaded and unloaded groups were significantly higher than those of wild - type groups . more importantly , unloading - induced bone loss was prevented in sost / mice , and wnt/-catenin signaling , which was shown to be involved in response to mechanical unloading , was not altered in unloaded sost / mice . the findings suggest that sclerostin play an important role in development of disuse osteoporosis via its action in wnt/-catenin signaling . physical disability , advancing age and/or exposure to microgravity increase the risk of suffering from osteoporosis , and such disuse osteoporosis is associated with huge economic and health burden . on the other hand understanding the pathology and the underlying mechanisms of disuse osteoporosis is important for the development of new strategies on pharmaceuticals or treatment protocols for preventing or reducing disuse osteoporosis . in this paper , the effects of long - term bed rest , paralysis , and exposure to microgravity on skeletons were outlined . also , recent research works on underlying mechanisms of disuse osteoporosis certainly , the entire picture of the pathophysiology of disuse osteoporosis is still unclear , but further investigations on action mechanism of hormones such as igf - i and antagonists of sclerostin would provide insights on prospective research works . | osteoporosis is a multifactorial skeletal disorder characterized by decreased bone mass and deteriorated microarchitecture that lead to increased risk of fracture .
the disuse osteoporosis refers to bone mass decrements under conditions of decreased mechanical loading , including decreased ground force reaction , muscular contraction , and microgravity - related bone loss in astronauts after space flights .
although there are many effective treatments available for primary osteoporosis , there is a lack of effective treatments for disuse osteoporosis .
this is because that the aetiology , pathophysiology , and resultant pathology of disuse osteoporosis differ from those of primary osteoporosis .
the objective of this paper is to examine the unique pathology and underlying pathophysiology of disuse osteoporosis . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
we captured 150 apparently healthy adult bats from 2 caves in yunnan province , china : 1 in jinghong city in november 2009 , and 1 in mengla county in december 2015 ( table 1 ; figure 1 ) . the bat species we collected were hipposideros armiger , aselliscus stoliczkanus , myotis ricketti , rhinolophus monoceros , miniopterus fuscus , ia io , eonycteris spelaea , and rousettus sp . we humanely killed all bats and collected their hearts , intestines , lungs , spleens , kidneys , livers , brains , and blood for testing . we used 2 methods to analyze bat lung tissues for presence of filovirus rna : first , we used nested pcr with the primers fv f1/r1 and fv f2/r2 ( 10 ) , and next , we used quantitative pcr ( qpcr ) with 3 groups of qpcr with primers and probes designed from viral sequences obtained in this study ( technical appendix table ) . elisa and western blot results for samples collected in 2009 were from a previous study ( 14 ) . bat collection sites for a study on genetically diverse filoviruses in rousettus and eonycteris spp . triangles indicate jinghong city and mengla county , yunnan province , where 150 apparently healthy adult bats were collected from 2 caves in november 2009 ( jinghong city ) and december 2015 ( mengla county ) . nested pcr , we detected filovirus rna in 15 fruit bat specimens ( e. spelaea and rousettus sp . ) ; the specimens comprised 10 ( 23.3% ) of 43 e. spelaea and rousettus sp . collected in 2009 and 5 ( 11.9% ) of 42 collected in 2015 . using qpcr , we detected filovirus rna in 20 specimens from e. spelaea ( n = 4 ) and rousettus sp . ( n = 16 ) bats : 10 ( 23.3% ) of the bats were collected in 2009 and 10 ( 23.8% ) in 2015 . kx371873kx371890 ) exhibited 65%99% nt identity among themselves and 61%99% nt identity with known filoviruses . phylogenetic analysis showed that the sequences from the bats formed 3 independent groups , groups 13 . groups 1 and 2 comprised 6 and 11 sequences , respectively , all of which were obtained in this study ( figure 2 ) . group 3 comprised 2 sequences , 1 from this study and 1 previously published ( 10 ) . pairwise distance analysis indicated that sequences in group 1 share the highest nucleotide identity ( 75%78% ) with marv and those in group 2 share the highest identity ( 69%74% ) with ravn virus . the 2 sequences in group 3 are highly similar and share 66%70% nt identity with other filovirus species . of note , 1 bat specimen ( no . 9447 ) was co - infected with 4 different filovirus strains ( btfiloyn94471 to 94474 ) with high divergence ( figure 2 ; technical appendix figure 1 ) . to further determine the phylogenetic relationship of these viruses with known filoviruses , we amplified more l gene sequence ( 1,475 bp ) for strains btfiloyn2162 and btfiloyn94471 . similar to the 310-bp sequences , the 1,475-bp sequence of the btfiloyn2162 shared 99% identity with btdh04 at the nucleotide level , the 1,475-bp sequence of btfiloyn94471 shared 62%71% with known filoviruses . phylogenetic analysis of filovirus isolates collected in study of genetically diverse filoviruses in rousettus and eonycteris spp . the maximum - likelihood tree was constructed based on the 310-bp segment with 1,000 bootstrap replicates . the sequences obtained in this study are marked with a triangle ( group 1 ) , black dot ( group 2 ) , or rectangle [ group 3 ) . sequences from genbank are listed by their accession numbers , followed by the virus name , collection year , and host . scale bar indicates nucleotide substitutions per site . to determine the tissue tropism of these viruses , we performed qpcr with primers and probes designed for each of the 3 different groups ( online technical appendix table ) . results showed that filoviruses were mainly located in the lung and that genome copy numbers ranged from 29 to 523,582/mg of tissue ( table 2 ) . only 2 bat blood samples ( nos . 2202 , 2188 , 9434 , 9442 , and 9447 ) contained filoviruses with more widespread tissue tropism . we were unable to isolate virus from pcr - positive samples by using vero - e6 cells . * these represent primers and probes designed based on partial sequences of the virus l gene obtained in this study . sequence information is provided in the online technical appendix ( https://wwwnc.cdc.gov/eid/article/23/3/16-1119-techapp1.pdf ) . to detect filovirus igg and igm , we expressed his - tagged truncated nucleoproteins from restv or zebov in escherichia coli and used them as antigens ( online technical appendix ) . in this experiment , we used 25 bat samples from 2015 that had enough serum volume for testing ; 14 samples showed a strong cross - reaction with the zebov nucleoprotein , and among them , 7 showed a weak cross - reaction with restv nucleoprotein . we used western blotting to confirm these results ; 11 of the 25 samples were positive for zebov nucleoprotein and 4 for restv nucleoprotein ( table 1 ; technical appendix figure 2 ) . no samples overlapped between those identified as positive by pcr and those identified as positive by serologic testing . results of a serum neutralization assay with hiv pseudovirus carrying the zebov glycoprotein showed that the elisa - positive samples had no cross - neutralization activity to the pseudovirus ( 14 ) . phylogenetic analysis of partial sequences suggested that at least 3 distinct groups of filovirus are circulating in fruit bats in china . the distances between these sequences indicates that the 3 groups may represent 3 novel species or genera . of interest , we detected antibodies reacting more strongly to zebov than restv nucleoprotein in some filovirus rna negative samples , suggesting that the bats were infected with another / other filovirus(es ) cross - reactive with zebov nucleoprotein or that nucleoproteins of the novel filoviruses were cross - reactive with zebov and restv nucleoproteins . the bat samples in this study were collected from 2 caves in 2009 and 2015 , respectively ; the caves are 200 km metric apart . across the 2 different years and locations , we detected closely related viruses and found 1 bat that was acutely co - infected by 4 different filoviruses ; this finding suggests that these viruses have been circulating in the 2 bat species and that densely populated bat caves provide opportunity for cross - infection with different viruses . however , considering the migration ability of the fruit bat , we can not exclude the possibility that there are exchanges of virus between the bats in these two caves . longitudinal surveillance with tracking tags may help to better understand the spatial temporal distribution of these viruses in bat populations . in previous reports , filoviruses were primarily detected in liver and spleen tissues ( 4,15 ) . in our study , we primarily detected filoviruses in the lung . thus , these filoviruses may have the potential to be transmitted through the respiratory tract . these results will be helpful in providing a better understanding of the distribution and diversity of filoviruses , which may have implications for public health . considering their feeding habitats , fruit bats are often in close contact with domestic animals and human populations . it is therefore necessary to establish long - term and proactive surveillance of these viruses and related diseases . detailed methods and primer sequences used in a study of genetically diverse filoviruses in rousettus and eonycteris spp . | genetically divergent filoviruses detected in rousettus and eonycteris spp .
bats in china exhibited 61%99% nt identity with reported filoviruses , based on partial replicase sequences , and they demonstrated lung tropism .
co - infection with 4 different filoviruses was found in 1 bat .
these results demonstrate that fruit bats are key reservoirs of filoviruses . |
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the empa - reg outcome trial ( clinicaltrials.gov identifier , nct01131676 ) recently announced the effects of empagliflozin on the cardiovascular outcomes ( cvo ) and mortality in persons with type 2 diabetes mellitus ( t2 dm ) . in this seminal cvo trial ( cvot ) , 7020 patients with t2 dm with coexisting cardiovascular disease ( cvd ; myocardial infarction [ mi ] , stroke , or peripheral arterial disease ) were randomized to either 10 mg empagliflozin , 25 mg empagliflozin , or placebo , over and above standard of care . the primary composite outcome was a total of three endpoints ( death from cv causes , non - fatal mi , and non - fatal stroke ) , while the key secondary endpoint included a fourth endpoint the results of this study revealed a statistically significant reduction in the primary endpoint with empagliflozin use ( 10.5% in the empagliflozin group vs. 12.1% in the placebo group ; hazard ratio 0.86 ; relative risk reduction ( rrr ) 14% , 95% confidence interval 0.740.99 ; p = 0.04 ) . similarly , a rrr reduction is noted in death from cv causes ( rrr 38% ; 3.7% vs. 5.9% ) , hospitalization for heart failure ( rrr 35% ; 2.7% vs. 4.1% ) , and all - cause death ( rrr 32% ; 5.7% vs. 8.3% ) . however , the difference in rates of non - fatal mi and non - fatal stroke did not reach statistical significance . this article is based on previously conducted studies and does not involve any new studies of human or animal subjects performed by the author . the university group diabetes program ( ugdp ) study , published nearly half a century ago , highlighted the negative effects of tolbutamide on cv health . the united kingdom prospective diabetes study ( ukpds ) , a large study with multiple arms , unearthed the beneficial impact of metformin in contrast to other drugs such as chlorpropamide in improving cvo in t2 dm . the stop - niddm trial , breaking new ground , suggested cvo improvement when acarbose was administered to persons with impaired glucose tolerance . similarly , the proactive trial on pioglitazone reported a reduction in the composite of all - cause mortality , non - fatal mi , and stroke in patients with t2 dm who have a high risk of macrovascular events . however , all of these studies were not designed as a cvot , and their primary aim was to assess glucose - lowering efficacy rather than improvement in cv health . advances in the understanding of the interlink between diabetes and cv disease ( cvd ) , an appreciation of the need to improve cvo in diabetes care , and the realization that effective glucose - lowering drugs could end up worsening cvo ( e.g. , muraglitazar ) , have led to a mandatory requirement for cvots in anti - diabetic drugs pending regulatory approval . it then became imperative for all newly developed glucose - lowering molecules to undergo cv safety analysis by performing a cvot . the first drug to report such an analysis , after institution of new regulatory requirements , was quick - release bromocriptine , which demonstrated cv safety in a trial which recruited 3070 subjects for a mean follow up of 52 weeks . since then , various data on dipeptidyl - peptidase 4 inhibitors [ 810 ] and insulins have been published from large - scale cvots which have suggested their cv safety . while these cvots follow similar trial designs and protocols , subtle variations are present [ 811 ] . differences in inclusion / exclusion criteria , the duration of follow up , and the choice of primary / secondary endpoints can be easily discerned . though debate around the validity of various trial designs is never ending , there is broad consensus that modern cvots are well designed and have good reliability . all recent cvots have been designed to demonstrate cv safety ( non - inferiority ) , rather than superiority , as requested by regulators . over the past few decades the standard of care for cv prevention has improved markedly across the globe . use of medical interventions such as aspirin , statins , angiotensin - converting - enzyme inhibitors ( acei ) , angiotensin receptor blockers ( arbs ) , and beta blockers has helped to enhance cvo , especially in persons with t2 dm . though widespread use of these standard of care drugs allow easier demonstration of cv safety , their use makes it difficult for a new add - on therapy to demonstrate improvement in cvo . this is exactly what empagliflozin has achieved . in a high - risk cohort of subjects , with over 75% usage of statins , over 80% use of acei / arbs , and over 60% beta - blocker therapy , administration of empagliflozin was able to demonstrate added benefit in terms of cvo . this speaks for the strength of the molecule being studied , as well as the robust quality of trial methodology followed by the authors of the study . inclusion of a wide variety of high - risk subjects , from 42 different nationalities , enhances the global applicability of these results . the benefits of empagliflozin were noted early on in the empa - reg outcome trial , and continued throughout the study . this finding makes empagliflozin stand apart from other cvd preventive drugs , such as statins and ramipril , which demonstrated a cv benefit after a longer duration of therapy . whether this means that empagliflozin is an effective drug for secondary prevention of cvd , that is , the prevention of progression of cvd to death is open to debate . detractors point to the lack of statistical significance noted with respect to occurrence of non - fatal mi and stroke . however , this finding may be thought to reinforce the preventive power of empagliflozin : it may even be used in tertiary cvd prevention , as it helps improve outcomes in persons with t2 dm who experience an mi or stroke , ensuring that they survive the acute illness . thus , empagliflozin may be useful for tertiary prevention , rather than secondary prevention , of cvd . the number needed to treat , ( for all - cause mortality ) , which was 39 for empagliflozin over a period of 3 years , is much lower than that reported for other drugs , including ramipril and simvastatin [ 12 , 13 ] . the highly significant benefit of empagliflozin ( p = 0.002 ) in preventing hospitalization for heart failure raises hope for its use in this clinical situation . this finding is especially welcome considering the controversy related to the increased risk of heart failure hospitalization with other glucose - lowering therapies . empagliflozin , therefore , gets evidence - backed justification for use not only as a glucose - lowering therapy , but also raises hope for its potential as adjunctive therapy for cvd prevention ( table 1).table 1impact of empa - reg outcome study strengths robust methodology / clinical design multinational coverage ( 42 countries ) applicability across age , gender , cv phenotype clinically relevant inclusion / exclusion criteria clear - cut answers from straightforward statistical analysispositive answers empagliflozin is safe in high - risk cv patients with t2 dm empagliflozin improves cv outcomes in high - risk cv patients with t2 dm empagliflozin can be used safely in combination with other vascular - tropic drugs over an extended period of time empagliflozin can be used for prevention of cvd empagliflozin does not increase the risk of diabetic ketoacidosis or bone fracturesunanswered questions is the beneficial effect of empagliflozin a class effect , or a property unique to this molecule ? what are the mechanisms that account for the potential of empagliflozin to reduce cv outcomes ? is the beneficial cv effect of empagliflozin relevant to patients with t2 dm and low cv risk , and to patients with type 1 diabetes mellitus ? can empagliflozin be prescribed in acute coronary settings ? can empagliflozin be considered an adjuvant preventive therapy for cvd ? cv cardiovascular , cvd cardiovascular disease , t2 dm type 2 diabetes mellitus impact of empa - reg outcome study
cv cardiovascular , cvd cardiovascular disease , t2 dm type 2 diabetes mellitus the empa - reg outcome results should be carefully interpreted to assess their impact on diabetes care and outcomes . while the findings for different subsets of patients need to be studied separately , the empa - reg outcome study also throws up a few interesting questions ( table 1 ) . will these beneficial results be relevant to persons with t2 dm and low cv risk , or to persons with type 1 diabetes mellitus ? whether the findings of empagliflozin can be extrapolated to other sodium - glucose co - transporter 2 inhibitors ( sglt2i ) are open to discussion . cvots are underway for both canagliflozin and dapagliflozin , and their results will decide whether the cv benefits of empagliflozin are a class effect or not . it is also uncertain if empagliflozin can be initiated , or continue to be used , in acute coronary settings , including unstable angina , mi , and heart failure . however , while translating cvot evidence to practice one should not lose sight of good clinical sense . this therapy should be accompanied by appropriate medication counseling and should not be prescribed to persons at risk of ketoacidosis or recurrent genital infections . while the empa - reg outcome study findings should be interpreted carefully to assess their impact on diabetes care and outcomes , the results go far beyond proving the superiority of empagliflozin in improving cvo . the seminal importance of these findings will ensure that this trial acts as a milestone in the fields of diabetology and cv medicine . the empa - reg outcome study raises the bar for future glucose - lowering drugs . such a development will help improve not only cvo , but also diabetes outcomes overall . the empa - reg outcome study may be just a small step for empagliflozin , but is a giant leap for diabetes care . | this article discusses the recently published empa - reg outcome trial , which assessed cardiovascular outcomes with empagliflozin therapy in persons with type 2 diabetes mellitus and coexisting cardiovascular disease .
the article describes the background and challenges of modern cardiovascular outcome trials , points out the strengths of the empa - reg outcome study , and places the results in perspective .
it highlights the significant impact that these results will have on cardiovascular preventive pharmacotherapy , and on future drug development in diabetes . at the same time
, it reminds readers of the limitations of the results , and lists the questions raised by , or left unanswered by , the trial . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
the study protocol was approved by the animal care and use committee of juntendo university . male c57bl/6 mice ( 7 weeks old ) were purchased from oriental yeast ( tokyo , japan ) and housed in specific pathogen deficient ( apoe ) mice ( 6 weeks old ) were purchased from the jackson laboratory ( bar harbor , me ) and housed in specific pathogen mice were maintained under 12-h light / dark cycle , fed a standard rodent diet ( from clea japan at nihon bioresearch ) , and provided with water ad libitum , except where noted . mice were treated with either high - dose ( 24 nmol kg body wt day ) or low - dose ( 300 pmol kg body wt day ) exendin-4 ( sigma - aldrich , tokyo , japan ) , or with saline through a mini - osmotic pump ( alzest , model 1004 ; durect , cupertino , ca ) that delivered the solution continuously for up to 28 days . at the age of 8 weeks , the osmotic pump was implanted under the skin of the back of each mouse after local anesthesia . islets were isolated by a standard collagenase digestion method as described previously ( 16 ) . mouse aortic vascular endothelial cells were isolated and cultured as described previously ( 17 ) . von willebrand factor antibody ( dako , carpinteria , ca ) and negative immunostaining with anti-smooth muscle actin ( sigma - aldrich ) . mouse aortic vascular smooth muscle cells were isolated and cultured as described previously ( 18 ) . the cultured cells were verified as smooth muscle cells by immunostaining using anti-smooth muscle actin . peritoneal macrophages were harvested from the mice with cold pbs at 3 days after intraperitoneal injection of 3% thioglycolate media . the pooled macrophages from each mouse were cultured in rpmi 1640 , supplemented with 0.2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c to allow cell adhesion . human peripheral blood mononuclear cells were isolated from whole blood collected from overnight - fasted healthy volunteers , with mono - poly resolving medium ( ds pharma biomedical , osaka , japan ) after heparinization . monocytes were isolated from peripheral blood mononuclear cells by positive selection using macs cd14 microbeads ( miltenyi biotec , bergisch gladbach , germany ) . we confirmed that 95% of the isolated cells were cd14 monocytes by flow cytometry analysis . cells were cultured in rpmi 1640 , supplemented with 2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol at 37c under a humidified atmosphere with 5% co2 . thp-1 cells were cultured in cultured in rpmi 1640 , supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c . human umbilical vein endothelial cells ( huvecs ) were cultured in 500-ml bottle of endothelial cell basal medium-2 and the following growth supplements : 0.2 ml hydrocortisone , 2 ml human fibroblast growth factor basic , 0.5 ml vascular endothelial growth factor , 0.5 ml r3-igf-1 , 0.5 ml ascorbic acid , 0.5 ml heparin , 10 ml fbs , 0.5 ml human epidermal growth factor , and 0.5 ml ga-1000 ( cambrex bioscience walkersville , charles city , ia ) under 95% relative humidity and 5% co2 at 37c . all samples were sonicated on ice and centrifuged at 15,000 g at 4c for 20 min . the supernatants were collected and western blot analysis was performed using anti glp-1r antibody ( ab3907 ; abcam , cambridge , u.k . ) or rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody ( cell signaling technology , beverly , ma ) as described previously ( 16 ) . the intraperitoneal glucose tolerance test ( ipgtt ) was performed at the age of 12 weeks ( 4 weeks after implantation of the osmotic pump ) . briefly , 1.0 g / kg body wt glucose was injected intraperitoneally after overnight fasting . blood glucose level was measured with a glucometer ( one - touch ultra ; life scan , burnaby , canada ) . plasma insulin levels were measured using an elisa kit ( morinaga , kanagawa , japan ) . the insulin tolerance test was performed at the age of 12 weeks with 0.75 units / kg body wt insulin ( humalin ; blood samples were collected from the retro - orbital venous plexus in awake mice to measure blood glucose and plasma insulin concentrations . after mice were killed by intraperitoneal injection of sodium pentobarbital ( 1 mg / kg ; abbott laboratories ) , tissue preparation was performed by systemic perfusion with saline and 10% buffered formalin . fixation was performed by immersion of the isolated thoracic aorta with 10% buffered formalin at 4c . for en face immunohistochemistry of the endothelial surface , the thoracic aorta was cut open longitudinally along the ventral side with scissors and placed on a glass slide . then immunohistochemistry was performed using anti - mouse mac-2 monoclonal antibody ( dako , cederlane , burlington , on , canada ) . next , each specimen was placed on a slide glass with the intimal side up , and covered with a coverslip . specimens were viewed under a microscope ( e800 ; nikon , tokyo , japan ) connected to an xyz controller and a digital camera ( sony , tokyo , japan ) . to count the number of endothelium - adherent monocytes , we set a rectangular area with sides that were twice the length of the long and short diameters of the vessel opening of the intracostal arteries , respectively , and that were centered on the opening . the total number of mac-2immunopositive cells within the entire rectangular areas were counted in each aorta . the cell density in each area was then calculated as the cell count ( determined by an examiner blinded to the treatment regimen ) divided by the total area ( 1921 ) . for fluorescent staining , the samples were embedded in optimal cutting temperature compound , then sectioned , air dried , and washed in pbs . after immersion in blocking solution of 10% goat serum in pbs for 30 min at room temperature , the sections were incubated overnight at 4c in a humidified chamber for labeling with rabbit polyclonal anti - glp1 receptor antibody ( 1:50 , ls - a1205 ; mbl international , woburn , ma ) , and rabbit anti mac-2 monoclonal antibody ( 1:200 ; dako ) . the specimens were placed in the appropriate goat secondary antibody conjugated with alexa fluor dyes ( invitrogen , carlsbad , ca ) and diluted 1:300 in pbs for 30 min at room temperature . the specimen was placed on a glass slide , dapi - containing mounting medium ( vector laboratories , burlingame , ca ) was added , and then the tissue was covered with a cover glass . samples were viewed by confocal laser scanning microscopy ( fluoview fv1000 ; olympus , tokyo , japan ) . the heart and the aorta were flushed with normal saline followed by 10% buffered formalin as described previously ( 20 ) . for quantitative analysis of arteriosclerotic lesions in the aortic sinus , the heart was cut in two halves and the top half was embedded in optimal cutting temperature compound , then cross - sectioned at 4-m thickness at 50-m interval with a cryostat . twelve consecutive sections were taken sequentially from just above the aortic valve throughout the aortic sinus and allowed to dry at room temperature for 30 min . the mean lesion area of those 12 sections was calculated and expressed in square millimeters . the isolated macrophages were washed once and then incubated with or without 0.03 , 0.3 , and 3 nmol / l exendin-4 or 10 mol / l forskolin ( an adenylate cyclase activator ; sigma - aldrich ) for 1 h , followed by incubation with or without lipopolysaccharide ( lps , 1 g / ml ; sigma - aldrich ) for 1 h. to inhibit the exenatide signal , macrophages were incubated with 5 mol / l mdl-12330a ( sigma - aldrich ) , a specific adenylate cyclase inhibitor , and 10 mol / l pki14 - 22 ( sigma - aldrich ) , a protein kinase a ( pka ) inhibitor , for 30 min before adding exendin-4 . control macrophages were incubated with the vehicle ( dmso , final concentration < 0.1% ) . after treatment , nuclear protein extracts were isolated from peritoneal macrophages and the content of nuclear factor-b ( nf-b ) p65 was determined using a specific elisa kit using the method recommended by the manufacturer ( imgenex , san diego , ca ) ( 22 ) . total rna was extracted from peritoneal macrophages using the rna easymicro kit ( qiagen , tokyo , japan ) and the instructions provided by the manufacturer . first - strand cdna was synthesized using 1 g of total rna with oligo - dt primers and superscript reverse transcriptase ( invitrogen ) as described previously ( 23 ) . the resulting cdnas were amplified using the sybr green pcr kit ( applied biosystems , foster city , ca ) . quantitative pcr was performed with an abi prism 7700 sequence detection system ( perkin elmer life sciences , boston , ma ) . the relative abundances of mrnas were calculated by the comparative cycle of threshold method with tata box after collection and stabilization for 6 h , monocytes were cultured in the presence or absence of different concentrations of exendin-4 for 24 h. the culture supernatants were removed and cells washed with pbs to remove nonadherent cells . the fc receptors were blocked by clear back ( human fc receptor blocking reagent ; mbl , nagoya , japan ) for 5 min at room temperature . then the cells were stained with phycoerythrin labeled antibodies to anti - human cd11b or the corresponding isotype control ( nonspecific mouse igg - phycoerythrin ) ( bd biosciences , cologne , germany ) and incubated at 4c for 15 min . bonferroni multiple comparison test was used for comparisons among multiple treatment groups and the control group . the study protocol was approved by the animal care and use committee of juntendo university . male c57bl/6 mice ( 7 weeks old ) were purchased from oriental yeast ( tokyo , japan ) and housed in specific pathogen deficient ( apoe ) mice ( 6 weeks old ) were purchased from the jackson laboratory ( bar harbor , me ) and housed in specific pathogen mice were maintained under 12-h light / dark cycle , fed a standard rodent diet ( from clea japan at nihon bioresearch ) , and provided with water ad libitum , except where noted . mice were treated with either high - dose ( 24 nmol kg body wt day ) or low - dose ( 300 pmol kg body wt day ) exendin-4 ( sigma - aldrich , tokyo , japan ) , or with saline through a mini - osmotic pump ( alzest , model 1004 ; durect , cupertino , ca ) that delivered the solution continuously for up to 28 days . at the age of 8 weeks , the osmotic pump was implanted under the skin of the back of each mouse after local anesthesia . islets were isolated by a standard collagenase digestion method as described previously ( 16 ) . mouse aortic vascular endothelial cells were isolated and cultured as described previously ( 17 ) . von willebrand factor antibody ( dako , carpinteria , ca ) and negative immunostaining with anti-smooth muscle actin ( sigma - aldrich ) . mouse aortic vascular smooth muscle cells were isolated and cultured as described previously ( 18 ) . the cultured cells were verified as smooth muscle cells by immunostaining using anti-smooth muscle actin . peritoneal macrophages were harvested from the mice with cold pbs at 3 days after intraperitoneal injection of 3% thioglycolate media . the pooled macrophages from each mouse were cultured in rpmi 1640 , supplemented with 0.2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c to allow cell adhesion . human peripheral blood mononuclear cells were isolated from whole blood collected from overnight - fasted healthy volunteers , with mono - poly resolving medium ( ds pharma biomedical , osaka , japan ) after heparinization . monocytes were isolated from peripheral blood mononuclear cells by positive selection using macs cd14 microbeads ( miltenyi biotec , bergisch gladbach , germany ) . we confirmed that 95% of the isolated cells were cd14 monocytes by flow cytometry analysis . cells were cultured in rpmi 1640 , supplemented with 2% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol at 37c under a humidified atmosphere with 5% co2 . thp-1 cells were cultured in cultured in rpmi 1640 , supplemented with 10% fcs , 10 mmol / l hepes , 1 mmol / l sodium pyruvate , 2 mmol / l l - glutamine , 100 units / ml penicillin , 100 g / ml streptomycin , and 50 mol / l 2-mercaptoethanol under 95% relative humidity and 5% co2 at 37c . human umbilical vein endothelial cells ( huvecs ) were cultured in 500-ml bottle of endothelial cell basal medium-2 and the following growth supplements : 0.2 ml hydrocortisone , 2 ml human fibroblast growth factor basic , 0.5 ml vascular endothelial growth factor , 0.5 ml r3-igf-1 , 0.5 ml ascorbic acid , 0.5 ml heparin , 10 ml fbs , 0.5 ml human epidermal growth factor , and 0.5 ml ga-1000 ( cambrex bioscience walkersville , charles city , ia ) under 95% relative humidity and 5% co2 at 37c . all samples were sonicated on ice and centrifuged at 15,000 g at 4c for 20 min . the supernatants were collected and western blot analysis was performed using anti glp-1r antibody ( ab3907 ; abcam , cambridge , u.k . ) or rabbit anti glyceraldehyde-3-phosphate dehydrogenase antibody ( cell signaling technology , beverly , ma ) as described previously ( 16 ) . the intraperitoneal glucose tolerance test ( ipgtt ) was performed at the age of 12 weeks ( 4 weeks after implantation of the osmotic pump ) . briefly , 1.0 g / kg body wt glucose was injected intraperitoneally after overnight fasting . blood glucose level was measured with a glucometer ( one - touch ultra ; life scan , burnaby , canada ) . plasma insulin levels were measured using an elisa kit ( morinaga , kanagawa , japan ) . the insulin tolerance test was performed at the age of 12 weeks with 0.75 units / kg body wt insulin ( humalin ; eli lilly , indianapolis , in ) after 6 h of fasting . blood samples were collected from the retro - orbital venous plexus in awake mice to measure blood glucose and plasma insulin concentrations . after mice were killed by intraperitoneal injection of sodium pentobarbital ( 1 mg / kg ; abbott laboratories ) , tissue preparation was performed by systemic perfusion with saline and 10% buffered formalin . fixation was performed by immersion of the isolated thoracic aorta with 10% buffered formalin at 4c . for en face immunohistochemistry of the endothelial surface , the thoracic aorta was cut open longitudinally along the ventral side with scissors and placed on a glass slide . then immunohistochemistry was performed using anti - mouse mac-2 monoclonal antibody ( dako , cederlane , burlington , on , canada ) . next , each specimen was placed on a slide glass with the intimal side up , and covered with a coverslip . specimens were viewed under a microscope ( e800 ; nikon , tokyo , japan ) connected to an xyz controller and a digital camera ( sony , tokyo , japan ) . to count the number of endothelium - adherent monocytes , we set a rectangular area with sides that were twice the length of the long and short diameters of the vessel opening of the intracostal arteries , respectively , and that were centered on the opening . the total number of mac-2immunopositive cells within the entire rectangular areas were counted in each aorta . the cell density in each area was then calculated as the cell count ( determined by an examiner blinded to the treatment regimen ) divided by the total area ( 1921 ) . for fluorescent staining , the samples were embedded in optimal cutting temperature compound , then sectioned , air dried , and washed in pbs . after immersion in blocking solution of 10% goat serum in pbs for 30 min at room temperature , the sections were incubated overnight at 4c in a humidified chamber for labeling with rabbit polyclonal anti - glp1 receptor antibody ( 1:50 , ls - a1205 ; mbl international , woburn , ma ) , and rabbit anti mac-2 monoclonal antibody ( 1:200 ; dako ) . the specimens were placed in the appropriate goat secondary antibody conjugated with alexa fluor dyes ( invitrogen , carlsbad , ca ) and diluted 1:300 in pbs for 30 min at room temperature . the specimen was placed on a glass slide , dapi - containing mounting medium ( vector laboratories , burlingame , ca ) was added , and then the tissue was covered with a cover glass . samples were viewed by confocal laser scanning microscopy ( fluoview fv1000 ; olympus , tokyo , japan ) . the heart and the aorta were flushed with normal saline followed by 10% buffered formalin as described previously ( 20 ) . for quantitative analysis of arteriosclerotic lesions in the aortic sinus , the heart was cut in two halves and the top half was embedded in optimal cutting temperature compound , then cross - sectioned at 4-m thickness at 50-m interval with a cryostat . twelve consecutive sections were taken sequentially from just above the aortic valve throughout the aortic sinus and allowed to dry at room temperature for 30 min . the mean lesion area of those 12 sections was calculated and expressed in square millimeters . the isolated macrophages were washed once and then incubated with or without 0.03 , 0.3 , and 3 nmol / l exendin-4 or 10 mol / l forskolin ( an adenylate cyclase activator ; sigma - aldrich ) for 1 h , followed by incubation with or without lipopolysaccharide ( lps , 1 g / ml ; sigma - aldrich ) for 1 h. to inhibit the exenatide signal , macrophages were incubated with 5 mol / l mdl-12330a ( sigma - aldrich ) , a specific adenylate cyclase inhibitor , and 10 mol / l pki14 - 22 ( sigma - aldrich ) , a protein kinase a ( pka ) inhibitor , for 30 min before adding exendin-4 . control macrophages were incubated with the vehicle ( dmso , final concentration < 0.1% ) . nuclear protein extracts were isolated from peritoneal macrophages and the content of nuclear factor-b ( nf-b ) p65 was determined using a specific elisa kit using the method recommended by the manufacturer ( imgenex , san diego , ca ) ( 22 ) . total rna was extracted from peritoneal macrophages using the rna easymicro kit ( qiagen , tokyo , japan ) and the instructions provided by the manufacturer . first - strand cdna was synthesized using 1 g of total rna with oligo - dt primers and superscript reverse transcriptase ( invitrogen ) as described previously ( 23 ) . the resulting cdnas were amplified using the sybr green pcr kit ( applied biosystems , foster city , ca ) . quantitative pcr was performed with an abi prism 7700 sequence detection system ( perkin elmer life sciences , boston , ma ) . the relative abundances of mrnas were calculated by the comparative cycle of threshold method with tata box after collection and stabilization for 6 h , monocytes were cultured in the presence or absence of different concentrations of exendin-4 for 24 h. the culture supernatants were removed and cells washed with pbs to remove nonadherent cells . the fc receptors were blocked by clear back ( human fc receptor blocking reagent ; mbl , nagoya , japan ) for 5 min at room temperature . then the cells were stained with phycoerythrin labeled antibodies to anti - human cd11b or the corresponding isotype control ( nonspecific mouse igg - phycoerythrin ) ( bd biosciences , cologne , germany ) and incubated at 4c for 15 min . bonferroni multiple comparison test was used for comparisons among multiple treatment groups and the control group . as a first step to elucidate the antiatherosclerotic effects of exendin-4 , we first investigated the expression of glp-1 receptor in cells associated with atherosclerogenesis . similar to lung and pancreatic -cells ( 24 ) , mice peritoneal macrophages and vascular smooth muscle cells abundantly expressed glp-1 receptor protein , and the expression level was higher than in freshly isolated endothelial cells . similar to the expression level in macrophages , glp-1 receptor was abundantly expressed in thp-1 cells , which are derived from human monocytes , and freshly isolated human monocytes . in contrast to the freshly isolated endothelial cells , abundant expression of glp-1 receptor was detected in huvecs ( fig . in addition , immunohistochemical staining showed glp-1 receptor expression in cells that expressed mac-2 , a marker of macrophages located in the atherosclerotic lesions of the aortic valve of apoe mice ( fig . these results may suggest that glp-1 can directly act on monocytes or macrophages and affect the progression of atherosclerosis . a : expression of glp-1 receptor in murine lung and liver , isolated murine islets , isolated murine macrophages ( ms ) , cultured murine endothelial cells ( ecs ) , cultured murine smooth muscle cells ( smcs ) , human monocyte derived line , thp-1 cells , and huvecs . c : immunohistochemical staining of glp-1 receptor ( green ) and mac-2 , a marker of macrophages ( red ) in atherosclerotic lesions of apoe mice . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the effect of glp-1 receptor activation on atherosclerosis , c57bl/6 mice received continuous infusion of 300 pmol kg day ( low dose ) or 24 nmol kg day ( high dose ) exendin-4 for 28 days . during the treatment period , neither dose affected body weight ( fig . 2a ) . after the 24-day treatment , both doses of exendin-4 improved glucose tolerance without affecting insulin secretion ( fig . the results of the insulin tolerance test were similar in the two groups ( fig . treatment with high - dose exendin-4 , but not the low dose , slightly increased total cholesterol and hdl cholesterol compared with control group ( table 1 ) . the density of monocytes that adhered to the endothelial cells of the thoracic aorta was markedly suppressed in both the low- and high - dose treatment groups , compared with control ( fig . exendin-4 reduced monocytic adhesion to the endothelium in c57bl/6 mice . a : changes in body weight during treatment with exendin-4 in c57bl/6 mice ( n = 6 each ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 each ) . e : the density of adherent mac-2positive cells on endothelial cells at branching areas in each group of mice after 28-day treatment ( n = 6 ) with representative en face views of immunohistologic staining with mac-2 antibody . data are mean sem . * p < 0.05 versus high - dose group , + p < 0.05 versus low - dose group . ( a high - quality digital representation of this figure is available in the online issue . ) results of laboratory tests in c57bl/6 mice and apoe mice after 28-day treatment with exendin-4 data are mean se . blood samples were collected from c57bl/6j mice ( n = 6 ) and apoe mice ( n = 13 except for hba1c , n = 6 for hba1c ) in the fasting state after 28-day treatment with exendin-4 . * p < 0.05 vs. control group . p < 0.05 for low ex4 vs. high ex4 . low ex4 , low - dose exendin-4 ; high ex4 , high - dose exendin-4 ; tg , triglycerides ; cm , chylomicron ; sd - ldl , small , dense ldl ; hba1c , a1c ; na , not applicable . to explore the role of glp-1 receptor activation on the progression of atherosclerosis treatment with high - dose exendin-4 modestly reduced body weight gain and glucose tolerance and decreased serum total cholesterol level without affecting ldl cholesterol level ( fig . on the other hand , treatment with low - dose exendin-4 only modestly reduced glucose level at 30 min after glucose injection without affecting other parameters investigated ( fig . the density of monocytes that adhered to endothelial cells of the thoracic aorta was significantly lower in the low- and high - dose groups than the control group ( fig . quantification of mrna expression in the thoracic aorta showed that exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 ( icam-1 ) and tended to downregulate vcam-1 ( fig . the oil red o positive area at the aortic valve level was significantly reduced in the high - dose group compared with the control group ( fig . the area of the atherosclerotic lesions in the low - dose group also tended to be smaller than the control group , however the difference was not significant . the metabolic effect of exendin-4 in apoe mice . a : changes in body weight during exendin-4 treatment in apoe mice ( n = 13 ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 ) . data are mean sem . * p < 0.01 versus high - dose group , + p < 0.01 versus low - dose group . exendin-4 reduced monocyte adhesion to the endothelium and atherosclerotic lesions in apoe mice . a : en face immunohistochemical staining with mac-2 antibody of the aorta of each group . the density of adherent mac-2positive cells on the endothelium at branching areas in each group of mice after 28-day treatment ( n = 7 ) and representative en face views of immunohistologic staining with mac-2 antibody . b : aortas harvested from each group of mice after 28-day treatment were used for isolation of total rna . the mrna expression levels of icam-1 and vcam-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in the test mice relative to that in the control group ( set at 1.0 , n = 57 ) . c : representative histologic sections of the aortic sinuses stained with oil red o after 28-day treatment . the mean area of oil red o positive lesions was determined ( n = 20 ) . ( a high - quality digital representation of this figure is available in the online issue . ) the data obtained from apoe mice and c57bl/6 mice suggested that exendin-4 could have beneficial effects against atherosclerosis without affecting the metabolic parameters and that it could potentially prevent the progression of atherosclerosis by its direct action on the cells involved in atherosclerogenesis . the abundant expression of glp-1 receptor in monocytes / macrophages and the inhibitory effects of exendin-4 on monocyte adhesion on endothelial cells encouraged us to investigate the effects of exendin-4 on the inflammatory response . indeed , incubation with 1 g / ml of lps for 1 h induced 10-fold increases in the expression levels of tnf- and monocyte chemoattractant protein-1 ( mcp-1 ) , a representative cytokine and a chemokine in isolated macrophages , respectively ( data not shown ) . thus , we investigated the effects of various concentrations of exendin-4 ( 0.033 nmol / l ) in counteracting this response . exendin-4 at all concentrations significantly suppressed lps - induced increases in the expression levels of tnf- and mcp-1 in macrophages ( fig . glp-1 receptor is well - known gs - protein coupled receptor , thus the activation of glp-1 receptor results in increased camp concentration due to activation of adenylate cyclase ( 25 ) . to explore the mechanism of exendin-4induced suppression of tnf- and mcp-1 expression in macrophages , we preincubated peritoneal macrophages with mdl-12330a , a specific adenylyl cyclase inhibitor , or forskolin , an adenylyl cyclase activator . the addition of mdl-12330a completely suppressed the inhibitory effect of exendin-4 on the expression levels of tnf- and mcp-1 ( fig . on the other hand , forskolin significantly suppressed lps - induced tnf- and mcp-1 expression in macrophages , and the levels of suppression by forskolin were similar to those of exendin-4 ( fig . these results suggest that the inhibitory effects of exendin-4 on the expression of tnf- and mcp-1 are largely dependent on the activation of adenylate cyclase . next , we investigated the downstream pathway of camp using pki14 - 22 , a specific pka inhibitor . similar to mdl-12330a , the inhibitory effect of exendin-4 was significantly reversed by pki14 - 22 ( fig . 5d ) , suggesting the involvement of pka in the anti - inflammatory effect of exendin-4 . exendin-4 reduced the inflammatory response through camp signaling pathway in macrophages , and reduced the expression of cd11b in human monocytes . a : peritoneal macrophages isolated from 8-week - old c57bl/6 mice were incubated with various concentrations of exendin-4 ( 0.033 nmol / l ) for 1 h followed by treatment with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna . the mrna expression levels of tnf- and mcp-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in peritoneal macrophages without the addition of exendin-4 set at 1.0 ( n = 45 ) . b : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 46 ) . / l exendin-4 or 10 mol / l forskolin for 1 h followed by lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l pki14 - 22 for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of nuclear protein extracts . the nuclear level of nf-b p65 was determined by enzyme - linked immunosorbent assay ( elisa ) ( n = 34 ) . f : human monocytes isolated from healthy volunteers were incubated without or with various concentrations of exendin-4 ( 0.033 nmol / l ) for 24 h. then , the surface expression of cd11b was assessed by flow cytometry . although nf-b is a major regulator of the expression of tnf- and mcp-1 , vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide are known to inhibit nf-b dependent gene activation by activation of pka in cultured monocytic cell line thp-1 ( 26 ) . thus , we investigated the effect of exendin-4 on lps - induced nuclear translocation of nf-b p65 in macrophages . without any stimulation , nuclear nf-b p65 was not detected in peritoneal macrophages , however , stimulation with lps robustly induced nuclear translocation of nf-b p65 ( data not shown ) . such translocation was markedly suppressed by exendin-4 , and this inhibitory effect was completely abolished by mdl-12330a ( fig . these results indicate that exendin-4 inhibits nuclear translocation of nf-b p65 by activating camp , in parallel with the expression of tnf- and mcp-1 . finally , we investigated the effect of exendin-4 on human monocytes . thus , we investigated the effects of exendin-4 on their counterreceptors , cd11b , in isolated human peripheral monocytes . exposure to 0.3 and 3 nmol / l , but not 0.03 nmol / l , exendin-4 for 24 h significantly reduced the surface expression of cd11b , as assessed by flow cytometry ( fig . these results suggest that activation of the glp-1 receptor has the antiatherogenic effects on human circulating monocytes . as a first step to elucidate the antiatherosclerotic effects of exendin-4 , we first investigated the expression of glp-1 receptor in cells associated with atherosclerogenesis . similar to lung and pancreatic -cells ( 24 ) , mice peritoneal macrophages and vascular smooth muscle cells abundantly expressed glp-1 receptor protein , and the expression level was higher than in freshly isolated endothelial cells . similar to the expression level in macrophages , glp-1 receptor was abundantly expressed in thp-1 cells , which are derived from human monocytes , and freshly isolated human monocytes . in contrast to the freshly isolated endothelial cells , abundant expression of glp-1 receptor was detected in huvecs ( fig . in addition , immunohistochemical staining showed glp-1 receptor expression in cells that expressed mac-2 , a marker of macrophages located in the atherosclerotic lesions of the aortic valve of apoe mice ( fig . these results may suggest that glp-1 can directly act on monocytes or macrophages and affect the progression of atherosclerosis . a : expression of glp-1 receptor in murine lung and liver , isolated murine islets , isolated murine macrophages ( ms ) , cultured murine endothelial cells ( ecs ) , cultured murine smooth muscle cells ( smcs ) , human monocyte derived line , thp-1 cells , and huvecs . c : immunohistochemical staining of glp-1 receptor ( green ) and mac-2 , a marker of macrophages ( red ) in atherosclerotic lesions of apoe mice . ( a high - quality digital representation of this figure is available in the online issue . ) to investigate the effect of glp-1 receptor activation on atherosclerosis , c57bl/6 mice received continuous infusion of 300 pmol kg day ( low dose ) or 24 nmol kg day ( high dose ) exendin-4 for 28 days . during the treatment period , neither dose affected body weight ( fig . 2a ) . after the 24-day treatment , both doses of exendin-4 improved glucose tolerance without affecting insulin secretion ( fig . the results of the insulin tolerance test were similar in the two groups ( fig . treatment with high - dose exendin-4 , but not the low dose , slightly increased total cholesterol and hdl cholesterol compared with control group ( table 1 ) . the density of monocytes that adhered to the endothelial cells of the thoracic aorta was markedly suppressed in both the low- and high - dose treatment groups , compared with control ( fig . exendin-4 reduced monocytic adhesion to the endothelium in c57bl/6 mice . a : changes in body weight during treatment with exendin-4 in c57bl/6 mice ( n = 6 each ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 each ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 each ) . e : the density of adherent mac-2positive cells on endothelial cells at branching areas in each group of mice after 28-day treatment ( n = 6 ) with representative en face views of immunohistologic staining with mac-2 antibody . data are mean sem . * p < 0.05 versus high - dose group , + p < 0.05 versus low - dose group . ( a high - quality digital representation of this figure is available in the online issue . ) results of laboratory tests in c57bl/6 mice and apoe mice after 28-day treatment with exendin-4 data are mean se . blood samples were collected from c57bl/6j mice ( n = 6 ) and apoe mice ( n = 13 except for hba1c , n = 6 for hba1c ) in the fasting state after 28-day treatment with exendin-4 . * p < 0.05 vs. control group . p < 0.05 for low ex4 vs. high ex4 . low ex4 , low - dose exendin-4 ; high ex4 , high - dose exendin-4 ; tg , triglycerides ; cm , chylomicron ; sd - ldl , small , dense ldl ; hba1c , a1c ; na , not applicable . to explore the role of glp-1 receptor activation on the progression of atherosclerosis , we treated apoe mice with low- or high - dose exendin-4 . treatment with high - dose exendin-4 modestly reduced body weight gain and glucose tolerance and decreased serum total cholesterol level without affecting ldl cholesterol level ( fig . 3 , table 1 ) . on the other hand , treatment with low - dose exendin-4 only modestly reduced glucose level at 30 min after glucose injection without affecting other parameters investigated ( fig . the density of monocytes that adhered to endothelial cells of the thoracic aorta was significantly lower in the low- and high - dose groups than the control group ( fig . quantification of mrna expression in the thoracic aorta showed that exendin-4 treatment significantly downregulated intercellular adhesion molecule-1 ( icam-1 ) and tended to downregulate vcam-1 ( fig . the oil red o positive area at the aortic valve level was significantly reduced in the high - dose group compared with the control group ( fig . the area of the atherosclerotic lesions in the low - dose group also tended to be smaller than the control group , however the difference was not significant . the metabolic effect of exendin-4 in apoe mice . a : changes in body weight during exendin-4 treatment in apoe mice ( n = 13 ) . b : blood glucose concentrations during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . c : plasma insulin levels during ipgtt after 24-day treatment with exendin-4 ( n = 6 ) . d : results of insulin tolerance test in each group after 24-day treatment with exendin-4 ( n = 6 ) . * p < 0.01 versus high - dose group , + p < 0.01 versus low - dose group . exendin-4 reduced monocyte adhesion to the endothelium and atherosclerotic lesions in apoe mice . a : en face immunohistochemical staining with mac-2 antibody of the aorta of each group . the density of adherent mac-2positive cells on the endothelium at branching areas in each group of mice after 28-day treatment ( n = 7 ) and representative en face views of immunohistologic staining with mac-2 antibody . b : aortas harvested from each group of mice after 28-day treatment were used for isolation of total rna . the mrna expression levels of icam-1 and vcam-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in the test mice relative to that in the control group ( set at 1.0 , n = 57 ) . c : representative histologic sections of the aortic sinuses stained with oil red o after 28-day treatment . the mean area of oil red o positive lesions was determined ( n = 20 ) . ( a high - quality digital representation of this figure is available in the online issue . ) the data obtained from apoe mice and c57bl/6 mice suggested that exendin-4 could have beneficial effects against atherosclerosis without affecting the metabolic parameters and that it could potentially prevent the progression of atherosclerosis by its direct action on the cells involved in atherosclerogenesis . the abundant expression of glp-1 receptor in monocytes / macrophages and the inhibitory effects of exendin-4 on monocyte adhesion on endothelial cells encouraged us to investigate the effects of exendin-4 on the inflammatory response . indeed , incubation with 1 g / ml of lps for 1 h induced 10-fold increases in the expression levels of tnf- and monocyte chemoattractant protein-1 ( mcp-1 ) , a representative cytokine and a chemokine in isolated macrophages , respectively ( data not shown ) . thus , we investigated the effects of various concentrations of exendin-4 ( 0.033 nmol / l ) in counteracting this response . exendin-4 at all concentrations significantly suppressed lps - induced increases in the expression levels of tnf- and mcp-1 in macrophages ( fig . glp-1 receptor is well - known gs - protein coupled receptor , thus the activation of glp-1 receptor results in increased camp concentration due to activation of adenylate cyclase ( 25 ) . to explore the mechanism of exendin-4induced suppression of tnf- and mcp-1 expression in macrophages , we preincubated peritoneal macrophages with mdl-12330a , a specific adenylyl cyclase inhibitor , or forskolin , an adenylyl cyclase activator . the addition of mdl-12330a completely suppressed the inhibitory effect of exendin-4 on the expression levels of tnf- and mcp-1 ( fig . on the other hand , forskolin significantly suppressed lps - induced tnf- and mcp-1 expression in macrophages , and the levels of suppression by forskolin were similar to those of exendin-4 ( fig . these results suggest that the inhibitory effects of exendin-4 on the expression of tnf- and mcp-1 are largely dependent on the activation of adenylate cyclase . next , we investigated the downstream pathway of camp using pki14 - 22 , a specific pka inhibitor . similar to mdl-12330a , the inhibitory effect of exendin-4 was significantly reversed by pki14 - 22 ( fig . 5d ) , suggesting the involvement of pka in the anti - inflammatory effect of exendin-4 . exendin-4 reduced the inflammatory response through camp signaling pathway in macrophages , and reduced the expression of cd11b in human monocytes . a : peritoneal macrophages isolated from 8-week - old c57bl/6 mice were incubated with various concentrations of exendin-4 ( 0.033 nmol / l ) for 1 h followed by treatment with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna . the mrna expression levels of tnf- and mcp-1 were determined by quantitative rt - pcr . relative gene expression is displayed as the level of expression in peritoneal macrophages without the addition of exendin-4 set at 1.0 ( n = 45 ) . b : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 46 ) . c : peritoneal macrophages were incubated with 0.3 nmol / l exendin-4 or 10 mol / l forskolin for 1 h followed by lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . mol / l pki14 - 22 for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of total rna ( n = 45 ) . e : peritoneal macrophages were preincubated with 5 mol / l mdl-12330a for 30 min before the addition of 0.3 nmol / l exendin-4 and then incubated with lps ( 1 g / ml ) for 1 h. then , macrophages were used for isolation of nuclear protein extracts . the nuclear level of nf-b p65 was determined by enzyme - linked immunosorbent assay ( elisa ) ( n = 34 ) . f : human monocytes isolated from healthy volunteers were incubated without or with various concentrations of exendin-4 ( 0.033 nmol / l ) for 24 h. then , the surface expression of cd11b was assessed by flow cytometry . although nf-b is a major regulator of the expression of tnf- and mcp-1 , vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide are known to inhibit nf-b dependent gene activation by activation of pka in cultured monocytic cell line thp-1 ( 26 ) . thus , we investigated the effect of exendin-4 on lps - induced nuclear translocation of nf-b p65 in macrophages . without any stimulation , nuclear nf-b p65 was not detected in peritoneal macrophages , however , stimulation with lps robustly induced nuclear translocation of nf-b p65 ( data not shown ) . such translocation was markedly suppressed by exendin-4 , and this inhibitory effect was completely abolished by mdl-12330a ( fig . these results indicate that exendin-4 inhibits nuclear translocation of nf-b p65 by activating camp , in parallel with the expression of tnf- and mcp-1 . finally , we investigated the effect of exendin-4 on human monocytes . thus , we investigated the effects of exendin-4 on their counterreceptors , cd11b , in isolated human peripheral monocytes . exposure to 0.3 and 3 nmol / l , but not 0.03 nmol / l , exendin-4 for 24 h significantly reduced the surface expression of cd11b , as assessed by flow cytometry ( fig . these results suggest that activation of the glp-1 receptor has the antiatherogenic effects on human circulating monocytes . in the present study , we provide evidence that exendin-4 , a glp-1 receptor agonist , prevents the progression of atherosclerosis in apoe mice without major effects on metabolic parameters . our data suggest that exendin-4 markedly reduced the accumulation of monocytes / macrophages in the vascular wall at least in part by suppressing the inflammatory response in macrophages through the activation of the camp / pka pathway . the results showed that the exendin-4 decreased monocyte adhesion to endothelial cells in two nondiabetic mice , c57bl6 and apoe mice . in both mouse strains , exendin-4 reduced glucose level during ipgtt , however , because the two strains are nondiabetic , the effect of exendin-4 on glucose level should play only a minor effect on its antiatherosclerogenetic properties . in this study , we confirmed the expression of glp-1 receptor in endothelial cells , smooth muscle cells , macrophages , and monocytes . because these cells play critical roles in the progression of atherosclerosis , glp-1 receptor activation in these cells may be involved in atherosclerogenesis . indeed , several groups reported the beneficial effects of glp-1 receptor activation on endothelial cells ( 1013 ) . on the other hand , we confirmed in the present study the direct anti - inflammatory effect of glp-1 on monocytes / macrophages . indeed , treatment with exendin-4 at concentrations observed during treatment of humans ( 27,28 ) reduced the expression of inflammatory mediators tnf- and mcp-1 in activated macrophages . tnf- and mcp-1 are among the important cytokines and chemokines whose atherogenic effect has been established . both tnf- and furthermore , forced expression of tnf- and mcp-1 in leukocytes promotes advanced atherosclerotic lesions ( 29,31 ) . thus , in addition to the effect of glp-1 on endothelial cells , its effect on monocytes / macrophages may also have a major impact on the attenuation of atherosclerosis . it was reported previously that the main effects of glp-1 are mediated through the activation of adenylate cyclase and the production of camp ( 25 ) . using adenylate cyclase inhibitor and activator , we also demonstrated in this study that stimulation of camp by exendin-4 is critical for the attenuated production of proinflammatory mediators from macrophages . this result is consistent with previous studies in which increased intracellular levels of camp inhibited tnf- production and its transcription in macrophage ( 3234 ) . these data suggest that exendin-4 regulates inflammatory response of macrophages via the camp / pka pathway , which inhibits proinflammatory cytokine production as reported recently ( 35,36 ) . our results showed that pka activation and inhibition of nf-b p65 translocation mediate overexpression of inflammatory cytokines by increased camp level elicited by glp-1 receptor activation . the adhesion of circulating monocytes to the intimal endothelial cells is thought to be one of the earliest events in the complex pathologic process of atherosclerogenesis ( 14,15 ) . this can be mediated by the interaction of specific adhesion molecules on vascular endothelial cells with their integrin counterreceptors on monocytes . activation of monocytes by cytokines , chemokines , hypercholesterolemia , and hyperglycemia leads to increased expression of this integrin , and increased monocyte expression of cd11b correlates with adhesion of these cells to the endothelium in patients with hypercholesterolemia ( 37 ) . our results showed a potential suppressive effect of exendin-4 on the surface expression of cd11b on human monocytes . on the other hand , we demonstrated that exendin-4 decreased the expression of icam-1 , which interacts with cd11b on monocytes in apoe mice . these effects may contribute at least in part to the reduced monocyte adhesion to the endothelium in vivo . however , additional experiments are required to clarify the mechanism of exendin-4induced inhibition of cd11b expression . in conclusion , our data suggest that glp-1 receptor activation significantly reduced the accumulation of monocytes / macrophages in the vascular wall and eventually inhibited atherosclerogenesis by regulating inflammation in macrophages via the camp / pka pathway and the integrin - related gene expression on monocytes . these unique effects of glp-1 receptor activation may help design new therapies for cardiovascular disease in patients with type 2 diabetes . | objectiveexogenous administration of glucagon - like peptide-1 ( glp-1 ) or glp-1 receptor agonists such as an exendin-4 has direct beneficial effects on the cardiovascular system .
however , their effects on atherosclerogenesis have not been elucidated .
the aim of this study was to investigate the effects of glp-1 on accumulation of monocytes / macrophages on the vascular wall , one of the earliest steps in atherosclerogenesis.research design and methodsafter continuous infusion of low ( 300 pmol
kg1
day1 ) or high ( 24 nmol kg1
day1 ) dose of exendin-4 in c57bl/6 or apolipoprotein e
deficient mice ( apoe/ ) , we evaluated monocyte adhesion to the endothelia of thoracic aorta and arteriosclerotic lesions around the aortic valve .
the effects of exendin-4 were investigated in mouse macrophages and human monocytes.resultstreatment with exendin-4 significantly inhibited monocytic adhesion in the aortas of c57bl/6 mice without affecting metabolic parameters . in apoe/ mice , the same treatment reduced monocyte adhesion to the endothelium and suppressed atherosclerogenesis . in vitro treatment of mouse macrophages with
exendin-4 suppressed lipopolysaccharide - induced mrna expression of tumor necrosis factor- and monocyte chemoattractant protein-1 , and suppressed nuclear translocation of p65 , a component of nuclear factor-b .
this effect was reversed by either mdl-12330a , a camp inhibitor or pki14 - 22 , a protein kinase a specific inhibitor . in human monocytes , exendin-4 reduced the expression of cd11b.conclusionsour data suggested that glp-1 receptor agonists reduced monocyte / macrophage accumulation in the arterial wall by inhibiting the inflammatory response in macrophages , and that this effect may contribute to the attenuation of atherosclerotic lesion by exendin-4 . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
singleton pregnancy is the desired outcome of any assisted reproductive technique ( art ) . with advances in the field of embryology and laboratory practices , great advances have been made such that the average pregnancy rate per cycle has increased . even though transfer of a single good quality embryo is advocated by most of the guidelines ( 1 ) , it has not become a routine practice globally . sometimes , in certain circumstances , as in repeated failure cases or , owing to a limited ability to select the embryos with the highest chance for implantation , a large cohort of embryos is transferred deliberately . in all these cases , though the chance of multifetal pregnancy is increased , singleton pregnancies are also seen . limited implantation potential of the transferred embryo or of the uterus itself different embryo transfer strategies have resulted in different multiple pregnancy rates ranging from 38.3% in siberia to 5.7% in sweden ( 2 ) . therefore , as the number of children born by art is significant , knowledge of factors affecting perinatal outcome in art singletons is crucial . it is known that singletons conceived by art are at increased risk of adverse pregnancy outcomes ( 24 ) whether this outcome is related to infertility itself , art technology or the embryo ( 4 ) . this study aimed to find if the obstetric outcomes are also affected by implantation rate . many previous studies have considered aspects which alter or influence implantation rate and thereby predict / determine the number of embryos to be transferred ( 1 , 5 ) . the purpose was to evaluate the perinatal outcome in singleton pregnancies as a function of different implantation rates . this may help understand two aspects of conception : 1 ) does embryonic crowding initially affect the implanted embryo ? and 2 ) does higher implantation rate translates into better reproductive and perinatal performance ? to the best of our knowledge , there have been no studies evaluating the possible effects of implantation rate on singleton pregnancies resulting from transfer of 4 or less cleavage stage embryos . the purpose of this study was to compare the perinatal outcomes of singleton pregnancies according to implantation rate . this is a retrospective analysis done in the department of reproductive medicine and surgery at sri aurobindo institute of medical sciences from the year 2012 to 2014 . women who conceived after in vitro fertilization ( ivf ) or intracytoplasmic sperm injection ( icsi ) with a single gestation sac visible on ultrasound ( usg ) at 6 weeks ( approximately 4 weeks after embryo transfer ) were included in the study . records of the patients were studied to exclude patients with elective single embryo transfers , blastocyst transfers , with donor oocyte , surrogacies and monochorionic gestation . as this was a retrospective study , informed consent for all cases in the study , less than 4 cleavage stage embryos transfer was done at a time . the following data was recorded from case file of patients included in this study age , previous conception ( parity ) , cause of infertility , type of transfer ( fresh or frozen ) , number of embryos transferred , pregnancy outcome in terms of abortion , live birth and birth weight at delivery . patients who had chronic diseases like diabetes mellitus , hypertension , thyroiditis and autoimmune disorders were excluded from the study . for purpose of stimulation , antagonist protocol was used in all patients included in the study ( 6 ) . the stimulation started with recombinant gonadotropins from day 2 or 3 of cycle when estradiol levels were < 50 pg / ml and ovarian quiescence seen on usg . the dose of recombinant gonadotropin gonal f ( merck serono spa , italy ) was decided as per patient s age , antral follicle count , bmi and previous response to stimulation if any . gonadotropin releasing hormone antagonist ( cetrorelix 0.25 mg , serum institute of india ltd , india ) was initiated as per flexible protocol when at least 1 follicle reached the average diameter of 14 mm till the day of trigger . ovulation trigger was given using either 10,000 iu ( 250 g ) recombinant hcg ( sanzyme ltd . india ) 2 mg when 3 or more follicles reached an average diameter of 17 mm . the choice of trigger depended on pre - trigger estradiol values and risk of ovarian hyperstimulation syndrome . patients given agonist trigger underwent frozen - thawed cycle in subsequent months wherein , down regulation was done using 3.75 mg leuprolide ( bdr pharmaceutical international pvt ltd . endometrial preparation was done with estradiol valerate ( in incremental doses ) ( 7 ) . all patients were given luteal phase support from day of oocyte retrieval till 14 weeks of gestation in the form of micronized progesterone vaginal gel once daily ( naturogest- east sikkim ) once daily . concentration of vaginal gel was 8% ( 1.35 gr in a pack which delivers 90 mg progesterone daily ) ( 8) . based on semen quality , the oocytes were inseminated 36 hr after oocyte retrieval with either icsi or conventional insemination . fertilization was indicated by the appearance of two distinct pronuclei and two polar bodies 1618 hr after insemination . embryonic development was assessed on day 2 ( 4143 hr ) and on day 3 ( 6567 hr ) after insemination . cleavage stage embryo transfer was performed using embryos with the highest number of blastomeres with the highest embryo score . all embryo transfers were performed by the same person under ultrasound guidance and a serum beta - hcg pregnancy test was performed 16 days after embryo transfer . as per departmental protocol , all patients had conceived after embryo transfer and had transvaginal usg done at around 6 weeks ( 4 weeks after embryo transfer ) . all patients were followed up every 2 to 4 weeks in the department till delivery / abortion and records were periodically updated . missed abortion early abortion was defined as loss of cardiac activity or disappearance of gestational sac before 12 weeks after documentation of live fetus at 6 weeks . loss of fetus between 13 to 28 weeks was defined as late abortion . any birth after 28 completed weeks of gestation ( period of viability for present study ) was considered as live birth with those between 2932 weeks being very preterm , 33 to 36 weeks as preterm and after 37 weeks as term birth . implantation rate was calculated by dividing number of gestational sacs seen ( 1 ) by the number of embryo transferred ( 4 , 3 or 2 ) . the basic characteristics of pregnancy ( gestational age , birth weight ) were compared using analysis of variance ( continuous variables ) , and categorical variables were evaluated with chi - squared tests and t test as applicable . as per the inclusion criteria , 195 patients conceived with singleton after art and delivered / aborted by the completion of study period . depending on the number of cleavage stage embryos , patients were divided into 3 groups as per calculated implantation rate ( 25% , 33.33% , and 50% ) . the age and previous conception were similar in all three groups , and the age of patients with an implantation rate of 50% was lesser than the other two groups ( table 1 ) though the difference was not statistically significant . patients characteristics in different implantation rate groups numbers in parenthesis denote percentage out of 195 patients included in the study , a total of 157 had a live birth ( table 2 ) . a majority , 68.15% ( 107/157 ) had term delivery ( at or beyond 37 weeks of gestation ) . the number of preterm and term deliveries between the three groups was not statistically significant . the average gestational age in live births showed an increasing trend as the implantation rate increased ; however , this does not appear to be statistically significant ( p=0.66 ) . when the average gestational age of the entire cohort was considered ( live births and abortions ) , a statistically higher probability of delivering later in gestation was found when the implantation rate was 50% ( p=0.04 ) . implantation rate and delivery outcome numbers in parenthesis denote percentage out of 195 patients , a total of 38 patients aborted or delivered at or before 28 weeks of gestation . the rate of abortion gestation did not appear to be statistically related to the implantation rate ( p= 0.37 ) ( table 3 ) . in the group of 25% implantation rate , 26% patients ( 13/50 ) had undergone spontaneous abortion as against 11.11% ( 7/63 ) in the group of 50% implantation though this result did not reach statistical significance . implantation rate and spontaneous miscarriage numbers in parenthesis denote percentage in the present study , 157 patients had a live birth with a majority 61.78% ( 97/157 ) with a birth weight of over 2.6 kg ( table 4 ) . no statistical significance was found in the birth weight amongst the 3-implantation groups . however , the percentage of babies with birth weight of more than 2.6 kg ( 66.07% ) and the average birth weight ( 2.71 kg ) was the highest in the 50% implantation group . implantation rate and birth weight in live births numbers in parenthesis denote percentage in the present study , out of 195 patients , 110 had frozen - thawed embryo transfers while 85 had fresh transfers . the type of embryo transfer ( fresh or frozen thawed ) does not appear to be statistically associated with the 3-implantation groups ( table 5 ) . embryo quality and endometrial receptivity are two determinants for pregnancy success in patients undergoing ivf - et program . with respect to embryos , clinical data show that 70% of embryos selected for transfer fail to implant . to increase pregnancy rates , multiple embryos are transferred , which is responsible for complications due to multiple gestation pregnancy ( 9 ) ; therefore , it is essential to develop better understanding of implantation and factors determining its rate . in an elaborate study done on rodent uteri ( 10 ) , embryo implantation at unfavorable site the authors also suggested that one cause of vanishing twin syndrome seen in ivf conceptions might be due to embryo crowding at implantation and subsequent resorption of one embryo as the result of competition for space and nutrient supply . it is an open but intriguing possibility that maternal stress at the time of embryo transfer and/or implantation may cause similar sympathetic activation of uterine 2-ar ( beta 2 adrenergic receptor ) in humans , resulting in sub - optimal embryo location and pregnancy complications ( 10 ) . oren et al . ( 3 ) have demonstrated that embryo quality had no effect on perinatal outcomes but it does affect pregnancy rate and implantation rate ( 11 ) . also , in our study , no significant association was found between live birth rate and birth weight and implantation rate in singleton conception / deliveries . however , regarding gestational age at termination of pregnancy ( abortion or delivery ) , a decreasing age with lower implantation rate was found . therefore , it is believed that early pregnancy events may depend on implantation rate though once the singleton progresses beyond the luteo - placental shift , number of initial embryos may not be relevant anymore . ( 12 ) have suggested that with increasing fetal / embryo number , the obstetric outcome of surviving singleton is adversely affected . they too have supported the hypothesis that early implantation site crowding leads to first trimester losses . a meta - analysis ( 2 ) found high - grade evidence that singleton ivf babies have higher risk of worse perinatal outcome in comparison to singleton of unselected spontaneously conceived women . also , in this study , it was shown that number of embryos transferred does not affect the outcome of a singleton pregnancy conceived consequently . this in turn indicates that there may be factors inherent to art technique or the infertile patient herself that may lead to poorer outcome . this meta - analysis also looked into the evidence comparing perinatal outcomes of singleton pregnancies after single or double embryo transfer . however , a slightly higher average birth weight and gestational age at delivery ( of live born ) were found when implantation rate was higher ( 50% ) as against lower implantation rates ( 25% and 33% ) . even the percentage of abortion was higher in lower implantation rate group though none of these results reached a statistical significance . a retrospective cohort study ( 13 ) showed that in older women , the chance of multiple sacs leading to delivery of multiple babies is low and likely reflects aneuploidy . therefore , they have advocated a more aggressive embryo transfer strategy to maximize the chance of actual live birth . in cases of multiple embryo transfer , especially in absence of pre implantation genetic screening , implantation rate will depend on the quality of the embryos themselves . presuming that only the best embryo implants in a singleton pregnancy , it can be further concluded that this naturally selected embryo will have similar obstetric outcome irrespective of its initial competition . in other words , correct measure of uterine receptivity and embryo quality will help pre select the one embryo that will implant and thereby reduces the potential for multiple pregnancies . asrm guidelines ( 1 ) have defined the number of embryos transferred on basis of patient age , embryo quality , the opportunity for cryopreservation , and results of previous ivf cycles if any . transfer of multiple embryos ( 2 or more ) has been suggested in cases with relatively poor prognosis secondary to age , previous failure or embryo quality . for patients with previous ivf failures , however , our study shows that as the implantation rate decreases , chances of live birth also decrease . therefore , in view of our own study , it is believed that the best outcome in terms of live birth may be achieved by increasing the implantation rate by transferring better quality embryo itself . in a large retrospective cohort analysis , over 35000 et after oocyte donation ( 14 ) , the authors concluded that as the number of embryos transferred per cycle increased , so did the total number of cycles required to achieve a newborn . this study also showed that percentage of miscarriage also increased along with total number of embryo transferred . this study , however , did not evaluate single- , double- , or triple - embryo transfers per se and the researchers did not take into consideration the quality of embryos transferred . despite these differences , the result of our own study appears to be similar . previous studies have demonstrated a poorer neonatal outcome in singleton pregnancies of art especially in fresh transfers ( 2 , 3 ) . an improvement in the outcome with frozen transfers ( 15 ) is attributed to better endometrial receptivity . our study shows that as the implantation potential improves , the possibility of better outcome especially in terms of live birth also increases . hsu et al . ( 16 ) , in their retrospective controlled clinical study , found an inverse relation between implantation rate and number of embryos transferred irrespective of their quality . a major strength of our study is that it was a single center study with close follow up by a single physician in all conceived patients . our results are limited by the retrospective nature of the study and smaller numbers . due to the study design itself , nature of endometrium at time of transfer and our results are limited by the retrospective nature of the study and smaller numbers . due to the study design itself , nature of endometrium at time of transfer and previously failed ivf cycles our study shows that in patients with singleton pregnancies conceived after multiple embryo transfer , there is higher chance of missed abortion which is significant if more than 2 embryos are transferred . however , there is a correlation between the number of embryos transferred with gestational age at delivery and birth weight . | background : singleton pregnancy with a live birth beyond 37 weeks of gestation is the ultimate goal of any assisted reproductive technology .
however , singletons conceived after art are found to have a poor perinatal outcome in comparison to naturally conceived singletons .
it was hypothesized that the outcome of singleton conceived after transfer of two or more embryos may be dependent on the sharing of uterine space with other embryos.methods:patients who had single gestational sac visualized at 6 weeks after transfer of 4 , 3 or 2 embryos were considered for the study .
195 singleton pregnancies were selected for final evaluation such that as per implantation rates of 25% , 33% , and 50% , they were divided into 3 groups of 50 , 82 and 63 cases , respectively .
the basic characteristics of pregnancy ( gestational age , birth weight ) were compared using analysis of variance ( continuous variables ) , and categorical variables were evaluated with chi - squared test .
the p<0.05 was considered statistically significant.results:among the various variables including maternal age , conception , type of infertility , type of abortion , total live birth , gestational age in live birth , birth weight , kind of embryo transfer and gestational age , there was not significant statistical differences between groups except gestational age that it was higher in group with 50% ( p<0.04 ) implantation rate .
therefore , higher number of initial embryos may affect the perinatal outcome of singleton conceived subsequently.conclusion:there is higher chance of missed abortion in patients with singleton pregnancies conceived after multiple embryo transfer .
gestational age at delivery and birth weight were correlated with number of embryo transfered . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
pseudomyxoma peritonei is an uncommon condition with an estimated incidence of one to two per million ( worldwide ) per year . it is characterized by the peritoneal deposition of mucinous tumors , most commonly of the appendix , and occasionally from the ovary , coupled by mucinous ascites . we report the case of a 76-year - old woman who presented with increased abdominal girth and dyspnea for 2 weeks . she was diagnosed as a case of pseudomyxoma peritonei . two years and 8 months later , she refers with postprandial fullness and has moderate ascites . pseudomyxoma peritonei ( pmp ) is a tumor with an estimated incidence of one to two million per year.1 it is a poorly understood disease that is known for its production of mucin in the abdominal cavity and mucinous implants , diffusely involving the peritoneal surfaces.2 the term pmp has been applied broadly and includes a heterogeneous group of pathological lesions from the benign to the borderline to the frankly malignant lesion.3,4 ronnett et al have suggested a classification based on tumor pathology in which they place all pmp in three groups in order of decreasing prognosis : disseminated peritoneal adenomucinosis , peritoneal mucinous carcinoma with intermediate or discordant features , and peritoneal mucinous carcinoma.4 a definitive diagnosis of pmp requires the presence both of mucinous neoplastic cells / epithelium and of mucinous ascites and diffuse intra - abdominal mucin . viable epithelial glandular cells must be identified within the mucin pools by histological analysis to diagnose pmp . a 76-year - old woman presented to our emergency department ( hospital professor doutor fernando fonseca , amadora , portugal ) with dyspnea , increased abdominal girth , and peripheral edema for 2 weeks . she was medicated with ramipril , furosemide , aspirin , simvastatin , rabeprazole , and lorazepam . the physical examination revealed tachypnea , a blood pressure of 144/82 mmhg , a pulse rate of 83 beats per minute ( bpm ) , and apyrexia . expiratory time was increased , and there were rales at the base of the lungs . there was collateral venous circulation on the abdomen wall , the wall was slightly depressible , and percussion revealed tympanic resonance in the center of the abdomen and dullness in both lumbar regions . laboratory investigations showed an absence of leukocytosis ( 8,400 leukocytes/l ) with neutrophilia ( 6,100 neutrophils/l ) and a c - reactive protein value of 6.21 mg / dl . the patient s erythrocyte sedimentation rate was 88 mm , her carcinoembryonic antigen level was 177 ng / ml ( 03 ng / ml ) , her cancer antigen 153 level was 55 u / ml ( 030 u / ml ) , her cancer antigen 125 level was 57.5 u / ml ( 035 u / ml ) , and her cancer antigen 19.9 level was lower than 2.5 u / ml ( 037 u / ml ) . abdomen ultrasonography and computed tomography showed images that were suggestive of pseudomyxoma and moderate , multiseptate ascites ( figure 1 ) . the patient was submitted to laparotomy with right oophorectomy , omentectomy , and pseudomyxoma debulking ( figure 2 ) . at surgery , the histological examination of biopsies ( from small intestine , ovary , peritoneum , and epiploon ) revealed mucinous neoplastic cells and diffuse intra - abdominal mucin , characteristics that allowed the diagnosis of pmp . the morphological ( figure 4 ) and immunophenotypic ( cytokeratin 7 focally positive [ figure 5 ] , cytokeratin 20 diffusedly positive [ figure 6 ] , and p16 negative [ figure 7 ] ) patterns were compatible with colorectal / appendicular origin . in the postoperative course , the patient had hemodynamic instability that reverted with fluid replacement and amines for a short period . she was discharged in a satisfactory condition with follow - up at the oncology division . two years and 8 months later , she has referred occasionally with postprandial fullness and moderate ascites . her carcinoembryonic antigen and cancer antigen 125 levels are 133 ng / ml and 139 u / ml , respectively . the last image exam ( abdomen ultrasonography ) showed a small volume of perihepatic , perisplenic , and interloop fluid with peritoneal leaflets thickening . it was possible to identify a mass with 58 30 mm in the left parietocolic groove , indicative of peritoneal carcinomatosis ( figure 8) . enlarged ganglia were also seen , the biggest of which was at the hepatic hilum , at 32 mm ( figure 9 ) . pmp was first described in 1842 by karl f rokitansky,6 but the term was used in 1884 in association with a mucinous carcinoma of the ovary.7 later , in 1901 , franckel described pmp in association with an appendiceal cystic tumor.8 pmp is a disease of the muc ( a gel - forming mucin that forms strong bonds with the surrounding stroma and is also believed to have tumor - suppressor activities)9 expressing goblet cells that are specific for mucin production . extracellular mucin accumulates dramatically and has no place to drain.4 a copious amount of mucinous fluid gradually fills the peritoneal cavity , resulting in the so - called jelly belly abdomen.5 the origin of pmp is a controversial subject.10 most acknowledge that pmp predominantly originates in the appendix in men , and increasingly , evidence suggests a similar origin in women . in women , however , immunohistochemistry and molecular genetic techniques support the hypothesis that in the majority of women , the ovarian tumor is metastatic from a perforated appendiceal mucinous tumor.1 this hypothesis is illustrated by the presented case , as histological analysis of the oophorectomy piece revealed a pattern compatible with colorectal / appendicular origin . the reported sex distribution is contradictory in the literature : some authors have noted a significant preponderance of women5,8,10 whereas others found no difference.3 however , as some authors highlight , it is improbable that the female preponderance found in some studies has a biological explanation ; instead , the effect is more likely the result of some referral bias , possibly resulting from the use of iterative surgery in secondary care hospitals to treat male patients with pmp and not referring them to a tertiary unit . female patients were more likely to be referred because of presumed ovarian malignancy requiring complex procedures available only in tertiary care units.1,10 the symptoms of this uncommon disease are nonspecific ; the most commonly recorded are abdominal pain10 resembling acute appendicitis , distension,10,11 or mass.12 nausea , vomiting , and asthenia have also been described.13 in rare cases , the mucinous collection can lead to signs of raised intra - abdominal pressure , such as uterine prolapse or abdominal hernia.10 in addition to symptoms being nonspecific , routine laboratory studies also are seldom helpful in making this diagnosis.5 an accurate preoperative diagnosis of pmp can be aided by radiological imaging,5 with a computed tomography scan being the currently optimal imaging modality for the diagnosis and staging of this disease . computed tomography scan findings may be pathognomonic . typical appearances include areas of low attenuation , with islands of higher attenuation resulting from solid elements within mucinous material . classically , scalloping of visceral surfaces , particularly of the liver and spleen , distinguishes mucinous from fluid ascites.1 however , diagnosis of pmp is challenging because of the mimicking nature of the disease . in the study conducted by jrvinen and lepist , only 28% of patients underwent initial surgery for presumed pmp.10 preoperative diagnosis is often appendicitis,1 gynecological cancer1/ovarian tumor,10 or peritonitis,1 which did not happen in our case , as imaging suggested from the beginning a diagnosis of pmp . a treatment strategy for pmp should pursue complete cytoreduction and prevention of recurrence or progression . combined modality treatment , consisting of cytoreductive surgery with hyperthermic intraperitoneal chemotherapy , seems very efficient in this regard.1417 this approach is currently carried out in many centers throughout the world , with promising results,18 and seems to win ground as the standard treatment approach.14 however , not all cases are suitable for this approach.10 patients must be medically fit to safely undergo these treatments , and patients with peritoneal carcinomatosis with an eastern cooperative oncology group ( ecog ) performance score of 2 to 3 have significantly poorer overall survival after the treatment compared with those with an ecog score of 1.1 in the presented case , the patient had an ecog performance status of 3 ; therefore , we considered her to be not medically fit to be submitted to the referred approach . unfortunately , the majority of patients develop recurrence after treatment,19 which requires repeated surgical intervention.6 elevated tumor markers such as carcinoembryonic antigen level , cancer antigen 19 - 9 level , and cancer antigen 125 level indicate advanced disease and may rise in association with recurrent disease.12,20 surgical reintervention is usually difficult because adhesions and fibrosis greatly increase the risk of unintentional enterotomies with subsequent leaks and fistulae formation ; it is , however , widely accepted that recurrences should be investigated vigorously and treated with further surgical debulking , with or without adjuvant chemotherapy , in the expectation that many patients will enjoy substantial additional symptom - free survival.6 the reported prognostic factors of pmp are age,21 histology,11 residual tumor volume,11 and intraperitoneal chemotherapy.13 an interesting fact is that chemotherapy may improve survival only if administered intraperitoneally,13,19 as reported by the mayo clinic , gough db.13 systemic chemotherapy seems not to affect prognosis.19 in addition , survival rates differ greatly according to histology : ronnett et al observed 5- and 10-year survival rates of 75% and 68% , respectively , for cases of disseminated peritoneal adenomucinosis ; 50% and 21% , respectively , for cases of intermediate malignancy ; and 14% and 3% , respectively , for peritoneal mucinous carcinoma.22 we described a case of a female patient who developed pmp , a rare and heterogeneous condition for which the diagnosis is often misleading in the preoperative period . in the light of current knowledge , awareness of this condition , although it is rare , is an important prerequisite for appropriate management , as the long - term outcomes for cytoreductive surgery with hyperthermic intraperitoneal chemotherapy are impressive for patients with low - grade histology susceptible to complete cytoreduction . | backgroundpseudomyxoma peritonei is an uncommon condition with an estimated incidence of one to two per million ( worldwide ) per year .
it is characterized by the peritoneal deposition of mucinous tumors , most commonly of the appendix , and occasionally from the ovary , coupled by mucinous ascites.case presentationwe report the case of a 76-year - old woman who presented with increased abdominal girth and dyspnea for 2 weeks .
she was diagnosed as a case of pseudomyxoma peritonei .
she was submitted to right oophorectomy , omentectomy , and pseudomyxoma debulking .
the histology was compatible with a mucinous tumor of colorectal / appendicular origin .
chemotherapy was not administered because of her functional status .
two years and 8 months later , she refers with postprandial fullness and has moderate ascites . |
You are an expert at summarizing long articles. Proceed to summarize the following text:
in addition , the accurate initial position determination of the lips is essential for planning of proper treatment . understanding the correlation between hard tissue and soft tissue before treatment is also important in order to predict the changes that may result from orthodontic intervention . placing the teeth according to accepted cephalometric criteria does not necessarily ensure that the overlying soft tissue will drape in a harmonious manner,1 or guarantee that the soft tissue profile will directly follow the underlying skeletal profile.2 this is because the soft tissue covering the teeth and bone can vary so greatly that the dentoskeletal pattern may be inadequate in evaluating facial disharmony.3 according to kasai , the relationship between the hard tissue and soft tissue profile is variable because some soft tissue structures are closely related to hard tissue while others are influenced by their length , thickness , and function.4 saxby and freer found that the position of the upper and lower incisors and angulation of the upper incisors are very important determinants of the associated soft tissues.5 in a study by sodagar et al , the ratio of maxillary incisor to upper lip retraction was 2:1 , and in another study a significant correlation was found between the retraction of the lower incisors and the retrusion of the lower lip.6,7 these correlations have also been established for the incisors and the lips,8 and for alveolar bone landmarks and the overlying soft tissues.9 lower facial height was also found to be an important determinant of soft tissue morphology . kasai reported that longer lower facial height and protruding lower incisors were associated with a thicker upper lip,4 while saxby and freer found correlations between lower facial height and soft tissue form in the horizontal and vertical planes.5 although the sagittal jaw relationship was found to be strongly related to the overlying soft tissue outline,5 soft tissue can mask or exaggerate the jaw discrepancy.10 racial and ethnic characteristics were found to be another factor influencing the soft tissue configuration . thin and minimal lip protrusion was found in white europeans and more protrusion in those of middle eastern origin , while greater thickness and protrusion were found among orientals and africans.11 several studies have indicated that there is a tendency for bimaxillary protrusion among the saudi population with more lip protrusion than the caucasian individuals.7,1214 facial soft tissue structure affects the perception of profile esthetics . african americans prefer a straighter profile than what has been measured as normal for their race.15,16 more recently , marques et al concluded that society perceives straight profiles as esthetically more pleasing in comparison with protruding profiles.17 canonical correlation analysis is a powerful statistical technique that enables examination of multiple predictor and multiple criterion variables.18 it is useful in assessing relationships and summarizing dependence between two groups of variables.19,20 in the dental literature , canonical analysis has served as an instrument for obtaining new knowledge about how important factors interact with each other.4,18,2024 when compared with multiple r - tests , simultaneous multivariate statistical methods are more robust.22 the present study evaluated the correlation between facial hard and soft tissues in saudi adults utilizing canonical correlation analysis to provide a basis for normal hard and soft tissue correlation in this population . the study sample included 60 lateral cephalometric radiographs from saudi adults ( 30 males and 30 females ) with normal occlusion . the study sample radiographs were taken from the archive of cephalometric radiographs for fourth year dental students ( mean age 221 years ) that were taken as a part of their undergraduate orthodontic course requirements . formal approval to conduct the study was obtained from the ethical subcommittee at king saud university , college of dentistry research center ( # nf 2101 ) . the selection criteria included : saudi citizenship , age over 18 years , average skeletal relationship with anb angle ranging between 14.5 ( based on the normal anb angle range in saudi individuals),2528 with no previous orthodontic treatment , a class i molar relationship , overjet 24 mm , and overbite one third to one half of the lower incisor crown height . a perfection 4990 photo scanner ( seiko epson corporation , nagano , japan ) linked to a dell computer running on microsoft windows xp was used to scan the cephalometric radiographs , which were then captured using dolphin imaging version 10.0 software ( dolphin imaging and management solutions , chatsworth , ca , usa ) . the landmarks and measurements used in the cephalometric analysis the soft tissue measurements included evaluation of the position of the upper and lower lips to different reference lines , and the upper and lower lip thickness and length , as follows :
the e - line extends from tip of the nose to soft tissue pogonion ( pog ) . ul - e is the distance from most anterior point of the upper lip to e - plane while the distance from the most anterior point of the lower lip to the e - plane is ll-e.the s - line extends from the soft tissue contour of the chin to the middle of an s formed by the lower border of the nose . the position of the upper and lower lips to this line was evaluated.the distances from the most anterior point of the upper and lower lips to the line connecting the soft tissue subnasale with soft tissue pog were also measured.upper and lower lip thickness represents the distance between the lip inside and the lip outside.the upper lip length extends from the subnasale to upper lip stomion superius point , while lower lip length extends from the lower lip stomion inferius to menton . the e - line extends from tip of the nose to soft tissue pogonion ( pog ) . ul - e is the distance from most anterior point of the upper lip to e - plane while the distance from the most anterior point of the lower lip to the e - plane is ll - e . the s - line extends from the soft tissue contour of the chin to the middle of an s formed by the lower border of the nose . the distances from the most anterior point of the upper and lower lips to the line connecting the soft tissue subnasale with soft tissue pog were also measured . upper and lower lip thickness represents the distance between the lip inside and the lip outside . the upper lip length extends from the subnasale to upper lip stomion superius point , while lower lip length extends from the lower lip stomion inferius to menton . the data were analyzed using statistical package for the social sciences version 16 software ( spss inc . , an evaluation of the error of the method was carried out using the coefficient of reliability , while canonical correlation was used to evaluate the correlation between sets of hard and soft tissue variables . canonical correlation evaluates the correlation of two canonical variables , one representing a set of independent variables and the other a set of dependent variables . in the present study , the following were the two sets :
set 1 ( hard tissue variables ) u1-l1 , u1-sn , u1-pp , u1-na , u1-na ( mm ) , u1-apog , u1-apog ( mm ) , l1-nb , l1-nb ( mm ) , l1-apog , l1-apog ( mm ) , l1-mp.set 2 ( soft tissue variables ) ul - snpog , ll - snpog , ul - e , ll - e , ul - s , ll - s , nla , nb - h , sn - uli , lls - me , ul thickness , ll thickness , a-tvl , ula - tvl , lla - tvl , b-tvl , ulc . set 1 ( hard tissue variables ) u1-l1 , u1-sn , u1-pp , u1-na , u1-na ( mm ) , u1-apog , u1-apog ( mm ) , l1-nb , l1-nb ( mm ) , l1-apog , l1-apog ( mm ) , l1-mp . set 2 ( soft tissue variables ) ul - snpog , ll - snpog , ul - e , ll - e , ul - s , ll - s , nla , nb - h , sn - uli , lls - me , ul thickness , ll thickness , a-tvl , ula - tvl , lla - tvl , b-tvl , ulc . all variables in the 15 cephalometric radiographs were remeasured by one examiner ( ms ) to evaluate the error of the method . the means and standard deviations of the skeletal , dental , and soft tissue variables are listed in table 1 . table 2 shows the canonical correlation results , and indicates that the correlations ranged from 0.917 to 0.196 . the percentage of the squared value of the first , second , and third canonical variates was found to be 84% , 74% , and 67% , respectively . the loading and standardized canonical coefficients between set 1 ( hard tissue variables ) and set 2 ( soft tissue variables ) for the canonical variates are presented in table 3 . it was found that the first canonical variate for hard tissue variables has a heavy positive loading with u1-apog ( mm ; 0.966 ) , l1-nb ( mm ; 0.887 ) , and a moderate positive loading with l1-nb ( 0.627 ) . the first canonical variate for soft tissue variables has a moderate positive loading with ll - e ( 0.756 ) and a moderate loading with sn - uli ( 0.459 ) . the second canonical variate for set 1 is related to u1-na ( 0.706 ) . in set 2 this retrospective study was designed to assess the correlation between hard and soft tissues in saudi adults . the age of the subjects selected was over 18 years , as recommended by subtelny and sakuda to exclude the effect of growth.29 the cephalographs of the saudi males and females were selected from the dental files of fourth year dental students ; these are usually taken as part of their training in the preclinical orthodontic course . the relationship between the soft tissue profile and the underlining hard tissue structure was evaluated by using canonical correlation analysis , which measures the strength of the overall relationships between the linear composites ( canonical variates ) of the independent and dependent variables . in general , the number of canonical dimensions ( canonical variates ) is equal to the number of variables in the smaller set ; however , the number of significant dimensions may be even smaller.30 canonical loadings are used as a basis for interpretation because of the deficiencies inherent in canonical weights . canonical loadings , also called canonical structure correlations , measure the simple linear correlation between an original observed variable in the dependent or independent set and the set s canonical variate . the larger the coefficient , the more important it is in deriving the canonical variate . the percentage of the squared canonical correlation of the first correlation in the study group indicated that 84% of the variation in soft tissue is explained by variation in hard tissue . analysis of this group of saudi adults with normal occlusion showed that the variation in upper lip length and lower lip position could be explained by variation in the position of the upper incisors and the position and inclination of the lower incisors . saxby and freer detected a correlation between the upper and lower incisors horizontal position and the upper incisors angulation to lip position.5 the second squared canonical correlation of the control group indicated that 74% of the variation in soft tissue variables could be accounted for by variation in hard tissue variables . the lower lip length was associated with the inclination of the upper incisors in the present study . the current findings agree with those of yogosawa , who found that the lower lip shortens about 80% of the amount of the maxillary incisors retraction while the upper lip shortens about 40% of that amount in patients with bimaxillary protrusion.31 in the same study , the influence of the maxillary incisors on lower lip position was detected . kasai detected an association between the position of the lower incisors and upper lip thickness.4 the horizontal position of both lips was found also to be correlated with the position of the lower incisors by some researchers.32 based on the previously described correlations , we can conclude that there is a correlation between hard tissue and soft tissue topography in normal saudi individuals . although many studies have focused on the importance of the teeth support on the upper lip , the present study shows that the upper and lower teeth influence the lower lip position . clinically , these results emphasize the possible effects of extraction and nonextraction orthodontic treatment on the upper and lower lips . while retraction of the lips has been observed following retraction of the incisors to a variable degree , changes in upper and lower lip length are another outcome that warrants consideration by clinicians . in addition , this study specified dentoalveolar and soft tissue measurements that can demonstrate the correlation between hard and soft tissues . among the 29 dentoalveolar and soft tissue variables studied , the current study identifies the seven most influential cephalometric measurements that can aid the clinician in diagnosis and assessment of posttreatment changes . the position of the upper and lower incisors and inclination of the lower incisors influence upper lip length and lower lip position . | backgroundthe purpose of this study was to analyze the relationship between facial hard and soft tissues in normal saudi individuals by studying the canonical correlation between specific hard tissue landmarks and their corresponding soft tissue landmarks.methodsa retrospective , cross - sectional study was designed , with a sample size of 60 saudi adults ( 30 males and 30 females ) who had a class i skeletal and dental relationship and normal occlusion .
lateral cephalometric radiographs of the study sample were investigated using a series of 29 linear and angular measurements of hard and soft tissue features .
the measurements were calculated electronically using dolphin software , and the data were analyzed using canonical correlation.resultseighty-four percent of the variation in the soft tissue was explained by the variation in hard tissue.conclusionthe position of the upper and lower incisors and inclination of the lower incisors influence upper lip length and lower lip position .
the inclination of the upper incisors is associated with lower lip length . |
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technological developments have simplified some tasks for human beings , but these
developments have also decreased the physical activity level of the average person . exercise
is a planned activity , and recurrent physical activity helps to protect or improve one or
more components of physical suitability2 . when modern city life began at the start of the 20th century , the importance of exercise was
better understood . in the past , the terms exercise were used interchangeably , but now
exercise is defined as a low category of physical activity . the effects of exercise on human
health can be divided into two main groups , namely physical and psychological effects . clinical pilates exercises were chosen in this study because of the holistic character of
pilates , which has both physical and psychological effects on human health . the positive
effects of the clinical pilates exercises can be seen in a short time , and the individual s
feelings about this improves the sustainability of the exercise behaviour . different
exercise forms and concepts are defined in the literature1 , 3 . in recent years , clinical
pilates has been gaining popularity , and physical therapists are using this exercise form
more frequently4 , 5 . pilates , which was introduced by joseph hubertus pilates , is a
physical fitness program that uses mat exercise equipment to reshape body , and increase
flexibility , strength , balance , and coordination6 . clinical pilates exercises have a low intensity compared with other
aerobic exercise forms ; however , it provides many benefits to human health . it reduces
cardiac disease risks , osteoporosis , reshapes the body , and develops balance and
flexibility . clinical pilates exercises have different levels for different conditions7 , 8 .
also , the pilates method is a functional form of exercise , because it utilizes a combination
of movements in different coordinates , planes , and positions9 . pilates exercises include a breathing component , which has many
positive effects on aerobic capacity and is also beneficial for psychological health . this
exercise form emphasizes the effect of mental concentration on the physical body by
incorporating a focus on breathing10 . on the other hand thus , we performed a study with a group of
people who received verbal education about exercise . we hypothesized that if we could understand exercise beliefs , we could find a way to
increase the exercise level of the community , so we sought to analyze the determinants of
exercise level . the theory of planned behavior ( tpb ) , which introduces a model about human
behavior , was first defined by icek ajzen11 , 12 . many people have positive intentions to
participate in an exercise program , but they do not follow through . the theory gives a road
map to work on the problems and present appropriate attempts to resolve them13 . according to the theory , human behavior
is guided by three kinds of considerations : beliefs about the likely outcomes of the
behavior and the evaluations of these outcomes ( behavioral beliefs ) , beliefs about the
normative expectations of others and motivation to comply with these expectations ( normative
beliefs ) , and beliefs about the presence of factors that may facilitate or impede
performance of the behavior and the perceived power of these factors ( control beliefs ) . in
combination , attitude toward the behavior , subjective norm , and perception of behavioral
control lead to the formation of a behavioral intention . as a general rule , the more
favorable the attitude and subjective norm , and the greater the perceived control , the
stronger should be a person s intention to perform the behavior in question . finally , given
a sufficient degree of actual control over the behavior the validity and reliability of the theory of planned
behavior were shown in previous exercise investigations15,16,17,18,19 . when considered objectively , it is clear that the tpb has been one
of the most cited and impressive models since it was first published by ajzen in 1985 . from this standpoint this study was performed to analyze the exercise beliefs of both sedentary and active
healthy women , in addition to psychosocial factors , and to observe the changes in these
parameters resulting from clinical pilates exercises and verbal education . subjects who had a
systemic , neurological or metabolic disease , had undergone any spinal surgical procedure , or
had a bmi over 29.9 kg / m were excluded from the study . seventhy healthy women between the ages of 2045 years participated in this study . however ,
four subjects declined to participate in the study because of health problems ( fig . flowchart of the study the clinical pilates ( n=21 ) and verbal education ( n=25 ) groups consisted of sedentary
subjects divided according to their intentions . all analyses were done in these groups
before the study and after eight weeks . the clinical pilates group performed clinical
pilates exercises with a trained physical therapist , three times a week for eight weeks at
the pamukkale university physical therapy and rehabilitation high school exercise saloon . the verbal education group was told about exercise principles and benefits by the researcher
at the beginning of the study and also had individual exercise programs and guidelines for
homework . the exercise programs consisted of postural , stretching , and strengthening
exercises , in addition to aerobic exercises like walking and cycling . the verbal education
group was not called or supervised by the researcher with regard to exercises during the
eight - week period . the control group was composed of healthy subjects who were already walking regularly for
one hour three or four times a week for at least one year without supervision ( n=20 ) ( fig . the control group was chosen from active
subjects to compare the physical and psychosocial effects of regular and long - term exercise
with the effects in the study groups . all the subjects understood the purpose of this study , agreed to participate in this
research , and signed an informed consent form . the study was approved by the hacettepe
university non - invasive clinical research ethics committee ( lut 12/58 - 04 ) . demographic information and physical characteristics like age , body mass index ( bmi ) , and
waist and hip circumference measurements were collected from all the subjects at the
beginning of the study . moreover , all the subjects answered the exercise beliefs
questionnaire , physical activity index , and psychosocial factors ( sf-36 , rosenberg
self - esteem scale , body cathexis index , beck depression scale , and visual analog scale for
tiredness ) forms . the exercise beliefs questionnaire was constructed according to the tpb manual for health
services researchers after performing a literature review ( table 1table 1.evaluation and suitability category of activity index ratingscoreevaluationsuitability category>100very active lifestylehigh80100active and healthyvery good4060acceptablenot enough2040not good enoughlow<20sedentaryvery low)20 . participants were asked
to complete the questionnaire on their own in terms with no guidance from the practitioner . each question was answered on a 6-point likert scale ( 1 , strongly disagree ; 2 , largely
disagree ; 3 , partially disagree ; 4 , partially agree ; 5 , largely agree on ; 6 , strongly
agree ) . attitudes , subjective norms , and perceived behavioral control were assessed with
control questions , and scoring was done according to a previously reported method15 , 19 . the questionnaire was divided into the following subscales :
advantages , subjective norms , and disadvantages . the exercise beliefs questionnaire was analyzed for reliability ( test - retest ) with a pilot
group ( n=15 ) . the cronbach s alpha values for the advantages and disadvantages was found to
be between 0.800.89 for this study . in addition , the subjects were asked about the turkish
language suitability , and the form was revised according to this feedback . the exercise levels of the subjects were analyzed by sharkey s physical activity index . physical activity level was calculated as intensity time frequency and categorized as
shown in table 2table 2.characteristics of the study participantscpg ( n=21 ) veg ( n=25 ) cg ( n=20 ) ( 31.82%)(37.88%)(30.30%)age ( years)36.041.8630.881.5334.702.10body mass index ( kg / m)25.300.8322.920.6121.950.49waist circumference ( cm)782.2173.962.1270.851.77hip circumference ( cm)105.572.45981.73960.96physical activity level3.003.003.002.0069.607.82kruskal - wallis signed - rank test , one - way anova ,
cpg : clinical pilates group ; veg : verbal education group ; cg : control
group21 . kruskal - wallis signed - rank test , one - way anova ,
cpg : clinical pilates group ; veg : verbal education group ; cg : control
group the sf-36 is a multipurpose , short - form health survey with only 36 questions that evaluate
quality of life . it yields an 8-scale profile of functional health and well - being scores as
well as psychometrically - based physical and mental health summary measures and a
preference - based health utility index22,23,24 . the rosenberg self - esteem scale is a 10-item scale that measures global self - worth by
measuring both positive and negative feelings about the self . all items are answered using a 4-point likert scale format ranging from
strongly agree to strongly disagree25 , 26 . body cathexis scale which was developed in 1953 by secord and jourard was used in order to
assess body image satisfaction levels . the body cathexis scale evaluates how the individual
perceives the body parts in a detailed manner . scores between 40200 are obtained from the
scale , and higher scores mean higher satisfaction . the beck depression scale is a widely used 21-item self - report inventory measuring the
severity of depression in adolescents and adults . the beck depression scale was revised in
1996 to be more consistent with dsm - iv criteria for depression . the results were categorized
as 09 , nothing / minimal depression ; 1018 , low depression ; 1929 , medium depression ; 3063 ,
severe depression28 . tiredness was analyzed with a visual analog scale ( vas ) of 0 to 10 . for statistical analyses , spss 16.00 was used , and descriptive data were expressed as the
mean standard deviation and number . the analysis techniques used were the wilcoxon
two - sample paired signed rank , two identical significance between test ( paired samples
t - test ) , mann - whitney u test , two average differences between the materiality test
( independent samples t test or student s t - test ) , kruskal - wallis analysis of variance ,
one - way analysis of variance ( anova ) , and chi - square analysis . to determine the reliability
of the exercise beliefs questionnaire , the data obtained from the test - retest analysis were
evaluated by intraclass reliability coefficient ( p<0.05 ) . the mean age ,
bmi , waist circumference , and hip circumference of the women in this study were
33.688.67 years , 23.383.37 kg / m , 74.3010.00 cm , 99.839.38 cm , respectively . twenty subjects ( 30.3% ) were smokers , and 9 subjects ( 13.6% ) were on medications such as
contraception pills . the clinical pilates group ( cpg ) contained 21 women ( 31.82% ) , the
verbal education group ( veg ) contained 25 women ( 37.88% ) , and the control group ( cg )
contained 20 women ( 30.30% ) ( table 3table 3.comparison of groups with each other before the studycpg ( n=21)veg ( n=25)cg ( n=20)exercise beliefsadvantages51.142.5158.723.5276.600.41subjective normpositive3.230.563.400.525.650.33negative5.250.994.960.491.000.23disadvantages29.422.8434.922.6213.700.55psychosocialfactorsvas5.610.505.960.441.950.34rosenberg self - esteemscale22.381.0923.601.0626.650.91beck depressionscale 12.141.8510.201.401.900.36body cathexis index126.617.53140.327.71171.204.46quality of lifephysical function78.092.8178.802.401000role limitations - physical78.574.2678.603.8796.501.66social function92.382.1777.783.2895.251.96pain802.7770.402.5996.001.12mental health75.042.6471.646.9498.201.10role limitations - emotional83.804.4760.803.111000vitality66.902.1463.402.8598.501.09general health perception78.382.5070.822.4396.601.06kruskal - wallis signed - rank test ) . kruskal - wallis signed - rank test at the beginning of the study , the exercise beliefs , physical activity index and
psychosocial factors of the participants were compared , and statistically meaningful
differences were found between the cpg , veg , and cg . in the veg , the disadvantages subscale ,
vas , and beck depression scale scores were meaningfully higher compared with the cg . on the
other hand , the advantages subscale , rosenberg self - esteem scale , and body cathexis scale
scores were meaningfully low in the cpg and veg ( p<0.05 ) ( table 4table 4.comparison of cpg data before and after the studyvariablesbefore after exercise beliefsadvantages51.142.5175.900.73*subjective normpositive3.230.564.280.56*negative5.250.993.400.89disadvantages29.422.8417.331.24*psychosocialfactorsvas5.612.312.381.90*beck depression scale12.148.483.953.89*rosenberg self - esteem scale22.385.0327.143.65*body cathexis scale126.617.53155.574.12*quality of lifephysicalfunction782.8199.281.79*role limitations - physical78.574.2686.603.19*social function92.3812.321000*pain80.002.7794.952.32*mental health75.042.6497.901.42*role limitations - emotional83.804.471000*vitality66.909.8096.668.56*general health perception78.3811.4797.427.18*bmi ( kg / m)5.300.8324.480.70*waist circumference ( cm)8.0010.1575.149.18*hip circumference ( cm)105.5711.23101.2810.03*statistically meaningful difference ) . statistically meaningful difference the cpg and veg data and the cg data were also compared . in the cg , the scores for positive
subjective norm , body image scale , and the sf-36 were higher and those for the bmi and waist
circumference were lower , and these differences were statistically meaningful ( p < 0.05 ) . the result of these analyzes showed that advantages subscale , rosenberg self - esteem scale ,
and sf-36 scores veg , that the disadvantages , fatigue , and depression scores were higher ,
and that these differences were statistically meaningful ( p < 0.05 ) . a statistically meaningful reduction was observed in bmi , waist and hip circumference ,
tiredness score , beck depression scale score , and disadvantages subscale score in the cpg
after the study . on the other hand , a statistically meaningful increase was found using the
rosenberg self - esteem scale , body cathexis scale , sf-36 , and advantages subscale scores
( p<0.05 ) ( table 5table 5.comparison of the differences between the study groups before and after the
studycpg ( n=21 ) veg ( n=25 ) dsdd sdexercise beliefsadvantages1.900.050.920.50subjective normpositive1.040.91.441.00negative 0.660.781.241.02disadvantages1.000.900.920.93psychosocialfactorsvas 3.232.342.401.23beck depression scale8.197.903.524.45rosenberg self - esteem scale4.763.431.801.23body cathexis index28.9523.3210.365.45quality of lifephysical function214.455.803.42role limitations- physical21.4214.48.006.67social function14.956.9010.809.08pain19.0412.237.163.45mental health29.7634.2314.6012.23role limitations- emotional7.616.5010.827.90vitality16.2013.238.335.69general health perception22.8523.2117.1214.31bmi ( kg / m)0.820.780.220.12waist circumference ( cm)2.851.230.881.09hip circumference ( cm)4.283.231.243.43mann - whitney u test , t - test , difference between
the first and last measurements ) . mann - whitney u test , t - test , difference between
the first and last measurements we found a statistically meaningful reduction in the disadvantages subscale score , waist
and hip circumference , tiredness score , and beck depression scale score in the veg . on the
other side , there was a meaningful increase in the rosenberg self - esteem scale and sf-36
scores ( p<0.05 ) . in this study , the differences between before and after clinical pilates and verbal
education were compared . the analyses showed that there were statistically meaningful
differences in the cpg compared with the veg in bmi , waist and hip circumference , and the
beck depression scale , rosenberg self - esteem scale , body cathexis scale , sf-36 , and exercise
beliefs advantages subscale scores in favor of the cpg ( p<0.05 ) . on the other hand , the
disadvantages subscale score did not differ groups ( p > 0.05 ) . the cpg showed significant
differences compared with the veg after training . although these individuals were initially
sedentary , their later parameters approached those of the cg , who already had exercise
habits . changes in physical activity and
exercise level also lead to changes in beliefs , and this paves the path for permanent
changes . by replacing intentions or behavior , we made changes in the level of exercise to understand the changes in
beliefs . our study demonstrates that exercise beliefs can be changed and that behavioral aspects are
gained from this change . since our goal was to change behavior of the cpg and veg , which
were composed of sedentary individuals , we selected individuals who already had a regular
exercise habit for the cg . the purpose of the cg , which was composed of active individuals ,
was to enable comparison of sedentary and active individuals during the same periods . to predict exercise behavior in korean americans , intention and perceived behavioral control statistically determined leisure
activities29 . from other aspects , tpb
has been applied to explain physical activity in canadian adults in a population consisting
of 4,073 people , and it was descriptive30 . positive changes in attitudes and intentions regarding exercise as
exercise time increased was shown in a study by chan et al . in our study , the changes in the
cpg before and after the study were similar to those in the cg . with an increase in the
duration of physical exercise , exercise habits may lead to a permanent change in behavior , and this is possible with a
systematic training program . , physiotherapists use these approaches when designing and
recommending exercise programs for patients as well as healthy individuals32 . in the present study , the veg was
informed about benefits of exercise through a program , and moreover an exercise program was
designed to meet each participant s individual needs . the other study group , the cpg ,
participated in therapist - led clinical pilates exercises . similarly , in another study , the
effects of behavior changes on exercise beliefs were examined with the tpb . the study was
designed with sedentary individuals to investigate if an increase in physical activity led
to an increase in positive beliefs about exercise . at baseline , after the sixth and twelfth
months , 365 adults were asked to complete forms regarding their physical activity and
expectations for a more active life in the next year . objectively , these adults reached the
activity levels that the targeted at the beginning by the twelfth month33 . in our study , an individual s intention to exercise were
not questioned before training ; but they were allowed to pick their own training group . therefore , the significant increase in level of
physical activity in the cpg may be associated with the form of instruction the group
received as well as the initial intentions of the subjects . when we compared the groups in
terms of impact after training , significant differences were observed in the cpg compared
with the veg , but both groups of individuals , which were initially sedentary , later
developed exercise habits and approached the cg in terms of many parameters . in previous
study by sirur et al . conducted a home exercise program and physical activity program and
demonstrated that treatment was ineffective . their study revealed the need to increase the
commitment to exercise for an effective program of physiotherapy , and there are five
theories that suggest a physiotherapistis necessary for this purpose . the five theories
consist of a health belief model , protection motivation theory , self - efficacy theory , the
tpb and the social cognitive theory34 . in
connection with this search , it is also thought to be possible to ensure the functioning of
the protection motivation and social cognitive theories in cpg , while individuals in veg may
only be able to perform the health belief model and induced impact theory . most of the
changes in the cpg brought their data closer to those of the cg . it is thought that this was
the result of the 8-week clinical pilates program , which has often been reported in the
literatre35,36,37,38 . however , significant differences in the cg , like those in body
image scale , sf-36 role limitations , emotions , bmi , and waist circumference , which are
parameters of a physically active lifestyle could be the result of the long - term and lasting
effects of physical activity . in our study , significant positive changes were observed in quality of life in the cpg
before and after measurements were made . in a study similar to ours , by leopoldino and
colleagues on sedentary individuals , the the effects of the pilates matwork method on sleep
quality and quality of life were examined . when sleep quality and quality of life levels
were compared before and after the study , significant improvements were found35 . in this sense , the results of our study
seem to be consistent with the literature . this study was planned and implemented with women to exclude differences in gender - specific
muscle mass , hormonal changes , etc . the importance of exercise in women for prevention of
cancer , diabetes , and hypertension , in addition to the benefits for overall health , has been
revealed in several studies36 , 37 . our study found that an increase in the level of exercise
has a positive effect on bmi , waist hip circumference , self - esteem , body image , and quality
of life . one of the most important results of our study is the indispensability of pilates
exercises for the female population , which also supports findings in the literature . oliveira and colleagues investigated the effects of pilates on muscle strength , postural
balance , and quality of life in older adults with a randomized , controlled , clinical trial . the 16 participants were assigned to a working group and control group randomly in their
study . they concluded that pilates exercises led to significant improvement in isokinetic
torque of the knee extensors and flexors , postural balance , and aspects of the
health - related quality of life of older adults38 . in the cpg in our study , reductions in parameters such
as the bmi and waist circumference revealed the positive effects of clinical pilates on the
physical properties of individuals . the positive effects of the pilates method are supported
by our study in terms of improving quality of life . in a study conducted by mcgrath et al . , the relationship between health - related quality of
life and group exercises was investigated in healthy individuals . the participants were divided into two groups , namely those who
participated in pilates exercise and those who participated in strengthening exercise . at
the end of the study , the pilates group was found to have significant differences in favor
of energy and fatigue components of the sf-36 . it was also revealed that the type of group
exercises affect health - related quality of life39 . as shown in our study and others , the analyses showed that clinical pilates causes an increase in all
parameters of the sf-36 . clinical pilates seems to be an effective physical fitness program
to improve the quality of life . in a study conducted in portugal that was similar to ours ,
the , life satisfaction , the impact on physical health and self - confidence levels of pilates
mat exercise positive significant changes were
found in parameters such as life satisfaction , the perception of acceptance by other people ,
physical appearance perception , and functionality perception in the working group at the
beginning and after 6 months40 . in the
present study , in the cpg , the positive changes obtained in the cpg in parameters such as
self - esteem , body image , and quality of life seem to be consistent with the results of the
portuguese study . | [ purpose ] exercise is one of the most important components of a healthy life .
the purpose
of this study was to analyze exercise beliefs and psychosocial factors in sedentary and
active healthy women and observe the changes in these parameters resulting from clinical
pilates exercises and verbal education in healthy women .
[ subjects and methods ] sixty - six
healthy women were included in the study .
participants were divided into clinical pilates
( n=21 ) , verbal education ( n=25 ) , and control groups ( n=20 ) . prior to and at the end of the
study , demographic information , body mass index , waist - hip circumference , exercise
beliefs , physical activity index , and
psychosocial factors ( rosenberg self - esteem scale ,
body cathexis index , sf-36 quality of life , beck depression scale , visual analog scale for
tiredness ) of the subjects were recorded .
[ results ] meaningful changes for all the
parameters took place in the clinical pilates and verbal education groups .
our analyses
indicated that the changes in the clinical pilates group were more meaningful than those
in the verbal education group . when the data of the study groups were compared with those
of the control group , the clinical pilates group showed meaningful differences .
[ conclusion ] the result of this study indicate that both clinical pilates and verbal
education are effective in changing exercise beliefs and physical and psychosocial
parameters . |
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